G2Cdb::Gene report

Gene id
G00000031
Gene symbol
HRAS (HGNC)
Species
Homo sapiens
Description
v-Ha-ras Harvey rat sarcoma viral oncogene homolog
Orthologue
G00000006 (Mus musculus)

Databases (8)

Gene
ENSG00000174775 (Ensembl human gene)
3265 (Entrez Gene)
61 (G2Cdb plasticity & disease)
HRAS (GeneCards)
Literature
190020 (OMIM)
Marker Symbol
HGNC:5173 (HGNC)
Protein Expression
2015 (human protein atlas)
Protein Sequence
P01112 (UniProt)

Diseases (22)

Disease Nervous effect Mutations Found Literature Mutations Type Genetic association?
D00000060: Wilms tumour N Y (1861864) No mutation found (N) N
D00000173: Unipolar affective disorder Y Y (1973904) No mutation found (N) N
D00000138: Multiple endocrine neoplasia type I N Y (2565762) Deletion (D) Y
D00000094: Acute myelomonocytic leukaemia N Y (2713271) Translocation (T) N
D00000035: Malignant melanoma N Y (2894003) TaqI polymorphism (TaP) Y
D00000302: Beckwith-Wiedemann syndrome N Y (2905880) Duplication (Du) N
D00000086: Malignant lymphoma N Y (8104094) Deletion (D) Y
D00000028: Lung cancer N Y (8336750) Unknown (?) Y
D00000057: Prostate cancer N Y (8336750) Unknown (?) Y
D00000085: Non-Hodgkin's lymphoma N Y (8336750) Unknown (?) Y
D00000049: Ovarian cancer N Y (8589723) Unknown (?) Y
D00000238: Long QT syndrome N Y (8751140) Single nucleotide polymorphism (SNP) N
D00000238: Long QT syndrome N Y (8751140) Insertion/deletion (I/D) N
D00000177: Autism Y Y (8832771) Repeat polymorphism (RP) Y
D00000048: Nonmucinous epithelial ovarian carcinoma N Y (9012461) Deletion (D) Y
D00000114: Spitz nevi N Y (10980135) Microinsertion (MI) ?
D00000064: Urothelial carcinoma N Y (15897885) Microinsertion (MI) Y
D00000322: Costello syndrome N Y (16170316) Single nucleotide polymorphism (SNP) Y
D00000322: Costello syndrome N Y (16170316) Dinucleotide polymorphism (DNP) Y
D00000322: Costello syndrome N Y (16329078) Microinsertion (MI) Y
D00000322: Costello syndrome N Y (16372351) Microinsertion (MI) Y
D00000322: Costello syndrome N Y (16443854) Microinsertion (MI) Y
D00000089: Childhood acute lymphoblastic leukaemia N Y (16533526) Unknown (?) Y
D00000322: Costello syndrome N Y (16835863) Microinsertion (MI) Y
D00000322: Costello syndrome N Y (16881968) Single nucleotide polymorphism (SNP) Y
D00000322: Costello syndrome N Y (16969868) Microinsertion (MI) Y
D00000322: Costello syndrome N Y (17054105) Microinsertion (MI) Y
D00000305: Noonan syndrome N Y (17054105) Microinsertion (MI) N
D00000303: Cardiofaciocutaneous syndrome Y Y (17054105) Microinsertion (MI) N
D00000321: Cancer N Y (17088437) Microinsertion (MI) Y
D00000322: Costello syndrome N Y (17164262) Microinsertion (MI) Y
D00000322: Costello syndrome N Y (17250658) Microinsertion (MI) Y
D00000042: Breast cancer N Y (17314276) Single nucleotide polymorphism (SNP) Y
D00000042: Breast cancer N Y (17314276) Microinsertion (MI) Y

References

  • Diversity, parental germline origin, and phenotypic spectrum of de novo HRAS missense changes in Costello syndrome.

    Zampino G, Pantaleoni F, Carta C, Cobellis G, Vasta I, Neri C, Pogna EA, De Feo E, Delogu A, Sarkozy A, Atzeri F, Selicorni A, Rauen KA, Cytrynbaum CS, Weksberg R, Dallapiccola B, Ballabio A, Gelb BD, Neri G and Tartaglia M

    Istituto di Clinica Pediatrica, Università Cattolica del Sacro Cuore, Rome, Italy.

    Activating mutations in v-Ha-ras Harvey rat sarcoma viral oncogene homolog (HRAS) have recently been identified as the molecular cause underlying Costello syndrome (CS). To further investigate the phenotypic spectrum associated with germline HRAS mutations and characterize their molecular diversity, subjects with a diagnosis of CS (N = 9), Noonan syndrome (NS; N = 36), cardiofaciocutaneous syndrome (CFCS; N = 4), or with a phenotype suggestive of these conditions but without a definitive diagnosis (N = 12) were screened for the entire coding sequence of the gene. A de novo heterozygous HRAS change was detected in all the subjects diagnosed with CS, while no lesion was observed with any of the other phenotypes. While eight cases shared the recurrent c.34G>A change, a novel c.436G>A transition was observed in one individual. The latter affected residue, p.Ala146, which contributes to guanosine triphosphate (GTP)/guanosine diphosphate (GDP) binding, defining a novel class of activating HRAS lesions that perturb development. Clinical characterization indicated that p.Gly12Ser was associated with a homogeneous phenotype. By analyzing the genomic region flanking the HRAS mutations, we traced the parental origin of lesions in nine informative families and demonstrated that de novo mutations were inherited from the father in all cases. We noted an advanced age at conception in unaffected fathers transmitting the mutation.

    Funded by: NHLBI NIH HHS: HL71207; NICHD NIH HHS: HD01294, HD048502; Telethon: GGP04172

    Human mutation 2007;28;3;265-72

  • Uniparental disomy at chromosome 11p15.5 followed by HRAS mutations in embryonal rhabdomyosarcoma: lessons from Costello syndrome.

    Kratz CP, Steinemann D, Niemeyer CM, Schlegelberger B, Koscielniak E, Kontny U and Zenker M

    Division of Pediatric Hematology and Oncology, Department of Pediatrics and Adolescent Medicine, University of Freiburg, Mathildenstrasse 1, 79106 Freiburg, Germany. christian.kratz@uniklinik-freiburg.de

    Costello syndrome (CS; MIM 218040) is characterized by short stature, facial dysmorphism, cardiac defects and predisposition to embryonal rhabdomyosarcoma (CS/ERMS) and other neoplasias. CS is caused by germline mutations in the HRAS gene on chromosome 11p15.5, a region showing allelic imbalances in sporadic ERMS and CS/ERMS. The critical gene for ERMS development in this region is unknown. The association of CS and ERMS as well as previous reports illustrating that somatic HRAS mutations are found in a proportion of these tumors prompted us to clarify the significance and a possible correlation of HRAS mutations and genomic rearrangements at 11p15.5 in sporadic ERMS. We screened for somatic HRAS mutations and 11p15.5 imbalances in six sporadic ERMS samples. This analysis uncovered five ERMS samples with uniparental disomy (UPD) at the HRAS locus, two of which harbored HRAS mutations. By analyzing informative genetic variations in or at the HRAS gene locus, we show that one HRAS allele is entirely lost in specimens with UPD at 11p15.5. Notably, in both cases with UPD and HRAS mutations these mutations were heterozygous. Therefore, they must have succeeded the emergence of UPD. In contrast, HRAS germline mutations are the first step in CS/ERMS. Subsequent development of UPD at 11p15.5 may explain previous observations that CS/ERMS express mutant HRAS only. These data implicate that in sporadic ERMS, UPD at 11p15.5 is not driven by HRAS mutations and that imbalances at 11p15.5 and HRAS mutations represent independent but cooperating events during ERMS development.

    Human molecular genetics 2007;16;4;374-9

  • HRAS and the Costello syndrome.

    Rauen KA

    Department of Pediatrics, Division of Medical Genetics, University of California, San Francisco, CA 94115, USA. rauen@cc.ucsf.edu

    Costello syndrome (CS) is a complex developmental disorder involving characteristic craniofacial features, failure to thrive, developmental delay, cardiac and skeletal anomalies and a predisposition to develop neoplasia, both benign and malignant. CS is caused by activating germline mutations in HRAS and belongs to an exciting class of genetic syndromes that are caused by perturbation of function through the Ras pathway. Some of these other syndromes include Noonan syndrome, LEOPARD syndrome, neurofibromatosis 1 and cardio-facio-cutaneous syndrome. Ras is a critical signaling hub in the cell and is activated by receptor tyrosine kinases, G-protein-coupled receptors, cytokine receptors and extracellular matrix receptors. The downstream effectors of Ras are many and control vital cellular functions including cell cycle progression, cell survival, motility, transcription, translation and membrane trafficking. Understanding the genetic etiology of CS is the first step in gaining insight to the role Ras plays in human development, cellular signaling and cancer pathogenesis.

    Funded by: NICHD NIH HHS: HD048502

    Clinical genetics 2007;71;2;101-8

  • Phosphatidylinositol-3-OH kinase or RAS pathway mutations in human breast cancer cell lines.

    Hollestelle A, Elstrodt F, Nagel JH, Kallemeijn WW and Schutte M

    Department of Medical Oncology, Josephine Nefkens Institute Be414, Erasmus MC, P.O. Box 1738, 3000 DR Rotterdam, the Netherlands.

    Constitutive activation of the phosphatidylinositol-3-OH kinase (PI3K) and RAS signaling pathways are important events in tumor formation. This is illustrated by the frequent genetic alteration of several key players from these pathways in a wide variety of human cancers. Here, we report a detailed sequence analysis of the PTEN, PIK3CA, KRAS, HRAS, NRAS, and BRAF genes in a collection of 40 human breast cancer cell lines. We identified a surprisingly large proportion of cell lines with mutations in the PI3K or RAS pathways (54% and 25%, respectively), with mutants for each of the six genes. The PIK3CA, KRAS, and BRAF mutation spectra of the breast cancer cell lines were similar to those of colorectal cancers. Unlike in colorectal cancers, however, mutational activation of the PI3K pathway was mutually exclusive with mutational activation of the RAS pathway in all but 1 of 30 mutant breast cancer cell lines (P = 0.001). These results suggest that there is a fine distinction between the signaling activators and downstream effectors of the oncogenic PI3K and RAS pathways in breast epithelium and those in other tissues.

    Molecular cancer research : MCR 2007;5;2;195-201

  • Mutation analysis of 24 known cancer genes in the NCI-60 cell line set.

    Ikediobi ON, Davies H, Bignell G, Edkins S, Stevens C, O'Meara S, Santarius T, Avis T, Barthorpe S, Brackenbury L, Buck G, Butler A, Clements J, Cole J, Dicks E, Forbes S, Gray K, Halliday K, Harrison R, Hills K, Hinton J, Hunter C, Jenkinson A, Jones D, Kosmidou V, Lugg R, Menzies A, Mironenko T, Parker A, Perry J, Raine K, Richardson D, Shepherd R, Small A, Smith R, Solomon H, Stephens P, Teague J, Tofts C, Varian J, Webb T, West S, Widaa S, Yates A, Reinhold W, Weinstein JN, Stratton MR, Futreal PA and Wooster R

    Cancer Genome Project, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, United Kingdom. mrs@sanger.ac.uk.

    The panel of 60 human cancer cell lines (the NCI-60) assembled by the National Cancer Institute for anticancer drug discovery is a widely used resource. The NCI-60 has been characterized pharmacologically and at the molecular level more extensively than any other set of cell lines. However, no systematic mutation analysis of genes causally implicated in oncogenesis has been reported. This study reports the sequence analysis of 24 known cancer genes in the NCI-60 and an assessment of 4 of the 24 genes for homozygous deletions. One hundred thirty-seven oncogenic mutations were identified in 14 (APC, BRAF, CDKN2, CTNNB1, HRAS, KRAS, NRAS, SMAD4, PIK3CA, PTEN, RB1, STK11, TP53, and VHL) of the 24 genes. All lines have at least one mutation among the cancer genes examined, with most lines (73%) having more than one. Identification of those cancer genes mutated in the NCI-60, in combination with pharmacologic and molecular profiles of the cells, will allow for more informed interpretation of anticancer agent screening and will enhance the use of the NCI-60 cell lines for molecularly targeted screens.

    Funded by: Intramural NIH HHS; Wellcome Trust: 077012

    Molecular cancer therapeutics 2006;5;11;2606-12

  • Somatic mosaicism for an HRAS mutation causes Costello syndrome.

    Gripp KW, Stabley DL, Nicholson L, Hoffman JD and Sol-Church K

    Division of Medical Genetics, A. I. duPont Hospital for Children, Wilmington, Delaware 19899, USA. kgripp@nemours.org

    De novo heterozygous HRAS point mutations have been reported in more than 81 patients with Costello syndrome (CS), but genotype/phenotype correlation remains incomplete because the majority of patients share a common mutation, G12S, seen in 65/81 (80%). Somatic HRAS mutations have previously been identified in solid tumors, and mutation hot spots related to a gain-of-function effect of the gene product are known. The germline mutations causing CS occur at these hot spots and convey a gain-of-function effect, thus accounting for the greatly increased cancer risk. Diagnostic testing for HRAS mutations is now available and the identification of a mutation in a patient with consistent clinical findings confirms a diagnosis of CS. It is not clear yet if the absence of an HRAS mutation precludes a diagnosis of CS. Because there is a significant overlap in the clinical findings of Costello, cardio-facio-cutaneous, and Noonan syndromes, diagnostic uncertainty remains in patients lacking an HRAS mutation. We report here on a female with findings suggestive of CS in whom mutation analysis performed with standard techniques on white blood cell derived DNA did not show an HRAS mutation. However, analysis of DNA derived from three independently collected buccal swabs showed a sequence change qualitatively consistent with the G12S mutation. Allelic quantitation showed the presence of the mutation in approximately 25%-30% of the sampled buccal cells. In this patient, standard technology failed to identify the disease causing mutation on DNA derived from a blood sample, highlighting the potential pitfalls in the interpretation of negative mutation studies. This is the first reported CS patient mosaic for the common HRAS mutation, likely due to a somatic mutation occurring very early in fetal development.

    Funded by: NCRR NIH HHS: 1 P20 RR020173-01

    American journal of medical genetics. Part A 2006;140;20;2163-9

  • PTPN11, RAS and FLT3 mutations in childhood acute lymphoblastic leukemia.

    Yamamoto T, Isomura M, Xu Y, Liang J, Yagasaki H, Kamachi Y, Kudo K, Kiyoi H, Naoe T and Kojma S

    Departments of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan. tomotomo@med.nagoya-u.ac.jp

    PTPN11, the gene which encodes protein tyrosine phosphatase SHP-2, plays an important role in regulating intracellular signaling. Germline mutations in PTPN11 were first observed in Noonan syndrome, while somatic mutations were identified in hematological myeloid malignancies. Recently, PTPN11 mutations have been reported in children with acute lymphoblastic leukemia (ALL). In the present study, we investigated the prevalence of mutations in PTPN11, RAS and FLT3 in samples from 95 Japanese children with ALL. We observed exon 3 and 8 missense mutations of PTPN11 in 6 children with B precursor ALL. One patient with Down syndrome and ALL had PTPN11 mutation. We also identified RAS mutations in ten patients and FLT3 internal tandem duplication (FLT3/ITD) in one patient. None of the patients had simultaneous mutations in PTPN11 and RAS, while one patient had both PTPN11 and FLT3 mutations. These data suggest that PTPN11 mutation may play an important role for leukemogenesis in a proportion of children with ALL, particularly B precursor ALL.

    Leukemia research 2006;30;9;1085-9

  • Recurring HRAS mutation G12S in Dutch patients with Costello syndrome.

    van Steensel MA, Vreeburg M, Peels C, van Ravenswaaij-Arts CM, Bijlsma E, Schrander-Stumpel CT and van Geel M

    Department of Dermatology, University Hospital Maastricht, Maastricht, The Netherlands. mvst@sder.azm.nl

    Costello syndrome (CS) is a rare multiple congenital anomaly/mental retardation syndrome characterized by coarse face, loose skin and cardiomyopathy. It is often associated with benign and malignant tumors. Several groups have now demonstrated that CS is caused by recurring mutations in the HRAS gene in different ethnic groups. Here, we describe three unrelated Dutch patients and show that they all have the same mutation, G12S, in HRAS. To our knowledge, our patients are the first Dutch to be analysed. The syndrome seems to be genetically homogeneous. We discuss the pertinent nosology of the syndrome.

    Experimental dermatology 2006;15;9;731-4

  • Paternal bias in parental origin of HRAS mutations in Costello syndrome.

    Sol-Church K, Stabley DL, Nicholson L, Gonzalez IL and Gripp KW

    Department of Biomedical Research, Nemours' Children's Clinic, Wilmington, Delaware, USA. ksolchur@nemours.org

    Costello syndrome (CS) is a rare congenital condition caused by heterozygous de novo missense mutations affecting the codon for glycine 12 or 13 of the HRAS gene. We have identified 39 CS patients harboring the p.Gly12Ser mutation (NM_005343.2:c.34 G > A), two patients with c.35G > C mutations resulting in p.Gly12Ala substitutions, and one patient carrying the p.Gly13Cys substitution (c.37G > A). We analyzed the region flanking the mutated sites in 42 probands and 59 parents, and used four polymorphic markers to trace the parental origin of the germline mutations: one highly polymorphic hexanucleotide (GGGCCT) repeat region, defining three alleles with different numbers of repeat units (two, three, or four), and three SNPs. One of the SNPs, rs12628 (c.81T > C), was found in strong linkage disequilibrium with the hexanucleotide repeat region. Out of a total of 24 probands with polymorphic markers, 16 informative families were tested and the paternal origin of the germline mutation was found in 14 CS probands; a distribution that is neither consistent with an equal likelihood of mutations arising in either parent (P = 0.0018), nor with exclusive paternal origin.

    Funded by: NCRR NIH HHS: 1 P20 RR020173-01

    Human mutation 2006;27;8;736-41

  • Genotype-phenotype correlation in Costello syndrome: HRAS mutation analysis in 43 cases.

    Kerr B, Delrue MA, Sigaudy S, Perveen R, Marche M, Burgelin I, Stef M, Tang B, Eden OB, O'Sullivan J, De Sandre-Giovannoli A, Reardon W, Brewer C, Bennett C, Quarell O, M'Cann E, Donnai D, Stewart F, Hennekam R, Cavé H, Verloes A, Philip N, Lacombe D, Levy N, Arveiler B and Black G

    Regional Genetic Service, Central Manchester University Hospital NHS Trust, Manchester, UK. bronwyn.kerr@cmmc.nhs.uk

    Background: Costello syndrome (CS) is a rare multiple congenital abnormality syndrome, associated with failure to thrive and developmental delay. One of the more distinctive features in childhood is the development of facial warts, often nasolabial and in other moist body surfaces. Individuals with CS have an increased risk of malignancy, suggested to be about 17%. Recently, mutations in the HRAS gene on chromosome 11p13.3 have been found to cause CS.

    Methods: We report here the results of HRAS analysis in 43 individuals with a clinical diagnosis of CS.

    Results: Mutations were found in 37 (86%) of patients. Analysis of parental DNA samples was possible in 16 cases for both parents and in three cases for one parent, and confirmed the mutations as de novo in all of these cases. Three novel mutations (G12C, G12E, and K117R) were found in five cases.

    Conclusions: These results confirm that CS is caused, in most cases, by heterozygous missense mutations in the proto-oncogene HRAS. Analysis of the major phenotypic features by mutation suggests a potential correlation between malignancy risk and genotype, which is highest for patients with an uncommon (G12A) substitution. These results confirm that mutation testing for HRAS is a reliable diagnostic test for CS.

    Journal of medical genetics 2006;43;5;401-5

  • HRAS mutation analysis in Costello syndrome: genotype and phenotype correlation.

    Gripp KW, Lin AE, Stabley DL, Nicholson L, Scott CI, Doyle D, Aoki Y, Matsubara Y, Zackai EH, Lapunzina P, Gonzalez-Meneses A, Holbrook J, Agresta CA, Gonzalez IL and Sol-Church K

    Division of Medical Genetics, A. I. duPont Hospital for Children, Wilmington, DE 19899, USA. kgripp@nemours.org

    Costello syndrome is a rare condition comprising mental retardation, distinctive facial appearance, cardiovascular abnormalities (typically pulmonic stenosis, hypertrophic cardiomyopathy, and/or atrial tachycardia), tumor predisposition, and skin and musculoskeletal abnormalities. Recently mutations in HRAS were identified in 12 Japanese and Italian patients with clinical information available on 7 of the Japanese patients. To expand the molecular delineation of Costello syndrome, we performed mutation analysis in 34 North American and 6 European (total 40) patients with Costello syndrome, and detected missense mutations in HRAS in 33 (82.5%) patients. All mutations affected either codon 12 or 13 of the protein product, with G12S occurring in 30 (90.9%) patients of the mutation-positive cases. In two patients, we found a mutation resulting in an alanine substitution in position 12 (G12A), and in one patient, we detected a novel mutation (G13C). Five different HRAS mutations have now been reported in Costello syndrome, however genotype-phenotype correlation remains incomplete.

    Funded by: NCRR NIH HHS: 1P20 RR 020173-01

    American journal of medical genetics. Part A 2006;140;1;1-7

  • HRAS mutations in Costello syndrome: detection of constitutional activating mutations in codon 12 and 13 and loss of wild-type allele in malignancy.

    Estep AL, Tidyman WE, Teitell MA, Cotter PD and Rauen KA

    Comprehensive Cancer Center, Cancer Research Institute, University of California-San Francisco, 2340 Sutter Street, San Francisco, CA 94115, USA.

    Costello syndrome (CS) is a complex developmental disorder involving characteristic craniofacial features, failure to thrive, developmental delay, cardiac and skeletal anomalies, and a predisposition to develop neoplasia. Based on similarities with other cancer syndromes, we previously hypothesized that CS is likely due to activation of signal transduction through the Ras/MAPK pathway [Tartaglia et al., 2003]. In this study, the HRAS coding region was sequenced for mutations in a large, well-characterized cohort of 36 CS patients. Heterogeneous missense point mutations predicting an amino acid substitution were identified in 33/36 (92%) patients. The majority (91%) had a 34G --> A transition in codon 12. Less frequent mutations included 35G --> C (codon 12) and 37G --> T (codon 13). Parental samples did not have an HRAS mutation supporting the hypothesis of de novo heterogeneous mutations. There is phenotypic variability among patients with a 34G --> A transition. The most consistent features included characteristic facies and skin, failure to thrive, developmental delay, musculoskeletal abnormalities, visual impairment, cardiac abnormalities, and generalized hyperpigmentation. The two patients with 35G --> C had cardiac arrhythmias whereas one patient with a 37G --> T transversion had an enlarged aortic root. Of the patients with a clinical diagnosis of CS, neoplasia was the most consistent phenotypic feature for predicating an HRAS mutation. To gain an understanding of the relationship between constitutional HRAS mutations and malignancy, HRAS was sequenced in an advanced biphasic rhabdomyosarcoma/fibrosarcoma from an individual with a 34G --> A mutation. Loss of the wild-type HRAS allele was observed, suggesting tumorigenesis in CS patients is accompanied by additional somatic changes affecting HRAS. Finally, due to phenotypic overlap between CS and cardio-facio-cutaneous (CFC) syndromes, the HRAS coding region was sequenced in a well-characterized CFC cohort. No mutations were found which support a distinct genetic etiology between CS and CFC syndromes.

    Funded by: NCI NIH HHS: CA 107300, CA 90571; NICHD NIH HHS: HD 048502

    American journal of medical genetics. Part A 2006;140;1;8-16

  • Germline mutations in HRAS proto-oncogene cause Costello syndrome.

    Aoki Y, Niihori T, Kawame H, Kurosawa K, Ohashi H, Tanaka Y, Filocamo M, Kato K, Suzuki Y, Kure S and Matsubara Y

    Department of Medical Genetics, Tohoku University School of Medicine, 1-1 Seiryo-machi, Sendai 980-8574, Japan. aokiy@mail.tains.tohoku.ac.jp

    Costello syndrome is a multiple congenital anomaly and mental retardation syndrome characterized by coarse face, loose skin, cardiomyopathy and predisposition to tumors. We identified four heterozygous de novo mutations of HRAS in 12 of 13 affected individuals, all of which were previously reported as somatic and oncogenic mutations in various tumors. Our observations suggest that germline mutations in HRAS perturb human development and increase susceptibility to tumors.

    Funded by: Telethon: GTF04002

    Nature genetics 2005;37;10;1038-40

  • FGFR3 and Ras gene mutations are mutually exclusive genetic events in urothelial cell carcinoma.

    Jebar AH, Hurst CD, Tomlinson DC, Johnston C, Taylor CF and Knowles MA

    Cancer Research UK Clinical Centre, St James's University Hospital, Beckett Street, Leeds LS9 7TF, UK.

    Fibroblast growth factor receptor 3 (FGFR3) mutations are frequent in superficial urothelial cell carcinoma (UCC). Ras gene mutations are also found in UCC. As oncogenic activation of both FGFR3 and Ras is predicted to result in stimulation of the mitogen-activated protein kinase (MAPK) pathway, we hypothesized that these might be mutually exclusive events. HRAS mutation has been widely studied in UCC, but all three Ras gene family members have not been screened for mutation in the same sample series. We screened 98 bladder tumours and 31 bladder cell lines for mutations in FGFR3, HRAS, NRAS and KRAS2. FGFR3 mutations were present in 54 tumours (55%) and three cell lines (10%), and Ras gene mutations in 13 tumours (13%) and four cell lines (13%). These included mutations in all three Ras genes; ten in HRAS, four in KRAS2 and four in NRAS and these were not associated with either tumour grade or stage. In no cases were Ras and FGFR3 mutation found together. This mutual exclusion suggests that FGFR3 and Ras gene mutation may represent alternative means to confer the same phenotype on UCC cells. If these events have biological equivalence, Ras mutant invasive UCC may represent a novel subgroup.

    Oncogene 2005;24;33;5218-25

  • Mutations and copy number increase of HRAS in Spitz nevi with distinctive histopathological features.

    Bastian BC, LeBoit PE and Pinkel D

    Departments of Dermatology and Pathology and UCSF Comprehensive Cancer Center, University of California San Francisco, San Francisco, California 94143-0808, USA. bastian@cc.ucsf.edu

    Spitz nevus is a benign melanocytic neoplasm that can be difficult or impossible to histologically distinguish from melanoma. We have recently described copy number increases of chromosome 11p in a subset of Spitz nevi. To study the molecular and histological features of this group, we studied 102 Spitz nevi for 11p copy number increases using fluorescence in situ hybridization (FISH) on tissue arrays. Copy number increases of at least threefold were found in 12 cases (11.8%) and involved the HRAS gene on chromosome 11p. Sequence analysis of HRAS showed frequent oncogenic mutations in cases with copy number increase (8/12 or 67%), contrasting with rare HRAS mutations in cases with normal HRAS copy numbers (1/21 or 5%, P: < 0.0001). Tumors with 11p copy number increases were larger, predominantly intradermal, had marked desmoplasia, characteristic cytological features, and had an infiltrating growth pattern. Proliferation rates in the majority of these cases were low to absent. HRAS activation by either mutation or copy number increase alone could explain several of the histological features that overlap with those of melanoma. We speculate that HRAS activation in the absence of co-operating additional genetic alterations drives the partially transformed melanocytes of these Spitz nevi into senescence or a stable growth arrest. Although there is no data suggesting that Spitz nevi with HRAS activation are at risk for progression to melanoma, future studies are warranted to assess their biological behavior more accurately.

    The American journal of pathology 2000;157;3;967-72

  • A novel 4-cM minimally deleted region on chromosome 11p15.1 associated with high grade nonmucinous epithelial ovarian carcinomas.

    Lu KH, Weitzel JN, Kodali S, Welch WR, Berkowitz RS and Mok SC

    Department of Obstetrics, Gynecology, and Reproductive Biology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.

    Prior cytogenetic and restriction fragment length polymorphism studies have demonstrated that allelic deletion of chromosome 11p is common in human invasive epithelial ovarian tumors. To construct a highly detailed deletion map of chromosome 11p, we used 13 polymorphic microsatellite CA repeat primers to identify regions harboring potential tumor suppressor genes. Twenty-three of 48 samples (48%) of invasive epithelial ovarian cancer showed LOH involving at least one locus, consistent with prior studies. None of the five mucinous tumors showed allelic deletion at any of the 13 primers, suggesting that loss of heterozygosity at chromosome 11p may not be involved in the pathogenesis of mucinous ovarian cancer. Two separate minimally deleted regions were identified in nonmucinous ovarian cancer. The first is an 11-cM region on chromosome 11pl5.5-15.3 that extends from D11S2071 to D11S988 and includes the HRAS locus. The second is a novel 4-cM region on 11p15.1, defined by marker D11S1310. Deletion of both regions at 11p15.5-15.3 and 11p15.1 is strongly associated with high grade nonmucinous epithelial ovarian cancer.

    Funded by: NCI NIH HHS: 1 KO8 CAO1574, R01CA63381

    Cancer research 1997;57;3;387-90

  • Studies of the c-Harvey-Ras gene in psychiatric disorders.

    Comings DE, Wu S, Chiu C, Muhleman D and Sverd J

    Department of Medical Genetics, City of Hope National Medical Center, Duarte, CA 91010-0269, USA.

    Hérault et al. (1993) previously reported a significant association between autism and the larger fragments of the c-Harvey-Ras (HRAS) Bam H1 polymorphism. We have sought to verify this finding and determine if there was any evidence for an association with other psychiatric disorders. Because of its greater sensitivity, we have examined the HRAS Msp 1 polymorphism. We found a just significant increase in the prevalence of the > 2.1 kb alleles in 48 subjects with autism versus 50 control subjects. There was no increase in the prevalence of the > 2.1 kb alleles in 164 probands with Tourette's syndrome. Examination of 16 preselected symptom clusters, however, showed a significant trend toward higher scores for obsessive-compulsive and phobic symptoms in > 2.1 kb homozygotes. While this locus requires further study, in conjunction with the results of Hérault et al., the present findings suggest that genetic defects in HRAS, and possibly other components of the G protein secondary messenger system, may play a role in some psychiatric disorders.

    Psychiatry research 1996;63;1;25-32

  • Ovarian cancer risk in BRCA1 carriers is modified by the HRAS1 variable number of tandem repeat (VNTR) locus.

    Phelan CM, Rebbeck TR, Weber BL, Devilee P, Ruttledge MH, Lynch HT, Lenoir GM, Stratton MR, Easton DF, Ponder BA, Cannon-Albright L, Larsson C, Goldgar DE and Narod SA

    Division of Medical Genetics, Montreal General Hospital, Quebec, Canada.

    Women who carry a mutation in the BRCA1 gene (on chromosome 17q21), have an 80% risk of breast cancer and a 40% risk of ovarian cancer by the age of 70 (ref. 1). The variable penetrance of BRCA1 suggests that other genetic and non-genetic factors play a role in tumourigenesis in these individuals. The HRAS1 variable number of tandem repeats (VNTR) polymorphism, located 1 kilobase (kb) downstream of the HRAS1 proto-oncogene (chromosome 11p15.5) is one possible genetic modifier of cancer penetrance. Individuals who have rare alleles of the VNTR have an increased risk of certain types of cancers, including breast cancer (2-4). To investigate whether the presence of rare HRAS1 alleles increases susceptibility to hereditary breast and ovarian cancer, we have typed a panel of 307 female BRCA1 carriers at this locus using a PCR-based technique. The risk for ovarian cancer was 2.11 times greater for BRCA1 carriers harbouring one or two rare HRAS1 alleles, compared to carriers with only common alleles (P = 0.015). The magnitude of the relative risk associated with a rare HRAS1 allele was not altered by adjusting for the other known risk factors for hereditary ovarian cancer (5). Susceptibility to breast cancer did not appear to be affected by the presence of rare HRAS1 alleles. This study is the first to show the effect of a modifying gene on the penetrance of an inherited cancer syndrome.

    Funded by: NCI NIH HHS: CA55914

    Nature genetics 1996;12;3;309-11

  • Molecular analysis at the Harvey Ras-1 gene in patients with long QT syndrome.

    Schulze-Bahr E, Haverkamp W, Wiebusch H, Schulte H, Hördt M, Borggrefe M, Breithardt G, Assmann G and Funke H

    Medizinische Klinik und Poliklinik, Westälische Wilhelms-Universität Münster, Germany.

    Patients with long QT syndrome (LQTS; MIM 1921500) frequently suffer from syncope and are threatened by sudden cardiac death due to ventricular arrhythmias, typically of the torsade de pointes type. Initial progress in revealing the molecular basis of the disease was made by the observation of genetic linkage of the disease locus to the Harvey Ras-1 gene (HRAS 1) on chromosome 11p15.5. More recently loci on chromosomes 3, 4, and 7 have also been found to be linked to LQTS, thus demonstrating heterogeneity in the causes for this disease. The present study performed sequence analysis on the HRAS 1 gene in patients with congenital and acquired LQTS to determine the frequency of HRAS 1 mutations in patients with this disease. In neither group were no mutations identified in the coding regions or in the splice donor and acceptor sites. Alleles characterized by a T to C transition in exon 1 and an insertion/deletion polymorphism upstream of exon 1 showed no significant difference in their frequencies between LQTS patients and normal controls. No quantitative influence of the such characterized genotypes on the QT duration was observed. These results demonstrate that structural mutations in the HRAS 1 gene are not a frequent cause of LQTS. Also, since there was no association of different alleles at the HRAS 1 locus with changes in QT duration, it appears unlikely that this gene is a major contributor to this disease.

    Journal of molecular medicine (Berlin, Germany) 1995;73;11;565-9

  • An association between the risk of cancer and mutations in the HRAS1 minisatellite locus.

    Krontiris TG, Devlin B, Karp DD, Robert NJ and Risch N

    Department of Medicine, Tufts University School of Medicine, Boston, MA.

    Background: The role of mutations in protooncogenes and their regulatory sequences in the pathogenesis of cancer is under close scrutiny. Minisatellites are unstable repetitive sequences of DNA that are present throughout the human genome. The highly polymorphic HRAS1 minisatellite locus just downstream from the protooncogene H-ras-1 consists of four common progenitor alleles and several dozen rare alleles, which apparently derive from mutations of the progenitors. We previously observed an association of the rare mutant alleles with many forms of cancer, and we undertook the present study to pursue this observation further.

    Methods: We conducted a case-control study, typing 736 HRAS1 alleles from patients with cancer and 652 from controls by Southern blotting of leukocyte DNA. We also carried out a meta-analysis of this study and 22 other published studies, estimating the relative risk of cancer (such as bladder, breast, or colorectal cancer) when one of the rare HRAS1 alleles was present.

    Results: Both the present case-control study (odds ratio, 1.83; 95 percent confidence interval, 1.28 to 2.67; P = 0.002) and the present study combined with our previous study (odds ratio, 2.07; 95 percent confidence interval, 1.47 to 2.92; P < 0.001), as well as the meta-analysis of all 23 studies (odds ratio, 1.93; 95 percent confidence interval, 1.63 to 2.30; chi-square = 57.58; P < 0.001), replicated our original finding and demonstrated a significant association of rare HRAS1 alleles with cancer. We found significant associations for four types of cancer: carcinomas of the breast, colorectum, and urinary bladder and acute leukemia. We also identified suggestive but not statistically significant associations for cancers of the lung and prostate and for non-Hodgkin's lymphoma.

    Conclusions: Mutant alleles of the HRAS1 minisatellite locus represent a major risk factor for common types of cancer. Although the relative risk associated with the presence of one rare allele is moderate, the aggregate prevalence of one rare allele is moderate, the aggregate prevalence of this class of mutant alleles implies an extremely important attributable risk: 1 in 11 cancers of the breast, colorectum, and bladder.

    Funded by: NCI NIH HHS: CA-45052

    The New England journal of medicine 1993;329;8;517-23

  • Rare HRAS1 alleles outside the VTR region in lymph nodes from patients with malignant lymphoma.

    Stul M, Baens M, Mecucci C, Van den Berghe H and Cassiman JJ

    Center for Human Genetics, University of Leuven, Belgium.

    We found a new type of rare HRAS1 alleles arising de novo in the lymph node of lymphoma patients. Rare alleles of 6.2 kb (a0.1) and 7.3 kb (a2.3) are displayed in the presence of common alleles of 6.6 kb (a1) and 7.6 kb (a3), respectively. These rare alleles lack nucleotide sequences, present in the common alleles in the 5' part between the BamHI and XmaIII site. In addition, the rare alleles may have the classic A-G mutation in the intron at position 2719. In contrast to what is observed for rare constitutional alleles, the VTR region of these rare alleles has the same size as the common alleles. At present the significance of this new type of alleles is not clear.

    Cancer genetics and cytogenetics 1993;69;1;60-4

  • Loss of heterozygosity for chromosome region 11p15 in Wilms' tumours is not related to HRAS gene transforming mutations.

    Baird P, Wadey R and Cowell J

    Department of Haematology and Oncology, Institute of Child Health, London, UK.

    Although a candidate Wilms' tumour gene--WT1--has been identified in chromosome region 11p13, there is strong evidence from loss of heterozygosity studies suggesting that a second relevant gene is present in region 11p15. The Harvey-Ras proto-oncogene also lies in this region. In other types of tumours mutations in RAS genes have been associated with the development and/or progression of a number of tumour types. We therefore analysed the sequence of the Ras oncogene for possible mutations in six Wilms' tumours showing loss of heterozygosity for chromosome region 11p15. No tumour analysed showed HRAS sequence mutations. We conclude that loss of heterozygosity at 11p15 does not implicate HRAS mutations in the molecular pathogenesis of Wilms' tumour.

    Oncogene 1991;6;7;1147-9

  • Evidence against close linkage of unipolar affective illness to human chromosome 11p markers HRAS1 and INS and chromosome Xq marker DXS52.

    Neiswanger K, Slaugenhaupt SA, Hughes HB, Frank E, Frankel DR, McCarty MJ, Chakravarti A, Zubenko GS, Kupfer DJ and Kaplan BB

    Department of Psychiatry, Western Psychiatric Institute and Clinic, Pittsburgh, PA 15213.

    The genetic basis of various subtypes of the affective disorders has been investigated by family, twin, and adoption studies, as well as by segregation and linkage analysis. Linkage analyses of bipolar disorder with the chromosome 11p15 DNA markers HRAS1 and INS, and the chromosome Xq28 markers for color blindness and G6PD have been reported. We have used restriction fragment length polymorphisms as markers to examine linkage in three extended families with unipolar affective illness, ascertained through probands with either recurrent unipolar or bipolar II illness. Using an inclusive definition of the affected phenotype, linkage could be excluded up to 28cM around the HRAS1-INS linkage group on chromosome 11p15, and up to 5 cM around the DNA marker DXS52 on Xq28. Negative linkage results were also obtained for two more restrictive definitions of affective illness. Thus, we find no evidence for the involvement of the chromosomal regions 11p15 and Xq28 with unipolar affective disorder in these three families.

    Funded by: NIGMS NIH HHS: GM33771; NIMH NIH HHS: MH29618, MH30915; ...

    Biological psychiatry 1990;28;1;63-72

  • Loss of the same alleles of HRAS1 and D11S151 in two independent pancreatic cancers from a patient with multiple endocrine neoplasia type 1.

    Yoshimoto K, Iizuka M, Iwahana H, Yamasaki R, Saito H, Saito S and Sekiya T

    Oncogene Division, National Cancer Center Research Institute, Tokyo, Japan.

    The DNAs from two independent pancreatic cancers (tumors 1 and 2) in a patient with multiple endocrine neoplasia type 1 were analyzed. No amplification or gross rearrangement of 19 protooncogenes was observed. However, Southern blot analysis using polymorphic DNA probes revealed loss of heterozygosity at loci on chromosome 11p in both tumors. In tumor 1, an extensive region including the HRAS1, PTH, CALCA, and D11S151 loci was deleted, while in tumor 2 loss of heterozygosity was limited at the HRAS1 and D11S151 loci. Because loss of heterozygosity at other chromosomal loci in the two tumors was quite rare, loss of genes on 11p might be nonrandom. It is noteworthy that the same allele at the HRAS1 locus and also the same allele at the D11S151 locus were lost in the two independent tumors. These results suggest that loss of genes at the HRAS1 and/or D11S151 loci plays an important role unmasking the remaining sequences probably having a recessive mutation.

    Cancer research 1989;49;10;2716-21

  • HRAS1 and INS genes are relocated but not structurally altered as a result of the t(7;11)(p15;p15) in a clone from a patient with acute myeloid leukaemia (M4).

    Morris CM, Fitzgerald PH, Kennedy MA, Hollings PE, Garry M and Corbett GM

    Cytogenetic and Molecular Oncology Unit, Christchurch Hospital, New Zealand.

    A patient whose leukaemic cells carried the rare t(7;11)(p15;p15) was diagnosed as having acute myelomonocytic leukaemia (AML-M4), and supports the association of this specific translocation with forms of acute myeloid leukaemia showing differentiation. Blast phase chronic myeloid leukaemia was excluded by lack of involvement of the ABL and BCR genes. Chromosome in situ hybridization studies showed that both the HRAS1 and INS genes were present on the terminal part of chromosome 11p which was translocated to chromosome 7p. Neither HRAS1 nor INS were structurally rearranged. Field inversion gel electrophoresis showed that a 400 kb fragment encompassing HRAS1 was structurally entire in leukaemic DNA. Because the INS gene, which was also translocated, is probably located proximal to HRAS1 on chromosome 11p, it is unlikely that HRAS1 was near the chromosome 11 breakpoint or involved in this leukaemia.

    British journal of haematology 1989;71;4;481-6

  • Duplication of HRAS1, INS, and IGF2 is not a common event in Beckwith-Wiedemann syndrome.

    Henry I, Jeanpierre M, Barichard F, Serre JL, Mallet J, Turleau C, de Grouchy J and Junien C

    INSERM U 73, Paris.

    A few cases of Beckwith-Wiedemann syndrome (BWS) have in common a duplication of 11p15. Among the genes located in 11p15, c-Ha-ras 1 (HRAS1), insulin (INS), and insulin-like growth factor II (IGF2) may account for the clinical features and the increased risk for malignancy. Using eight 11p15 markers including HRAS1, INS and IGF2 we have studied eight sporadic and hereditary cases of BWS whether or not associated with a nephroblastoma. By gene dosage determination and family studies, we have shown the following: the eight patients examined had an apparent diploid representation of all of the eight markers studied, thus indicating that a microduplication of these markers or of the region characterized by these markers is not a common event in BWS; in a family with three affected sibs the genes for HRAS1 and INS/IGF2 did not cosegregate with BWS and therefore may not participate in the pathogenic processes here observed.

    Annales de genetique 1988;31;4;216-20

  • HRAS1 proto-oncogene polymorphisms in human malignant melanoma: TaqI defined alleles significantly associated with the disease.

    Radice P, Pierotti MA, Borrello MG, Illeni MT, Rovini D and Della Porta G

    Division of Experimental Oncology, Istituto Nazionale Tumori, Milan, Italy.

    We have analysed the DNA of peripheral blood leukocytes (PBL) from 55 melanoma patients and 53 healthy individuals and failed to find any significant association between melanoma and rare HRAS1 alleles defined by MspI/HpaII digestion. However, the analysis of the same DNAs for a different polymorphism based on the presence of additional TaqI sites in the variable tandem repeat region of HRAS1 showed that the total frequency of a group of allelic variants, named Tp, was significantly higher in melanoma patients than in normal donors.

    Oncogene 1987;2;1;91-5

Literature (409)

Pubmed - human_disease

  • HRAS and the Costello syndrome.

    Rauen KA

    Department of Pediatrics, Division of Medical Genetics, University of California, San Francisco, CA 94115, USA. rauen@cc.ucsf.edu

    Costello syndrome (CS) is a complex developmental disorder involving characteristic craniofacial features, failure to thrive, developmental delay, cardiac and skeletal anomalies and a predisposition to develop neoplasia, both benign and malignant. CS is caused by activating germline mutations in HRAS and belongs to an exciting class of genetic syndromes that are caused by perturbation of function through the Ras pathway. Some of these other syndromes include Noonan syndrome, LEOPARD syndrome, neurofibromatosis 1 and cardio-facio-cutaneous syndrome. Ras is a critical signaling hub in the cell and is activated by receptor tyrosine kinases, G-protein-coupled receptors, cytokine receptors and extracellular matrix receptors. The downstream effectors of Ras are many and control vital cellular functions including cell cycle progression, cell survival, motility, transcription, translation and membrane trafficking. Understanding the genetic etiology of CS is the first step in gaining insight to the role Ras plays in human development, cellular signaling and cancer pathogenesis.

    Funded by: NICHD NIH HHS: HD048502

    Clinical genetics 2007;71;2;101-8

  • Mutation analysis of 24 known cancer genes in the NCI-60 cell line set.

    Ikediobi ON, Davies H, Bignell G, Edkins S, Stevens C, O'Meara S, Santarius T, Avis T, Barthorpe S, Brackenbury L, Buck G, Butler A, Clements J, Cole J, Dicks E, Forbes S, Gray K, Halliday K, Harrison R, Hills K, Hinton J, Hunter C, Jenkinson A, Jones D, Kosmidou V, Lugg R, Menzies A, Mironenko T, Parker A, Perry J, Raine K, Richardson D, Shepherd R, Small A, Smith R, Solomon H, Stephens P, Teague J, Tofts C, Varian J, Webb T, West S, Widaa S, Yates A, Reinhold W, Weinstein JN, Stratton MR, Futreal PA and Wooster R

    Cancer Genome Project, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, United Kingdom. mrs@sanger.ac.uk.

    The panel of 60 human cancer cell lines (the NCI-60) assembled by the National Cancer Institute for anticancer drug discovery is a widely used resource. The NCI-60 has been characterized pharmacologically and at the molecular level more extensively than any other set of cell lines. However, no systematic mutation analysis of genes causally implicated in oncogenesis has been reported. This study reports the sequence analysis of 24 known cancer genes in the NCI-60 and an assessment of 4 of the 24 genes for homozygous deletions. One hundred thirty-seven oncogenic mutations were identified in 14 (APC, BRAF, CDKN2, CTNNB1, HRAS, KRAS, NRAS, SMAD4, PIK3CA, PTEN, RB1, STK11, TP53, and VHL) of the 24 genes. All lines have at least one mutation among the cancer genes examined, with most lines (73%) having more than one. Identification of those cancer genes mutated in the NCI-60, in combination with pharmacologic and molecular profiles of the cells, will allow for more informed interpretation of anticancer agent screening and will enhance the use of the NCI-60 cell lines for molecularly targeted screens.

    Funded by: Intramural NIH HHS; Wellcome Trust: 077012

    Molecular cancer therapeutics 2006;5;11;2606-12

  • PTPN11, RAS and FLT3 mutations in childhood acute lymphoblastic leukemia.

    Yamamoto T, Isomura M, Xu Y, Liang J, Yagasaki H, Kamachi Y, Kudo K, Kiyoi H, Naoe T and Kojma S

    Departments of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan. tomotomo@med.nagoya-u.ac.jp

    PTPN11, the gene which encodes protein tyrosine phosphatase SHP-2, plays an important role in regulating intracellular signaling. Germline mutations in PTPN11 were first observed in Noonan syndrome, while somatic mutations were identified in hematological myeloid malignancies. Recently, PTPN11 mutations have been reported in children with acute lymphoblastic leukemia (ALL). In the present study, we investigated the prevalence of mutations in PTPN11, RAS and FLT3 in samples from 95 Japanese children with ALL. We observed exon 3 and 8 missense mutations of PTPN11 in 6 children with B precursor ALL. One patient with Down syndrome and ALL had PTPN11 mutation. We also identified RAS mutations in ten patients and FLT3 internal tandem duplication (FLT3/ITD) in one patient. None of the patients had simultaneous mutations in PTPN11 and RAS, while one patient had both PTPN11 and FLT3 mutations. These data suggest that PTPN11 mutation may play an important role for leukemogenesis in a proportion of children with ALL, particularly B precursor ALL.

    Leukemia research 2006;30;9;1085-9

  • Paternal bias in parental origin of HRAS mutations in Costello syndrome.

    Sol-Church K, Stabley DL, Nicholson L, Gonzalez IL and Gripp KW

    Department of Biomedical Research, Nemours' Children's Clinic, Wilmington, Delaware, USA. ksolchur@nemours.org

    Costello syndrome (CS) is a rare congenital condition caused by heterozygous de novo missense mutations affecting the codon for glycine 12 or 13 of the HRAS gene. We have identified 39 CS patients harboring the p.Gly12Ser mutation (NM_005343.2:c.34 G > A), two patients with c.35G > C mutations resulting in p.Gly12Ala substitutions, and one patient carrying the p.Gly13Cys substitution (c.37G > A). We analyzed the region flanking the mutated sites in 42 probands and 59 parents, and used four polymorphic markers to trace the parental origin of the germline mutations: one highly polymorphic hexanucleotide (GGGCCT) repeat region, defining three alleles with different numbers of repeat units (two, three, or four), and three SNPs. One of the SNPs, rs12628 (c.81T > C), was found in strong linkage disequilibrium with the hexanucleotide repeat region. Out of a total of 24 probands with polymorphic markers, 16 informative families were tested and the paternal origin of the germline mutation was found in 14 CS probands; a distribution that is neither consistent with an equal likelihood of mutations arising in either parent (P = 0.0018), nor with exclusive paternal origin.

    Funded by: NCRR NIH HHS: 1 P20 RR020173-01

    Human mutation 2006;27;8;736-41

  • Genotype-phenotype correlation in Costello syndrome: HRAS mutation analysis in 43 cases.

    Kerr B, Delrue MA, Sigaudy S, Perveen R, Marche M, Burgelin I, Stef M, Tang B, Eden OB, O'Sullivan J, De Sandre-Giovannoli A, Reardon W, Brewer C, Bennett C, Quarell O, M'Cann E, Donnai D, Stewart F, Hennekam R, Cavé H, Verloes A, Philip N, Lacombe D, Levy N, Arveiler B and Black G

    Regional Genetic Service, Central Manchester University Hospital NHS Trust, Manchester, UK. bronwyn.kerr@cmmc.nhs.uk

    Background: Costello syndrome (CS) is a rare multiple congenital abnormality syndrome, associated with failure to thrive and developmental delay. One of the more distinctive features in childhood is the development of facial warts, often nasolabial and in other moist body surfaces. Individuals with CS have an increased risk of malignancy, suggested to be about 17%. Recently, mutations in the HRAS gene on chromosome 11p13.3 have been found to cause CS.

    Methods: We report here the results of HRAS analysis in 43 individuals with a clinical diagnosis of CS.

    Results: Mutations were found in 37 (86%) of patients. Analysis of parental DNA samples was possible in 16 cases for both parents and in three cases for one parent, and confirmed the mutations as de novo in all of these cases. Three novel mutations (G12C, G12E, and K117R) were found in five cases.

    Conclusions: These results confirm that CS is caused, in most cases, by heterozygous missense mutations in the proto-oncogene HRAS. Analysis of the major phenotypic features by mutation suggests a potential correlation between malignancy risk and genotype, which is highest for patients with an uncommon (G12A) substitution. These results confirm that mutation testing for HRAS is a reliable diagnostic test for CS.

    Journal of medical genetics 2006;43;5;401-5

  • HRAS mutations in Costello syndrome: detection of constitutional activating mutations in codon 12 and 13 and loss of wild-type allele in malignancy.

    Estep AL, Tidyman WE, Teitell MA, Cotter PD and Rauen KA

    Comprehensive Cancer Center, Cancer Research Institute, University of California-San Francisco, 2340 Sutter Street, San Francisco, CA 94115, USA.

    Costello syndrome (CS) is a complex developmental disorder involving characteristic craniofacial features, failure to thrive, developmental delay, cardiac and skeletal anomalies, and a predisposition to develop neoplasia. Based on similarities with other cancer syndromes, we previously hypothesized that CS is likely due to activation of signal transduction through the Ras/MAPK pathway [Tartaglia et al., 2003]. In this study, the HRAS coding region was sequenced for mutations in a large, well-characterized cohort of 36 CS patients. Heterogeneous missense point mutations predicting an amino acid substitution were identified in 33/36 (92%) patients. The majority (91%) had a 34G --> A transition in codon 12. Less frequent mutations included 35G --> C (codon 12) and 37G --> T (codon 13). Parental samples did not have an HRAS mutation supporting the hypothesis of de novo heterogeneous mutations. There is phenotypic variability among patients with a 34G --> A transition. The most consistent features included characteristic facies and skin, failure to thrive, developmental delay, musculoskeletal abnormalities, visual impairment, cardiac abnormalities, and generalized hyperpigmentation. The two patients with 35G --> C had cardiac arrhythmias whereas one patient with a 37G --> T transversion had an enlarged aortic root. Of the patients with a clinical diagnosis of CS, neoplasia was the most consistent phenotypic feature for predicating an HRAS mutation. To gain an understanding of the relationship between constitutional HRAS mutations and malignancy, HRAS was sequenced in an advanced biphasic rhabdomyosarcoma/fibrosarcoma from an individual with a 34G --> A mutation. Loss of the wild-type HRAS allele was observed, suggesting tumorigenesis in CS patients is accompanied by additional somatic changes affecting HRAS. Finally, due to phenotypic overlap between CS and cardio-facio-cutaneous (CFC) syndromes, the HRAS coding region was sequenced in a well-characterized CFC cohort. No mutations were found which support a distinct genetic etiology between CS and CFC syndromes.

    Funded by: NCI NIH HHS: CA 107300, CA 90571; NICHD NIH HHS: HD 048502

    American journal of medical genetics. Part A 2006;140;1;8-16

  • FGFR3 and Ras gene mutations are mutually exclusive genetic events in urothelial cell carcinoma.

    Jebar AH, Hurst CD, Tomlinson DC, Johnston C, Taylor CF and Knowles MA

    Cancer Research UK Clinical Centre, St James's University Hospital, Beckett Street, Leeds LS9 7TF, UK.

    Fibroblast growth factor receptor 3 (FGFR3) mutations are frequent in superficial urothelial cell carcinoma (UCC). Ras gene mutations are also found in UCC. As oncogenic activation of both FGFR3 and Ras is predicted to result in stimulation of the mitogen-activated protein kinase (MAPK) pathway, we hypothesized that these might be mutually exclusive events. HRAS mutation has been widely studied in UCC, but all three Ras gene family members have not been screened for mutation in the same sample series. We screened 98 bladder tumours and 31 bladder cell lines for mutations in FGFR3, HRAS, NRAS and KRAS2. FGFR3 mutations were present in 54 tumours (55%) and three cell lines (10%), and Ras gene mutations in 13 tumours (13%) and four cell lines (13%). These included mutations in all three Ras genes; ten in HRAS, four in KRAS2 and four in NRAS and these were not associated with either tumour grade or stage. In no cases were Ras and FGFR3 mutation found together. This mutual exclusion suggests that FGFR3 and Ras gene mutation may represent alternative means to confer the same phenotype on UCC cells. If these events have biological equivalence, Ras mutant invasive UCC may represent a novel subgroup.

    Oncogene 2005;24;33;5218-25

  • Mutations and copy number increase of HRAS in Spitz nevi with distinctive histopathological features.

    Bastian BC, LeBoit PE and Pinkel D

    Departments of Dermatology and Pathology and UCSF Comprehensive Cancer Center, University of California San Francisco, San Francisco, California 94143-0808, USA. bastian@cc.ucsf.edu

    Spitz nevus is a benign melanocytic neoplasm that can be difficult or impossible to histologically distinguish from melanoma. We have recently described copy number increases of chromosome 11p in a subset of Spitz nevi. To study the molecular and histological features of this group, we studied 102 Spitz nevi for 11p copy number increases using fluorescence in situ hybridization (FISH) on tissue arrays. Copy number increases of at least threefold were found in 12 cases (11.8%) and involved the HRAS gene on chromosome 11p. Sequence analysis of HRAS showed frequent oncogenic mutations in cases with copy number increase (8/12 or 67%), contrasting with rare HRAS mutations in cases with normal HRAS copy numbers (1/21 or 5%, P: < 0.0001). Tumors with 11p copy number increases were larger, predominantly intradermal, had marked desmoplasia, characteristic cytological features, and had an infiltrating growth pattern. Proliferation rates in the majority of these cases were low to absent. HRAS activation by either mutation or copy number increase alone could explain several of the histological features that overlap with those of melanoma. We speculate that HRAS activation in the absence of co-operating additional genetic alterations drives the partially transformed melanocytes of these Spitz nevi into senescence or a stable growth arrest. Although there is no data suggesting that Spitz nevi with HRAS activation are at risk for progression to melanoma, future studies are warranted to assess their biological behavior more accurately.

    The American journal of pathology 2000;157;3;967-72

  • A novel 4-cM minimally deleted region on chromosome 11p15.1 associated with high grade nonmucinous epithelial ovarian carcinomas.

    Lu KH, Weitzel JN, Kodali S, Welch WR, Berkowitz RS and Mok SC

    Department of Obstetrics, Gynecology, and Reproductive Biology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.

    Prior cytogenetic and restriction fragment length polymorphism studies have demonstrated that allelic deletion of chromosome 11p is common in human invasive epithelial ovarian tumors. To construct a highly detailed deletion map of chromosome 11p, we used 13 polymorphic microsatellite CA repeat primers to identify regions harboring potential tumor suppressor genes. Twenty-three of 48 samples (48%) of invasive epithelial ovarian cancer showed LOH involving at least one locus, consistent with prior studies. None of the five mucinous tumors showed allelic deletion at any of the 13 primers, suggesting that loss of heterozygosity at chromosome 11p may not be involved in the pathogenesis of mucinous ovarian cancer. Two separate minimally deleted regions were identified in nonmucinous ovarian cancer. The first is an 11-cM region on chromosome 11pl5.5-15.3 that extends from D11S2071 to D11S988 and includes the HRAS locus. The second is a novel 4-cM region on 11p15.1, defined by marker D11S1310. Deletion of both regions at 11p15.5-15.3 and 11p15.1 is strongly associated with high grade nonmucinous epithelial ovarian cancer.

    Funded by: NCI NIH HHS: 1 KO8 CAO1574, R01CA63381

    Cancer research 1997;57;3;387-90

  • Studies of the c-Harvey-Ras gene in psychiatric disorders.

    Comings DE, Wu S, Chiu C, Muhleman D and Sverd J

    Department of Medical Genetics, City of Hope National Medical Center, Duarte, CA 91010-0269, USA.

    Hérault et al. (1993) previously reported a significant association between autism and the larger fragments of the c-Harvey-Ras (HRAS) Bam H1 polymorphism. We have sought to verify this finding and determine if there was any evidence for an association with other psychiatric disorders. Because of its greater sensitivity, we have examined the HRAS Msp 1 polymorphism. We found a just significant increase in the prevalence of the > 2.1 kb alleles in 48 subjects with autism versus 50 control subjects. There was no increase in the prevalence of the > 2.1 kb alleles in 164 probands with Tourette's syndrome. Examination of 16 preselected symptom clusters, however, showed a significant trend toward higher scores for obsessive-compulsive and phobic symptoms in > 2.1 kb homozygotes. While this locus requires further study, in conjunction with the results of Hérault et al., the present findings suggest that genetic defects in HRAS, and possibly other components of the G protein secondary messenger system, may play a role in some psychiatric disorders.

    Psychiatry research 1996;63;1;25-32

  • Ovarian cancer risk in BRCA1 carriers is modified by the HRAS1 variable number of tandem repeat (VNTR) locus.

    Phelan CM, Rebbeck TR, Weber BL, Devilee P, Ruttledge MH, Lynch HT, Lenoir GM, Stratton MR, Easton DF, Ponder BA, Cannon-Albright L, Larsson C, Goldgar DE and Narod SA

    Division of Medical Genetics, Montreal General Hospital, Quebec, Canada.

    Women who carry a mutation in the BRCA1 gene (on chromosome 17q21), have an 80% risk of breast cancer and a 40% risk of ovarian cancer by the age of 70 (ref. 1). The variable penetrance of BRCA1 suggests that other genetic and non-genetic factors play a role in tumourigenesis in these individuals. The HRAS1 variable number of tandem repeats (VNTR) polymorphism, located 1 kilobase (kb) downstream of the HRAS1 proto-oncogene (chromosome 11p15.5) is one possible genetic modifier of cancer penetrance. Individuals who have rare alleles of the VNTR have an increased risk of certain types of cancers, including breast cancer (2-4). To investigate whether the presence of rare HRAS1 alleles increases susceptibility to hereditary breast and ovarian cancer, we have typed a panel of 307 female BRCA1 carriers at this locus using a PCR-based technique. The risk for ovarian cancer was 2.11 times greater for BRCA1 carriers harbouring one or two rare HRAS1 alleles, compared to carriers with only common alleles (P = 0.015). The magnitude of the relative risk associated with a rare HRAS1 allele was not altered by adjusting for the other known risk factors for hereditary ovarian cancer (5). Susceptibility to breast cancer did not appear to be affected by the presence of rare HRAS1 alleles. This study is the first to show the effect of a modifying gene on the penetrance of an inherited cancer syndrome.

    Funded by: NCI NIH HHS: CA55914

    Nature genetics 1996;12;3;309-11

  • Molecular analysis at the Harvey Ras-1 gene in patients with long QT syndrome.

    Schulze-Bahr E, Haverkamp W, Wiebusch H, Schulte H, Hördt M, Borggrefe M, Breithardt G, Assmann G and Funke H

    Medizinische Klinik und Poliklinik, Westälische Wilhelms-Universität Münster, Germany.

    Patients with long QT syndrome (LQTS; MIM 1921500) frequently suffer from syncope and are threatened by sudden cardiac death due to ventricular arrhythmias, typically of the torsade de pointes type. Initial progress in revealing the molecular basis of the disease was made by the observation of genetic linkage of the disease locus to the Harvey Ras-1 gene (HRAS 1) on chromosome 11p15.5. More recently loci on chromosomes 3, 4, and 7 have also been found to be linked to LQTS, thus demonstrating heterogeneity in the causes for this disease. The present study performed sequence analysis on the HRAS 1 gene in patients with congenital and acquired LQTS to determine the frequency of HRAS 1 mutations in patients with this disease. In neither group were no mutations identified in the coding regions or in the splice donor and acceptor sites. Alleles characterized by a T to C transition in exon 1 and an insertion/deletion polymorphism upstream of exon 1 showed no significant difference in their frequencies between LQTS patients and normal controls. No quantitative influence of the such characterized genotypes on the QT duration was observed. These results demonstrate that structural mutations in the HRAS 1 gene are not a frequent cause of LQTS. Also, since there was no association of different alleles at the HRAS 1 locus with changes in QT duration, it appears unlikely that this gene is a major contributor to this disease.

    Journal of molecular medicine (Berlin, Germany) 1995;73;11;565-9

  • An association between the risk of cancer and mutations in the HRAS1 minisatellite locus.

    Krontiris TG, Devlin B, Karp DD, Robert NJ and Risch N

    Department of Medicine, Tufts University School of Medicine, Boston, MA.

    Background: The role of mutations in protooncogenes and their regulatory sequences in the pathogenesis of cancer is under close scrutiny. Minisatellites are unstable repetitive sequences of DNA that are present throughout the human genome. The highly polymorphic HRAS1 minisatellite locus just downstream from the protooncogene H-ras-1 consists of four common progenitor alleles and several dozen rare alleles, which apparently derive from mutations of the progenitors. We previously observed an association of the rare mutant alleles with many forms of cancer, and we undertook the present study to pursue this observation further.

    Methods: We conducted a case-control study, typing 736 HRAS1 alleles from patients with cancer and 652 from controls by Southern blotting of leukocyte DNA. We also carried out a meta-analysis of this study and 22 other published studies, estimating the relative risk of cancer (such as bladder, breast, or colorectal cancer) when one of the rare HRAS1 alleles was present.

    Results: Both the present case-control study (odds ratio, 1.83; 95 percent confidence interval, 1.28 to 2.67; P = 0.002) and the present study combined with our previous study (odds ratio, 2.07; 95 percent confidence interval, 1.47 to 2.92; P < 0.001), as well as the meta-analysis of all 23 studies (odds ratio, 1.93; 95 percent confidence interval, 1.63 to 2.30; chi-square = 57.58; P < 0.001), replicated our original finding and demonstrated a significant association of rare HRAS1 alleles with cancer. We found significant associations for four types of cancer: carcinomas of the breast, colorectum, and urinary bladder and acute leukemia. We also identified suggestive but not statistically significant associations for cancers of the lung and prostate and for non-Hodgkin's lymphoma.

    Conclusions: Mutant alleles of the HRAS1 minisatellite locus represent a major risk factor for common types of cancer. Although the relative risk associated with the presence of one rare allele is moderate, the aggregate prevalence of one rare allele is moderate, the aggregate prevalence of this class of mutant alleles implies an extremely important attributable risk: 1 in 11 cancers of the breast, colorectum, and bladder.

    Funded by: NCI NIH HHS: CA-45052

    The New England journal of medicine 1993;329;8;517-23

  • Rare HRAS1 alleles outside the VTR region in lymph nodes from patients with malignant lymphoma.

    Stul M, Baens M, Mecucci C, Van den Berghe H and Cassiman JJ

    Center for Human Genetics, University of Leuven, Belgium.

    We found a new type of rare HRAS1 alleles arising de novo in the lymph node of lymphoma patients. Rare alleles of 6.2 kb (a0.1) and 7.3 kb (a2.3) are displayed in the presence of common alleles of 6.6 kb (a1) and 7.6 kb (a3), respectively. These rare alleles lack nucleotide sequences, present in the common alleles in the 5' part between the BamHI and XmaIII site. In addition, the rare alleles may have the classic A-G mutation in the intron at position 2719. In contrast to what is observed for rare constitutional alleles, the VTR region of these rare alleles has the same size as the common alleles. At present the significance of this new type of alleles is not clear.

    Cancer genetics and cytogenetics 1993;69;1;60-4

  • Loss of heterozygosity for chromosome region 11p15 in Wilms' tumours is not related to HRAS gene transforming mutations.

    Baird P, Wadey R and Cowell J

    Department of Haematology and Oncology, Institute of Child Health, London, UK.

    Although a candidate Wilms' tumour gene--WT1--has been identified in chromosome region 11p13, there is strong evidence from loss of heterozygosity studies suggesting that a second relevant gene is present in region 11p15. The Harvey-Ras proto-oncogene also lies in this region. In other types of tumours mutations in RAS genes have been associated with the development and/or progression of a number of tumour types. We therefore analysed the sequence of the Ras oncogene for possible mutations in six Wilms' tumours showing loss of heterozygosity for chromosome region 11p15. No tumour analysed showed HRAS sequence mutations. We conclude that loss of heterozygosity at 11p15 does not implicate HRAS mutations in the molecular pathogenesis of Wilms' tumour.

    Oncogene 1991;6;7;1147-9

  • Evidence against close linkage of unipolar affective illness to human chromosome 11p markers HRAS1 and INS and chromosome Xq marker DXS52.

    Neiswanger K, Slaugenhaupt SA, Hughes HB, Frank E, Frankel DR, McCarty MJ, Chakravarti A, Zubenko GS, Kupfer DJ and Kaplan BB

    Department of Psychiatry, Western Psychiatric Institute and Clinic, Pittsburgh, PA 15213.

    The genetic basis of various subtypes of the affective disorders has been investigated by family, twin, and adoption studies, as well as by segregation and linkage analysis. Linkage analyses of bipolar disorder with the chromosome 11p15 DNA markers HRAS1 and INS, and the chromosome Xq28 markers for color blindness and G6PD have been reported. We have used restriction fragment length polymorphisms as markers to examine linkage in three extended families with unipolar affective illness, ascertained through probands with either recurrent unipolar or bipolar II illness. Using an inclusive definition of the affected phenotype, linkage could be excluded up to 28cM around the HRAS1-INS linkage group on chromosome 11p15, and up to 5 cM around the DNA marker DXS52 on Xq28. Negative linkage results were also obtained for two more restrictive definitions of affective illness. Thus, we find no evidence for the involvement of the chromosomal regions 11p15 and Xq28 with unipolar affective disorder in these three families.

    Funded by: NIGMS NIH HHS: GM33771; NIMH NIH HHS: MH29618, MH30915; ...

    Biological psychiatry 1990;28;1;63-72

  • Loss of the same alleles of HRAS1 and D11S151 in two independent pancreatic cancers from a patient with multiple endocrine neoplasia type 1.

    Yoshimoto K, Iizuka M, Iwahana H, Yamasaki R, Saito H, Saito S and Sekiya T

    Oncogene Division, National Cancer Center Research Institute, Tokyo, Japan.

    The DNAs from two independent pancreatic cancers (tumors 1 and 2) in a patient with multiple endocrine neoplasia type 1 were analyzed. No amplification or gross rearrangement of 19 protooncogenes was observed. However, Southern blot analysis using polymorphic DNA probes revealed loss of heterozygosity at loci on chromosome 11p in both tumors. In tumor 1, an extensive region including the HRAS1, PTH, CALCA, and D11S151 loci was deleted, while in tumor 2 loss of heterozygosity was limited at the HRAS1 and D11S151 loci. Because loss of heterozygosity at other chromosomal loci in the two tumors was quite rare, loss of genes on 11p might be nonrandom. It is noteworthy that the same allele at the HRAS1 locus and also the same allele at the D11S151 locus were lost in the two independent tumors. These results suggest that loss of genes at the HRAS1 and/or D11S151 loci plays an important role unmasking the remaining sequences probably having a recessive mutation.

    Cancer research 1989;49;10;2716-21

  • HRAS1 and INS genes are relocated but not structurally altered as a result of the t(7;11)(p15;p15) in a clone from a patient with acute myeloid leukaemia (M4).

    Morris CM, Fitzgerald PH, Kennedy MA, Hollings PE, Garry M and Corbett GM

    Cytogenetic and Molecular Oncology Unit, Christchurch Hospital, New Zealand.

    A patient whose leukaemic cells carried the rare t(7;11)(p15;p15) was diagnosed as having acute myelomonocytic leukaemia (AML-M4), and supports the association of this specific translocation with forms of acute myeloid leukaemia showing differentiation. Blast phase chronic myeloid leukaemia was excluded by lack of involvement of the ABL and BCR genes. Chromosome in situ hybridization studies showed that both the HRAS1 and INS genes were present on the terminal part of chromosome 11p which was translocated to chromosome 7p. Neither HRAS1 nor INS were structurally rearranged. Field inversion gel electrophoresis showed that a 400 kb fragment encompassing HRAS1 was structurally entire in leukaemic DNA. Because the INS gene, which was also translocated, is probably located proximal to HRAS1 on chromosome 11p, it is unlikely that HRAS1 was near the chromosome 11 breakpoint or involved in this leukaemia.

    British journal of haematology 1989;71;4;481-6

  • Duplication of HRAS1, INS, and IGF2 is not a common event in Beckwith-Wiedemann syndrome.

    Henry I, Jeanpierre M, Barichard F, Serre JL, Mallet J, Turleau C, de Grouchy J and Junien C

    INSERM U 73, Paris.

    A few cases of Beckwith-Wiedemann syndrome (BWS) have in common a duplication of 11p15. Among the genes located in 11p15, c-Ha-ras 1 (HRAS1), insulin (INS), and insulin-like growth factor II (IGF2) may account for the clinical features and the increased risk for malignancy. Using eight 11p15 markers including HRAS1, INS and IGF2 we have studied eight sporadic and hereditary cases of BWS whether or not associated with a nephroblastoma. By gene dosage determination and family studies, we have shown the following: the eight patients examined had an apparent diploid representation of all of the eight markers studied, thus indicating that a microduplication of these markers or of the region characterized by these markers is not a common event in BWS; in a family with three affected sibs the genes for HRAS1 and INS/IGF2 did not cosegregate with BWS and therefore may not participate in the pathogenic processes here observed.

    Annales de genetique 1988;31;4;216-20

  • HRAS1 proto-oncogene polymorphisms in human malignant melanoma: TaqI defined alleles significantly associated with the disease.

    Radice P, Pierotti MA, Borrello MG, Illeni MT, Rovini D and Della Porta G

    Division of Experimental Oncology, Istituto Nazionale Tumori, Milan, Italy.

    We have analysed the DNA of peripheral blood leukocytes (PBL) from 55 melanoma patients and 53 healthy individuals and failed to find any significant association between melanoma and rare HRAS1 alleles defined by MspI/HpaII digestion. However, the analysis of the same DNAs for a different polymorphism based on the presence of additional TaqI sites in the variable tandem repeat region of HRAS1 showed that the total frequency of a group of allelic variants, named Tp, was significantly higher in melanoma patients than in normal donors.

    Oncogene 1987;2;1;91-5

Pubmed - other

  • Loss of genes on the short arm of chromosome 11 in bladder cancer.

    Fearon ER, Feinberg AP, Hamilton SH and Vogelstein B

    Recent studies have shown that normal cellular sequences on chromosome 13 are lost during the development of retinoblastomas and that sequences on chromosome 11 are similarly lost during the development of Wilms' kidney tumours and embryonal tumours. Cells from these tumors have been found to contain either the paternal or maternal copies of loci on the affected chromosome, but not both. Thus, the somatic loss of heterozygosity for sequences on chromosome 13 or 11 is hypothesized to result in homozygosity for a recessive mutant allele on these chromosomes, and in this way the chromosomal loss may contribute to the development of these tumours. We sought to investigate whether similar losses of heterozygosity for chromosome 11 sequences occurred in a common adult tumour. We chose to analyse bladder cancers, since such cancers are common in the adult population and are derived from urogenital tissue, as are Wilms' tumours. We examined constitutional and tumour genotypes at loci on the short arm of chromosome 11 (11p) in 12 patients with transitional cell carcinomas. In five tumours, we observed the somatic loss of genes on 11p resulting in homozygosity or hemizygosity of the non-deleted alleles in the tumour cells. Our results show that the frequency of loss of 11p sequences in bladder cancer approaches that seen in Wilms' tumour (42% compared with 55%), and suggest that recessive genetic changes involving sequences on 11p may contribute to the development of bladder neoplasms.

    Funded by: NCI NIH HHS: CA35494; NIGMS NIH HHS: GM 07309

    Nature 

  • Modeling the effect of the RB tumor suppressor on disease progression: dependence on oncogene network and cellular context.

    Dean JL, McClendon AK, Stengel KR and Knudsen ES

    Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA.

    The retinoblastoma tumor suppressor, RB, is a key regulator of cellular proliferation that is functionally inactivated at high frequency in human cancer. Although RB has been extensively studied with regard to tumor etiology, loss of tumor-suppressor function often occurs relatively late in tumor progression. Therefore, inactivation of RB could have a profound impact on the behavior of tumors driven by discrete oncogenes. Here, collaboration between Ras or c-Myc deregulation and RB functional state was investigated in a model of conditional genetic deletion to decipher the effects related to disease progression. These studies showed that RB loss had a robust impact on mitogen dependence, anchorage dependence and overall survival, which was significantly modified by oncogene activation. Specifically, RB deficiency predisposed c-Myc-expressing cells to cell death and reduced overall tumorigenic proliferation. In contrast, RB deficiency exacerbated the tumorigenic behavior of Ras-transformed cells in both the model system and human tumor cell lines. As these tumors exhibited highly aggressive behavior, the possibility of exploiting the intrinsic sensitivity to cell death with RB loss was evaluated. Particularly, although Ras-transformed, RB-deficient cells bypassed the G1-checkpoint elicited by pharmacological activation of the p53 pathway, they were also highly sensitized to cell death. Altogether, these data suggest that the impact of RB deletion is dependent on the oncogene milieu, and can directly contribute to transformed phenotypes and response to therapeutic intervention.

    Oncogene 2010;29;1;68-80

  • RalA suppresses early stages of Ras-induced squamous cell carcinoma progression.

    Sowalsky AG, Alt-Holland A, Shamis Y, Garlick JA and Feig LA

    Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, MA 02111, USA.

    Ras proteins activate Raf and PI-3 kinases, as well as exchange factors for RalA and RalB GTPases. Many previous studies have reported that the Ral-signaling cascade contributes positively to Ras-mediated oncogenesis. Here, using a bioengineered tissue model of early steps in Ras-induced human squamous cell carcinoma of the skin, we found the opposite. Conversion of Ras-expressing keratinocytes from a premalignant to malignant state induced by decreasing E-cadherin function was associated with and required an approximately two to threefold decrease in RalA expression. Moreover, direct knockdown of RalA to a similar degree by shRNA expression in these cells reduced E-cadherin levels and also induced progression to a malignant phenotype. Knockdown of the Ral effector, Exo84, mimicked the effects of decreasing RalA levels in these engineered tissues. These phenomena can be explained by our finding that the stability of E-cadherin in Ras-expressing keratinocytes depends upon this RalA signaling cascade. These results imply that an important component of the early stages in squamous carcinoma progression may be a modest decrease in RalA gene expression that magnifies the effects of decreased E-cadherin expression by promoting its degradation.

    Funded by: NIGMS NIH HHS: R01 GM047717, R01 GM047717-16, R01 GM047717-16S1, R56 GM047717, R56 GM047717-17A1; NIMH NIH HHS: R01 MH083324, R01 MH083324-21A2

    Oncogene 2010;29;1;45-55

  • Phosphorylation by Cdk2 is required for Myc to repress Ras-induced senescence in cotransformation.

    Hydbring P, Bahram F, Su Y, Tronnersjö S, Högstrand K, von der Lehr N, Sharifi HR, Lilischkis R, Hein N, Wu S, Vervoorts J, Henriksson M, Grandien A, Lüscher B and Larsson LG

    Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE-171 77 Stockholm, Sweden.

    The MYC and RAS oncogenes are frequently activated in cancer and, together, are sufficient to transform rodent cells. The basis for this cooperativity remains unclear. We found that although Ras interfered with Myc-induced apoptosis, Myc repressed Ras-induced senescence, together abrogating two main barriers of tumorigenesis. Inhibition of cellular senescence required phosphorylation of Myc at Ser-62 by cyclin E/cyclin-dependent kinase (Cdk) 2. Cdk2 interacted with Myc at promoters, where it affected Myc-dependent regulation of genes, including Bmi-1, p16, p21, and hTERT, which encode proteins known to control senescence. Repression of senescence by Myc was abrogated by the Cdk inhibitor p27Kip1, which is induced by antiproliferative signals like IFN-gamma or by pharmacological inhibitors of Cdk2 but not by inhibitors of other Cdks. In contrast, a phospho-mimicking Myc-S62D mutant was resistant to these manipulations. Inhibition of cyclin E/Cdk2 reversed the senescence-associated gene expression pattern imposed by Myc/cyclin E/Cdk2. This indicates a role of Cdk2 as a transcriptional cofactor and activator of the antisenescence function of Myc and provides mechanistic insight into the Myc-p27Kip1 antagonism. Finally, our findings highlight that pharmacological inhibition of Cdk2 activity is a potential therapeutical principle for cancer therapy, in particular for tumors with activated Myc or Ras.

    Proceedings of the National Academy of Sciences of the United States of America 2010;107;1;58-63

  • Methylation associated inactivation of RASSF1A and its synergistic effect with activated K-Ras in nasopharyngeal carcinoma.

    Wang T, Liu H, Chen Y, Liu W, Yu J and Wu G

    Cancer Center of Wuhan Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China. wangtaowh@yahoo.com.cn

    Background: Epigenetic silencing of tumor suppressor genes associated with promoter methylation is considered to be a hallmark of oncogenesis. RASSF1A is a candidate tumor suppressor gene which was found to be inactivated in many human cancers. Although we have had a preliminary cognition about the function of RASSF1A, the exact mechanisms about how RASSF1A functions in human cancers were largely unknown. Moreover, the effect of mutated K-Ras gene on the function of RASSF1A is lacking. The aim of this study was to investigate the expression profile and methylation status of RASSF1A gene, and to explore its concrete mechanisms as a tumor suppressor gene in Nasopharyngeal Carcinoma.

    Methods: We examined the expression profile and methylation status of RASSF1A in two NPC cell lines, 38 primary nasopharyngeal carcinoma and 14 normal nasopharyngeal epithelia using RT-PCR and methylated specific PCR(MSP) respectively. 5-aza-dC was then added to confirm the correlation between hypermethylation status and inactivation of RASSF1A. The NPC cell line CNE-2 was transfected with exogenous pcDNA3.1(+)/RASSF1A plasmid in the presence or absence of mutated K-Ras by liposome-mediated gene transfer method. Flow cytometry was used to examine the effect of RASSF1A on cell cycle modulation and apoptosis. Meanwhile, trypan blue dye exclusion assays was used to detect the effect of RASSF1A transfection alone and the co-transfection of RASSF1A and K-Ras on cell proliferation.

    Results: Promoter methylation of RASSF1A could be detected in 71.05% (27/38) of NPC samples, but not in normal nasopharyngeal epithelia. RASSF1A expression in NPC primary tumors was lower than that in normal nasopharyngeal epithelial (p < 0.01). Expression of RASSF1A was down-regulated in two NPC cell lines. Loss of RASSF1A expression was greatly restored by the methyltransferase inhibitor 5-aza-dC in CNE-2. Ectopic expression of RASSF1A in CNE-2 could increase the percentage of G0/G1 phase cells (p < 0.01), inhibit cell proliferation and induce apoptosis (p < 0.001). Moreover, activated K-Ras could enhance the growth inhibition effect induced by RASSF1A in CNE-2 cells (p < 0.01).

    Conclusion: Expression of RASSF1A is down-regulated in NPC due to the hypermethylation of promoter. Exogenous expression of RASSF1A is able to induce growth inhibition effect and apoptosis in tumor cell lines, and this effect could be enhanced by activated K-Ras.

    Journal of experimental & clinical cancer research : CR 2009;28;160

  • P19 H-ras induces G1/S phase delay maintaining cells in a reversible quiescence state.

    Camats M, Kokolo M, Heesom KJ, Ladomery M and Bach-Elias M

    Unidad de Splicing, Instituto de Investigaciones Biomédicas de Barcelona-Consejo Superior de Investigaciones Científicas, Barcelona, Spain.

    Background: Three functional c-ras genes, known as c-H-ras, c-K-ras, and c-N-ras, have been largely studied in mammalian cells with important insights into normal and tumorigenic cellular signal transduction events. Two K-Ras mRNAs are obtained from the same pre-mRNA by alternative splicing. H-Ras pre-mRNA can also be alternatively spliced in the IDX and 4A terminal exons, yielding the p19 and p21 proteins, respectively. However, despite the Ras gene family's established role in tumorigenic cellular signal transduction events, little is known about p19 function. Previous results showed that p19 did not interact with two known p21 effectors, Raf1 and Rin1, but was shown to interact with RACK1, a scaffolding protein that promotes multi-protein complexes in different signaling pathways (Cancer Res 2003, 63 p5178). This observation suggests that p19 and p21 play differential and complementary roles in the cell.

    We found that p19 regulates telomerase activity through its interaction with p73alpha/beta proteins. We also found that p19 overexpression induces G1/S phase delay; an observation that correlates with hypophosphorylation of both Akt and p70SK6. Similarly, we also observed that FOXO1 is upregulated when p19 is overexpressed. The three observations of (1) hypophosphorylation of Akt, (2) G1/S phase delay and (3) upregulation of FOXO1 lead us to conclude that p19 induces G1/S phase delay, thereby maintaining cells in a reversible quiescence state and preventing entry into apoptosis. We then assessed the effect of p19 RNAi on HeLa cell growth and found that p19 RNAi increases cell growth, thereby having the opposite effect of arrest of the G1/S phase or producing a cellular quiescence state.

    Significance: Interestingly, p19 induces FOXO1 that in combination with the G1/S phase delay and hypophosphorylation of both Akt and p70SK6 leads to maintenance of a reversible cellular quiescence state, thereby preventing entry into apoptosis.

    PloS one 2009;4;12;e8513

  • Silencing of the Lats2 tumor suppressor overrides a p53-dependent oncogenic stress checkpoint and enables mutant H-Ras-driven cell transformation.

    Aylon Y, Yabuta N, Besserglick H, Buganim Y, Rotter V, Nojima H and Oren M

    Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel.

    The Lats2 tumor suppressor protein has been implicated earlier in promoting p53 activation in response to mitotic apparatus stress, by preventing Mdm2-driven p53 degradation. We now report that Lats2 also has a role in an ATR-Chk1-mediated stress check point in response to oncogenic H-Ras. Activated mutant H-Ras triggers the translocation of Lats2 from centrosomes into the nucleus, coupled with an increase in Lats2 protein levels. This leads to the induction of p53 activity, upregulation of proapoptotic genes, downregulation of antiapoptotic genes and eventually apoptotic cell death. Many of the cells that survive apoptosis undergo senescence. However, a fraction of the cells escape this checkpoint mechanism, despite maintaining a high mutant H-Ras expression. These escapers display increased genome instability, as evidenced by a substantial fraction of cells with micronuclei and cells with polyploid genomes. Interestingly, such cells show markedly reduced levels of Lats2, in conjunction with enhanced hypermethylation of the Lats2 gene promoter. Our findings suggest that Lats2 might have an important role in quenching H-Ras-induced transformation, whereas silencing of Lats2 expression might serve as a mechanism to enable tumor progression.

    Funded by: NCI NIH HHS: R37 CA040099, R37 CA040099-25, R37 CA40099

    Oncogene 2009;28;50;4469-79

  • Polycythemia vera erythroid precursors exhibit increased proliferation and apoptosis resistance associated with abnormal RAS and PI3K pathway activation.

    Laubach JP, Fu P, Jiang X, Salter KH, Potti A and Arcasoy MO

    Department of Medicine, Duke University School of Medicine, Durham, NC 27710, USA.

    Objective: Polycythemia vera (PV) is characterized by erythrocytosis associated with the presence of the activating JAK2(V617F) mutation in a variable proportion of hematopoietic cells. JAK2(V617F) is detected in other myeloproliferative neoplasms, does not appear to be the PV-initiating event, and its specific role in deregulated erythropoiesis in PV is incompletely understood. We investigated the pathogenesis of PV to characterize abnormal proliferation and apoptosis responses and aberrant oncogenic pathway activation in primary PV erythroid precursors.

    Peripheral blood CD34(+) cells isolated from PV patients and healthy controls were grown in liquid culture to expand a population of primary erythroblasts for experiments designed to analyze cellular proliferation, apoptosis, JAK2(V617F) mutation status, cytokine-dependent protein phosphorylation and gene expression profiling using Affymetrix microarrays.

    Results: The survival and proliferation of PV erythroblasts were growth factor-dependent under strict serum-free conditions requiring both erythropoietin (EPO) and stem cell factor. PV erythroblasts exhibited EPO hypersensitivity and enhanced cellular proliferation associated with increased EPO-mediated extracellular signal-regulated kinases 1 and 2 phosphorylation. EPO-induced AKT phosphorylation was observed in PV but not normal erythroblasts, an effect associated with apoptosis resistance in PV erythroblasts. Analysis of gene expression and oncogenic pathway activation signatures revealed increased RAS (p<0.01) and phosphoinositide-3 kinase (p<0.05) pathway activation in PV erythroblasts.

    Conclusion: Deregulated erythropoiesis in PV involves EPO hypersensitivity and apoptosis resistance of erythroid precursor cells associated with abnormally increased activation of RAS-ERK and phosphoinositide-3 kinase-AKT pathways. These data suggest that investigation of the mechanisms of abnormal RAS and phosphoinositide-3 kinase pathway activation in erythroblasts may contribute to our understanding of the molecular pathogenesis of PV.

    Funded by: NIDDK NIH HHS: K08 DK002566-06, USA) K08 DK02566

    Experimental hematology 2009;37;12;1411-22

  • Activating mutations in FGFR3 and HRAS reveal a shared genetic origin for congenital disorders and testicular tumors.

    Goriely A, Hansen RM, Taylor IB, Olesen IA, Jacobsen GK, McGowan SJ, Pfeifer SP, McVean GA, Rajpert-De Meyts E and Wilkie AO

    Genes mutated in congenital malformation syndromes are frequently implicated in oncogenesis, but the causative germline and somatic mutations occur in separate cells at different times of an organism's life. Here we unify these processes to a single cellular event for mutations arising in male germ cells that show a paternal age effect. Screening of 30 spermatocytic seminomas for oncogenic mutations in 17 genes identified 2 mutations in FGFR3 (both 1948A>G, encoding K650E, which causes thanatophoric dysplasia in the germline) and 5 mutations in HRAS. Massively parallel sequencing of sperm DNA showed that levels of the FGFR3 mutation increase with paternal age and that the mutation spectrum at the Lys650 codon is similar to that observed in bladder cancer. Most spermatocytic seminomas show increased immunoreactivity for FGFR3 and/or HRAS. We propose that paternal age-effect mutations activate a common 'selfish' pathway supporting proliferation in the testis, leading to diverse phenotypes in the next generation including fetal lethality, congenital syndromes and cancer predisposition.

    Funded by: Wellcome Trust: 078666

    Nature genetics 2009;41;11;1247-52

  • Association of H-ras polymorphisms and susceptibility to sporadic colon cancer.

    Catela Ivkovic T, Loncar B, Spaventi R and Kapitanovic S

    Rudjer Boskovic Institute, Bijenicka c. 54, Zagreb, Croatia. tcatela@irb.hr

    High incidence of colon cancer worldwide indicates the importance of studying genetic alterations that lead to its carcinogenesis. Two polymorphisms in H-ras gene, hexanucleotide tandem repeats in the first intron and SNP 81T>C in the first exon, that might be connected with susceptibility to cancer have been described. The aim of our study was to investigate these loci in Croatian population and to determine if any of them is connected with susceptibility to colon cancer in our population. Two hundred healthy volunteers and 200 colon cancer patients were genotyped using PCR and RFLP methods. We noted statistically significant difference in genotype distribution at hexanucleotide locus between healthy population and colon cancer patients (p=0.013) but not in allelic distribution. At SNP 81 T>C statistically significant difference in distribution of both genotypes (p=9.15x10(-6)) and alleles (p=2.77x10(-6)) was found. No differences were found between genders.

    International journal of oncology 2009;35;5;1169-73

  • Frequent and significant K-ras mutation in the pancreas, the bile duct, and the gallbladder in autoimmune pancreatitis.

    Kamisawa T, Tsuruta K, Okamoto A, Horiguchi S, Hayashi Y, Yun X, Yamaguchi T and Sasaki T

    Department of Internal Medicine, Tokyo Metropolitan Komagome Hospital, Tokyo, Japan. kamisawa@cick.jp

    Objectives: To assess the relationship between autoimmune pancreatitis (AIP) and pancreatic cancer, we analyzed K-ras mutation in the pancreatobiliary tissues of patients with AIP.

    Methods: An analysis of K-ras mutation and an immunohistochemical study were performed on the pancreas of 8 patients with AIP and 10 patients with chronic alcoholic pancreatitis and on the common bile duct and the gallbladder of 9 patients with AIP. K-ras mutation was analyzed in the pure pancreatic juice from 3 patients with AIP.

    Results: High-frequency K-ras mutation (2+ or 3+) was detected in the pancreas of all the 8 patients and in the pancreatic juice of the other 2 patients. The mutation in codon 12 of the ras gene was GAT in all the 10 patients. High-frequency K-ras mutation was detected in the common bile duct of 5 patients with AIP and in the gallbladder epithelium of 4 patients with AIP. The K-ras mutation was detected in the fibroinflammatory pancreas, the bile duct, and the gallbladder, with abundant infiltrating IgG4-positive plasma and Foxp3-positive cells of patients with AIP with elevated serum IgG4 levels.

    Conclusions: Significant K-ras mutation occurs most frequently in the pancreatobiliary regions of patients with AIP. Autoimmune pancreatitis may be a risk factor of pancreatobiliary cancer.

    Pancreas 2009;38;8;890-5

  • Organophosphorous pesticides and estrogen induce transformation of breast cells affecting p53 and c-Ha-ras genes.

    Calaf GM, Echiburu-Chau C and Roy D

    Instituto de Alta Investigación, Universidad de Tarapacá, Arica, Chile. gmc24@columbia.edu

    Cancer progression has been associated with an increase in genomic instability indicated by inactivation of tumor suppressor genes and activation of oncogenes. Epidemiological and experimental evidence has implicated estrogens in the etiology of breast cancer. To study environmental organophosphorous pesticides is of interest since evidence indicate that pesticides may enhance cell division, increasing the risk of breast cancer. The aim was to evaluate the effects of these pesticides, such as parathion and malathion in the presence of estrogen on malignant transformation as well as on genomic instability, that is in the frequency of loss of heterozygosity (LOH) and microsatellite instability (MSI). The MCF-10F immortalized human breast epithelial cell line, that was treated with parathion or malathion alone and in combination with estrogen was used. These studies indicated that either pesticide alone or in combination with estrogen induced malignant transformation as shown by anchorage-independent growth capability and invasive characteristics in comparison to control. Such malignant phenotypic characteristics were corroborated by significant (P<0.05) increase in p53 and c-Ha-ras protein expression. Results indicated different degrees of allelic imbalance in the form of LOH or MSI with different microsatellite markers. MSI was found in malathion and estrogen-treated cells with a marker used for p53 tumor suppressor gene at loci 17p13.1. The same combination of substances presented MSI with a marker used for c-Ha-ras mapped in chromosome 11p14.1, as well as mutations in c-Ha-ras for codons 12 and 61. LOH was observed in codon 12 in the presence of estrogen or malathion alone. Parathion alone and combined with estrogen induced MSI in codon 61. It can be concluded that the organophosphorous pesticides parathion and malathion induced malignant transformation of breast cells through genomic instability altering p53 and c-Ha-ras, considered pivotal to cancer process.

    International journal of oncology 2009;35;5;1061-8

  • PI3K/Akt pathway mutations in retinoblastoma.

    Cohen Y, Merhavi-Shoham E, Avraham-Lubin BC, Savetsky M, Frenkel S, Pe'er J and Goldenberg-Cohen N

    Department of Gynecology, Sheba Medical Center, Tel Hashomer, Israel.

    Purpose: Many malignancies are known to be associated with abnormal activation of the PI3K-AKT pathway. Recently, a somatic mutation in the AKT1 gene (E17K) was identified in a small proportion of human tumors. This mutation activated AKT1 by means of abnormal membrane recruitment and stimulated downstream signaling. This study was designed to analyze AKT1 mutations in retinoblastoma and gain insights into the role PI3K-AKT pathway plays in the development of this tumor.

    Methods: Twenty-four samples of retinoblastoma from children were analyzed for mutations in the AKT1, PTEN and K-RAS genes, using a chip-based matrix-assisted laser desorption-time-of-flight (MALDI-TOF) mass spectrometer. Mutations in the PIK3CA gene were analyzed in 16 retinoblastoma samples using direct sequencing.

    Results: These results show that the mutation E17K/AKT1 was not detected in the 24 samples of retinoblastoma analyzed. K-RAS mutations were identified in two samples. There were no mutations in any of the other genes analyzed by a mass array system. On direct sequencing of 16 samples for the PIK3CA gene, one sample showed gain of function mutation in exon 9. In another sample, a genetic polymorphism of unknown significance (rs17849079) was detected in exon 20.

    Conclusions: Although the PI3K-AKT pathway is known to be dysregulated in retinoblastoma, the low frequency of oncogenic mutations in the AKT1, PIK3CA, and PTEN genes, suggests a different activating mechanism.

    Investigative ophthalmology & visual science 2009;50;11;5054-6

  • RAS signaling dysregulation in human embryonal Rhabdomyosarcoma.

    Martinelli S, McDowell HP, Vigne SD, Kokai G, Uccini S, Tartaglia M and Dominici C

    Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy. simone.martinelli@iss.it

    Rhabdomyosarcoma (RMS) is a common childhood solid tumor, resulting from dysregulation of the skeletal myogenesis program. Two major histological subtypes occur in childhood RMS, embryonal and alveolar. While chromosomal rearrangements account for the majority of alveolar tumors, the genetic defects underlying the pathogenesis of embryonal RMS remain largely undetermined. A few studies performed on small series of embryonal tumors suggest that dysregulation of RAS function may be relevant to disease pathogenesis. To explore further the biological and clinical relevance of mutations with perturbing consequences on RAS signaling in embryonal RMS, we investigated the prevalence of PTPN11, HRAS, KRAS, NRAS, BRAF, MEK1, and MEK2 mutations in a relatively large cohort of primary tumors. While HRAS and KRAS were found to be rarely mutated, we identified somatic NRAS lesions in 20% of cases. All mutations were missense and affected codon 61, with the introduction of a positive charged amino acid residue representing the most common event. PTPN11 was found mutated in one tumor specimen, confirming that somatic defects in this gene are relatively uncommon in RMS, while no mutation was observed in BRAF and MEK genes. Although no clear association of mutations with any clinical variable was observed, comparison of the outcome between mutation-positive and mutation-negative cases indicated a trend for a higher percentage of patients exhibiting a better outcome in the former. Our findings provide evidence that dysregulation of RAS signaling is a major event contributing to embryonal RMS pathogenesis.

    Funded by: Telethon: GGP07115

    Genes, chromosomes & cancer 2009;48;11;975-82

  • Detection of two novel mutations and relatively high incidence of H-RAS mutations in Vietnamese oral cancer.

    Murugan AK, Hong NT, Cuc TT, Hung NC, Munirajan AK, Ikeda MA and Tsuchida N

    Department of Molecular Cellular Oncology and Microbiology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan. amuruga1@jhmi.edu

    Oral squamous cell carcinoma is the sixth most common cancer in the world and the seventh most common cancer in Vietnam. The RAS and PI3K-AKT signaling pathways play an important role in oral carcinogenesis. Our previous study on PI3K signaling pathway showed the absence of PIK3CA and PTEN gene mutations in Vietnamese oral cancer. We thus hypothesized that the RAS could be more likely activated as an upstream effector. However, the status of RAS mutations in Vietnamese oral cancer had not been studied. In the present study, Fifty six primary tumor DNA samples were screened for mutations of hot spots in exons 1 and 2 of H-RAS and a part of the samples for exon 7 of ERK2 gene in which we previously reported a mutation in an OSCC cell line. The H-RAS mutations were detected in 10 of 56 tumors (18%). Two novel mutations were found, one was an insertion of three nucleotides (GGC) between codons 10 and 11 resulting in in-frame insertion of glycine (10(Gly)11) and the other was a missense mutation in codon 62 (GAG>GGG). We also found T81C single nucleotide polymorphism in 12 of 56 tumors (22%) and there was no mutation in exon 7 of ERK2 gene. The H-RAS mutation incidence showed significant association with advanced stages of the tumor and also with well-differentiated tumor grade. Our study is the first to report H-RAS mutation from Vietnamese ethnicity, with two novel mutations and relatively high incidence of H-RAS mutations. The results suggest that RAS is an important member in the PI3K-AKT signaling and could play an important role in the tumorigenesis of oral carcinoma.

    Oral oncology 2009;45;10;e161-6

  • Oncogenic Ras signals through activation of both phosphoinositide 3-kinase and Rac1 to induce c-Jun NH2-terminal kinase-mediated, caspase-independent cell death.

    Byun JY, Kim MJ, Yoon CH, Cha H, Yoon G and Lee SJ

    Laboratory of Molecular Biochemistry, Department of Chemistry, Hanyang University, Seoul 133-791, Korea.

    Cells avert the development of malignancy in response to deregulated oncogene expression by activating a regulated cell death pathway. However, the molecular mechanism underlying this oncogene-induced cellular death process remains unclear. Here, we show that retroviral expression of oncogenic H-ras induced cell death in a caspase-independent manner in normal cells. Inhibition of c-Jun NH2-terminal kinase (JNK) by pretreatment with SP600125 or a dominant-negative form of JNK blocked cell death. Rac1 and phosphoinositide 3-kinase (PI3K) were activated in cells overexpressing oncogenic H-ras. Inhibition of Rac1 with RacN17, a dominant-negative form of Rac1, attenuated oncogenic H-ras-induced JNK activation and subsequent cell death. Interestingly, inhibition of PI3K with LY294002 or by small interfering RNA-mediated knockdown of PI3K p85 or p110 subunits also clearly attenuated JNK activation and cell death. No cross talk was observed between Rac1 and PI3K, indicating that these pathways operate in parallel. Our findings show that JNK is necessary for oncogenic H-ras-induced, caspase-independent cell death, and that both PI3K and Rac1 activities are required for JNK activation and cell death. Determining the molecular mechanisms that mediate cell death responses to deregulated oncogenes provides a more refined understanding of cellular disposal processes in normal cells and increases our appreciation of these events as a mechanism for protecting against malignant progression.

    Molecular cancer research : MCR 2009;7;9;1534-42

  • BMI1 cooperates with H-RAS to induce an aggressive breast cancer phenotype with brain metastases.

    Hoenerhoff MJ, Chu I, Barkan D, Liu ZY, Datta S, Dimri GP and Green JE

    Transgenic Oncogenesis Group, Laboratory of Cancer Biology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20982, USA.

    B-lymphoma Moloney murine leukaemia virus insertion region-1 (BMI1) is a member of the polycomb group of transcription repressors, which functions in stem cell maintenance and oncogenesis through the inhibition of the INK4A/ARF tumour suppressor locus. Overexpression of BMI1 is associated with poor prognosis in several human cancers, including breast cancer. We have previously shown that BMI1 collaborates with H-RAS to induce transformation of MCF10A human mammary epithelial cells through dysregulation of multiple growth pathways independent of the INK4A/ARF locus. In this study, we show that BMI1 collaborates with H-RAS to promote increased proliferation, invasion and resistance to apoptosis in vitro, and an increased rate of spontaneous metastases from mammary fat pad xenografts including novel metastases to the brain. Furthermore, in collaboration with H-RAS, BMI1 induced fulminant metastatic disease in the lung using a tail vein model of haematogenous spread through accelerated cellular proliferation and inhibition of apoptosis. Finally, we show that knockdown of BMI1 in several established breast cancer cell lines leads to decreased oncogenic behaviour in vitro and in vivo. In summary, BMI1 collaborates with H-RAS to induce an aggressive and metastatic phenotype with the unusual occurrence of brain metastasis, making it an important target for diagnosis and treatment of aggressive breast cancer.

    Funded by: Intramural NIH HHS: Z01 BC005740-15; NCI NIH HHS: R01 CA094150, R01CA 094150

    Oncogene 2009;28;34;3022-32

  • Sorafenib inhibits non-small cell lung cancer cell growth by targeting B-RAF in KRAS wild-type cells and C-RAF in KRAS mutant cells.

    Takezawa K, Okamoto I, Yonesaka K, Hatashita E, Yamada Y, Fukuoka M and Nakagawa K

    Department of Medical Oncology, Kinki University School of Medicine, Sakai Hospital, Osaka, Japan.

    Sorafenib is a multikinase inhibitor whose targets include B-RAF and C-RAF, both of which function in the extracellular signal-regulated kinase (ERK) signaling pathway but which also have distinct downstream targets. The relative effects of sorafenib on B-RAF and C-RAF signaling in tumor cells remain unclear, however. We have now examined the effects of sorafenib as well as of B-RAF or C-RAF depletion by RNA interference on cell growth and ERK signaling in non-small cell lung cancer (NSCLC) cell lines with or without KRAS mutations. Sorafenib inhibited ERK phosphorylation in cells with wild-type KRAS but not in those with mutant KRAS. Despite this difference, sorafenib inhibited cell growth and induced G(1) arrest in both cell types. Depletion of B-RAF, but not that of C-RAF, inhibited ERK phosphorylation as well as suppressed cell growth and induced G(1) arrest in cells with wild-type KRAS. In contrast, depletion of C-RAF inhibited cell growth and induced G(1) arrest, without affecting ERK phosphorylation, in cells with mutant KRAS; depletion of B-RAF did not induce G(1) arrest in these cells. These data suggest that B-RAF-ERK signaling and C-RAF signaling play the dominant roles in regulation of cell growth in NSCLC cells with wild-type or mutant KRAS, respectively. The G(1) arrest induced by either C-RAF depletion or sorafenib in cells with mutant KRAS was associated with down-regulation of cyclin E. Our results thus suggest that sorafenib inhibits NSCLC cell growth by targeting B-RAF in cells with wild-type KRAS and C-RAF in those with mutant KRAS.

    Cancer research 2009;69;16;6515-21

  • Tumor suppressor density-enhanced phosphatase-1 (DEP-1) inhibits the RAS pathway by direct dephosphorylation of ERK1/2 kinases.

    Sacco F, Tinti M, Palma A, Ferrari E, Nardozza AP, Hooft van Huijsduijnen R, Takahashi T, Castagnoli L and Cesareni G

    Department of Biology, University of Rome Tor Vergata, Via della Ricerca Scientifica, 00133 Rome, Italy.

    Density-enhanced phosphatase-1 (DEP-1) is a trans-membrane receptor protein-tyrosine phosphatase that plays a recognized prominent role as a tumor suppressor. However, the mechanistic details underlying its function are poorly understood because its primary physiological substrate(s) have not been firmly established. To shed light on the mechanisms underlying the anti-proliferative role of this phosphatase, we set out to identify new DEP-1 substrates by a novel approach based on screening of high density peptide arrays. The results of the array experiment were combined with a bioinformatics filter to identify eight potential DEP-1 targets among the proteins annotated in the MAPK pathway. In this study we show that one of these potential targets, the ERK1/2, is indeed a direct DEP-1 substrate in vivo. Pulldown and in vitro dephosphorylation assays confirmed our prediction and demonstrated an overall specificity of DEP-1 in targeting the phosphorylated tyrosine 204 of ERK1/2. After epidermal growth factor stimulation, the phosphorylation of the activation loop of ERK1/2 can be modulated by changing the concentration of DEP-1, without affecting the activity of the upstream kinase MEK. In addition, we show that DEP-1 contains a KIM-like motif to recruit ERK1/2 proteins by a docking mechanism mediated by the common docking domain in ERK1/2. ERK proteins that are mutated in the conserved docking domain become insensitive to DEP-1 de-phosphorylation. Overall this study provides novel insights into the anti-proliferative role of this phosphatase and proposes a new mechanism that may also be relevant for the regulation of density-dependent growth inhibition.

    The Journal of biological chemistry 2009;284;33;22048-58

  • BRAF, NRAS and HRAS mutations in spitzoid tumours and their possible pathogenetic significance.

    Da Forno PD, Pringle JH, Fletcher A, Bamford M, Su L, Potter L and Saldanha G

    Department of Histopathology, University Hospitals of Leicester NHS Trust, Leicester, UK.

    Background: The relationships between so-called spitzoid tumours have proven difficult to understand.

    Objectives: To address three questions: does spitzoid tumour morphological similarity reflect molecular similarity? Does Spitz naevus progress into spitzoid melanoma? Are ambiguous spitzoid tumours genuine entities?

    Methods: BRAF, NRAS and HRAS mutations were analysed using single-strand conformational polymorphism analysis and sequencing.

    Results: Both Spitz naevi and spitzoid melanoma had a lower combined BRAF and NRAS mutation frequency compared with common acquired naevi (P = 0.0001) and common forms of melanoma (P = 0.0072), respectively. To look for evidence of progression from Spitz naevi to spitzoid melanoma, HRAS was analysed in 21 spitzoid melanomas, with no mutations identified. The binomial probability of this was 0.03 based on an assumption of a 15% mutation frequency in Spitz naevi with unbiased progression. Under these assumptions, HRAS mutations must be rare/absent in spitzoid melanoma. Thus, Spitz naevi seem unlikely to progress into spitzoid melanoma, implying that ambiguous spitzoid tumours cannot be intermediate degrees of progression. In addition, the data suggest that HRAS mutation is a potential marker of benign behaviour, in support of which none of three HRAS mutant spitzoid cases metastasized.

    Conclusions: First, the morphological similarity of spitzoid tumours reflects an underlying molecular similarity, namely a relative lack of dependence on BRAF/NRAS mutations. Second, Spitz naevi do not appear to progress into spitzoid melanoma, and consequently ambiguous spitzoid tumours are likely to be unclassifiable Spitz naevi or spitzoid melanoma rather than genuine entities. Third, HRAS mutation may be a marker of Spitz naevus, raising the possibility that other molecular markers for discriminating Spitz naevi from spitzoid melanoma can be discovered.

    The British journal of dermatology 2009;161;2;364-72

  • Chronic NF-kappaB activation delays RasV12-induced premature senescence of human fibroblasts by suppressing the DNA damage checkpoint response.

    Batsi C, Markopoulou S, Vartholomatos G, Georgiou I, Kanavaros P, Gorgoulis VG, Marcu KB and Kolettas E

    Cell and Molecular Physiology Unit, Laboratory of Physiology, School of Medicine, University of Ioannina, Ioannina, Greece.

    Normal cells divide for a limited number of generations, after which they enter a state of irreversible growth arrest termed replicative senescence. While replicative senescence is due to telomere erosion, normal human fibroblasts can undergo stress-induced senescence in response to oncogene activation, termed oncogene-induced senescence (OIS). Both, replicative and OIS, initiate a DNA damage checkpoint response (DDR) resulting in the activation of the p53-p21(Cip1/Waf1) pathway. However, while the nuclear factor-kappaB (NF-kappaB) signaling pathway has been implicated in DDR, its role in OIS has not been investigated. Here, we show that oncogenic Ha-RasV12 promoted premature senescence of IMR-90 normal human diploid fibroblasts by activating DDR, hence verifying the classical model of OIS. However, enforced expression of a constitutively active IKKbeta T-loop mutant protein (IKKbetaca), significantly delayed OIS of IMR-90 cells by suppressing Ha-RasV12 instigated DDR. Thus, our experiments have uncovered an important selective advantage in chronically activating canonical NF-kappaB signaling to overcome the anti-proliferative OIS response of normal primary human fibroblasts.

    Funded by: NIGMS NIH HHS: GM066882, R01 GM066882-03

    Mechanisms of ageing and development 2009;130;7;409-19

  • Dominance of EGFR and insignificant KRAS mutations in prediction of tyrosine-kinase therapy for NSCLC patients stratified by tumor subtype and smoking status.

    Pesek M, Benesova L, Belsanova B, Mukensnabl P, Bruha F and Minarik M

    Departments of Pneumology, Faculty Hospital Pilsen, Pilsen, Czech Republic.

    Background: Therapy by tyrosine kinase inhibitors (TKI) has become inevitable in treatment of advanced NSCLC. Mutations in EGFR and KRAS genes have been identified as the main potential predictive and prognostic factors. Here the clinical implications of EGFR/KRAS mutations in patients from two separate trials treated with gefitinib or erlotinib are analysed.

    A total of 360 patients (269 gefitinib and 91 erlotinib) were evaluated. Mutations in EGFR (exon 19 and 21) and KRAS (codons 12 and 13) and their impact on response and survival with respect to tumor subtype and smoking status were assessed.

    Results: Adenocarcinomas revealed 399 days to progression (TTP) and 548 days overall survival (OS) for EGFR mutated vs. 119 days to progression and 137 days survival for non-mutated, p<0.0001 (TTP) and p=0.0001 (OS). No EGFR effect was recorded for squamous cell tumors. For smoking status, both EGFR-mutated smokers and non-smokers profited from TKI therapy. Smokers: 243 vs. 122 days (mutated vs. non-mutated), p=0.0284 (TTP) and 244 vs. 126 days, p=0.0396 (OS); non-smokers: 390 vs. 71 days, p<0.0001, (TTP) and 548 vs. 135 days, p<0.0001 (OS). KRAS mutation in tumors did not result in a poorer prognosis in the subtype-selected groups, nor did it present as a negative factor in smokers.

    Conclusion: EGFR mutations possess statistical significance for a better therapy response and longer survival in all patients with adenocarcinomas (smokers as well as non-smokers). KRAS does not seem an "a priori" negative factor for TKI-based treatment of NSCLC.

    Anticancer research 2009;29;7;2767-73

  • Follow-up study of K-ras mutations in the plasma of patients with pancreatic cancer: correlation with clinical features and carbohydrate antigen 19-9.

    Däbritz J, Preston R, Hänfler J and Oettle H

    Department of Medical Oncology and Hematology, Charité School of Medicine, Campus Virchow-Klinikum, Humboldt University of Berlin, Berlin, Germany. Jan.Daebritz@charite.de

    Objective: We followed up the presence of Kirsten rat sarcoma (K-ras) mutations in plasma DNA and assessed its clinical value in patients with pancreatic cancer.

    Methods: Plasma samples (N = 430) of 56 patients with pancreatic cancer and 13 patients with pancreatitis were analyzed by real-time polymerase chain reaction using peptide nucleic acid-mediated polymerase chain reaction clamping.

    Results: K-ras mutations could be detected in the plasma DNA of 20 patients with cancer (36%). No K-ras mutation was found in the plasma of patients with pancreatitis. In 7 (35%) of 20 patients with lowly or moderately elevated carbohydrate antigen 19.9 (CA 19-9) levels lower than 100 U/mL, the result of the assay was positive for K-ras mutation. The combination of K-ras and CA 19-9 level determination gave a sensitivity for the diagnosis of pancreatic cancer of 91% (40/44) of the patients. Thirteen of 35 patients with pancreatic cancer (102 plasma samples) with elevated CA 19-9 levels (>35 U/mL) and altered K-ras gene showed significant correlation with elevated CA 19-9 levels (P=0.048).

    Conclusions: The summary of our approach of noninvasive, convenient, extremely high-sensitive K-ras mutation analysis in plasma might provide diagnostic and prognostic information to clinicians but will not be sufficient in a standardized early diagnosis of pancreatic carcinoma. The combination with CA 19-9 assay is useful for detection and prognostic evaluation of pancreatic carcinoma.

    Pancreas 2009;38;5;534-41

  • Mutational profile of advanced primary and metastatic radioactive iodine-refractory thyroid cancers reveals distinct pathogenetic roles for BRAF, PIK3CA, and AKT1.

    Ricarte-Filho JC, Ryder M, Chitale DA, Rivera M, Heguy A, Ladanyi M, Janakiraman M, Solit D, Knauf JA, Tuttle RM, Ghossein RA and Fagin JA

    Human Oncology and Pathogenesis Program and Departments of Medicine and Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA.

    Patients with poorly differentiated thyroid cancers (PDTC), anaplastic thyroid cancers (ATC), and radioactive iodine-refractory (RAIR) differentiated thyroid cancers have a high mortality, particularly if positive on [(18)F]fluorodeoxyglucose (FDG)-positron emission tomography (PET). To obtain comprehensive genetic information on advanced thyroid cancers, we designed an assay panel for mass spectrometry genotyping encompassing the most significant oncogenes in this disease: 111 mutations in RET, BRAF, NRAS, HRAS, KRAS, PIK3CA, AKT1, and other related genes were surveyed in 31 cell lines, 52 primary tumors (34 PDTC and 18 ATC), and 55 RAIR, FDG-PET-positive recurrences and metastases (nodal and distant) from 42 patients. RAS mutations were more prevalent than BRAF (44 versus 12%; P = 0.002) in primary PDTC, whereas BRAF was more common than RAS (39 versus 13%; P = 0.04) in PET-positive metastatic PDTC. BRAF mutations were highly prevalent in ATC (44%) and in metastatic tumors from RAIR PTC patients (95%). Among patients with multiple metastases, 9 of 10 showed between-sample concordance for BRAF or RAS mutations. By contrast, 5 of 6 patients were discordant for mutations of PIK3CA or AKT1. AKT1_G49A was found in 9 specimens, exclusively in metastases. This is the first documentation of AKT1 mutation in thyroid cancer. Thus, RAIR, FDG-PET-positive metastases are enriched for BRAF mutations. If BRAF is mutated in the primary, it is likely that the metastases will harbor the defect. By contrast, absence of PIK3CA/AKT1 mutations in one specimen may not reflect the status at other sites because these mutations arise during progression, an important consideration for therapies directed at phosphoinositide 3-kinase effectors.

    Funded by: NCI NIH HHS: CA50706, P30 CA008748, R01 CA050706, R01 CA050706-20, R01 CA072597

    Cancer research 2009;69;11;4885-93

  • Lifetime history of alcohol consumption and K-ras mutations in pancreatic ductal adenocarcinoma.

    Crous-Bou M, Porta M, López T, Jariod M, Malats N, Morales E, Guarner L, Rifà J, Carrato A, Real FX and PANKRAS II Study Group

    Institut Municipal d'Investigació Mèdica, Barcelona, Spain.

    Background: In pancreatic ductal adenocarcinoma (PDA), evidence on the etiopathogenic role of alcohol consumption in the occurrence of K-ras mutations is scant, and the role of alcohol in pancreatic carcinogenesis is not well established. We analyzed the relation between lifetime consumption of alcohol and mutations in codon 12 of the K-ras oncogene in patients with PDA.

    Methods: Incident cases of PDA were prospectively identified and interviewed face-to-face during hospital admission about lifetime alcohol consumption and other lifestyle factors. Logistic regression was used to compare PDA cases (N = 107) with mutated and wild-type K-ras tumors (case-case study).

    Results: Mutated cases were moderate or heavy drinkers more frequently than wild-type cases: the odds ratio adjusted by age, sex, smoking, and history of pancreatitis (ORa) was 3.18 (95% confidence interval: 1.02-9.93; P = 0.046). Total grams of alcohol and years of consumption were higher in mutated than in wild-type cases: the ORa for lifetime alcohol consumption over 507,499 g was 3.35 (95% CI: 0.81-13.88); and for more than 40 years of alcohol consumption it was 4.47 (95% CI: 1.05-19.02). Age at onset of alcohol consumption and years of abstinence were also associated with the presence of K-ras mutations. There were no significant differences in alcohol dependency.

    Conclusions: Alcohol consumption is weakly associated with an increased risk of having a K-ras mutated PDA. To confirm or to refute the hypothesis that ethanol, acetaldehyde or other alcohol-related substances might influence the acquisition or persistence of K-ras mutations in the pancreatic epithelium, large and unselected studies are warranted.

    Funded by: PHS HHS: 04-C-N272

    Environmental and molecular mutagenesis 2009;50;5;421-30

  • Rho signaling, ROCK and mDia1, in transformation, metastasis and invasion.

    Narumiya S, Tanji M and Ishizaki T

    Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto, 606-8501, Japan. snaru@mfour.med.kyoto-u.ac.jp

    The Rho subgroup of the Rho GTPases consisting of RhoA, RhoB and RhoC induces a specific type of actin cytoskeleton and carry out a variety of functions in the cell. mDia and ROCK are downstream effectors of Rho mediating Rho action on the actin cytoskeleton; mDia produces actin filaments by nucleation and polymerization and ROCK activate myosin to cross-link them for induction of actomyosin bundles and contractility. mDia is potentially linked to Rac activation and membrane ruffle formation through c-Src-induced phosphorylation of focal adhesion proteins, and ROCK antagonizes this mDia action. Thus, cell morphogenesis, adhesion, and motility can be determined by the balance between mDia and ROCK activities. Though they are not oncogenes by themselves, overexpression of RhoA and RhoC are often found in clinical cancers, and RhoC has been repeatedly identified as a gene associated with metastasis. The Rho-ROCK pathway is implicated in Ras-mediated transformation, the amoeboid movement of tumor cells in the three-dimensional matrix, and transmigration of tumor cells through the mesothelial monolayer. On the other hand, the Rho-mDia1 pathway is implicated in Src-mediated remodeling of focal adhesions and migration of tumor cells. There is also an indication that the Rho pathway other than ROCK is involved in Src-mediated induction of podosome and regulation of matrix metalloproteases. Thus, Rho mediates various phenotypes of malignant transformation by Ras and Src through its effectors, ROCK and mDia.

    Cancer metastasis reviews 2009;28;1-2;65-76

  • p53, secreted by K-Ras-Snail pathway, is endocytosed by K-Ras-mutated cells; implication of target-specific drug delivery and early diagnostic marker.

    Lee SH, Lee SJ, Chung JY, Jung YS, Choi SY, Hwang SH, Choi D, Ha NC and Park BJ

    Department of Molecular Biology, Pusan National University, Busan, Republic of Korea.

    p53 is eliminated from K-Ras-mutated cancer cells through direct interaction with Snail. However, it is not achieved through proteasome-mediated degradation or transcriptional repression. Here we provide evidence that p53, binding with Snail, is exported from a K-Ras-mutated cell through a vesicle transport-like mechanism, independently using a p53-nuclear-exporting mechanism. Although we can detect p53 in culture media, a majority of p53 might be degraded by extracellular proteases. Thus, we can recover the secreted p53 in culture media by the inhibition of protease and endocytosis. In addition, a considerable amount of p53 is endocytosed by neighboring cells. As p53 resorption occurs in a K-Ras-dependent manner, treatment of recombinant p53 is detected in the whole-cell lysate of K-Ras-mutated cells, but not in that of wild-type cells. Using the property of p53, we can deliver the chemical (propidium iodine) into K-Ras mutated cells selectively. In contrast, Snail, a co-secreted protein with p53 in response to oncogenic K-Ras, shows resistance to endocytosis and protease, and results in remaining in the media. Thus, we can detect an autoantibody against Snail in the serum of a human cancer patient. Our finding can be used for a mutant K-Ras-specific anticancer drug delivery system and for the diagnosis of pancreatic, colon and lung cancers.

    Oncogene 2009;28;19;2005-14

  • Activation of RAS family genes in urothelial carcinoma.

    Boulalas I, Zaravinos A, Karyotis I, Delakas D and Spandidos DA

    Department of Urology, Asklipieio General Hospital, Voula, Athens, Greece.

    Purpose: Bladder cancer is the fifth most common malignancy in men in Western society. We determined RAS codon 12 and 13 point mutations and evaluated mRNA expression levels in transitional cell carcinoma cases.

    Samples from 30 human bladder cancers and 30 normal tissues were analyzed by polymerase chain reaction/restriction fragment length polymorphism and direct sequencing to determine the occurrence of mutations in codons 12 and 13 of RAS family genes. Moreover, we used real-time reverse transcriptase-polymerase chain reaction to evaluate the expression profile of RAS genes in bladder cancer specimens compared to that in adjacent normal tissues.

    Results: Overall H-RAS mutations in codon 12 were observed in 9 tumor samples (30%). Two of the 9 patients (22%) had invasive bladder cancer and 7 (77%) had noninvasive bladder cancer. One H-RAS mutation (11%) was homozygous and the remaining 89% were heterozygous. All samples were WT for K and N-RAS oncogenes. Moreover, 23 of 30 samples (77%) showed over expression in at least 1 RAS family gene compared to adjacent normal tissue. K and N-RAS had the highest levels of over expression in bladder cancer specimens (50%), whereas 27% of transitional cell carcinomas demonstrated H-RAS over expression relative to paired normal tissues.

    Conclusions: Our results underline the importance of H-RAS activation in human bladder cancer by codon 12 mutations. Moreover, they provide evidence that increased expression of all 3 RAS genes is a common event in bladder cancer that is associated with disease development.

    The Journal of urology 2009;181;5;2312-9

  • Micropapillary lung adenocarcinoma: EGFR, K-ras, and BRAF mutational profile.

    De Oliveira Duarte Achcar R, Nikiforova MN and Yousem SA

    Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA 15213-2582, USA.

    Micropapillary lung adenocarcinoma (MPA) has been reported as an aggressive variant of adenocarcinoma, frequently manifesting at high stage with a poor prognosis. We analyzed the clinical and molecular profile of 15 primary MPAs for K-ras, EGFR, and BRAF mutations and performed fluorescence in situ hybridization for EGFR amplification. In our study, 11 (73%) of 15 MPAs harbored mutually exclusive mutations: 5 (33%) K-ras, 3 (20%) EGFR, and 3 (20%) BRAF. Mutations in all 3 genes occurred in patients with a smoking history and tumors with mucinous differentiation and secondary lepidic, acinar, and solid growth, suggesting that in a Western population, cytomorphologic correlation with genetic mutations is more unpredictable than in Japanese cohorts. We conclude that K-ras, EGFR, and BRAF mutations are disproportionately seen in adenocarcinomas of lung with a dominant micropapillary growth pattern compared with conventional adenocarcinoma in our institutional experience.

    American journal of clinical pathology 2009;131;5;694-700

  • Mutation status of K-ras, p53 and allelic losses at 9p and 18q are not prognostic markers in patients with pancreatic cancer.

    Salek C, Minarikova P, Benesova L, Nosek V, Strnad R, Zavoral M and Minarik M

    Laboratory for Molecular Genetics and Oncology, Genomac International Ltd., Bavorska 856, 15541 Prague 5, Czech Republic.

    Background: K-ras mutations and allelic losses of tumor suppressors p16 and DPC4 are perceived as potential markers for screening of pancreatic malignancy. In this study, molecular data is compared with survival statistics of the patients and whether they correlate with patients' prognosis is questioned.

    Fifty three consecutive patients with advanced pancreatic cancer (stage III and IV) who underwent EUS-guided fine needle aspiration (FNA) were enrolled into the study (28 males, 25 females, 63+/-10.5 years). Samples were evaluated on-site for presence of malignant cells. DNA was extracted from Giemsa stained smears using laser microdissection, and mutation status of K-ras and p53 was tested by cycling-gradient capillary electrophoresis (CGCE). In addition, allelic losses of tumor suppressor genes p16 (INK4, CDKN2A) and DPC4 (MADH4, SMAD4) were detected by monitoring the loss of heterozygosity (LOH) at 9p and 18q loci. Molecular data were compared with survival statistics using Kaplan-Meier method.

    Results: The median survival in K-ras positive group was 7.0+/-2.4 months (95% CI 2.3-11.7) and in K-ras negative group was 10.0+/-0.6 months (95% CI 8.7-11.3). The median survival in p53 positive group was 10.0+/-2.2 months (95% CI 5.6-14.4) and in p53 negative group was 6.0+/-2.5 months (95% CI 1.1-10.9). The median survival in LOH 9p positive group was 9.0+/-5.1 months (95% CI 0-18.9), in LOH 9p negatives was 10.0+/-5.0 months (95% CI 0.2-19.8). The median survival in LOH 18q positive group was 10.0+/-4.2 months (95% CI 1.8-18.2) and in LOH 18q negative group was 3.0+/-1.3 months (95% CI 0.5-5.5). After the adjustment for age using Cox proportional hazards model, none of the evaluated molecular markers was shown to be an independent prognostic marker for survival of patients with pancreatic cancer.

    Conclusion: None of the studied molecular markers was identified as an independent factor determining survival prognosis.

    Anticancer research 2009;29;5;1803-10

  • Comparison of ras-responsive gene enhancers in pancreatic tumor cells that express either wild-type or mutant K-ras.

    Wilder PJ, Chakravarthy H, Hollingsworth MA and Rizzino A

    Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, 68198-6805, USA.

    There is a pressing need for new therapies to treat pancreatic cancer. In principle, this could be achieved by taking advantage of signaling pathways that are active in tumor, but not normal, cells. The work described in this study set out to determine whether the activities of three enhancers, which have been reported to be highly responsive to activated ras, differ in pancreatic tumor cells that express wild-type versus constitutively active mutant forms of K-ras. Surprisingly, the three enhancers are active in four different pancreatic tumor cell lines that express either normal K-ras gene or mutant K-ras. Moreover, reducing the concentration of serum in the growth medium from 10% to 0.5% had relatively little effect on the strength of any of the enhancers, although it drastically affected cell growth. Importantly, our studies also indicate that MEK is active in pancreatic tumor cells that possess wild-type as well as mutant K-ras, even when cultured in medium that severely limits cell growth. These findings support the hypothesis that the Ras/Raf/Mek/Erk pathway may be constitutively active even in pancreatic tumor cells that express wild-type K-ras.

    Funded by: NCI NIH HHS: CA36727

    Biochemical and biophysical research communications 2009;381;4;706-11

  • Genetic mutations associated with cigarette smoking in pancreatic cancer.

    Blackford A, Parmigiani G, Kensler TW, Wolfgang C, Jones S, Zhang X, Parsons DW, Lin JC, Leary RJ, Eshleman JR, Goggins M, Jaffee EM, Iacobuzio-Donahue CA, Maitra A, Klein A, Cameron JL, Olino K, Schulick R, Winter J, Vogelstein B, Velculescu VE, Kinzler KW and Hruban RH

    Department of Oncology, The Sol Goldman Pancreatic Cancer Research Center, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21231, USA.

    Cigarette smoking doubles the risk of pancreatic cancer, and smoking accounts for 20% to 25% of pancreatic cancers. The recent sequencing of the pancreatic cancer genome provides an unprecedented opportunity to identify mutational patterns associated with smoking. We previously sequenced >750 million bp DNA from 23,219 transcripts in 24 adenocarcinomas of the pancreas (discovery screen). In this previous study, the 39 genes that were mutated more than once in the discovery screen were sequenced in an additional 90 adenocarcinomas of the pancreas (validation screen). Here, we compared the somatic mutations in the cancers obtained from individuals who ever smoked cigarettes (n = 64) to the somatic mutations in the cancers obtained from individuals who never smoked cigarettes (n = 50). When adjusted for age and gender, analyses of the discovery screen revealed significantly more nonsynonymous mutations in the carcinomas obtained from ever smokers (mean, 53.1 mutations per tumor; SD, 27.9) than in the carcinomas obtained from never smokers (mean, 38.5; SD, 11.1; P = 0.04). The difference between smokers and nonsmokers was not driven by mutations in known driver genes in pancreatic cancer (KRAS, TP53, CDKN2A/p16, and SMAD4), but instead was predominantly observed in genes mutated at lower frequency. No differences were observed in mutations in carcinomas from the head versus tail of the gland. Pancreatic carcinomas from cigarette smokers harbor more mutations than do carcinomas from never smokers. The types and patterns of these mutations provide insight into the mechanisms by which cigarette smoking causes pancreatic cancer.

    Funded by: NCI NIH HHS: CA62924, P50 CA062924, P50 CA062924-08S30011, P50 CA062924-090011, P50 CA062924-160011, R01 CA039416, R37 CA043460

    Cancer research 2009;69;8;3681-8

  • BLNK binds active H-Ras to promote B cell receptor-mediated capping and ERK activation.

    Imamura Y, Oda A, Katahira T, Bundo K, Pike KA, Ratcliffe MJ and Kitamura D

    Division of Molecular Biology, Research Institute for Biological Sciences, Tokyo University of Science, 2669 Yamazaki, Noda, Chiba 278-0022, Japan.

    Cross-linked B cell receptor (BCR) aggregates on the cell surface, then assembles into the "cap" where Ras is co-localized, and transduces various intracellular signals including Ras-ERK activation. BCR signals induce proliferation, differentiation, or apoptosis of B cells depending on their maturational stage. The adaptor protein BLNK binds various signaling proteins and Igalpha, a signaling subunit of the BCR complex, and plays an important role in the BCR signal transduction. BLNK was shown to be required for activation of ERK, but not of Ras, after BCR cross-linking, raising a question how BLNK facilitates ERK activation. Here we demonstrate that BLNK binds the active form of H-Ras, and their binding is facilitated by BCR cross-linking. We have identified a 10-amino acid Ras-binding domain within BLNK that is necessary for restoration of BCR-mediated ERK activation in BLNK-deficient B cells and for anti-apoptotic signaling. The Ras-binding domain fused with a CD8alpha-Igalpha chimeric receptor could induce prolonged ERK phosphorylation, transcriptional activation of Elk1, as well as the capping of the receptor in BLNK-deficient B cells. These results indicate that BLNK recruits active H-Ras to the BCR complex, which is essential for sustained surface expression of BCR in the form of the cap and for the signal leading to functional ERK activation.

    The Journal of biological chemistry 2009;284;15;9804-13

  • EGFR and K-ras mutations along the spectrum of pulmonary epithelial tumors of the lung and elaboration of a combined clinicopathologic and molecular scoring system to predict clinical responsiveness to EGFR inhibitors.

    Sartori G, Cavazza A, Sgambato A, Marchioni A, Barbieri F, Longo L, Bavieri M, Murer B, Meschiari E, Tamberi S, Cadioli A, Luppi F, Migaldi M and Rossi G

    Section of Pathologic Anatomy, Azienda Ospedaliero-Universitaria Policlinico di Modena, Via del Pozzo, 71- 41100- Modena, Italy.

    We tested 418 neoplasms along the whole spectrum of primary lung tumor histotypes for epidermal growth factor receptor (EGFR) and K-ras mutations. Clinicopathologic data from 154 patients undergoing treatment with EGFR tyrosine kinase inhibitors (TKIs) were retrospectively studied. A scoring system assigning a score for each positive or negative characteristic (+1, female sex, nonsmoking status, adenocarcinoma histotype, Asian ethnicity, and EGFR mutation; -1, current smoker and K-ras mutation; and 0, male sex, ex-smoker, nonadenocarcinoma histotype, and no mutations) was elaborated and tested with EGFR-TKI response. Salivary gland-type, mucin-rich, and neuroendocrine tumors do not harbor EGFR mutations. A subset of nonmucinous adenocarcinomas, not necessarily of the bronchioloalveolar type, is related to EGFR mutations. Three probability groups significantly correlating with response to EGFR-TKIs were identified. Of note, the addition of molecular results did not significantly change the predictive value obtained by the combination of clinicopathologic characteristics alone in this scoring system. K-ras mutations, significantly associated with the mucin-secreting type of adenocarcinoma, consistently predict lack of response in white patients.

    American journal of clinical pathology 2009;131;4;478-89

  • PKCdelta survival signaling in cells containing an activated p21Ras protein requires PDK1.

    Xia S, Chen Z, Forman LW and Faller DV

    Cancer Research Center, Boston University School of Medicine, Boston, Massachusetts 02118, USA.

    Protein kinase C delta (PKCdelta) modulates cell survival and apoptosis in diverse cellular systems. We recently reported that PKCdelta functions as a critical anti-apoptotic signal transducer in cells containing activated p21(Ras) and results in the activation of AKT, thereby promoting cell survival. How PKCdelta is regulated by p21(Ras), however, remains incompletely understood. In this study, we show that PKCdelta, as a transducer of anti-apoptotic signals, is activated by phosphotidylinositol 3' kinase/phosphoinositide-dependent kinase 1 (PI(3)K-PDK1) to deliver the survival signal to Akt in the environment of activated p21(Ras). PDK1 is upregulated in cells containing an activated p21Ras. Knock-down of PDK1, PKCdelta, or AKT forces cells containing activated p21(Ras) to undergo apoptosis. PDK1 regulates PKCdelta activity, and constitutive expression of PDK1 increases PKCdelta activity in different cell types. Conversely, expression of a kinase-dead (dominant-negative) PDK1 significantly suppresses PKCdelta activity. p21(Ras)-mediated survival signaling is therefore regulated by via a PI(3)K-AKT pathway, which is dependent upon both PDK1 and PKCdelta, and PDK1 activates and regulates PKCdelta to determine the fate of cells containing a mutated, activated p21(Ras).

    Funded by: NCI NIH HHS: CA108100, CA112102, R01 CA112102, R01 CA112102-01A1, R01 CA112102-02, R01 CA112102-03, R01 CA112102-03S1, R01 CA112102-04, R01 CA112102-04S1, R41 CA108100, R41 CA108100-01

    Cellular signalling 2009;21;4;502-8

  • H-Ras is degraded by Wnt/beta-catenin signaling via beta-TrCP-mediated polyubiquitylation.

    Kim SE, Yoon JY, Jeong WJ, Jeon SH, Park Y, Yoon JB, Park YN, Kim H and Choi KY

    National Research Laboratory of Molecular Complex Control and Department of Biotechnology, BK21 project for Medical Science, Yonsei University, Seoul 120-749, Korea.

    Ras is an important proto-protein that is regulated primarily by GDP/GTP exchange. Here, we report a novel regulatory mechanism whereby turnover of both endogenous and overexpressed H-Ras protein is controlled by beta-TrCP-mediated ubiquitylation, proteasomal degradation and the Wnt/beta-catenin signaling pathway. The interaction of H-Ras with the WD40 domain of beta-TrCP targeted H-Ras for polyubiquitylation and degradation. This process was stimulated by Axin or adenomatous polyposis coli (Apc), and was inhibited by Wnt3a. Ras-mediated cellular transformation was also inhibited by the expression of beta-TrCP and/or Axin. In vivo regulation of Ras stability by Wnt/beta-catenin signaling was determined via measurements of the status of Ras in the intestines of mice stimulated with recombinant Wnt3a by intravenous tail vein injection. The regulation of Ras stability by Wnt/beta-catenin signaling provides a mechanical basis for crosstalk between the Wnt/beta-catenin and the Ras-ERK pathways involved in transformation.

    Journal of cell science 2009;122;Pt 6;842-8

  • Male-to-male transmission of Costello syndrome: G12S HRAS germline mutation inherited from a father with somatic mosaicism.

    Sol-Church K, Stabley DL, Demmer LA, Agbulos A, Lin AE, Smoot L, Nicholson L and Gripp KW

    Nemours Biomedical Research, Nemours Children's Clinic, Wilmington, Delaware 19803, USA. ksolchur@nemours.org

    Costello syndrome is a rare congenital anomaly syndrome associated with mental retardation and predisposition to benign and malignant tumors, caused by heterozygous missense mutations in the HRAS oncogene. Previously, all molecularly analyzed mutations appeared de novo, and most arose in the paternal germline. A single patient with somatic mosaicism for a Costello syndrome causing HRAS mutation has been reported. Here we describe the first documented transmission of an HRAS mutation from a parent with somatic mosaicism to a child with typical Costello syndrome. Prior to the identification of the underlying gene mutation in Costello syndrome, this family had been identified clinically. The proband was subsequently found to carry a G12S HRAS germline mutation. Testing of the parents for parental origin identified his father as mosaic for the same HRAS mutation. The mother was found not to carry an HRAS mutation. The causative familial mutation is identified as a c.34G > A, which is the most common mutation in the HRAS gene in patients with Costello syndrome. The father carries the mutation in 7-8% of his alleles. This is the second case of mosaicism observed in Costello syndrome and the first direct molecular evidence of father-to-son transmission of the disease-causing mutation. Our observation underlines the importance of parental evaluation, and may have implications for genetic counseling and clinical practice.

    Funded by: NCRR NIH HHS: 1 P20 RR020173, P20 RR020173, P20 RR020173-05

    American journal of medical genetics. Part A 2009;149A;3;315-21

  • NAT2 fast acetylator genotype and MGMT promoter methylation may contribute to gender difference in K-RAS mutation occurrence in Taiwanese colorectal cancer.

    Huang CC, Chien WP, Wong RH, Cheng YW, Chen MC and Lee H

    Colorectal Division, Department of Surgery, Chung Shan Medical University Hospital, Taichung, Taiwan, Republic of China.

    A recent study conducted by our group showed that the NAT2 fast acetylator genotype is associated with an increasing risk for the development of colorectal cancer (CRC), especially for females. We therefore examined whether a higher risk of CRC in females with the NAT2 fast acetylator genotype was associated with the occurrence of K-RAS mutation, and to further verify whether MGMT promoter methylation was linked to the occurrence of K-RAS mutation in patients with the NAT2 fast acetylator genotype. Herein, 151 CRC cases were examined for NAT2 genetic polymorphisms and MGMT promoter methylation by PCR-RFLP and methylation-specific PCR (MSP). The results of this study show that the NAT2 fast acetylator genotype is associated with the occurrence of K-RAS mutation in female cases (OR = 4.820, 95% CI = 1.113-20.873), but not associated with MGMT promoter methylation. Surprisingly, MGMT promoter methylation significantly deepens the impact of NAT2 fast acetylation on K-RAS mutation in the female cases (OR = 5.129, 95% CI = 1.092-24.105). In conclusion, Taiwanese women with the NAT2 fast acetylator genotype may exhibit a higher risk of CRC with increased occurrence of K-RAS mutation. Detection of NAT2 genotypes and MGMT promoter methylation may be useful in the risk assessment for CRC in Taiwanese women.

    Environmental and molecular mutagenesis 2009;50;2;127-33

  • Oncogenic HRAS mutations cause prolonged PI3K signaling in response to epidermal growth factor in fibroblasts of patients with Costello syndrome.

    Rosenberger G, Meien S and Kutsche K

    Institut für Humangenetik, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany.

    Costello syndrome (CS) is a rare congenital disorder characterized by failure to thrive, craniofacial dysmorphisms, cardiac and skin abnormalities, mental retardation, and predisposition to malignancies. CS is caused by heterozygous gain-of-function mutations in HRAS that also occur as somatic alterations in human tumors. HRAS is one of the three classical RAS proteins and cycles between an active, GTP- and an inactive, GDP-bound conformation. We used primary human skin fibroblasts from patients with CS as a model system to study the functional consequences of HRAS mutations on endogenous signaling pathways. The GTP-bound form of HRAS was significantly enriched in CS compared with normal fibroblasts. Active HRAS is known to stimulate both the RAF-MEK-ERK and the PI3K-AKT signaling cascade. Phosphorylation of MEK and ERK was normal in CS fibroblasts under basal conditions and slightly prolonged after epidermal growth factor (EGF) stimulation. Interestingly, basal phosphorylation of AKT was increased yet more in CS fibroblasts. Moreover, AKT phosphorylation was diminished in the early and enhanced in the late phase of EGF stimulation. Taken together, these results document that CS-associated HRAS mutations result in prolonged signal flux in a ligand-dependent manner. Our data suggest that altered cellular response to growth factors rather than constitutive activation of HRAS downstream signaling molecules may contribute to some of the clinical features in patients with CS.

    Human mutation 2009;30;3;352-62

  • Molecular determinants of response to matuzumab in combination with paclitaxel for patients with advanced non-small cell lung cancer.

    Schittenhelm MM, Kollmannsberger C, Oechsle K, Harlow A, Morich J, Honecker F, Kurek R, Störkel S, Kanz L, Corless CL, Wong KK, Bokemeyer C and Heinrich MC

    University Hospital Tübingen, Department of Hematology, Oncology, Rheumatology, Immunology and Pulmonology, Otfried-Müller-Strasse 10, 72074 Tübingen, Germany. marcus.schittenhelm@med.uni-tuebingen.de

    Antibodies targeting epidermal growth factor receptor (EGFR) have proven to be effective in patients with non-small cell lung cancer (NSCLC) that express EGFR. We recently published a phase I study of weekly matuzumab plus paclitaxel. This therapy was well tolerated and showed clinical responses in the majority of patients. Although matuzumab displays potent antitumor activity in some patients, not all patients respond well to treatment. Whether dysregulation of EGFR-mediated pathways precludes or sensitizes cells to paclitaxel is unknown. We sought to determine molecular predictive factors for therapy response in a phase I/II study patient cohort treated with matuzumab+/-paclitaxel. Twenty-three cases [including one complete response (CR), three partial responses (PR), 10 stable diseases (SD)] were screened using immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), PCR/sequencing and denaturing wave high performance liquid chromatography (D-HPLC) for expression, amplification, and mutation status of EGFR and downstream signaling pathways. All patients with PR or CR displayed an either high overall or single-cell EGFR expression in the majority of cells. In addition, all of the moderate responders, who achieved SD after at least two cycles of therapy, showed diffuse EGFR expression rates and/or strong single-cell EGFR expression. In contrast, 44% of the nonresponders showed low overall or single-cell EGFR expression levels. No low-expressing EGFR cases were present within the responder group. In addition, among patients with a gain-of-function mutation in KRAS primary therapy failure and/or short responses to therapy were observed. Our data suggest that EGFR expression and KRAS mutation status is predictive for clinical response to matuzumab +/- paclitaxel in patients with advanced NSCLC.

    Molecular cancer therapeutics 2009;8;3;481-9

  • High-throughput sequencing screen reveals novel, transforming RAS mutations in myeloid leukemia patients.

    Tyner JW, Erickson H, Deininger MW, Willis SG, Eide CA, Levine RL, Heinrich MC, Gattermann N, Gilliland DG, Druker BJ and Loriaux MM

    Division of Hematology and Medical Oncology, Oregon Health & Science University Knight Cancer Institute, Portland, OR 97239, USA

    Transforming mutations in NRAS and KRAS are thought to play a causative role in the development of numerous cancers, including myeloid malignancies. Although mutations at amino acids 12, 13, or 61 account for the majority of oncogenic Ras variants, we hypothesized that less frequent mutations at alternate residues may account for disease in some patients with cancer of unexplained genetic etiology. To search for additional, novel RAS mutations, we sequenced all coding exons in NRAS, KRAS, and HRAS in 329 acute myeloid leukemia (AML) patients, 32 chronic myelomonocytic leukemia (CMML) patients, and 96 healthy individuals. We detected 4 "noncanonical" point mutations in 7 patients: N-Ras(G60E), K-Ras(V14I), K-Ras(T74P), and K-Ras(A146T). All 4 Ras mutants exhibited oncogenic properties in comparison with wild-type Ras in biochemical and functional assays. The presence of transforming RAS mutations outside of positions 12, 13, and 61 reveals that alternate mechanisms of transformation by RAS may be overlooked in screens designed to detect only the most common RAS mutations. Our results suggest that RAS mutations may play a greater role in leukemogenesis than currently believed and indicate that high-throughput screening for mutant RAS alleles in cancer should include analysis of the entire RAS coding region.

    Funded by: Howard Hughes Medical Institute

    Blood 2009;113;8;1749-55

  • Hdm2 is regulated by K-Ras and mediates p53-independent functions in pancreatic cancer cells.

    Sui X, Shin S, Zhang R, Firozi PF, Yang L, Abbruzzese JL and Reddy SA

    Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

    There is emerging evidence that the oncogenic potential of hdm2 (human and/or murine double minute-2 protein) stems not only from its ability to counteract tumor suppressor p53 but also from its less understood p53-independent functions. Surprisingly, little is known about the role and regulation of hdm2 in pancreatic tumors, a large proportion (50-75%) of which contain mutant p53. In this study, we determined that hdm2 was expressed in a Ras-signaling-dependent manner in various pancreatic cancer cell lines. As p53 was mutated and inactive in these cells, the expression of hdm2 was seemingly redundant. Indeed, the proliferation and survival of cell lines such as Panc-1 and Panc-28 could be inhibited by PRIMA-1 (mutant p53 activator) but not by Nutlin-3 (inhibitor of the hdm2-p53 interaction). Unexpectedly, however, the proliferation of both cell lines was strongly inhibited by hdm2-specific RNAi. Our data also revealed cyclin D1, c-Jun and c-Myc to be novel targets of hdm2 and suggested that they might mediate hdm2's role in cellular proliferation and/or survival. We conclude from our results that hdm2 is expressed in pancreatic cancer cells as a result of activated Ras signaling, and that it regulates cellular proliferation and the expression of three novel target genes by p53-independent mechanisms.

    Funded by: NCI NIH HHS: CA016672, CA101936, P20 CA101936

    Oncogene 2009;28;5;709-20

  • Cognitive profile of disorders associated with dysregulation of the RAS/MAPK signaling cascade.

    Cesarini L, Alfieri P, Pantaleoni F, Vasta I, Cerutti M, Petrangeli V, Mariotti P, Leoni C, Ricci D, Vicari S, Selicorni A, Tartaglia M, Mercuri E and Zampino G

    Pediatric Neurology Unit, Catholic University, Rome, Italy.

    Mutations in genes coding for transducers participating in the RAS/MAPK pathway have been identified as the molecular cause underlying a group of clinically related developmental disorders with cognitive deficits of variable severity. To determine the spectrum of cognitive defects associated with dysregulation of this signal cascade, we studied the profile of cognitive abilities in patients with mutations affecting the PTPN11, SOS1, HRAS, KRAS, BRAF, RAF1, and MEK1 genes and phenotype-genotype correlations. Our findings support the observation that heterogeneity in cognitive abilities can be at least partially ascribed to the individual affected genes and type of mutation involved. While mutations affecting transducers upstream of RAS were less frequently associated with mental retardation, mutations in downstream components of the pathway were generally associated with a more severe cognitive impairment. Among patients with a heterozygous PTPN11 mutation, the T468M substitution was associated with a mean IQ significantly higher compared to that of individuals carrying the N308D change. Our study provides insights on the range of cognitive abilities in patients with gene mutations causing dysregulation of RAS signaling suggesting that the presence and severity of cognitive involvement can be predicted in part by the gene involved.

    Funded by: Telethon: GGP07115

    American journal of medical genetics. Part A 2009;149A;2;140-6

  • HTLV-1 Tax protein cooperates with Ras in protecting cells from apoptosis.

    Vajente N, Trevisan R and Saggioro D

    Department of Oncology and Surgical Sciences, Oncology Section, University of Padova, via Gattamelata 64, 35128 Padova, Italy.

    Tax protein of the human T-cell leukemia virus type 1 (HTLV-1) plays a critical role in HTLV-I-correlated diseases through its ability to deregulate the expression of a vast array of cellular genes. We have previously shown that Tax counteracts apoptosis induced by stimuli triggering mitochondria apoptotic pathway, most likely by activating CREB-mediated transcription and affecting the phosphorylation levels of CREB at Ser-133. Here, we report data that indicate the oncoprotein Ras as a possible mediator of Tax-induced apoptosis protection and suggest a possible role of Tax in Ras activation. In addition, using inhibitors of down stream effectors of Ras, we found that ERK signaling is the most relevant for Tax-mediated apoptosis protection. As a whole, our findings provide intriguing evidence of a possible link between Ras signaling and Tax capability to counteract apoptosis and to enhance P-CREB levels, and implicates a potential role for Ras in HTLV-1-induced diseases.

    Apoptosis : an international journal on programmed cell death 2009;14;2;153-63

  • Blocking of p53-Snail binding, promoted by oncogenic K-Ras, recovers p53 expression and function.

    Lee SH, Lee SJ, Jung YS, Xu Y, Kang HS, Ha NC and Park BJ

    Department of Molecular Biology, College of Natural Science, Pusan National University, Geumjeong-gu, Busan, South Korea.

    Differentially from other kinds of Ras, oncogenic K-Ras, which is mutated approximately 30% of human cancer, does not induce apoptosis and senescence. Here, we provide the evidence that oncogenic K-Ras abrogates p53 function and expression through induction of Ataxia telangiectasia-mutated and Rad3-related mediated Snail stabilization. Snail directly binds to DNA binding domain of p53 and diminishes the tumor-suppressive function of p53. Thus, elimination of Snail through si-RNA can induce p53 in K-Ras-mutated cells, whereas Snail and mutant K-Ras can suppress p53 in regardless of K-Ras status. Chemicals, isolated from inhibitor screening of p53-Snail binding, can block the Snail-mediated p53 suppression and enhance the expression of p53 as well as the transcriptional activity of p53 in an oncogenic K-Ras-dependent manner. Among the chemicals, two are very similar in structure. These results can answer why K-Ras can coexist with wild type p53 and propose the Snail-p53 binding as the new therapeutic target for K-Ras-mutated cancers including pancreatic, lung, and colon cancers.

    Neoplasia (New York, N.Y.) 2009;11;1;22-31, 6p following 31

  • LDLs stimulate p38 MAPKs and wound healing through SR-BI independently of Ras and PI3 kinase.

    Bulat N, Waeber G and Widmann C

    Department of Physiology and Department of Cell Biology and Morphology, Lausanne University, 1005 Lausanne, Switzerland.

    Intracellular signals elicited by LDLs are likely to play a role in the pathogenesis associated with increased LDL blood levels. We have previously determined that LDL stimulation of human skin fibroblasts, used as a model system for adventitial fibroblasts, activates p38 mitogen-activated protein kinases (MAPKs), followed by IL-8 production and increased wound-healing capacity of the cells. The proximal events triggering these responses had not been characterized, however. Here we show that MAPK kinases MKK3 and MKK6, but not MKK4, are the upstream kinases responsible for the activation of the p38 MAPKs and stimulation of wound closure in response to LDLs. Phosphoinositide 3 kinases (PI3Ks) and Ras have been suggested to participate in lipoprotein-induced MAPK activation. However, specific PI3K inhibitors or expression of a dominant-negative form of Ras failed to blunt LDL-induced p38 MAPK activation. The classical LDL receptor does not participate in LDL signaling, but the contribution of other candidate lipoprotein receptors has not been investigated. Using cells derived from scavenger receptor class B type I (SR-BI) knockout mice or the BLT-1 SR-BI inhibitor, we now show that this receptor is required for LDLs to stimulate p38 MAPKs and to promote wound healing. Identification of MKK3/6 and SR-BI as cellular relays in LDL-mediated p38 activation further defines the signaling events that could participate in LDL-mediated pathophysiological responses.

    Journal of lipid research 2009;50;1;81-9

  • Membrane binding of lipidated Ras peptides and proteins--the structural point of view.

    Brunsveld L, Waldmann H and Huster D

    Max Planck Institute of Molecular Physiology, Dortmund, Germany.

    Biological membranes are interesting interfaces, at which important biological processes occur. In addition to integral membrane proteins, a number of proteins bind to the membrane surface and associate with it. Posttranslational lipid modification is one important mechanism, by which soluble molecules develop a propensity towards the membrane and reversibly bind to it. Membrane binding by insertion of hydrophobic lipid moieties is relevant for up to 10% of all cellular proteins. A particular interesting lipid-modified protein is the small GTPase Ras, which plays a key role in cellular signal transduction. Until recently, the structural basis for membrane binding of Ras was not well-defined. However, with the advent of new synthesis techniques and the advancement of several biophysical methods, a number of structural and dynamical features about membrane binding of Ras proteins have been revealed. This review will summarize the chemical biology of Ras and discuss in more detail the biophysical and structural features of the membrane bound C-terminus of the protein.

    Biochimica et biophysica acta 2009;1788;1;273-88

  • Analysis of candidate genes in occurrence and growth of colorectal adenomas.

    Olschwang S, Vernerey D, Cottet V, Pariente A, Nalet B, Lafon J, Faivre J, Laurent-Puig P, Bonithon-Kopp C and Bonaiti-Pellié C

    Centre de Recherche en Cancérologie de Marseille, INSERM U891, 13009 Marseille, France.

    Predisposition to sporadic colorectal tumours is influenced by genes with minor phenotypic effects. A case-control study was set up on 295 patients treated for a large adenoma matched with polyp-free individuals on gender, age, and geographic origin in a 1 : 2 proportion. A second group of 302 patients treated for a small adenoma was also characterized to distinguish effects on adenoma occurrence and growth. We focussed the study on 38 single nucleotide polymorphisms (SNPs) encompassing 14 genes involved in colorectal carcinogenesis. Effect of SNPs was tested using unconditional logistic regression. Comparisons were made for haplotypes within a given gene and for biologically relevant genes combinations using the combination test. The APC p.Glu1317Gly variant appeared to influence the adenoma growth (P = .04, exact test) but not its occurrence. This result needs to be replicated and genome-wide association studies may be necessary to fully identify low-penetrance alleles involved in early stages of colorectal tumorigenesis.

    Journal of oncology 2009;2009;306786

  • Cell type-specific importance of ras-c-raf complex association rate constants for MAPK signaling.

    Kiel C and Serrano L

    European molecular biology laboratory-centre for genomic regulation (CRG), systems biology unit, university pompeu fabra (UPF), Universitat Pompeu Fabra, Dr. Aiguader 88, 08003 Barcelona, Spain. christina.kiel@crg.es

    We generated 17 c-Raf (RAF proto-oncogene serine-threonine protein kinase) mutants with altered Ras-Raf association and dissociation rates to investigate the role of electrostatically driven Ras-Raf association rates on epidermal growth factor (EGF)-activated mitogen-activated protein kinase (MAPK) signal transduction. Some of these mutants had compensating changes in association and dissociation rates, enabling the effects of changes in association rate to be distinguished from those of changes in affinity. In rabbit kidney (RK13) cells, these mutants affected downstream signaling, with changes in Ras-c-Raf association rates having a greater effect on MAPK signaling than did similar changes in dissociation rates. Mutants with compensating decreases in both association and dissociation rates stimulated less extracellular signal-regulated kinase (ERK)-dependent reporter activity than did wild-type c-Raf, whereas the converse was true for mutants with increased association and dissociation rates. In marked contrast, the mutants had little or no effect on signaling in human embryonic kidney (HEK) 293 cells. These two cell lines also showed distinct patterns of EGF-dependent ERK phosphorylation and signaling: ERK activation and signaling were transient in HEK293 cells and sustained in RK13 cells, with the difference resulting from the lack of negative feedback from ERK to Sos (Son of Sevenless) in the latter. Computer simulation revealed that, in the presence of negative feedback, changes in the rate of Ras-c-Raf binding have little effect on ERK activation. Thus, EGF-MAPK activation kinetics and feedback regulation is cell type specific and depends on the network topology.

    Science signaling 2009;2;81;ra38

  • Multiple genetic variants along candidate pathways influence plasma high-density lipoprotein cholesterol concentrations.

    Lu Y, Dollé ME, Imholz S, van 't Slot R, Verschuren WM, Wijmenga C, Feskens EJ and Boer JM

    Division of Human Nutrition, Wageningen University, Wageningen, The Netherlands. kevin.lu@wur.nl

    The known genetic variants determining plasma HDL cholesterol (HDL-C) levels explain only part of its variation. Three hundred eighty-four single nucleotide polymorphisms (SNPs) across 251 genes based on pathways potentially relevant to HDL-C metabolism were selected and genotyped in 3,575 subjects from the Doetinchem cohort, which was examined thrice over 11 years. Three hundred fifty-three SNPs in 239 genes passed the quality-control criteria. Seven SNPs [rs1800777 and rs5882 in cholesteryl ester transfer protein (CETP); rs3208305, rs328, and rs268 in LPL; rs1800588 in LIPC; rs2229741 in NRIP1] were associated with plasma HDL-C levels with false discovery rate (FDR) adjusted q values (FDR_q) < 0.05. Five other SNPs (rs17585739 in SC4MOL, rs11066322 in PTPN11, rs4961 in ADD1, rs6060717 near SCAND1, and rs3213451 in MBTPS2 in women) were associated with plasma HDL-C levels with FDR_q between 0.05 and 0.2. Two less well replicated associations (rs3135506 in APOA5 and rs1800961 in HNF4A) known from the literature were also observed, but their significance disappeared after adjustment for multiple testing (P = 0.008, FDR_q = 0.221 for rs3135506; P = 0.018, FDR_q = 0.338 for rs1800961, respectively). In addition to replication of previous results for candidate genes (CETP, LPL, LIPC, HNF4A, and APOA5), we found interesting new candidate SNPs (rs2229741 in NRIP1, rs3213451 in MBTPS2, rs17585739 in SC4MOL, rs11066322 in PTPN11, rs4961 in ADD1, and rs6060717 near SCAND1) for plasma HDL-C levels that should be evaluated further.

    Journal of lipid research 2008;49;12;2582-9

  • Polymorphism in the IL18 gene and epithelial ovarian cancer in non-Hispanic white women.

    Palmieri RT, Wilson MA, Iversen ES, Clyde MA, Calingaert B, Moorman PG, Poole C, Anderson AR, Anderson S, Anton-Culver H, Beesley J, Hogdall E, Brewster W, Carney ME, Chen X, Chenevix-Trench G, Chang-Claude J, Cunningham JM, Dicioccio RA, Doherty JA, Easton DF, Edlund CK, Gayther SA, Gentry-Maharaj A, Goode EL, Goodman MT, Kjaer SK, Hogdall CK, Hopkins MP, Jenison EL, Blaakaer J, Lurie G, McGuire V, Menon U, Moysich KB, Ness RB, Pearce CL, Pharoah PD, Pike MC, Ramus SJ, Rossing MA, Song H, Terada KY, Vandenberg D, Vierkant RA, Wang-Gohrke S, Webb PM, Whittemore AS, Wu AH, Ziogas A, Berchuck A, Schildkraut JM, Ovarian Cancer Association Consortium, Australian Cancer Study (Ovarian Cancer Group) and Australian Ovarian Cancer Study Group

    Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

    Over 22,000 cases of ovarian cancer were diagnosed in 2007 in the United States, but only a fraction of them can be attributed to mutations in highly penetrant genes such as BRCA1. To determine whether low-penetrance genetic variants contribute to ovarian cancer risk, we genotyped 1,536 single nucleotide polymorphisms (SNP) in several candidate gene pathways in 848 epithelial ovarian cancer cases and 798 controls in the North Carolina Ovarian Cancer Study (NCO) using a customized Illumina array. The inflammation gene interleukin-18 (IL18) showed the strongest evidence for association with epithelial ovarian cancer in a gene-by-gene analysis (P = 0.002) with a <25% chance of being a false-positive finding (q value = 0.240). Using a multivariate model search algorithm over 11 IL18 tagging SNPs, we found that the association was best modeled by rs1834481. Further, this SNP uniquely tagged a significantly associated IL18 haplotype and there was an increased risk of epithelial ovarian cancer per rs1834481 allele (odds ratio, 1.24; 95% confidence interval, 1.06-1.45). In a replication stage, 12 independent studies from the Ovarian Cancer Association Consortium (OCAC) genotyped rs1834481 in an additional 5,877 cases and 7,791 controls. The fixed effects estimate per rs1834481 allele was null (odds ratio, 0.99; 95% confidence interval, 0.94-1.05) when data from the 12 OCAC studies were combined. The effect estimate remained unchanged with the addition of the initial North Carolina Ovarian Cancer Study data. This analysis shows the importance of consortia, like the OCAC, in either confirming or refuting the validity of putative findings in studies with smaller sample sizes. (Cancer Epidemiol Biomarkers Prev 2008;17(12):3567-72).

    Funded by: Cancer Research UK: 10118, A10119; Department of Health; NCI NIH HHS: 1-R01-CA122443, 1-R01-CA76016, CA-58860, CA14089, CA16056, CA17054, CA61132, CA63464, CA71766, K07 CA092044, N01 CN025403, N01 PC067010, N01-CN-25403, N01-CN-67001, P01 CA017054, P30 CA014089, P30 CA016056, R01 CA058598, R01 CA058860, R01 CA063464, R01 CA076016, R01 CA076016-06A1, R01 CA076016-07, R01 CA076016-08, R01 CA076016-09, R01 CA076016-10, R01 CA095023, R01 CA112523, R01 CA122443, R01-CA-58598, R01CA095023, R01CA112523, R03 CA113148, R03-CA113148, T32 CA009330, T32 CA009330-26, U01 CA058860, U01 CA063464; PHS HHS: 01 GB 9401, 050E8709

    Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 2008;17;12;3567-72

  • Restoration of E-cadherin cell-cell junctions requires both expression of E-cadherin and suppression of ERK MAP kinase activation in Ras-transformed breast epithelial cells.

    Li Q and Mattingly RR

    Department of Pharmacology, Wayne State University, Detroit, MI 48201, USA.

    E-cadherin is a main component of the cell-cell adhesion junctions that play a principal role in maintaining normal breast epithelial cell morphology. Breast and other cancers that have up-regulated activity of Ras are often found to have down-regulated or mislocalized E-cadherin expression. Disruption of E-cadherin junctions and consequent gain of cell motility contribute to the process known as epithelial-to-mesenchymal transition (EMT). Enforced expression of E-cadherin or inhibition of Ras-signal transduction pathway has been shown to be effective in causing reversion of EMT in several oncogene-transformed and cancer-derived cell lines. In this study, we investigated MCF10A human breast epithelial cells and derivatives that were transformed with either activated H-Ras or N-Ras to test for the reversion of EMT by inhibition of Ras-driven signaling pathways. Our results demonstrated that inhibition of mitogen-activated protein kinase (MAPK) kinase, but not PI3-kinase, Rac, or myosin light chain kinase, was able to completely restore E-cadherin cell-cell junctions and epithelial morphology in cell lines with moderate H-Ras expression. In MCF10A cells transformed by a high-level expression of activated H-Ras or N-Ras, restoration of E-cadherin junction required both the enforced reexpression of E-cadherin and suppression of MAPK kinase. Enforced expression of E-cadherin alone did not induce reversion from the mesenchymal phenotype. Our results suggest that Ras transformation has at least two independent actions to disrupt E-cadherin junctions, with effects to cause both mislocalization of E-cadherin away from the cell surface and profound decrease in the expression of E-cadherin.

    Funded by: NCI NIH HHS: P30 CA22453, R01 CA131990, R01 CA131990-05; NIEHS NIH HHS: P30 ES06639

    Neoplasia (New York, N.Y.) 2008;10;12;1444-58

  • Secondary structure of lipidated Ras bound to a lipid bilayer.

    Güldenhaupt J, Adigüzel Y, Kuhlmann J, Waldmann H, Kötting C and Gerwert K

    Lehrstuhl für Biophysik, Ruhr-Universität Bochum, Germany.

    Ras proteins are small guanine nucleotide binding proteins that regulate many cellular processes, including growth control. They undergo distinct post-translational lipid modifications that are required for appropriate targeting to membranes. This, in turn, is critical for Ras biological function. However, most in vitro studies have been conducted on nonlipidated truncated forms of Ras proteins. Here, for the first time, attenuated total reflectance-FTIR studies of lipid-modified membrane-bound N-Ras are performed, and compared with nonlipidated truncated Ras in solution. For these studies, lipidated N-Ras was prepared by linking a farnesylated and hexadecylated N-Ras lipopeptide to a truncated N-Ras protein (residues 1-181). It was then bound to a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer tethered on an attenuated total reflectance crystal. The structurally sensitive amide I absorbance band in the IR was detected and analysed to determine the secondary structure of the protein. The NMR three-dimensional structure of truncated Ras was used to calibrate the contributions of the different secondary structural elements to the amide I absorbance band of truncated Ras. Using this novel approach, the correct decomposition was selected from several possible solutions. The same parameter set was then used for the membrane-bound lipidated Ras, and provided a reliable decomposition for the membrane-bound form in comparison with truncated Ras. This comparison indicates that the secondary structure of membrane-bound Ras is similar to that determined for the nonlipidated truncated Ras protein for the highly conserved G-domain. This result validates the multitude of investigations of truncated Ras without anchor in vitro. The novel attenuated total reflectance approach opens the way for detailed studies of the interaction network of the membrane-bound Ras protein.

    The FEBS journal 2008;275;23;5910-8

  • Binding of calcium ions to Ras promotes Ras guanine nucleotide exchange under emulated physiological conditions.

    Liao JM, Mo ZY, Wu LJ, Chen J and Liang Y

    State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China.

    Both Ras protein and calcium play significant roles in various cellular processes via complex signaling transduction networks. However, it is not well understood whether and how Ca(2+) can directly regulate Ras function. Here we demonstrate by isothermal titration calorimetry that Ca(2+) directly binds to the H-Ras.GDP.Mg(2+) complex with moderate affinity at the first binding site followed by two weak binding events. The results from limited proteinase degradation show that Ca(2+) protects the fragments of H-Ras from being further degraded by trypsin and by proteinase K. HPLC studies together with fluorescence spectroscopic measurements indicate that binding of Ca(2+) to the H-Ras.GDP.Mg(2+) complex remarkably promotes guanine nucleotide exchange on H-Ras under emulated physiological Ca(2+) concentration conditions. Addition of high concentrations of either of two macromolecular crowding agents, Ficoll 70 and dextran 70, dramatically enhances H-Ras guanine nucleotide exchange extent in the presence of Ca(2+) at emulated physiological concentrations, and the nucleotide exchange extent increases significantly with the concentrations of crowding agents. Together, these results indicate that binding of calcium ions to H-Ras remarkably promotes H-Ras guanine nucleotide exchange under emulated physiological conditions. We thus propose that Ca(2+) may activate Ras signaling pathway by interaction with Ras, providing clues to understand the role of calcium in regulating Ras function in physiological environments.

    Biochimica et biophysica acta 2008;1784;11;1560-9

  • [Molecular-genetic analysis of clonal intratumoral heterogeneity on colorectal adenocarcinomas].

    Nemtsova MN, Pal'tseva EM, Babaian AIu, Mikhaĭlenko DS, Babenko OV, Samofalova OIu, Tsar'kov PV and Zaletaev DV

    We have examined the existence of intratumoral genetic heterogeneity for LOH on chromosomes 9p21 (p16, p15, p19), 13p14 (RB1), 10q23 (PTEN), 17p (TP53), microsatellite instability and K-RAS point mutations on four different segments of sporadic colorectal cancers. The intratumoral genetic heterogenity was detected in 9/11 (81%) colorectal adenocarcinomas and morphologically validated. These results show that colorectal cancer is highly heterogeneous for these molecular markers. Furthermore, the analysis has shown the order (succession) of the appearance of these molecular anomalies during tumorigenesis on sporadic CRC, and supposed, that K-RAS point mutations, and anomalies of p16-RB1-cyclin D pathway could occur before LOH on 10q23 (PTEN) and microsatellite instability during tumor progression.

    Molekuliarnaia biologiia 2008;42;6;1040-7

  • Loss of human Scribble cooperates with H-Ras to promote cell invasion through deregulation of MAPK signalling.

    Dow LE, Elsum IA, King CL, Kinross KM, Richardson HE and Humbert PO

    Research Division, Cell Cycle and Cancer Genetics, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.

    Activating mutations in genes of the Ras-mitogen-activated protein kinase (MAPK) pathway occur in approximately 30% of all human cancers; however, mutation of Ras alone is rarely sufficient to induce tumour development. Scribble is a polarity regulator recently isolated from a Drosophila screen for events that cooperate with Ras mutation to promote tumour progression and cell invasion. In mammals, Scribble regulates directed cell migration and wound healing in vivo; however, no role has been identified for mammalian Scribble in oncogenic transformation. Here we show that in human epithelial cells expressing oncogenic Ras or Raf, loss of Scribble promotes invasion of cells through extracellular matrix in an organotypic culture system. Further, we show that the mechanism by which this occurs is in the regulation of MAPK signalling by Scribble. The suppression of MAPK signalling is a highly conserved function of Scribble as it also prevents Raf-mediated defects in Drosophila wing development. Our data identify Scribble as an important mediator of MAPK signalling and provide a molecular basis for the observation that Scribble expression is decreased in many invasive human cancers.

    Oncogene 2008;27;46;5988-6001

  • A homologous promoterless K-ras cDNA targeting endogenous K-ras expression inhibits human pancreatic cancer cell growth in vitro and in vivo.

    Ren XY, Liang ZY, Shi XH and Liu TH

    Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, PR China.

    It has been reported that the local introduction of a promoter-less DNA containing the complementary DNA (cDNA) sequence of a gene could induce gene-specific silencing in plants. The feasibility of this kind of silencing in human cancer cells is as yet unknown. The current study was designed to investigate the anti-tumor effects of a homologous promoterless K-ras cDNA system on pancreatic cancer. A full-length K-ras cDNA fragment was cloned into the promoterless plasmid puc19 to yield puc-K-ras. This construct was then transfected into pancreatic cancer cells. Our results demonstrated that the transfection of a promoterless K-ras cDNA resulted in a significant decrease in endogenous K-ras in a dose- and time-dependent manner and induced pancreatic cell apoptosis. Furthermore, stable puc-K-ras transfection decreased the endogenous protein level of K-ras and inhibited cell proliferation, clone formation and tumorigenicity in vivo. These findings indicate a promising application of this homologous promoterless cDNA silencing system in pancreatic cancer gene therapy.

    Cancer biology & therapy 2008;7;10;1641-7

  • Extremely weak tumor-promoting effect of troglitazone on splenic hemangiosarcomas in rasH2 mice induced by urethane.

    Jin M, Matsumoto S, Dewa Y, Nishimura J, Saekusa Y, Hasumi K and Mitsumori K

    Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan. meilan@cc.tuat.ac.jp

    To examine the tumor-promoting effect of troglitazone (TRG), a novel thiazolidinedione insulin-sensitizing agent, on splenic hemangiosarcomas in rasH2 mice, histopathological and molecular analyses were performed in the spleen of female rasH2 mice fed a diet containing 6,000 or 0 ppm TRG for 16 weeks after 1,000 or 0 mg/kg urethane (UR) initiation. Histopathologically, splenic hemangiosarcomas were observed in the UR-alone and UR + TRG groups, but there was no significant difference in the incidence of splenic hemangiosarcomas between the UR-alone and UR+TRG groups. There were increasing tendencies in the number of positive cells for anti-PCNA antibody and gene expression in the UR + TRG group, but such a change was not statistically significant as compared to that in the UR-alone group. The gene expressions of VEGF, VEGFR1, VEGFC, VEGFR2 and Tie2 related to angiogenesis; c-fos related to MAPK cascade activation; and cyclin D1 related to cell cycle in the UR-alone and UR + TRG groups were significantly higher than those in the untreated control group. However, only the Tie2 gene in the UR + TRG group was significantly increased as compared to that in the UR-alone group. These results suggest that the vascular tumor-promoting activity of TRG in rasH2 mice is extremely low in the present experimental condition and a part of the gene related to angiogenesis probably contributes to the promotion of splenic hemangiosarcomas in rasH2 mice given TRG.

    Archives of toxicology 2008;82;10;771-7

  • Global gene expression profiling unveils S100A8/A9 as candidate markers in H-ras-mediated human breast epithelial cell invasion.

    Moon A, Yong HY, Song JI, Cukovic D, Salagrama S, Kaplan D, Putt D, Kim H, Dombkowski A and Kim HR

    College of Pharmacy, Duksung Women's University, Seoul, Korea. armoon@duksung.ac.kr

    The goal of the present study is to unveil the gene expression profile specific to the biological processes of human breast epithelial cell invasion and migration using an MCF10A model genetically engineered to constitutively activate the H-ras or N-ras signaling pathway. We previously showed that H-Ras, but not N-Ras, induces MCF10A cell invasion/migration, whereas both H-Ras and N-Ras induce cell proliferation and phenotypic transformation. Thus, these cell lines provide an experimental system to separate the gene expression profile associated with cell invasion apart from cell proliferation/transformation. Analysis of whole human genome microarray revealed that 412 genes were differentially expressed among MCF10A, N-Ras MCF10A, and H-Ras MCF10A cells and hierarchical clustering separated 412 genes into four clusters. We then tested whether S100A8 and S100A9, two of the genes which are most highly up-regulated in an H-Ras-specific manner, play a causative role for H-Ras-mediated MCF10A cell invasion and migration. Importantly, small interfering RNA-mediated knockdown of S100A8/A9 expression significantly reduced H-Ras-induced invasion/migration. Conversely, the induction of S100A8/A9 expression conferred the invasive/migratory phenotype to parental MCF10A cells. Furthermore, we provided evidence of signaling cross-talk between S100A8/A9 and the mitogen-activated protein kinase signaling pathways essential for H-Ras-mediated cell invasion and migration. Taken together, this study revealed S100A8/A9 genes as candidate markers for metastatic potential of breast epithelial cells. Our gene profile data provide useful information which may lead to the identification of additional potential targets for the prognosis and/or therapy of metastatic breast cancer.

    Funded by: NCI NIH HHS: R41 CA105578; NIEHS NIH HHS: P30 ES06639

    Molecular cancer research : MCR 2008;6;10;1544-53

  • p21 Ras/impedes mitogenic signal propagation regulates cytokine production and migration in CD4 T cells.

    Czyzyk J, Chen HC, Bottomly K and Flavell RA

    Departments of Pathology and Immunobiology.

    The propensity of T cells to generate coordinated cytokine responses is critical for the host to develop resistance to pathogens while maintaining the state of immunotolerance to self-antigens. The exact mechanisms responsible for preventing the overproduction of proinflammatory cytokines including interferon (IFN)-gamma are not fully understood, however. In this study, we examined the role of a recently described Ras GTPase effector and repressor of the Raf/MEK/ERK cascade called impedes mitogenic signal propagation (Imp) in limiting the induction of T-cell cytokines. We found that stimulation of the T cell receptor complex leads to the rapid development of a physical association between Ras and Imp. Consistent with the hypothesis that Imp inhibits signal transduction, we also found that disengagement of this molecule by the Ras(V12G37) effector loop mutant or RNA interference markedly enhances the activation of the NFAT transcription factor and IFN-gamma secretion. A strong output of IFN-gamma is responsible for the distinct lymphocyte traffic pattern observed in vivo because the transgenic or retroviral expression of Ras(V12G37) caused T cells to accumulate preferentially in the lymph nodes and delayed their escape from the lymphoid tissue, respectively. Together, our results describe a hitherto unrecognized negative regulatory role for Imp in the production of IFN-gamma in T cells and point to Ras-Imp binding as an attractive target for therapeutic interventions in conditions involving the production of this inflammatory cytokine.

    Funded by: PHS HHS: R01 5R37A102679-1

    The Journal of biological chemistry 2008;283;34;23004-15

  • p19(ras) amplifies p73beta-induced apoptosis through mitochondrial pathway.

    Kim JW, Kim WH, Jeong MH, Jang SM, Song KH, Park SI, Song PI, Kang KH and Choi KH

    Department of Life Science, College of Natural Sciences, Chung-Ang University, 221 Heuksuk Dong, Dongjak Ku, Seoul 156-756, Republic of Korea.

    p73 and p53 have been known to play an important role in cellular damage responses such as apoptosis. Although p73 is a structural and functional homolog of p53 tumor suppressor gene, much less is known about the mechanism of p73-induced apoptotic cell death. In this study, we demonstrate that p19(ras) interaction with p73beta amplifies p73beta-induced apoptotic signaling responses including Bax mitochondrial translocation, cytochrome c release, increased production of reactive oxygen species (ROS) and loss of mitochondrial transmembrane potential (DeltaPsi(m)). Furthermore, endogenous expression of p19(ras) and p73beta is significantly increased by Taxol treatment, and Taxol-enhanced endogenous p73beta transcriptional activities are further amplified by p19(ras), which markedly increased cellular apoptosis in p53-null SAOS2 cancer cell line. These results have important implications for understanding the molecular events of p19(ras) to p73 functions in cancer cells.

    Biochemical and biophysical research communications 2008;373;1;146-50

  • K-Ras mutations and treatment outcome in colorectal cancer patients receiving exclusive fluoropyrimidine therapy.

    Etienne-Grimaldi MC, Formento JL, Francoual M, François E, Formento P, Renée N, Laurent-Puig P, Chazal M, Benchimol D, Delpero JR, Letoublon C, Pezet D, Seitz JF and Milano G

    Oncopharmacology Department, Centre Antoine Lacassagne, Université René Descartes, Paris, France.

    Purpose: K-Ras mutations predict resistance to anti-epidermal growth factor receptor (EGFR) monoclonal antibodies. Because combinations of anti-EGFR with 5-fluorouracil (5-FU)-based chemotherapy are promising treatments, we analyzed the effect of K-Ras mutations in patients having received exclusive 5-FU therapy.

    This study was conducted on 93 stage IV colorectal cancer patients with unresectable measurable liver metastasis receiving 5-FU-leucovorin (56 men and 37 women; 77 cancer deaths). Liver metastases (n = 93) along with primary tumors (n = 48) were analyzed for K-Ras mutations (codons 12 and 13), p53 mutations (exons 4-9), p53 polymorphism (codon 72), thymidylate synthase (TS) polymorphism (28-bp repeats including G>C mutation), methylenetetrahydrofolate reductase polymorphism (677C>T, 1298A>C), thymidylate synthase (TS) activity, dihydropyrimidine dehydrogenase activity, folylpolyglutamate synthase activity, and p53 protein expression.

    Results: Thirty-six of 93 (38.7%) metastases were K-Ras mutated (30 at codon 12 and 6 at codon 13). Mutated primary tumors (16 of 48) matched perfectly with mutated metastases. The additional analyzed tumor markers were not different between K-Ras mutated and wild-type tumors. The objective response rate was 37%: 44.4% in K-Ras mutated versus 32.1% in wild-type K-Ras metastasis (P = 0.27). Low TS activity in metastasis was the only significant predictor of tumor response (P = 0.047). K-Ras status did not influence specific survival.

    Conclusions: The present data indicate a perfect concordance of K-Ras mutations between primary and liver metastasis and suggest that any predictive and/or prognostic value of K-Ras mutations in treatments combining anti-EGFR monoclonal antibodies with 5-FU should be exclusively linked to the anti-EGFR agent.

    Clinical cancer research : an official journal of the American Association for Cancer Research 2008;14;15;4830-5

  • Low-penetrant RB allele in small-cell cancer shows geldanamycin instability and discordant expression with mutant ras.

    Park Y, Kubo A, Komiya T, Coxon A, Beebe K, Neckers L, Meltzer PS and Kaye FJ

    Genetics Branch, Center for Cancer Research, National Cancer Institute and National Naval Medical Center, Bethesda, Maryland 20889, USA.

    Certain kindreds with low-penetrant (lp) retinoblastoma carry mutant alleles which retain partial tumor suppressor activity and we previously showed that these alleles exhibit defective, temperature-sensitive binding in yeast. To investigate the molecular basis for incomplete penetrance, we studied three recurrent lp alleles and observed approximately 50% of wildtype activity measured by (i) phosphorylation at key regulatory sites, S780, S795, S807/S811, (ii) transcriptional co-activation, and (iii) 'flat-cell' differentiation in mammalian cells in vivo. In addition, we studied a small-cell carcinoma that is homozygous for the R661W allele providing the first analysis of the effect of a naturally occurring lp allele in a human tumor. While we detected abundant expression of the R661W protein, we noted marked instability of both endogenous and recombinant R661W following treatment in vivo with the Hsp90 inhibitor, geldanamycin and stabilization of R661W following heat shock. In addition, we observed a discordant phenotype in the tumor cells with induction of p16 and loss of cyclin D1 consistent with a null RB status combined with homozygous expression of mutant ras which had not been reported previously for RB (-) small-cell cancer. These findings show that a recurrent missense lp allele retains greater functional activity in vivo than predicted from earlier in vitro assays, proposing a role for stabilizing chaperone-like activity in vivo. In addition, these data suggest that reversible protein instability and the requirement for a cooperating mutation may provide a stochastic explanation for the molecular basis of incomplete penetrance in kindreds carrying these alleles.

    Funded by: NCI NIH HHS: Z01 SC007256-19

    Cell cycle (Georgetown, Tex.) 2008;7;15;2384-91

  • P53 mutations in colorectal cancer from northern Iran: Relationships with site of tumor origin, microsatellite instability and K-ras mutations.

    Mahdavinia M, Bishehsari F, Verginelli F, Cumashi A, Lattanzio R, Sotoudeh M, Ansari R, Semeraro D, Hormazdi M, Fakheri H, Rakhshani N, De Lellis L, Curia MC, Cama A, Piantelli M, Malekzadeh R, Iacobelli S and Mariani-Costantini R

    Department of Oncology and Neurosciences, University G. d'Annunzio, and Center of Excellence on Aging (CeSI), G. d'Annunzio University Foundation, Chieti, Italy.

    CRC-associated P53 mutations have not been studied extensively in non-Western countries at relatively low CRC risk. We examined, for the first time, 196 paraffin-embedded CRC cases from Northern Iran for mutations in P53 exons 5-8 using PCR-direct sequencing. P53 status and mutation site/type were correlated with nuclear protein accumulation, clinicopathologic variables and data on K-ras mutations and high-level microsatellite instability (MSI-H). We detected 96 P53 mutations in 87 (44.4%) cases and protein accumulation in 84 cases (42.8%). P53 mutations correlated directly with stage and inversely with MSI-H. Distal CRCs were more frequently mutated at major CpG hotspot codons [248 (8/66, 12.1%), 175 (7/66, 10.6%), and 245 (7/66, 10.6%)], while in proximal tumors codon 213, emerged as most frequently mutated (5/28, 17.9% vs. 3/66, 4.5%, P = 0.048). Transitions at CpGs, the most common mutation type, were more frequent in non-mucinous (25% vs. 10.4% in mucinous, P = 0.032), and distal CRC (27% vs. 12.5% in proximal, P = 0.02), and correlated with K-ras transversions. Transitions at non-CpGs, second most common P53 mutation, were more frequent in proximal tumors (15.6% vs. 4.7% in distal, P = 0.01), and correlated with K-ras transitions and MSI-H. Overall frequency and types of mutations and correlations with P53 accumulation, stage and MSI-H were as reported for non-Iranian patients. However P53 mutation site/type and correlations between P53 and K-ras mutation types differed between proximal and distal CRC. The codon 213 P53 mutation that recurred in proximal CRC was previously reported as frequent in esophageal cancer from Northern Iran.

    Journal of cellular physiology 2008;216;2;543-50

  • Molecular detection (k-ras) of exfoliated tumour cells in the pelvis is a prognostic factor after resection of rectal cancer?

    Kristensen AT, Wiig JN, Larsen SG, Giercksky KE and Ekstrøm PO

    Section for Surgical Oncology, Rikshospitalet-Radiumhospitalet Medical Center, Montebello, Oslo, Norway. a.t.kristensen@medisin.uio.no

    Background: After total mesorectal excision (TME) for rectal cancer around 10% of patients develops local recurrences within the pelvis. One reason for recurrence might be spillage of cancer cells during surgery. This pilot study was conducted to investigate the incidence of remnant cancer cells in pelvic lavage after resection of rectal cancer. DNA from cells obtained by lavage, were analysed by denaturing capillary electrophoresis with respect to mutations in hotspots of the k-ras gene, which are frequently mutated in colorectal cancer.

    Results: Of the 237 rectal cancer patients analyzed, 19 had positive lavage fluid. There was a significant survival difference (p = 0.006) between patients with k-ras positive and negative lavage fluid.

    Conclusion: Patients with k-ras mutated cells in the lavage immediately after surgery have a reduced life expectation. Detection of exfoliated cells in the abdominal cavity may be a useful diagnostic tool to improve the staging and eventually characterize patients who may benefit from aggressive multimodal treatment of rectal cancer.

    BMC cancer 2008;8;213

  • Alphavirus production is inhibited in neurofibromin 1-deficient cells through activated RAS signalling.

    Kolokoltsova OA, Domina AM, Kolokoltsov AA, Davey RA, Weaver SC and Watowich SJ

    Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA. oakoloko@utmb.edu

    Virus-host interactions essential for alphavirus pathogenesis are poorly understood. To address this shortcoming, we coupled retrovirus insertional mutagenesis and a cell survival selection strategy to generate clonal cell lines broadly resistant to Sindbis virus (SINV) and other alphaviruses. Resistant cells had significantly impaired SINV production relative to wild-type (WT) cells, although virus binding and fusion events were similar in both sets of cells. Analysis of the retroviral integration sites identified the neurofibromin 1 (NF1) gene as disrupted in alphavirus-resistant cell lines. Subsequent analysis indicated that expression of NF1 was significantly reduced in alphavirus-resistant cells. Importantly, independent down-regulation of NF1 expression in WT HEK 293 cells decreased virus production and increased cell viability during SINV infection, relative to infected WT cells. Additionally, we observed hyperactive RAS signalling in the resistant HEK 293 cells, which was anticipated because NF1 is a negative regulator of RAS. Expression of constitutively active RAS (HRAS-G12V) in a WT HEK 293 cell line resulted in a marked delay in virus production, compared with infected cells transfected with parental plasmid or dominant-negative RAS (HRAS-S17N). This work highlights novel host cell determinants required for alphavirus pathogenesis and suggests that RAS signalling may play an important role in neuronal susceptibility to SINV infection.

    Funded by: NIAID NIH HHS: 2 T32 AI007536-06, AI48807, AI53551-01, R01 AI048807, R01 AI048807-01, R21 AI053551, R21 AI053551-01, T32 AI007536, T32 AI007536-06

    Virology 2008;377;1;133-42

  • The nitric oxide-sensitive p21Ras-ERK pathway mediates S-nitrosoglutathione-induced apoptosis.

    Tsujita M, Batista WL, Ogata FT, Stern A, Monteiro HP and Arai RJ

    Departamento de Bioquímica/Biologia Molecular, Centro Interdisciplinar de Terapia Gênica CINTERGEN, Universidade Federal de São Paulo, Escola Paulista de Medicina, São Paulo, Brazil.

    p21Ras protein plays a critical role in cellular signaling that induces either cell cycle progression or apoptosis. Nitric oxide (NO) has been consistently reported to activate p21Ras through the redox sensitive cysteine residue (118). In this study, we demonstrated that the p21Ras-ERK pathway regulates THP-1 monocyte/macrophage apoptosis induced by S-nitrosoglutathione (SNOG). This was apparent from studies in THP-1 cells expressing NO-insensitive p21Ras (p21Ras(C118S)) where the pro-apoptotic action of SNOG was almost abrogated. Three major MAP kinase pathways (ERK, JNK, and p38) that are downstream to p21Ras were investigated. It was observed that only the activation of ERK1/2 MAP kinases by SNOG in THP-1 cells was attributable to p21Ras. The inhibition of the ERK pathway by PD98059 markedly attenuated apoptosis in SNOG-treated THP-1 cells, but had a marginal effect on SNOG-treated THP-1 cells expressing NO-insensitive p21Ras. The inhibition of the JNK and p38 pathways by selective inhibitors had no marked effects on the percentage of apoptosis. The induction of p21Waf1 expression by SNOG was observed in THP-1 cells harboring mutant and wild-type p21Ras, however in cells expressing mutant Ras, the expression of p21Waf1 was significantly attenuated. The treatment of THP-1 cells expressing wild-type p21Ras with PD98059 resulted in significant attenuation of p21Waf1 expression. These results indicate that the redox sensitive p21Ras-ERK pathway plays a critical role in sensing and delivering the pro-apoptotic signaling mediated by SNOG.

    Biochemical and biophysical research communications 2008;369;4;1001-6

  • FAD24, a regulator of adipogenesis and DNA replication, inhibits H-RAS-mediated transformation by repressing NF-kappaB activity.

    Johmura Y, Suzuki M, Osada S, Nishizuka M and Imagawa M

    Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan.

    We have previously reported that a novel gene, factor for adipocyte differentiation (fad) 24, promotes adipogenesis. Moreover, our recent findings indicated that this regulation involves the control of DNA replication by fad24 during the early stage in adipogenesis. Considering that abnormal regulation of DNA replication is linked to transformation, we examined whether the over-expression of fad24 leads to the formation of colonies in soft agarose. The over-expression itself generated no colonies. Rather, it inhibited oncogenic H-ras-mediated formation of colonies. The over-expression of histone acetyltransferase binding to ORC1 (HBO1), a partner of FAD24, also inhibited the H-ras-mediated colony-forming process. Furthermore, we revealed that FAD24 interacts with p65, a subunit of NF-kappaB which is known to be activated by H-ras. The over-expression of fad24 repressed NF-kappaB-mediated promoter activity and gene expression. Taken together, these results reveal a novel role for fad24 in the repression of NF-kappaB activity and H-ras-mediated transformation.

    Biochemical and biophysical research communications 2008;369;2;464-70

  • Alterations of the K-ras and p53 genes in Tamoxifen-associated endometrial carcinoma.

    Fujiwara K, Enomoto T, Fujita M, Kanda T, Fujii S, Ito K, Wakasa K, Ishiko O, Ueda M, Yamaguchi S, Kimura T and Umesaki N

    Department of Obstetrics and Gynecology, Osaka University, Graduate School of Medicine, Faculty of Medicine, Osaka 565-0871, Japan.

    To better understand the molecular mechanisms of carcinogenesis induced in uterine endometrium by therapeutic anti-estrogenic Tamoxifen (TAM) exposure, 27 uterine tumors (4 benign endometrial polyps and 23 carcinomas) associated with TAM exposure were analyzed for the presence and spectrum of p53 and K-ras mutations. Although there was no significant difference between TAM-associated endometrial carcinomas and sporadic endometrial tumors in the frequency of these mutations, the spectrum of p53 mutations was characteristically unique to the TAM-associated tumors. The median duration of TAM exposure was significantly longer in patients with p53 mutations than those without p53 mutations (62 vs. 30 months, p=0.028). Our observation suggests that prolonged TAM exposure may directly inactivate the p53 gene by acting as a mutagen in a significant fraction of TAM-associated endometrial carcinomas.

    Oncology reports 2008;19;5;1293-8

  • Association of K-ras mutational status and clinical outcomes in patients with metastatic colorectal cancer receiving panitumumab alone.

    Freeman DJ, Juan T, Reiner M, Hecht JR, Meropol NJ, Berlin J, Mitchell E, Sarosi I, Radinsky R and Amado RG

    Department of Oncology Research, Amgen Inc, Thousand Oaks, CA 91320, USA. dfreeman@amgen.com

    Background: Identifying predictive biomarkers is important to optimally treat patients. This analysis evaluated the association of K-ras, BRAF, and PIK3CA gene mutations with tumor resistance to panitumumab alone.

    From 3 phase II panitumumab metastatic colorectal cancer (mCRC) studies, 62 of 533 patient samples were available. Mutations were identified from genomic DNA by sequencing.

    Results: Of the 62 samples, 24 (38.7%) harbored a K-ras mutation, and 38 (61.3%) were wild type. In the wild-type K-ras group, 11% of patients had a partial response (PR), 53% had stable disease (SD), and 37% had progressive disease (PD). In the mutant K-ras group, 21% of patients had SD, and 79% of patients had PD; there were no responses. The absence of a K-ras mutation was associated with response to panitumumab (PR vs. SD vs. PD; P = .0028). The hazard ratio for wild-type versus mutant K-ras was 0.4 (95% CI, 0.2-0.7) for progression-free survival and 0.5 (95% CI, 0.3-0.9) for overall survival. Four patients had a V600E BRAF mutation, and 2 patients had a PIK3CA mutation.

    Conclusion: These data suggest that patients with mCRC with activating K-ras mutations are less likely to respond to panitumumab alone. The small sample size limits us from defining a predictive role of PIK3CA and BRAF mutations for panitumumab treatment.

    Clinical colorectal cancer 2008;7;3;184-90

  • Membrane-dependent signal integration by the Ras activator Son of sevenless.

    Gureasko J, Galush WJ, Boykevisch S, Sondermann H, Bar-Sagi D, Groves JT and Kuriyan J

    Department of Molecular and Cell Biology, and Howard Hughes Medical Institute, QB3 Institute, 176 Stanley Hall, University of California, Berkeley, California 94720, USA.

    The kinetics of Ras activation by Son of sevenless (SOS) changes profoundly when Ras is tethered to membranes, instead of being in solution. SOS has two binding sites for Ras, one of which is an allosteric site that is distal to the active site. The activity of the SOS catalytic unit (SOS(cat)) is up to 500-fold higher when Ras is on membranes compared to rates in solution, because the allosteric Ras site anchors SOS(cat) to the membrane. This effect is blocked by the N-terminal segment of SOS, which occludes the allosteric site. We show that SOS responds to the membrane density of Ras molecules, to their state of GTP loading and to the membrane concentration of phosphatidylinositol-4,5-bisphosphate (PIP2), and that the integration of these signals potentiates the release of autoinhibition.

    Funded by: Howard Hughes Medical Institute; NIGMS NIH HHS: R01 GM078266, R01 GM078266-01A1, T32 GM008295, T32 GM008295-10

    Nature structural & molecular biology 2008;15;5;452-61

  • PIK3CA, HRAS and KRAS gene mutations in human penile cancer.

    Andersson P, Kolaric A, Windahl T, Kirrander P, Söderkvist P and Karlsson MG

    Division of Cell Biology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden. patan@ibk.liu.se <e-mail:patan@ibk.liu.se&gt;

    Purpose: The knowledge of somatic mutations that arise in penile cancer is limited. We examined the dysregulation of components in the phosphatidylinositol 3-kinase and Ras pathways.

    Using single stranded conformational analysis and direct sequencing we performed mutational analysis of the PIK3CA, PTEN, HRAS, KRAS, NRAS and BRAF genes in 28 penile tumors.

    Results: We identified somatic missense mutations in 11 of the 28 penile cancer samples (39%). In the PIK3CA gene 8 mutations (29%) were identified that were E542K or E545K. In the HRAS gene a G12S and a Q61L mutation were found (7%). The KRAS gene contained 1 mutation (3%), that is a G12S change. PIK3CA mutations were found in all grades and stages, whereas HRAS and KRAS mutations were found in larger and more advanced tumors. The mutations were mutually exclusive, suggesting that dysregulation of either pathway is sufficient for the development and progression of penile carcinoma.

    Conclusions: The high frequency of mutations in the PIK3CA, HRAS and KRAS genes leads us to believe that dysregulation of the phosphatidylinositol 3-kinase or Ras pathway is significant for the development and progression of penile carcinoma.

    The Journal of urology 2008;179;5;2030-4

  • Cigarette smoking and KRAS oncogene mutations in sporadic colorectal cancer: results from the Netherlands Cohort Study.

    Weijenberg MP, Aardening PW, de Kok TM, de Goeij AF and van den Brandt PA

    GROW-School for Oncology and Developmental Biology, University Maastricht, Department of Epidemiology, Maastricht University, Maastricht, The Netherlands. MP.Weijenberg@epid.unimaas.nl <MP.Weijenberg@epid.unimaas.nl&gt;

    Since a KRAS oncogene mutation is an early event in colorectal cancer development and cigarette smoking is thought to have an effect on early stages of colorectal tumorigenesis, smoking, especially long-term smoking, may be associated with the risk for colorectal cancer with KRAS oncogene mutations. In the Netherlands Cohort Study on diet and cancer (n=120,852 men and women), using a case-cohort design, adjusted incidence rate ratios (RR) and 95% confidence intervals (CI) were computed for colorectal tumors with wild-type and with mutated KRAS gene, and with specific G:C-->T:A or G:C-->A:T point mutations in KRAS, according to cigarette smoking status, frequency, duration, pack years, age at first exposure, years since cessation, inhalation and filter usage. After 7.3 years and excluding the first 2.3 years, 648 cases and 4083 sub-cohort members were included in the analyses. Ex-smokers, but not current smokers, were at increased risk for colorectal cancer with wild-type KRAS gene tumors when compared with never smokers, albeit not statistically significant (RR 1.26, 95% CI 0.96-1.66). This was not observed for KRAS mutated tumors when comparing ex-smokers with never smokers (RR 1.15, 95% CI 0.79-1.66). The highest category of smoking frequency (>20 cigarettes/day) and inhalation of smoke were associated with an increased risk for colorectal cancer with wild-type KRAS gene tumors, though not statistically significant, when compared with never smoking (frequency: RR 1.24, 95% CI 0.90-1.71 and inhalation: RR 1.25, 95% CI 0.94-1.67). These associations were strongest in men (ex-smokers: RR 1.79, 95% CI 1.00-3.20; frequency: RR 1.91, 95% CI 1.03-3.52; inhalation: RR 1.69, 95% CI 0.94-3.04). No associations were observed between any of the smoking characteristics and the risk for colorectal cancer with mutated KRAS gene tumors, nor where there any clear associations with tumors with specific G:C-->A:T transitions or G:C-->T:A transversions. These results suggest that, in contrast to the hypothesis, smoking does not increase the risk for colorectal tumors with a mutated KRAS gene. Some smoking characteristics, i.e. being an ex-smoker, frequency and inhalation, may be associated with risk for colorectal cancer characterized by the wild-type KRAS gene, especially in men.

    Mutation research 2008;652;1;54-64

  • Costello syndrome associated with novel germline HRAS mutations: an attenuated phenotype?

    Gripp KW, Innes AM, Axelrad ME, Gillan TL, Parboosingh JS, Davies C, Leonard NJ, Lapointe M, Doyle D, Catalano S, Nicholson L, Stabley DL and Sol-Church K

    Division of Medical Genetics, A. I. duPont Hospital for Children, Wilmington, Delaware 19899, USA. kgripp@nemours.org

    Costello syndrome is a rare congenital disorder typically characterized by severe failure-to-thrive, cardiac abnormalities including tachyarrhythmia and hypertrophic cardiomyopathy, distinctive facial features, a predisposition to papillomata and malignant tumors, neurologic abnormalities, developmental delay, and mental retardation. Its underlying cause is de novo germline mutations in the oncogene HRAS. Almost all Costello syndrome mutations affect one of the glycine residues in position 12 or 13 of the protein product. More than 80% of patients with Costello syndrome share the same underlying mutation, resulting in a G12S amino acid change. We report on two patients with novel HRAS mutations affecting amino acids 58 (T58I) and 146 (A146V), respectively. Despite facial features that appear less coarse than those typically seen in Costello patients, both patients show many of the physical and developmental problems characteristic for Costello syndrome. These novel HRAS mutations may be less common than the frequently reported G12S change, or patients with these changes may be undiagnosed due to their less coarse facial features. In addition to the findings previously known to occur in Costello syndrome, one of our patients had hypertrophic pyloric stenosis. This led us to review the medical histories on a cohort of proven HRAS mutation positive Costello syndrome patients, and we found a statistically significantly (P < 0.001) increased frequency of pyloric stenosis in Costello syndrome (5/58) compared to the general population frequency of 2-3/1,000. Thus we add hypertrophic pyloric stenosis to the abnormalities seen with increased frequency in Costello syndrome.

    Funded by: NCRR NIH HHS: 4P20 RR020173-01

    American journal of medical genetics. Part A 2008;146A;6;683-90

  • A unique platform for H-Ras signaling involving clathrin-independent endocytosis.

    Porat-Shliom N, Kloog Y and Donaldson JG

    Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.

    Trafficking of H-Ras was examined to determine whether it can enter cells through clathrin-independent endocytosis (CIE). H-Ras colocalized with the CIE cargo protein, class I major histocompatibility complex, and it was sequestered in vacuoles that formed upon expression of an active mutant of Arf6, Q67L. Activation of Ras, either through epidermal growth factor stimulation or the expression of an active mutant of Ras, G12V, induced plasma membrane ruffling and macropinocytosis, a stimulated form of CIE. Live imaging of cells expressing H-RasG12V and fluorescent protein chimeras with pleckstrin homology domains that recognize specific phosphoinositides showed that incoming macropinosomes contained phosphatidylinositol 4,5-bisphosphate (PIP(2)) and phosphatiylinositol 3,4,5-trisphosphate (PIP(3)). PIP(2) loss from the macropinosome was followed by the recruitment of Rab5, a downstream target of Ras, and then PIP(3) loss. Our studies support a model whereby Ras can signal on macropinosomes that pass through three distinct stages: PIP(2)/PIP(3), PIP(3)/Rab5, and Rab5. Vacuoles that form in cells expressing Arf6Q67L trap Ras signaling in the first stage, recruiting the active form of the Ras effectors extracellular signal-regulated kinase and protein kinase B (Akt) but not Rab5. Arf6 stimulation of macropinocytosis also involves passage through the distinct lipid phases, but recruitment of Akt is not observed.

    Funded by: Intramural NIH HHS

    Molecular biology of the cell 2008;19;3;765-75

  • Familial non-medullary thyroid carcinoma (FNMTC): analysis of fPTC/PRN, NMTC1, MNG1 and TCO susceptibility loci and identification of somatic BRAF and RAS mutations.

    Cavaco BM, Batista PF, Martins C, Banito A, do Rosário F, Limbert E, Sobrinho LG and Leite V

    Centro de Investigação de Patobiologia Molecular (CIPM) and Serviço de Endocrinologia, Instituto Português de Oncologia de Lisboa Francisco Gentil, Lisboa, Portugal. bcavaco@ipolisboa.min-saude.pt

    Linkage analysis has identified four familial non-medullary thyroid carcinoma (FNMTC) susceptibility loci: fPTC/PRN (1p13.2-1q22), NMTC1 (2q21), MNG1 (14q32) and TCO (19p13.2). To date, there is no evidence for the involvement of genes from the RAS/RAF signalling pathway in FNMTC. The aim of our study was to evaluate the role of the four susceptibility loci, and RAS/RAF signalling pathway genes, in FNMTC. In total, 8 FNMTC families, and 27 thyroid lesions from family members (22 papillary thyroid carcinomas (PTCs): 11 classic, 10 of the follicular variant and 1 of the mixed variant; 4 follicular thyroid adenomas (FTAs) and 1 nodular goitre (NG)), were evaluated for the involvement of the four susceptibility regions, using linkage and loss of heterozygosity (LOH) analyses. BRAF and H-, N- and K-RAS mutations were also screened in the 27 lesions and patients. Linkage analysis in seven informative families showed no evidence for the involvement of any of the four candidate regions, supporting a genetic heterogeneity for FNMTC. Twenty tumours (74%), of which 18 were PTCs, showed no LOH at the four susceptibility loci. The remaining seven tumours (four PTCs, two FTAs and one NG) showed variable patterns of LOH. Fourteen tumours (52%) had somatic mutations: BRAF-V600E mutation was observed in 9 out of the 22 PTCs (41%); and H-RAS and N-RAS mutations were detected in 5 out of the 22 PTCs (23%). Our data suggest that the four candidate regions are not frequently involved in FNMTC and that the somatic activation of BRAF and RAS plays a role in FNMTC tumourigenesis.

    Endocrine-related cancer 2008;15;1;207-15

  • Severe neonatal manifestations of Costello syndrome.

    Lo IF, Brewer C, Shannon N, Shorto J, Tang B, Black G, Soo MT, Ng DK, Lam ST and Kerr B

    Background: Costello syndrome (CS) is due to mutations in HRAS, with the most common mutation being c.34G>A (p.G12S), found in most patients in all the published series. A small number of less common mutations have been reported.

    HRAS mutation analysis has been undertaken in 74 predominantly British patients with a possible diagnosis of CS. A HRAS mutation was found in 27 patients, 15 of whom have been previously reported. PHENOTYPE ANALYSIS: Four cases had an unusually severe phenotype, associated in three cases with two unusual mutations, c.35G>A, p.G12D in two cases and c.34G>T, p.G12C in the other. Hypoglycaemia, renal abnormalities, severe early cardiomyopathy, congenital lung and airway abnormalities, pleural and pericardial effusion, chylous ascites and pulmonary lymphangectasia are confirmed as part of the clinical spectrum seen in CS. A lung pathology resembling alveolar capillary dysplasia is reported in one case.

    Conclusion: These cases illustrate that the diagnosis of CS may be difficult in the newborn period, and should be considered in the differential diagnosis of the sick newborn infant with multisystem disease. Study of more cases will be required to establish if there is a definite association between severe disease and less common mutations.

    Journal of medical genetics 2008;45;3;167-71

  • Mutation analysis in Costello syndrome: functional and structural characterization of the HRAS p.Lys117Arg mutation.

    Denayer E, Parret A, Chmara M, Schubbert S, Vogels A, Devriendt K, Frijns JP, Rybin V, de Ravel TJ, Shannon K, Cools J, Scheffzek K and Legius E

    Department of Human Genetics, Catholic University of Leuven, Leuven, Belgium.

    Costello syndrome is a mental retardation syndrome characterized by high birth weight, postnatal growth retardation, coarse face, loose skin, cardiovascular problems, and tumor predisposition. De novo heterozygous missense mutations in HRAS codon 12 and 13 disturbing the intrinsic GTP hydrolysis cause Costello syndrome. We report a patient with typical Costello syndrome and a novel heterozygous missense mutation in codon 117 (c.350A>G, p.Lys117Arg) of the HRAS gene, resulting in constitutive activation of the RAS/MAPK pathway similar to the typical p.Gly12Ser and p.Gly12Ala mutations. Recombinant HRAS p.Lys117Arg demonstrates normal intrinsic GTP hydrolysis and responsiveness to GTPase-activating proteins, but the nucleotide dissociation rate is increased 80-fold. Consistent with the biochemical data, the crystal structure of the p.Lys117Arg mutant indicates an altered interaction pattern of the side chain that is associated with unfavorable nucleotide binding properties. Together, these data show that a RAS mutation that only perturbs guanine nucleotide binding has similar functional consequences as mutations that impair GTP hydrolysis and causes human disease.

    Funded by: NCI NIH HHS: CA72614

    Human mutation 2008;29;2;232-9

  • Evidence that sprouty 2 is necessary for sarcoma formation by H-Ras oncogene-transformed human fibroblasts.

    Lito P, Mets BD, Kleff S, O'Reilly S, Maher VM and McCormick JJ

    Carcinogenesis Laboratory, Department of Microbiology & Molecular Genetics, Michigan State University, East Lansing, Michigan 48824-1302, USA.

    Sprouty 2 (Spry2) acts as an inhibitor of receptor tyrosine kinase signaling in various cellular contexts. Interestingly, Spry2 also prevents the c-Cbl-induced degradation of epidermal growth factor receptor (EGFR). We compared human fibroblasts malignantly transformed by overexpression of H-Ras(V12) oncogene to their nontransformed parental cells and found that the malignant cells express a high level of Spry2. These cells also exhibited an increase in the level of EGFR compared with their precursor cells. We found that intact EGFR was required if H-Ras-transformed cells were to grow in the absence of exogenous growth factors or form large colonies in agarose. When we decreased expression of Spry2, using a Spry2-specific shRNA, the H-Ras(V12)-transformed fibroblasts could no longer form large colonies in agarose, grow in reduced levels of serum, or form tumors in athymic mice. The level of active H-Ras in these cells remained unaltered. A similar, but less pronounced, effect in tumor formation was observed when Spry2 was down-regulated in human patient-derived fibrosarcoma cell lines. In H-Ras-transformed cells Spry2 sustained the level and the downstream signaling activity of EGFR. In the parental, non-H-Ras-transformed fibroblasts, expression of Spry2 resulted in the inhibition of H-Ras and ERK activation, suggesting that the positive effect of Spry2 in tumor formation is specific to H-Ras transformation. Co-immunoprecipitation studies with H-Ras-transformed cells revealed that Spry2 and H-Ras interact and that H-Ras interacts with Spry2-binding partners, c-Cbl and CIN85, in a Spry2-dependent manner. These data show that Spry2 plays a critical role in the ability of H-Ras-transformed cells to form tumors in athymic mice.

    Funded by: NCI NIH HHS: CA098305

    The Journal of biological chemistry 2008;283;4;2002-9

  • Brain cancer stem-like cell genesis from p53-deficient mouse astrocytes by oncogenic Ras.

    Lee JS, Gil JE, Kim JH, Kim TK, Jin X, Oh SY, Sohn YW, Jeon HM, Park HJ, Park JW, Shin YJ, Chung YG, Lee JB, You S and Kim H

    Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Anam-dong, Seongbuk-gu, Seoul 136-713, Republic of Korea.

    Here, we show that H-ras(V12) causes the p53-knockout mouse astrocytes (p53-/- astrocytes) to be transformed into brain cancer stem-like cells. H-ras(V12) triggers the p53-/- astrocytes to express a Nestin and a Cd133, which are expressed in normal and cancer neural stem cells. H-ras(V12) also induces the formation of a single cell-derived neurosphere under neural stem cell culture conditions. Furthermore, H-ras(V12)-overexpressing p53-/- astrocytes (p53-/-ast-H-ras(V12)) possess an in vitro self-renewal capacity, and are aberrantly differentiated into Tuj1-positve neurons both in vitro and in vivo. Amongst a variety of Ras-mediated canonical signaling pathways, we demonstrated that the MEK/ERK signaling pathway is responsible for neurosphere formation in p53-deficient astrocytes, whereas the PI3K/AKT signaling pathway is involved in oncogenic transformation in these cells. These findings suggest that the activation of Ras signaling pathways promotes the generation of brain cancer stem-like cells from p53-deficient mouse astrocytes by changing cell fate and transforming cell properties.

    Biochemical and biophysical research communications 2008;365;3;496-502

  • Genetic changes of p53, K-ras, and microsatellite instability in gallbladder carcinoma in high-incidence areas of Japan and Hungary.

    Nagahashi M, Ajioka Y, Lang I, Szentirmay Z, Kasler M, Nakadaira H, Yokoyama N, Watanabe G, Nishikura K, Wakai T, Shirai Y, Hatakeyama K and Yamamoto M

    Division of Molecular and Diagnostic Pathology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-dori, Chuo-ku, Niigata 951-8510, Japan.

    Aim: To disclose geographic differences in genetic changes involved in gallbladder carcinogenesis between two distinct high-incidence areas of Japan and Hungary.

    Methods: We examined 42 cases of gallbladder carcinoma: 22 Japanese and 20 Hungarian cases. p53 mutations at exons 5 to 8 and K-ras mutations at codon 12 were tested by direct sequencing. Microsatellite instability was determined from fluorescent dye-labeled PCR amplifications of five-microsatellite markers (BAT-25, BAT-26, D2S123, D5S346, and D17S250).

    Results: Mutations of p53 were detected in 11 of 22 Japanese cases and 6 of 18 Hungarian cases (11/22 vs 6/18, P = 0.348). Transition at CpG sites was found in none of 11 Japanese cases and 2 of 6 Hungarian cases; the difference was marginally significant (0/11 vs 2/6, P = 0.110). K-ras mutations were detected in only one of the Hungarian cases. Eight of 19 (42.1%) Japanese cases were MSI-high (presence of novel peaks in more than one of the five loci analyzed), whereas only 1 of 15 (6.7%) Hungarian cases was MSI-high (P = 0.047).

    Conclusion: It appears that the p53 mutations and MSI differ in patients with gallbladder carcinoma between two distinct high-incidence areas. Geographic variation might exist in the process of gallbladder carcinogenesis.

    World journal of gastroenterology : WJG 2008;14;1;70-5

  • BRAF and K-RAS mutation in a Greek papillary and medullary thyroid carcinoma cohort.

    Goutas N, Vlachodimitropoulos D, Bouka M, Lazaris AC, Nasioulas G and Gazouli M

    Histopathology Department, Evgenidion Hospital, University of Athens, Athens, Greece.

    Background: The genes RAS and BRAF have been shown to be frequently mutated in human thyroid carcinomas. The aim of this study was to genotype a cohort of 55 sporadic papillary thyroid carcinomas (PTC) and 44 sporadic medullary thyroid carcinomas (MTC) for the K-RAS codon 12 and BRAF codon 600 mutations.

    K-RAS and BRAF mutations were characterized by an enhanced polymerase chain reaction followed by restriction fragment length polymorphism analysis (PCR-RFLP).

    Results: The K-RAS codon 12 mutation was found in 54.5% of the PTC and 40.9% of the MTC cases tested. The BRAF V600E mutation was detected in 27.3% of the PTC and 68.2% of the MTC samples. No significant association between K-RAS and BRAF mutations and clinicopathological parameters was found.

    Conclusion: These data indicate that K-RAS and BRAF mutations were a frequent genetic event in our samples of sporadic PTC and MTC.

    Anticancer research 2008;28;1A;305-8

  • Mutation and phenotypic spectrum in patients with cardio-facio-cutaneous and Costello syndrome.

    Schulz AL, Albrecht B, Arici C, van der Burgt I, Buske A, Gillessen-Kaesbach G, Heller R, Horn D, Hübner CA, Korenke GC, König R, Kress W, Krüger G, Meinecke P, Mücke J, Plecko B, Rossier E, Schinzel A, Schulze A, Seemanova E, Seidel H, Spranger S, Tuysuz B, Uhrig S, Wieczorek D, Kutsche K and Zenker M

    Institut für Humangenetik, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany.

    Cardio-facio-cutaneous (CFC) and Costello syndrome (CS) are congenital disorders with a significant clinical overlap. The recent discovery of heterozygous mutations in genes encoding components of the RAS-RAF-MAPK pathway in both CFC and CS suggested a similar underlying pathogenesis of these two disorders. While CFC is heterogeneous with mutations in BRAF, MAP2K1, MAP2K2 and KRAS, HRAS alterations are almost exclusively associated with CS. We carried out a comprehensive mutation analysis in 51 CFC-affected patients and 31 individuals with CS. Twelve different BRAF alterations were found in twenty-four patients with CFC (47.0%), two MAP2K1 mutations in five (9.8%) and two MAP2K2 sequence variations in three CFC-affected individuals (5.9%), whereas three patients had a KRAS alteration (5.9%). We identified four different missense mutations of HRAS in twenty-eight cases with CS (90.3%), while KRAS mutations were detected in two infants with a phenotype meeting criteria for CS (6.5%). In 14 informative families, we traced the parental origin of HRAS alterations and demonstrated inheritance of the mutated allele exclusively from the father, further confirming a paternal bias in the parental origin of HRAS mutations in CS. Careful clinical evaluation of patients with BRAF and MAP2K1/2 alterations revealed the presence of slight phenotypic differences regarding craniofacial features in MAP2K1- and MAP2K2-mutation positive individuals, suggesting possible genotype-phenotype correlations.

    Clinical genetics 2008;73;1;62-70

  • Association between H-RAS T81C genetic polymorphism and gastrointestinal cancer risk: a population based case-control study in China.

    Zhang Y, Jin M, Liu B, Ma X, Yao K, Li Q and Chen K

    Department of Epidemiology & Health Statistics, Zhejiang University School of Medicine, Hangzhou 310058, Zhejiang, PR China.

    Background: Gastrointestinal cancer, such as gastric, colon and rectal cancer, is a major medical and economic burden worldwide. However, the exact mechanism of gastrointestinal cancer development still remains unclear. RAS genes have been elucidated as major participants in the development and progression of a series of human tumours and the single nucleotide polymorphism at H-RAS cDNA position 81 was demonstrated to contribute to the risks of bladder, oral and thyroid carcinoma. Therefore, we hypothesized that this polymorphisms in H-RAS could influence susceptibility to gastrointestinal cancer as well, and we conducted this study to test the hypothesis in Chinese population.

    Methods: A population based case-control study, including 296 cases with gastrointestinal cancer and 448 healthy controls selected from a Chinese population was conducted. H-RAS T81C polymorphism was genotyped by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) assay.

    Results: In the healthy controls, the TT, TC and CC genotypes frequencies of H-RAS T81C polymorphism, were 79.24%, 19.87% and 0.89%, respectively, and the C allele frequency was 10.83%. Compared with TT genotype, the TC genotype was significantly associated with an increased risk of gastric cancer (adjusted OR = 3.67, 95%CI = 2.21-6.08), while the CC genotype showed an increased risk as well (adjusted OR = 3.29, 95%CI = 0.54-19.86), but it was not statistically significant. In contrast, the frequency of TC genotype was not significantly increased in colon cancer and rectal cancer patients. Further analysis was performed by combining TC and CC genotypes compared against TT genotype. As a result, a statistically significant risk with adjusted OR of 3.65 (95%CI, 2.22-6.00) was found in gastric cancer, while no significant association of H-RAS T81C polymorphism with colon cancer and rectal cancer was observed.

    Conclusion: These findings indicate, for the first time, that there is an H-RAS T81C polymorphism existing in Chinese population, and this SNP might be a low penetrance gene predisposition factor for gastric cancer.

    BMC cancer 2008;8;256

  • Mutations of RAS gene family in specimens of bladder cancer.

    Karimianpour N, Mousavi-Shafaei P, Ziaee AA, Akbari MT, Pourmand G, Abedi A, Ahmadi A and Afshin Alavi H

    Institute of Biochemistry and Biophysics, Tehran University, Tehran, Iran.

    Introduction: Studies have shown different types of RAS mutations in human bladder tumors with a wide range of mutation frequencies in different patient populations. This study aimed to assess the frequency of specific-point mutations in the RAS gene family of a group of Iranian patients with bladder cancer.

    We examined the tumor specimens of 35 consecutive patients with transitional cell carcinoma. The DNA samples were evaluated for the occurrence of HRAS, KRAS, and NRAS activation using a polymerase chain reaction-restriction fragment length polymorphism technique.

    Results: None of the patients had mutations in the RAS gene family "hot spots" including codons 12, 13, and 61.

    Conclusion: We failed to find RAS mutations in our bladder tumor samples. These observations may reflect the involvement of different etiological factors in the induction of bladder tumor of which RAS mutation might not be present in all populations.

    Urology journal 2008;5;4;237-42

  • Oncogenic H-Ras V12 promotes anchorage-independent cytokinesis in human fibroblasts.

    Thullberg M, Gad A, Le Guyader S and Strömblad S

    Department of Biosciences and Nutrition, Karolinska Institutet, SE-141 57 Huddinge, Sweden. minna.thullberg@ki.se

    Cell anchorage is required for cell proliferation of untransformed cells, whereas anchorage-independent growth can be induced by oncogenes and is a hallmark of transformation. Whereas anchorage-dependent control of the progression of the G(1) phase of the cell cycle has been extensively studied, it is less clear whether and how anchorage may control other cell cycle phases and whether oncogenes may affect such controls. Here, we found that lack of cell anchorage did not influence progression through the cell cycle S phase, G(2) phase, or most of mitosis of primary human fibroblasts. However, unanchored fibroblasts could not complete cytokinesis. The cleavage furrow and central spindle were still formed in the absence of anchorage, but cells were unable to complete ingression, causing binucleation. Importantly, V12 H-Ras-transformed fibroblasts and two cancer cell lines progressed through the entire cell cycle without anchorage, including through cytokinesis. This indicates that oncogenic signaling may contribute to anchorage-independent growth and tumorigenesis by promoting the final cleavage furrow ingression during cytokinesis.

    Proceedings of the National Academy of Sciences of the United States of America 2007;104;51;20338-43

  • Bmi-1 cooperates with H-Ras to transform human mammary epithelial cells via dysregulation of multiple growth-regulatory pathways.

    Datta S, Hoenerhoff MJ, Bommi P, Sainger R, Guo WJ, Dimri M, Band H, Band V, Green JE and Dimri GP

    Division of Cancer Biology and Department of Medicine, Feinberg School of Medicine, Northwestern University, Evanston, IL 60201, USA.

    Elevated expression of Bmi-1 is associated with many cancers, including breast cancer. Here, we examined the oncogenic potential of Bmi-1 in MCF10A cells, a spontaneously immortalized, nontransformed strain of human mammary epithelial cells (HMEC). Bmi-1 overexpression alone in MCF10A cells did not result in oncogenic transformation. However, Bmi-1 co-overexpression with activated H-Ras (RasG12V) resulted in efficient transformation of MCF10A cells in vitro. Although early-passage H-Ras-expressing MCF10A cells were not transformed, late-passage H-Ras-expressing cells exhibited features of transformation in vitro. Early- and late-passage H-Ras-expressing cells also differed in levels of expression of H-Ras and Ki-67, a marker of proliferation. Subsets of early-passage H-Ras-expressing cells exhibited high Ras expression and were negative for Ki-67, whereas most late-passage H-Ras-expressing cells expressed low levels of Ras and were Ki-67 positive. Injection of late-passage H-Ras-expressing cells in severe combined immunodeficient mice formed carcinomas with leiomatous, hemangiomatous, and mast cell components; these tumors were quite distinct from those induced by late-passage cells co-overexpressing Bmi-1 and H-Ras, which formed poorly differentiated carcinomas with spindle cell features. Bmi-1 and H-Ras co-overexpression in MCF10A cells also induced features of epithelial-to-mesenchymal transition. Importantly, Bmi-1 inhibited senescence and permitted proliferation of cells expressing high levels of Ras. Examination of various growth-regulatory pathways suggested that Bmi-1 overexpression together with H-Ras promotes HMEC transformation and breast oncogenesis by deregulation of multiple growth-regulatory pathways by p16(INK4a)-independent mechanisms.

    Funded by: NCI NIH HHS: R01 CA094150, R01 CA094150-05, R01CA094150

    Cancer research 2007;67;21;10286-95

  • H-Ras mutation modulates the expression of major cell cycle regulatory proteins and disease prognosis in oral carcinoma.

    Sathyan KM, Nalinakumari KR and Kannan S

    Laboratory of Cell Cycle Regulation and Molecular Oncology, Division of Cancer Research, Regional Cancer Centre, Thiruvananthapuram, Kerala, India.

    Activating mutations of the Ras is a moderately frequent event in oral carcinogenesis in Indian patients. Ras pathway has essential roles in regulation of various phases of the cell cycle, especially at G1 phase. Despite a large body of in vitro evidence, the multidimensional interaction between mutated Ras pathway and G1 cell cycle regulatory proteins in tumours in vivo is poorly determined. In the present study, DNA samples were screened for mutations in hot spot exons of B-Raf and hot spot codons 12, 13 and 61 of H-, K- and N-Ras by PCR-SSCP. Mutations were confirmed by direct sequencing. Expression of G1 cell cycle regulatory proteins-cyclin D1, CDK4, Rb, p53, p16 and p21 and proliferation marker PCNA was analysed immunohistochemically. The results revealed the absence of B-Raf mutations in oral carcinoma in spite of 12.5% of the samples showing H-Ras mutation. The H-Ras mutant cases showed significantly low cyclin D1 (P=0.027) and CDK4 (P=0.046) expression and overexpression of Rb (P=0.011) and p16 (P=0.026). H-Ras mutant carriers also had significantly high recurrence-free survival (P=0.033). In summary the present study demonstrated an epistatic interaction between H-Ras mutation and G1 cell cycle regulatory proteins in vivo. H-Ras mutation, thus, defines a molecular subtype of oral carcinoma with favourable outcome and unique biology.

    Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 2007;20;11;1141-8

  • Mutations and differential expression of the ras family genes in human nasal polyposis.

    Zaravinos A, Bizakis J, Soufla G, Sourvinos G and Spandidos DA

    Laboratory of Virology, Medical School, University of Crete, Heraklion 71100, Crete, Greece.

    Although it is well established that ras genes contribute to tumourigenesis either through the accumulation of mutations or by aberrant expression in a wide range of human cancers, little is known regarding their involvement in human nasal polyps (NPs). In the present study, the occurrence of mutations in codons 11 and 12 of the ras family genes was examined by PCR/RFLP and direct sequencing in 23 human NPs and their adjacent turbinates, as well as in turbinates from 13 control subjects. Moreover, the expression pattern of ras mRNA levels was assessed in NP specimens and compared to adjacent and control tissues. K-ras codon 11 and 12 mutations were detected in 17 and 35% of NPs, respectively, and were found in the adjacent inferior turbinate (AIT) (22 and 16%, respectively) and adjacent middle turbinates (AMT) (16 and 26%, respectively). K- and H-ras expression levels were elevated, whereas N-ras mRNA levels were lower in NPs and adjacent turbinates as compared to the control tissues. K-ras mRNA levels were up-regulated in advanced-stage polyps (P=0.037), while N-ras levels were found elevated in small polyps (P=0.046). Statistically significant negative correlations between K- and N-ras expression profiles arose in NPs and AITs (P=0.009 and 0.003, respectively). This, to our knowledge, is the first report on ras mutations and expression analysis in NPs. Our findings suggest a potential key role for activated members of ras family genes in terms of their contribution to the development of NPs as well as to the hypertrophy of adjacent turbinates.

    International journal of oncology 2007;31;5;1051-9

  • The RAS-dependent ERF control of cell proliferation and differentiation is mediated by c-Myc repression.

    Verykokakis M, Papadaki C, Vorgia E, Le Gallic L and Mavrothalassitis G

    Medical School, University of Crete and Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Crete, 710 03, Greece.

    The ERF transcriptional repressor is a downstream effector of the RAS/ERK pathway that interacts with and is directly phosphorylated by ERKs in vivo and in vitro. This phosphorylation results in its cytoplasmic export and inactivation, although lack of ERK activity allows its immediate nuclear accumulation and repressor function. Nuclear ERFs arrest cell cycle progression in G(1) and can suppress ras-dependent tumorigenicity. Here we provide evidence that ERF function is mediated by its ability to repress transcription of c-Myc. Promoter reporter assays indicate a DNA binding-dependent and repressor domain-dependent Myc transcriptional repression. Chromatin immunoprecipitations in primary cells suggest that ERF specifically binds on the c-Myc promoter in an E2F4/5-dependent manner and only under conditions that the physiological c-Myc transcription is stopped. Cellular systems overexpressing nuclear ERF exhibit reduced c-Myc mRNA and tumorigenic potential. Elimination of Erf in animal models results in increased c-Myc expression, whereas Erf(-)(/)(-) primary fibroblasts fail to down-regulate Myc in response to growth factor withdrawal. Finally, elimination of c-Myc in primary mouse embryo fibroblasts negates the ability of nuclear ERF to suppress proliferation. Thus Erf provides a direct link between the RAS/ERK signaling and the transcriptional regulation of c-Myc and suggests that RAS/ERK attenuation actively regulates cell fate.

    The Journal of biological chemistry 2007;282;41;30285-94

  • A crosstalk between hSiah2 and Pias E3-ligases modulates Pias-dependent activation.

    Depaux A, Regnier-Ricard F, Germani A and Varin-Blank N

    Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France.

    Protein inhibitor of activated STAT (Pias) and human homologues of seven in absentia (hSiah) proteins both exhibit properties of ubiquitin-family peptides conjugating enzymes. Pias present E3-ligase activity for small ubiquitin-related modifiers (Sumo) covalent attachment to their targets. This post-translational modification is responsible for the activation of different transcription factors such as AP1. HSiah proteins possess ubiquitin-E3-ligase activity that triggers their partners to proteasomal-dependent degradation. The present study identifies Pias as a new hSiah2-interacting protein. We demonstrate that hSiah2 regulates specifically the proteasome-dependent degradation of Pias proteins. On reverse, Pias does not prevent hSiah2 degradation. We provide evidences for hSiah2-dependent degradation of Pias as being a mechanism in the regulation of c-jun N-terminal kinase-activating pathways. This report describes a new interconnection between sumoylation and ubiquitination pathways by regulating the levels of the E3-ligases available for these processes.

    Oncogene 2007;26;46;6665-76

  • Prevalence of RAS point mutations in papillary thyroid carcinoma; a novel mutation at codon 31 of K-RAS.

    Cyniak-Magierska A, Brzeziańska E, Januszkiewicz-Caulier J, Jarzab B and Lewiński A

    Department of Endocrinology and Metabolic Diseases, Medical University of Lodz, Polish Mother's Memorial Hospital - Research Institute, Lodz, Poland.

    The aim of this study was to assess the incidence of point mutations in RAS oncogenes of papillary thyroid carcinoma (PTC). Tumour specimens were obtained from 29 PTCs. The fragments of exons 1 and 2 of RAS oncogenes family (H- RAS, K- RAS, N- RAS) were amplified and then, point mutations were detected by SSCP and/or by RFLP analysis. Several DNA samples were directly sequenced to confirm the results. Two mutations were found in this study (GAA/CAA at codon 31 of K- RAS and CAA/CAC at codon 61 of N- RAS oncogene). These data confirm the results of previous studies, showing that RAS mutations are more rarely found in PTC than in follicular neoplasms. The influence of a novel mutation at codon 31 of K- RAS oncogene on the development of PTC needs further studies.

    Experimental and clinical endocrinology & diabetes : official journal, German Society of Endocrinology [and] German Diabetes Association 2007;115;9;594-9

  • B- and C-RAF display essential differences in their binding to Ras: the isotype-specific N terminus of B-RAF facilitates Ras binding.

    Fischer A, Hekman M, Kuhlmann J, Rubio I, Wiese S and Rapp UR

    Institut für Medizinische Strahlenkunde und Zellforschung, University of Wuerzburg, 97078 Wuerzburg, Germany.

    Recruitment of RAF kinases to the plasma membrane was initially proposed to be mediated by Ras proteins via interaction with the RAF Ras binding domain (RBD). Data reporting that RAF kinases possess high affinities for particular membrane lipids support a new model in which Ras-RAF interactions may be spatially restricted to the plane of the membrane. Although the coupling features of Ras binding to the isolated RAF RBD were investigated in great detail, little is known about the interactions of the processed Ras with the functional and full-length RAF kinases. Here we present a quantitative analysis of the binding properties of farnesylated and nonfarnesylated H-Ras to both full-length B- and C-RAF in the presence and absence of lipid environment. Although isolated RBD fragments associate with high affinity to both farnesylated and nonfarnesylated H-Ras, the full-length RAF kinases revealed fundamental differences with respect to Ras binding. In contrast to C-RAF that requires farnesylated H-Ras, cytosolic B-RAF associates effectively and with significantly higher affinity with both farnesylated and nonfarnesylated H-Ras. To investigate the potential farnesyl binding site(s) we prepared several N-terminal fragments of C-RAF and found that in the presence of cysteine-rich domain only the farnesylated form of H-Ras binds with high association rates. The extreme N terminus of B-RAF turned out to be responsible for the facilitation of lipid independent Ras binding to B-RAF, since truncation of this region resulted in a protein that changed its kinase properties and resembles C-RAF. In vivo studies using PC12 and COS7 cells support in vitro results. Co-localization measurements using labeled Ras and RAF documented essential differences between B- and C-RAF with respect to association with Ras. Taken together, these data suggest that the activation of B-RAF, in contrast to C-RAF, may take place both at the plasma membrane and in the cytosolic environment.

    The Journal of biological chemistry 2007;282;36;26503-16

  • BCR-tyrosine 177 plays an essential role in Ras and Akt activation and in human hematopoietic progenitor transformation in chronic myelogenous leukemia.

    Chu S, Li L, Singh H and Bhatia R

    Department of Hematopoietic Stem Cell and Leukemia Research, Division of Hematology and Hematopoietic Cell Transplantation, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, CA 91010, USA.

    Chronic myelogenous leukemia (CML) results from the transformation of a primitive hematopoietic cell by the BCR/ABL gene. BCR/ABL signaling has been studied in cell lines and murine models, but the transforming effects of BCR/ABL are highly dependent on cellular context, and mechanisms responsible for the transformation of primitive human hematopoietic cells remain poorly understood. Current targeted therapies fail to eliminate malignant CML progenitors, and improved understanding of crucial molecular mechanisms of progenitor transformation may facilitate the development of improved therapeutic approaches. We investigated the role of BCR/ABL tyrosine 177 (BCR/ABL-Y177) in CML progenitor transformation by comparing the effects of expression of Y177-mutated BCR/ABL, wild-type BCR/ABL, or green fluorescent protein alone on normal CD34(+) cells. We show that BCR/ABL-Y177 plays a critical role in CML progenitor expansion, proliferation, and survival. BCR/ABL expression results in enhanced Ras and Akt activity but reduced mitogen-activated protein kinase activity in human hematopoietic cells, which is reversed by BCR/ABL-Y177 mutation. Blocking BCR/ABL-Y177-mediated signaling enhances targeting of CML progenitors by imatinib mesylate. Our studies indicate that BCR/ABL-Y177 plays an essential role in Ras and Akt activation and in human hematopoietic progenitor transformation in CML.

    Funded by: NCI NIH HHS: R01 CA 95684; NCRR NIH HHS: 5M01 RR 00043; NHLBI NIH HHS: R01 HL 77847

    Cancer research 2007;67;14;7045-53

  • De novo HRAS and KRAS mutations in two siblings with short stature and neuro-cardio-facio-cutaneous features.

    Søvik O, Schubbert S, Houge G, Steine SJ, Norgård G, Engelsen B, Njølstad PR, Shannon K and Molven A

    Section for Pediatrics, Department of Clinical Medicine, University of Bergen, Bergen, Norway.

    Mutations in genes involved in Ras signalling cause Noonan syndrome and other disorders characterised by growth disturbances and variable neuro-cardio-facio-cutaneous features. We describe two sisters, 46 and 31 years old, who presented with dysmorphic features, hypotonia, feeding difficulties, retarded growth and psychomotor retardation early in life. The patients were initially diagnosed with Costello syndrome, and autosomal recessive inheritance was assumed. Remarkably, however, we identified a germline HRAS mutation (G12A) in one sister and a germline KRAS mutation (F156L) in her sibling. Both mutations had arisen de novo. The F156L mutant K-Ras protein accumulated in the active, guanosine triphosphate-bound conformation and affected downstream signalling. The patient harbouring this mutation was followed for three decades, and her cardiac hypertrophy gradually normalised. However, she developed severe epilepsy with hippocampal sclerosis and atrophy. The occurrence of distinct de novo mutations adds to variable expressivity and gonadal mosaicism as possible explanations of how an autosomal dominant disease may manifest as an apparently recessive condition.

    Funded by: NCI NIH HHS: R01 CA072614, R01 CA104282, R01 CA72614

    Journal of medical genetics 2007;44;7;e84

  • Myopathy caused by HRAS germline mutations: implications for disturbed myogenic differentiation in the presence of constitutive HRas activation.

    van der Burgt I, Kupsky W, Stassou S, Nadroo A, Barroso C, Diem A, Kratz CP, Dvorsky R, Ahmadian MR and Zenker M

    Background: Rare reports on patients with congenital myopathy with excess of muscle spindles (CMEMS), hypertrophic cardiomyopathy and variable features resembling Noonan syndrome have been published, but the genetic basis of this condition is so far unknown.

    We analysed PTPN11 and RAS genes in five unrelated patients with this phenotype, and found HRAS mutations in four of them. Two disease-associated mutations, G12V and G12S, have previously been observed in patients with Costello syndrome (CS), and two other mutations, E63K and Q22K, are novel. All four mutations are predicted to enhance downstream HRas signalling, suggesting that CMEMS is a developmental consequence of sustained HRas activation in skeletal muscle.

    Conclusion: This type of myopathy may represent a previously unrecognized manifestation of CS. However, some patients carrying HRAS mutations may exhibit prominent congenital muscular dysfunction, although features of CS may be less obvious, suggesting that germline HRAS mutations may underlie some cases of otherwise unclassified neonatal neuromuscular disorders.

    Journal of medical genetics 2007;44;7;459-62

  • Use of a novel coinfection system reveals a role for Rac1, H-Ras, and CrkII phosphorylation in Helicobacter pylori-induced host cell actin cytoskeletal rearrangements.

    Brandt S, Shafikhani S, Balachandran P, Jin S, Hartig R, König W, Engel J and Backert S

    Department of Medical Microbiology, Otto von Guericke University, Magdeburg, Germany.

    The Helicobacter pylori CagA protein induces profound morphological changes in the host cytoskeleton and cell scattering, but the signalling involved is poorly understood. Pseudomonas aeruginosa also affects host actin cytoskeleton in a variety of ways by injecting the ExoS and ExoT toxins which encode N-terminal GTPase activating protein and C-terminal ADP-ribosyltransferase (ADPRT) activities. In this study we developed a novel coinfection assay to gain new insights into CagA effector protein functions. We found that P. aeruginosa injecting either ExoT or ExoS efficiently prevented the H. pylori-induced scattering phenotype. Both the Rho-GAP and the ADPRT domains of ExoS were needed to block the H. pylori-induced actin cytoskeletal rearrangements, whereas either domain of ExoT was sufficient for this activity. This strategy revealed common pathways subverted by different pathogens, and aided in the definition of signalling cascades that control the CagA-mediated cell scattering and elongation. We identified Crk adapter proteins, Rac1 and H-Ras, but not RhoA or Cdc42, which are the ExoS and/or ExoT targets, as crucial components of the CagA-induced phenotype. In addition, we show that ADP-ribosylation of CrkII by ExoT blocks phosphorylation of CrkII at Y-221, which is also important for the CagA-induced signalling.

    Funded by: NIAID NIH HHS: F32 AI054056, R01 AI42806, R01 AI53914

    FEMS immunology and medical microbiology 2007;50;2;190-205

  • Binding of ras to phosphoinositide 3-kinase p110alpha is required for ras-driven tumorigenesis in mice.

    Gupta S, Ramjaun AR, Haiko P, Wang Y, Warne PH, Nicke B, Nye E, Stamp G, Alitalo K and Downward J

    Signal Transduction Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, UK.

    Ras proteins signal through direct interaction with a number of effector enzymes, including type I phosphoinositide (PI) 3-kinases. Although the ability of Ras to control PI 3-kinase has been well established in manipulated cell culture models, evidence for a role of the interaction of endogenous Ras with PI 3-kinase in normal and malignant cell growth in vivo has been lacking. Here we generate mice with mutations in the Pi3kca gene encoding the catalytic p110alpha isoform that block its interaction with Ras. Cells from these mice show proliferative defects and selective disruption of signaling from growth factors to PI 3-kinase. The mice display defective development of the lymphatic vasculature, resulting in perinatal appearance of chylous ascites. Most importantly, they are highly resistant to endogenous Ras oncogene-induced tumorigenesis. The interaction of Ras with p110alpha is thus required in vivo for certain normal growth factor signaling and for Ras-driven tumor formation.

    Cell 2007;129;5;957-68

  • Low prevalence of RAS-RAF-activating mutations in Spitz melanocytic nevi compared with other melanocytic lesions.

    Indsto JO, Kumar S, Wang L, Crotty KA, Arbuckle SM and Mann GJ

    Westmead Institute for Cancer Research, University of Sydney at Westmead Millennium Institute, Westmead, NSW, Australia. james_indsto@wmi.usyd.edu.au

    Melanocytic lesions, including Spitz nevi (SN), common benign nevi (CBN) and cutaneous metastatic melanoma (CMM), were analyzed for activating mutations in NRAS, HRAS and BRAF oncogenes, which induce cellular proliferation via the MAP kinase pathway. One of 22 (4.5%) SN tested showed an HRAS G61L mutation. Another lesion, a 'halo' SN, showed a BRAF V600E (T1796A) mutation. BRAF V600E mutations were found in two thirds (20/31) of CBN, while a further 19% (6/31) showed NRAS codon 61 mutations. One third of CMM (10/30) had various BRAF mutations of codon 600, and a further 6% (2/31) showed NRAS codon 61 mutations. Seventeen SN tested for loss of heterozygosity (LOH) at 9p and 10q regions, known to be frequently deleted in melanoma, showed LOH at the 9p loci D9S942 and IFNA. A further lesion was found with low-level microsatellite instability at one locus, D10S214. The low rate of RAS-RAF mutations (2/22, 9.1%) observed in SN suggests that these lesions harbor as yet undetected activating mutations in other components of the RAS-RAF-MEK-ERK-MAPK pathway. Germline DNA from members of 111 multiple-case melanoma families, representing a range of known (CDKN2A) and unknown predisposing gene defects, was analyzed for germline BRAF mutations, but none was found.

    Journal of cutaneous pathology 2007;34;6;448-55

  • The role of PKC and PDK1 in monocyte lineage specification by Ras.

    Pearn L, Fisher J, Burnett AK and Darley RL

    Department of Haematology, School of Medicine, Cardiff University, Cardiff, United Kingdom.

    Although hyperactivation of Ras is a common feature of myeloid malignancy, its role in subverting hematopoiesis is unclear. We have examined the influence of Ras on normal human uncommitted myeloid subsets and show that expression of this oncogene strongly favors monocyte lineage selection in bipotential granulocyte/macrophage progenitors while inhibiting colony formation in other uncommitted subsets. Ras also promoted monocytic differentiation but not the proliferation of these cells. The mechanism through which Ras drives monocyte lineage selection was dependent on PKC activity and Ras was found to promote the expression, membrane translocation, and phosphorylation of conventional and novel PKC isoforms. We further show that Ras promoted the expression of the AGC kinase master regulator, PDK1, which maintains the stability and activity of PKC isoforms. Consistent with this, overexpression of PDK1 itself promoted monocyte colony formation and translocation of PKC. Overexpression of PDK1 was found to be a common feature of acute myeloid leukemia (45% of patients) and was closely associated with hyperphosphorylation of PKC. These data demonstrate that Ras is able to promote monocyte lineage selection via PKC and show for the first time the involvement of the kinase master regulator, PDK1, in both lineage specification and in human leukemia.

    Blood 2007;109;10;4461-9

  • Statins synergistically potentiate 7-hydroxystaurosporine (UCN-01) lethality in human leukemia and myeloma cells by disrupting Ras farnesylation and activation.

    Dai Y, Khanna P, Chen S, Pei XY, Dent P and Grant S

    Department of Medicine, Virginia Commonwealth University/Massey Cancer Center, Richmond, VA 23298, USA.

    Interactions between UCN-01 and HMG-CoA reductase inhibitors (ie, statins) have been examined in human leukemia and myeloma cells. Exposure of U937 and U266 cells to minimally toxic concentrations of UCN-01 and various statins (eg, lovastatin, simvastatin, or fluvastatin) dramatically increased mitochondrial dysfunction, caspase activation, and apoptosis. Comparable effects were observed in other leukemia and myeloma cell lines as well as in primary acute myeloid leukemia (AML) blasts but not in normal hematopoietic cells. Potentiation of UCN-01 lethality by lovastatin was associated with disruption of Ras prenylation and activation. These events were significantly attenuated by farnesyl pyrophosphate (FPP) but not by geranylgeranyl pyrophosphate (GGPP), implicating perturbations in farnesylation rather than geranylgeranylation in synergistic interactions. Coexposure to statins and UCN-01 resulted in inactivation of ERK1/2 and Akt, accompanied by JNK activation. U266 cells ectopically expressing JNK1-APF, a dominant negative JNK1 mutant, displayed significantly reduced susceptibility to lovastatin/UCN-01-mediated lethality. Moreover, transfection of U266 cells with constitutively activated H-Ras (Q61L) attenuated ERK1/2 inactivation and dramatically diminished the lethality of this regimen. Collectively, these findings indicate that HMG-CoA reductase inhibitors act through a Ras farnesylation-associated mechanism to induce signaling perturbations, particularly prevention of Ras and ERK1/2 activation, in UCN-01-treated cells, resulting in the synergistic induction of cell death.

    Funded by: NCI NIH HHS: CA100866, CA63753, CA72955, CA88906, CA93738, P01 CA072955, R01 CA063753, R01 CA088906, R01 CA093738, R01 CA100866

    Blood 2007;109;10;4415-23

  • The tumor suppressor neurofibromin confers sensitivity to apoptosis by Ras-dependent and Ras-independent pathways.

    Shapira S, Barkan B, Friedman E, Fridman E, Kloog Y and Stein R

    Department of Neurobiochemistry, George S Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel.

    Neurofibromatosis type 1 (NF1) is characterized by a high incidence of benign and malignant tumors attributed to loss of function of Nf1, which encodes neurofibromin, a tumor suppressor with Ras-GAP activity. Neurofibromin deficiency typically causes chronic activation of Ras, considered the major contributor to manifestation of NF1. Resistance to radio- and chemotherapy are typical of NF1-associated tumors, but the underlying mechanism is unknown. Here, we investigated interrelationships between neurofibromin expression, Ras activity, and sensitivity to apoptosis. Neurofibromin-deficient mouse embryonic fibroblasts (MEFs) and human NF1 tumor cells were more resistant than neurofibromin-expressing cells to apoptosis. Moreover, Nf1(-/-), Nf1(+/-), and Nf1(+/+) MEFs exhibited gene-dosage-related resistance to apoptosis. Resistance of the Nf1-deficient cells was mediated by two survival pathways: a Ras-dependent pathway, and a Ras-independent pathway promoted by the lack of an NF1-GRD-independent proapoptotic action of neurofibromin. Therefore, besides its Ras-dependent growth inhibition, neurofibromin can exert tumor suppression via a proapoptotic effect.

    Cell death and differentiation 2007;14;5;895-906

  • Activated Ras induces cytoplasmic vacuolation and non-apoptotic death in glioblastoma cells via novel effector pathways.

    Kaul A, Overmeyer JH and Maltese WA

    Department of Biochemistry and Cancer Biology, University of Toledo College of Medicine, Toledo, Ohio 43614, USA.

    Expression of activated H-Ras induces a unique form of non-apoptotic cell death in human glioblastoma cells and other specific tumor cell lines. The major cytopathological features of this form of death are the accumulation of large phase-lucent, LAMP1-positive, cytoplasmic vacuoles. In this study we sought to determine if induction of cytoplasmic vacuolation a) depends on Ras farnesylation, b) is specific to H-Ras, and c) is mediated by signaling through the major known Ras effector pathways. We find that the unusual effects of activated H-Ras depend on farnesylation and membrane association of the GTPase. Both H-Ras(G12V) and K-Ras4B(G12V) stimulate vacuolation, but activated forms of Cdc42 and RhoA do not. Amino acid substitutions in the Ras effector domain, which are known to selectively impair its interactions with Raf kinase, class-I phosphatidylinositide 3-kinase (PI3K), or Ral nucleotide exchange factors, initially pointed to Raf as a possible mediator of cell vacuolation. However, the MEK inhibitor, PD98059, did not block the induction of vacuoles, and constitutively active Raf-Caax did not mimic the effects of Ras(G12V). Introduction of normal PTEN together with H-Ras(G12V) into U251 glioblastoma cells reduced the PI3K-dependent activation of Akt, but had no effect on vacuolation. Finally, co-expression of H-Ras(G12V) with a dominant-negative form of RalA did not suppress vacuolation. Taken together, the observations indicate that Ras activates non-conventional and perhaps unique effector pathways to induce cytoplasmic vacuolation in glioblastoma cells. Identification of the relevant signaling pathways may uncover specific molecular targets that can be manipulated to activate non-apoptotic cell death in this type of cancer.

    Funded by: NCI NIH HHS: R01 CA034569, R01 CA115495, R01 CA34569

    Cellular signalling 2007;19;5;1034-43

  • Electrical properties of plasma membrane modulate subcellular distribution of K-Ras.

    Gomez GA and Daniotti JL

    Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC, UNC-CONICET), Departamento de Química Biológica, Universidad Nacional de Córdoba, Argentina.

    K-Ras is a small G-protein, localized mainly at the inner leaflet of the plasma membrane. The membrane targeting signal of this protein consists of a polybasic C-terminal sequence of six contiguous lysines and a farnesylated cysteine. Results from biophysical studies in model systems suggest that hydrophobic and electrostatic interactions are responsible for the membrane binding properties of K-Ras. To test this hypothesis in a cellular system, we first evaluated in vitro the effect of electrolytes on K-Ras membrane binding properties. Results demonstrated the electrical and reversible nature of K-Ras binding to anionic lipids in membranes. We next investigated membrane binding and subcellular distribution of K-Ras after disruption of the electrical properties of the outer and inner leaflets of plasma membrane and ionic gradients through it. Removal of sialic acid from the outer plasma membrane caused a redistribution of K-Ras to recycling endosomes. Inhibition of polyphosphoinositide synthesis at the plasma membrane, by depletion of cellular ATP, resulted in a similar subcellular redistribution of K-Ras. Treatment of cells with ionophores that modify transmembrane potential caused a redistribution of K-Ras to cytoplasm and endomembranes. Ca2+ ionophores, compared to K+ ionophores, caused a much broader redistribution of K-Ras to endomembranes. Taken together, these results reveal the dynamic nature of interactions between K-Ras and cellular membranes, and indicate that subcellular distribution of K-Ras is driven by electrostatic interaction of the polybasic region of the protein with negatively charged membranes.

    The FEBS journal 2007;274;9;2210-28

  • Low prevalence of p53, p16(INK4a) and Ha-ras tumour-specific mutations in low-graded actinic keratosis.

    Nindl I, Gottschling M, Krawtchenko N, Lehmann MD, Röwert-Huber J, Eberle J, Stockfleth E and Forschner T

    Department of Dermatology, Charité, Skin Cancer Center Charité, University Hospital of Berlin, Berlin, Germany. ingo.nindl@charite.de

    Background: Ultraviolet radiation induces DNA damage and is the major risk factor for the development of non-melanoma skin cancer (NMSC). Different mutation rates of p53, p16(INK4a) and Ha-ras in cutaneous squamous cell carcinoma (SCC) and the earlier stage actinic keratosis (AK) have been reported.

    Objectives: To assess the presence of missense mutations in hotspot exons of p53, p16(INK4a) and Ha-ras in low-graded AK.

    Cryo-biopsies of 75 sun-exposed AK lesions and 75 sun-shielded areas of normal skin from 75 AK patients were analysed to identify mutations in p53 (exons 7 and 8), p16(INK4a) (exon 2) and Ha-ras (exon 1) using polymerase chain reaction (PCR) followed by direct sequencing. As a representative subset of the specimens, ten mutation-negative AK were also micro-dissected in order to exclude the possibility that additional mutations were undetected.

    Results: Eight missense and one nonsense point mutations were found in the 75 AK lesions examined (12%), of which seven (9%) were tumour-specific (i.e. present in AK lesions only) and two (3%) were p16(INK4a) mutations (i.e. also detected in normal skin). Three of the tumour-specific mutations (42%) were cytosine (C) to thymine (T) transitions at pyrimidine-rich sequences. Tumour-specific mutations were identified in 1% of p16(INK4a) (exon 2), 1% of Ha-ras (exon 1) and at a higher rate of 7% in p53 (exons 7 and 8), including one nonsense mutation.

    Conclusions: The evaluation of a large number of AK specimens in this study have found a low gene mutation rate in low-graded AK lesions. p53 mutations rather than p16(INK4a) and/or Ha-ras mutations may be an early event in the development of AK to cutaneous SCC.

    The British journal of dermatology 2007;156 Suppl 3;34-9

  • RIG1 suppresses Ras activation and induces cellular apoptosis at the Golgi apparatus.

    Tsai FM, Shyu RY and Jiang SY

    Graduate Institute of Life Sciences, National Defense Medical Center, and Department of Medical Education and Research, Buddhist Tzu Chi General Hospital, 289 Jianguo Road, Xindian City, Taipei, Taiwan, ROC.

    Retinoid-inducible gene 1 encodes RIG1 is a growth regulator, which inhibits the pathways of the RAS/mitogen-activated protein kinases by suppressing the activation of RAS. Confocal microscopic analysis demonstrated that RIG1 is localized in the endoplasmic reticulum (ER) and Golgi apparatus in HtTA cervical cancer cells. Carboxyterminal-deleted RIG1 targeted to the Golgi or ER was constructed and validated. The activation of HRAS was inhibited by 25.1% or 81.4% in cells cotransfected with wild-type or Golgi-targeted RIG1, respectively. Expression of wild-type or Golgi-targeted RIG1 for 24 h induced cellular apoptosis in HtTA cells, as assessed by MTT assay, the release of lactate dehydrogenase, and chromatin condensation. In contrast, ER-targeted RIG1 and carboxyterminal-deleted RIG1 (RIG1DeltaC) exhibited no activity. Caspase-2, -3, and -9 were activated following the expression of wild-type and Golgi-targeted RIG1. Although the caspase-3 inhibitor Z-DEVD-FMK partially or completely reversed the cell death induced by wild-type or Golgi-targeted RIG1, it did not prevent the anti-RAS effect of RIG1. In conclusion, the proapoptotic and anti-RAS activities of RIG1 are primarily associated with the Golgi localization of the protein. The proapoptotic activities of RIG1 are mediated through the activation of caspase-2 and -3 and are independent of its effect on RAS.

    Cellular signalling 2007;19;5;989-99

  • Selective role for RGS12 as a Ras/Raf/MEK scaffold in nerve growth factor-mediated differentiation.

    Willard MD, Willard FS, Li X, Cappell SD, Snider WD and Siderovski DP

    Department of Pharmacology, University of North Carolina, Chapel Hill, NC 27599-7365, USA.

    Regulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by heterotrimeric G-protein alpha subunits and thus inhibit signaling by many G protein-coupled receptors. Several RGS proteins have a multidomain architecture that adds further complexity to their roles in cell signaling in addition to their GTPase-accelerating activity. RGS12 contains a tandem repeat of Ras-binding domains but, to date, the role of this protein in Ras-mediated signal transduction has not been reported. Here, we show that RGS12 associates with the nerve growth factor (NGF) receptor tyrosine kinase TrkA, activated H-Ras, B-Raf, and MEK2 and facilitates their coordinated signaling to prolonged ERK activation. RGS12 is required for NGF-mediated neurite outgrowth of PC12 cells, but not outgrowth stimulated by basic fibroblast growth factor. siRNA-mediated knockdown of RGS12 expression also inhibits NGF-induced axonal growth in dissociated cultures of primary dorsal root ganglia neurons. These data suggest that RGS12 may play a critical, and receptor-selective, role in coordinating Ras-dependent signals that are required for promoting and/or maintaining neuronal differentiation.

    Funded by: NIGMS NIH HHS: GM062338, R01 GM062338, T32 GM007040; NINDS NIH HHS: R01 NS031768

    The EMBO journal 2007;26;8;2029-40

  • Further evidence for a somatic KRAS mutation in a pilocytic astrocytoma.

    Janzarik WG, Kratz CP, Loges NT, Olbrich H, Klein C, Schäfer T, Scheurlen W, Roggendorf W, Weiller C, Niemeyer C, Korinthenberg R, Pfister S and Omran H

    Department of Neurology, University Hospital Freiburg, Freiburg, Germany.

    Astrocytomas are the most common brain tumors of childhood. However, knowledge of the molecular etiology of astrocytomas WHO grade I and II is limited. Germline mutations in the Ras-guanosine triphosphatase-activating protein, neurofibromin, in individuals with neurofibromatosis type I predispose to pilocytic astrocytomas. This association suggests that constitutive activation of the Ras signaling pathway plays a fundamental role in astrocytoma development. We screened 25 WHO I and II astrocytomas for mutations of PTPN11, NRAS, KRAS, and HRAS genes and identified the somatic G12A KRAS mutation in one pilocytic astrocytoma. These data suggest that Ras is rarely mutated in these tumors. Analyzed astrocytomas without mutations in Ras or neurofibromin may harbor mutations in other proteins of this pathway leading to hyperactive Ras signaling.

    Neuropediatrics 2007;38;2;61-3

  • Distal phalangeal creases--a distinctive dysmorphic feature in disorders of the RAS signalling pathway?

    Ørstavik KH, Tangeraas T, Molven A and Prescott TE

    Department of Medical Genetics, Rikshospitalet-Radiumhospitalet Medical Centre, Oslo, Norway.

    We have observed unusual transverse distal phalangeal creases in two patients, one with Costello syndrome (G12S mutation in the HRAS gene) and one with cardio-facio-cutaneous (CFC) syndrome or possibly Noonan syndrome (Q22E mutation in the KRAS gene). This feature along with fetal pads was present in both children at birth and has persisted until age two years. Distal phalangeal creases, when present, may be a good diagnostic handle for syndromes belonging to the RAS signalling pathway.

    European journal of medical genetics 2007;50;2;155-8

  • Diversity, parental germline origin, and phenotypic spectrum of de novo HRAS missense changes in Costello syndrome.

    Zampino G, Pantaleoni F, Carta C, Cobellis G, Vasta I, Neri C, Pogna EA, De Feo E, Delogu A, Sarkozy A, Atzeri F, Selicorni A, Rauen KA, Cytrynbaum CS, Weksberg R, Dallapiccola B, Ballabio A, Gelb BD, Neri G and Tartaglia M

    Istituto di Clinica Pediatrica, Università Cattolica del Sacro Cuore, Rome, Italy.

    Activating mutations in v-Ha-ras Harvey rat sarcoma viral oncogene homolog (HRAS) have recently been identified as the molecular cause underlying Costello syndrome (CS). To further investigate the phenotypic spectrum associated with germline HRAS mutations and characterize their molecular diversity, subjects with a diagnosis of CS (N = 9), Noonan syndrome (NS; N = 36), cardiofaciocutaneous syndrome (CFCS; N = 4), or with a phenotype suggestive of these conditions but without a definitive diagnosis (N = 12) were screened for the entire coding sequence of the gene. A de novo heterozygous HRAS change was detected in all the subjects diagnosed with CS, while no lesion was observed with any of the other phenotypes. While eight cases shared the recurrent c.34G>A change, a novel c.436G>A transition was observed in one individual. The latter affected residue, p.Ala146, which contributes to guanosine triphosphate (GTP)/guanosine diphosphate (GDP) binding, defining a novel class of activating HRAS lesions that perturb development. Clinical characterization indicated that p.Gly12Ser was associated with a homogeneous phenotype. By analyzing the genomic region flanking the HRAS mutations, we traced the parental origin of lesions in nine informative families and demonstrated that de novo mutations were inherited from the father in all cases. We noted an advanced age at conception in unaffected fathers transmitting the mutation.

    Funded by: NHLBI NIH HHS: HL71207; NICHD NIH HHS: HD01294, HD048502; Telethon: GGP04172

    Human mutation 2007;28;3;265-72

  • Uniparental disomy at chromosome 11p15.5 followed by HRAS mutations in embryonal rhabdomyosarcoma: lessons from Costello syndrome.

    Kratz CP, Steinemann D, Niemeyer CM, Schlegelberger B, Koscielniak E, Kontny U and Zenker M

    Division of Pediatric Hematology and Oncology, Department of Pediatrics and Adolescent Medicine, University of Freiburg, Mathildenstrasse 1, 79106 Freiburg, Germany. christian.kratz@uniklinik-freiburg.de

    Costello syndrome (CS; MIM 218040) is characterized by short stature, facial dysmorphism, cardiac defects and predisposition to embryonal rhabdomyosarcoma (CS/ERMS) and other neoplasias. CS is caused by germline mutations in the HRAS gene on chromosome 11p15.5, a region showing allelic imbalances in sporadic ERMS and CS/ERMS. The critical gene for ERMS development in this region is unknown. The association of CS and ERMS as well as previous reports illustrating that somatic HRAS mutations are found in a proportion of these tumors prompted us to clarify the significance and a possible correlation of HRAS mutations and genomic rearrangements at 11p15.5 in sporadic ERMS. We screened for somatic HRAS mutations and 11p15.5 imbalances in six sporadic ERMS samples. This analysis uncovered five ERMS samples with uniparental disomy (UPD) at the HRAS locus, two of which harbored HRAS mutations. By analyzing informative genetic variations in or at the HRAS gene locus, we show that one HRAS allele is entirely lost in specimens with UPD at 11p15.5. Notably, in both cases with UPD and HRAS mutations these mutations were heterozygous. Therefore, they must have succeeded the emergence of UPD. In contrast, HRAS germline mutations are the first step in CS/ERMS. Subsequent development of UPD at 11p15.5 may explain previous observations that CS/ERMS express mutant HRAS only. These data implicate that in sporadic ERMS, UPD at 11p15.5 is not driven by HRAS mutations and that imbalances at 11p15.5 and HRAS mutations represent independent but cooperating events during ERMS development.

    Human molecular genetics 2007;16;4;374-9

  • GTP binding is essential to the protein kinase activity of LRRK2, a causative gene product for familial Parkinson's disease.

    Ito G, Okai T, Fujino G, Takeda K, Ichijo H, Katada T and Iwatsubo T

    Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyoku, Tokyo, 113-0033 Japan.

    Leucine-rich repeat kinase 2 (LRRK2), a product of a causative gene for the autosomal-dominant form of familial Parkinson's disease (PARK8), harbors a Ras-like small GTP binding protein-like (ROC) domain besides the kinase domain, although the relationship between these two functional domains remains elusive. Here we show by thin-layer chromatographic analysis that LRRK2 stably binds GTP but lacks a GTPase activity in HEK293 and Neuro-2a cells. A ROC domain mutation that converts LRRK2 to a guanine nucleotide-free form (T1348N) abolishes the kinase activity of LRRK2 as well as its phosphate incorporation upon metabolic labeling. The phosphorylation of LRRK2 was inhibited by potential inhibitors for cyclic AMP-dependent protein kinase. These data suggest that binding of GTP to the ROC domain regulates the kinase activity of LRRK2 as well as its phosphorylation by other kinase(s).

    Biochemistry 2007;46;5;1380-8

  • The B cell antigen receptor controls AP-1 and NFAT activity through Ras-mediated activation of Ral.

    de Gorter DJ, Vos JC, Pals ST and Spaargaren M

    Department of Pathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

    Signaling by the BCR involves activation of several members of the Ras superfamily of small GTPases, among which is Ras itself. Ras can control the activity of multiple effectors, including Raf, PI3K, and guanine nucleotide exchange factors for the small GTPase Ral. Ras, Raf, and PI3K have been implicated in a variety of processes underlying B cell development, differentiation, and function; however, the role of Ral in B lymphocytes remains to be established. In this study, we show that Ral is activated upon BCR stimulation in human tonsillar and mouse splenic B lymphocytes and in B cell lines. Using signaling molecule-deficient B cells, we demonstrate that this activation is mediated by Lyn and Syk, Btk, phospholipase C-gamma2, and inositol-1,4,5-trisphosphate receptor-mediated Ca(2+) release. In addition, although Ral can be activated by Ras-independent mechanisms, we demonstrate that BCR-controlled activation of Ral is dependent on Ras. By means of expression of the dominant-negative mutants RasN17 and RalN28, or of RalBPDeltaGAP, a Ral effector mutant which sequesters active Ral, we show that Ras and Ral mediate BCR-controlled transcription of c-fos. Furthermore, while not involved in NF-kappaB activation, Ras and Ral mediate BCR-controlled activation of JUN/ATF2 and NFAT transcription factors. Taken together, our data show that Ral is activated upon BCR stimulation and mediates BCR-controlled activation of AP-1 and NFAT transcription factors. These findings suggest that Ral plays an important role in B cell development and function.

    Journal of immunology (Baltimore, Md. : 1950) 2007;178;3;1405-14

  • Mechanisms of guanosine triphosphate hydrolysis by Ras and Ras-GAP proteins as rationalized by ab initio QM/MM simulations.

    Grigorenko BL, Nemukhin AV, Shadrina MS, Topol IA and Burt SK

    Department of Chemistry, M.V. Lomonosov Moscow State University, Moscow, 119992, Russian Federation.

    The hydrolysis reaction of guanosine triphosphate (GTP) by p21(ras) (Ras) has been modeled by using the ab initio type quantum mechanical-molecular mechanical simulations. Initial geometry configurations have been prompted by atomic coordinates of the crystal structure (PDBID: 1QRA) corresponding to the prehydrolysis state of Ras in complex with GTP. Multiple searches of minimum energy geometry configurations consistent with the hydrogen bond networks have been performed, resulting in a series of stationary points on the potential energy surface for reaction intermediates and transition states. It is shown that the minimum energy reaction path is consistent with an assumption of a two-step mechanism of GTP hydrolysis. At the first stage, a unified action of the nearest residues of Ras and the nearest water molecules results in a substantial spatial separation of the gamma-phosphate group of GTP from the rest of the molecule (GDP). This phase of hydrolysis process proceeds through the low barrier (16.7 kcal/mol) transition state TS1. At the second stage, the inorganic phosphate is formed in consequence of proton transfers mediated by two water molecules and assisted by the Gln61 residue from Ras. The highest transition state at this segment, TS3, is estimated to have an energy 7.5 kcal/mol above the enzyme-substrate complex. The results of simulations are compared to the previous findings for the GTP hydrolysis in the Ras-GAP (p21(ras)-p120(GAP)) protein complex. Conclusions of the modeling lead to a better understanding of the anticatalytic effect of cancer causing mutation of Gln61 from Ras, which has been debated in recent years.

    Funded by: NCI NIH HHS: N01-CO-12400

    Proteins 2007;66;2;456-66

  • Expansion of the genotypic and phenotypic spectrum in patients with KRAS germline mutations.

    Zenker M, Lehmann K, Schulz AL, Barth H, Hansmann D, Koenig R, Korinthenberg R, Kreiss-Nachtsheim M, Meinecke P, Morlot S, Mundlos S, Quante AS, Raskin S, Schnabel D, Wehner LE, Kratz CP, Horn D and Kutsche K

    Institute of Human Genetics, University of Erlangen-Nuremberg, Germany.

    Background: Noonan syndrome, cardio-facio-cutaneous syndrome (CFC) and Costello syndrome constitute a group of developmental disorders with an overlapping pattern of congenital anomalies. Each of these conditions can be caused by germline mutations in key components of the highly conserved Ras-MAPK pathway, possibly reflecting a similar pathogenesis underlying the three disorders. Germline mutations in KRAS have recently been identified in a small number of patients with Noonan syndrome and CFC.

    260 patients were screened for KRAS mutations by direct sequencing. Overall, we detected KRAS mutations in 12 patients, including three known and eight novel sequence alterations. All mutations are predicted to cause single amino acid substitutions. Remarkably, our cohort of individuals with KRAS mutations showed a high clinical variability, ranging from Noonan syndrome to CFC, and also included two patients who met the clinical criteria of Costello syndrome.

    Conclusion: Our findings reinforce the picture of a clustered distribution of disease associated KRAS germline alterations. We further defined the phenotypic spectrum associated with KRAS missense mutations and provided the first evidence of clinical differences in patients with KRAS mutations compared with Noonan syndrome affected individuals with heterozygous PTPN11 mutations and CFC patients carrying a BRAF, MEK1 or MEK1 alteration, respectively. We speculate that the observed phenotypic variability may be related, at least in part, to specific genotypes and possibly reflects the central role of K-Ras in a number of different signalling pathways.

    Journal of medical genetics 2007;44;2;131-5

  • p73 cooperates with Ras in the activation of MAP kinase signaling cascade.

    Fernandez-Garcia B, Vaqué JP, Herreros-Villanueva M, Marques-Garcia F, Castrillo F, Fernandez-Medarde A, León J and Marín MC

    Instituto de Biomedicina, Universidad de León, Campus de Vegazana, León 24071, Spain.

    The p73 gene is capable of inducing cell cycle arrest, apoptosis, senescence, differentiation and to cooperate with oncogenic Ras in cellular transformation. Ras can be considered as a branch point in signal transduction, where diverse extracellular stimuli converge. The intensity of the mitogen-activated protein kinase (MAPK) cascade activation influences the cellular response to Ras. Despite the fundamental role of p53 in Ras-induced growth arrest and senescence, it remains unclear how the Ras/MEK/ERK pathway induces growth arrest in the absence of p53. We report here that oncogenic Ras stabilizes p73 resulting in p73 accumulation and enhancement of its activity. p73, in turn, induces a sustained activation of the MAP kinase cascade synergizing with oncogenic Ras. We also found that inhibition of p73 function modifies the cellular outcome to Ras activation inhibiting Ras-dependent differentiation. Here, we show for the first time that there is a signaling loop between Ras-dependent MAPK cascade activation and p73 function.

    Cell death and differentiation 2007;14;2;254-65

  • Phosphatidylinositol-3-OH kinase or RAS pathway mutations in human breast cancer cell lines.

    Hollestelle A, Elstrodt F, Nagel JH, Kallemeijn WW and Schutte M

    Department of Medical Oncology, Josephine Nefkens Institute Be414, Erasmus MC, P.O. Box 1738, 3000 DR Rotterdam, the Netherlands.

    Constitutive activation of the phosphatidylinositol-3-OH kinase (PI3K) and RAS signaling pathways are important events in tumor formation. This is illustrated by the frequent genetic alteration of several key players from these pathways in a wide variety of human cancers. Here, we report a detailed sequence analysis of the PTEN, PIK3CA, KRAS, HRAS, NRAS, and BRAF genes in a collection of 40 human breast cancer cell lines. We identified a surprisingly large proportion of cell lines with mutations in the PI3K or RAS pathways (54% and 25%, respectively), with mutants for each of the six genes. The PIK3CA, KRAS, and BRAF mutation spectra of the breast cancer cell lines were similar to those of colorectal cancers. Unlike in colorectal cancers, however, mutational activation of the PI3K pathway was mutually exclusive with mutational activation of the RAS pathway in all but 1 of 30 mutant breast cancer cell lines (P = 0.001). These results suggest that there is a fine distinction between the signaling activators and downstream effectors of the oncogenic PI3K and RAS pathways in breast epithelium and those in other tissues.

    Molecular cancer research : MCR 2007;5;2;195-201

  • Feedback inhibition of calcineurin and Ras by a dual inhibitory protein Carabin.

    Pan F, Sun L, Kardian DB, Whartenby KA, Pardoll DM and Liu JO

    Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

    Feedback regulation of adaptive immunity is a fundamental mechanism for controlling the overall output of different signal transduction pathways, including that mediated by the T-cell antigen receptor (TCR). Calcineurin and Ras are known to have essential functions during T-cell activation. However, how the calcineurin signalling pathway is terminated in the process is still largely unknown. Although several endogenous inhibitors of calcineurin have been reported, none fulfils the criteria of a feedback inhibitor, as their expression is not responsive to TCR signalling. Here we identify an endogenous inhibitor of calcineurin, named Carabin, which also inhibits the Ras signalling pathway through its intrinsic Ras GTPase-activating protein (GAP) activity. Expression of Carabin is upregulated on TCR signalling in a manner that is sensitive to inhibitors of calcineurin, indicating that Carabin constitutes part of a negative regulatory loop for the intracellular TCR signalling pathway. Knockdown of Carabin by short interfering RNA led to a significant enhancement of interleukin-2 production by antigen-specific T cells in vitro and in vivo. Thus, Carabin is a negative feedback inhibitor of the calcineurin signalling pathway that also mediates crosstalk between calcineurin and Ras.

    Nature 2007;445;7126;433-6

  • Cervical keratinocytes containing stably replicating extrachromosomal HPV-16 are refractory to transformation by oncogenic H-Ras.

    Berger KL, Barriga F, Lace MJ, Turek LP, Zamba GJ, Domann FE, Lee JH and Klingelhutz AJ

    Department of Microbiology, Holden Comprehensive Cancer Research Center, University of Iowa, 375 Newton Road, 2202 MERF, Iowa City, IA 52242, USA.

    Ras expression in human epithelial cells with integrated HPV genomes has been shown to cause tumorigenic transformation. The effects of Ras in cells representing early stage HPV-associated disease (i.e., when HPV is extrachromosomal and the oncogenes are under control of native promoters) have not been examined. Here, we used human cervical keratinocyte cell lines containing stably replicating extrachromosomal HPV-16 and present the novel finding that these cells resist transformation by oncogenic H-Ras. Ras expression consistently diminished anchorage-independent growth (AI), reduced E6 and E7 expression, and caused p53 induction in these cells. Conversely, AI was enhanced or maintained in Ras-transduced cervical cells that were immortalized with a 16E6/E7 retrovirus, and minimal effects on E6 and E7 expression were observed. Ras expression with either episomal HPV-16 or LXSN-E6/E7 was insufficient for tumorigenic growth suggesting that other events are needed for tumorigenic transformation. In conclusion, our results indicate that Ras-mediated transformation depends on the context of HPV oncogene expression and that this is an important point to address when developing HPV tumor models.

    Funded by: NCI NIH HHS: CA66081, P01 CA066081; NIA NIH HHS: AG18265, R01 AG018265

    Virology 2006;356;1-2;68-78

  • Ductal origin of pancreatic adenocarcinomas induced by conditional activation of a human Ha-ras oncogene in rat pancreas.

    Ueda S, Fukamachi K, Matsuoka Y, Takasuka N, Takeshita F, Naito A, Iigo M, Alexander DB, Moore MA, Saito I, Ochiya T and Tsuda H

    Department of Molecular Toxicology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan.

    Pancreatic ductal adenocarcinoma is one of the most debilitating malignancies in humans. Currently, radiation and chemotherapy are ineffective, with median survival times after treatment of <12 months. Animal models that reflect the human condition and can be used to explore screening and therapeutic approaches are clearly desirable. One feature of human pancreatic adenocarcinoma is an exceedingly high frequency of K-ras mutation. The present study was conducted to determine if targeted activation of a human oncogenic-ras transgene in rat pancreas would induce carcinomas correspondent to human pancreatic ductal adenocarcinomas. We established transgenic (Hras250) rats in which expression of a human Ha-rasG12V oncogene is regulated by the Cre/lox system. Targeted pancreatic activation of the transgene was accomplished by injection of Cre-carrying adenovirus into the pancreatic ducts and acini through the common bile duct. Adenoviral infection of injected animals was exclusive to the pancreas; infected cells could be identified in duct, intercalated duct, centroacinar and, less frequently, acinar cells, but not in endocrine islet cells. Four weeks after injection, proliferative lesions in the duct epithelium, intercalated ducts and centroacinar cells, but not acinar cells, were widespread. Tumorigenesis in other tissues was not observed. Most lesions, including atypical duct proliferative lesions, PanIN-like lesions and carcinomas, were positive for cytokeratins 19 and 7, cyclooxygenase 2 and MMP-7 but negative for amylase and chymotrypsin. Many adenocarcinoma lesions were positive for EGF and EGFR. Duct epithelial and atypical duct proliferative lesions and carcinoma lesions were all positive for transduced Ha-rasG12V oncogene expression. The cytogenesis of pancreatic ductal type carcinoma was depicted. This model exhibits important similarities to the human disease and promises to advance our understanding of the behavior of pancreas adenocarcinomas and expedite screening and therapy.

    Carcinogenesis 2006;27;12;2497-510

  • [K-ras mutation analysis in ovarian serous borderline and malignant tumors].

    Ma L, Guo LN, Liang ZY, Ren XY and Wu SF

    Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China.

    Objective: To investigate the roles of K-ras gene in the tumorigenesis of ovarian serous borderline and malignant tumors.

    Methods: Fifty one tissue samples of ovarian serous tumors, including 18 conventional serous borderline tumors, 11 micropapillary serous borderline tumors, 12 invasive micropapillary serous carcinomas, and 10 conventional serous carcinomas were investigated for the presence of K-ras mutation. DNA was extracted after microdissection of the tumor tissue, the exon 1 of K-ras gene was amplified by PCR, and the presence of mutation at the codons 12 and 13 was evaluated by direct sequencing analysis.

    Results: GGT to GTT mutation at codon 12 of the K-ras gene was found in one conventional serous borderline tumors, resulting in valine to glycine substitution. All other 50 cases showed no K-ras mutation. All tumors had a wild-type codon 13.

    Conclusions: Mutations of K-ras at codons 12 and 13 in ovarian serous tumors are very rare in this series of patients, suggesting a difference present between the Chinese and Caucasian populations. K-ras mutations may play a less important role in the tumorigenesis of ovarian serous tumor of the Chinese patients.

    Zhonghua bing li xue za zhi = Chinese journal of pathology 2006;35;12;714-8

  • MIP-1alpha (CCL3) is a downstream target of FGFR3 and RAS-MAPK signaling in multiple myeloma.

    Masih-Khan E, Trudel S, Heise C, Li Z, Paterson J, Nadeem V, Wei E, Roodman D, Claudio JO, Bergsagel PL and Stewart AK

    Department of Medical Oncology, Princess Margaret Hospital, University Health Network, Toronto, ON, Canada.

    Overexpression of fibroblast growth factor receptor 3 (FGFR3) is a hallmark of t(4;14) multiple myeloma (MM). To dissect the mechanism of FGFR3 oncogenesis in MM, we used 3 FGFR selective kinase inhibitors-CHIR258, PD173074, and SU5402-and FGFR3-specific siRNA to modulate FGFR3 activity. Conversely, the ligand FGF was used to stimulate FGFR3 function in human MM cells. The transcriptional response to FGFR3 modification was recorded, and gene expression changes common to all 5 modifiers were documented. Ten genes were commonly regulated. Macrophage inflammatory protein-1 alpha (MIP-1alpha) was the single most differentially altered gene. MIP-1 alpha promoter function, gene expression, and protein secretion were each down-regulated following inhibition of FGFR3 signaling. Down-regulation of MIP-1 alpha was not, however, observed following FGFR3 inhibition in MM cells with RAS mutations implicating RAS-MAPK in MIP-1 alpha regulation. As confirmation, inhibition of ERK1 also down-regulated MIP-1 alpha in FGFR3 inhibitor-resistant cells harboring RAS mutations. MIP-1 alpha is implicated in the survival and proliferation of MM cells and the pathogenesis of MM bone disease. Our observation is the first to directly link an initiating IgH translocation not only to MM-cell growth and survival but also to the disease-associated bone disease.

    Blood 2006;108;10;3465-71

  • Ras triggers acidosis-induced activation of the extracellular-signal-regulated kinase pathway in cardiac myocytes.

    Haworth RS, Dashnyam S and Avkiran M

    Cardiovascular Division, King's College London, The Rayne Institute, St Thomas' Hospital, Lambeth Palace Road, London SE1 7EH, UK. robert.haworth@kcl.ac.uk

    In cardiac myocytes, sustained (3 min) intracellular acidosis activates the ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway and, through this pathway, increases sarcolemmal NHE (Na+/H+ exchanger) activity [Haworth, McCann, Snabaitis, Roberts and Avkiran (2003) J. Biol. Chem. 278, 31676-31684]. In the present study, we aimed to determine the time-dependence, pH-dependence and upstream signalling mechanisms of acidosis-induced ERK1/2 activation in ARVM (adult rat ventricular myocytes). Cultured ARVM were subjected to intracellular acidosis for up to 20 min by exposure to NH4Cl, followed by washout with a bicarbonate-free Tyrode solution containing the NHE1 inhibitor cariporide. After the desired duration of intracellular acidosis, the phosphorylation status of ERK1/2 and its downstream effector p90(RSK) (90 kDa ribosomal S6 kinase) were determined by Western blotting. This revealed a time-dependent transient phosphorylation of both ERK1/2 and p90(RSK) by intracellular acidosis (intracellular pH approximately 6.6), with maximum activation occurring at 3 min and a return to basal levels by 20 min. When the degree of intracellular acidosis was varied from approximately 6.8 to approximately 6.5, maximum ERK1/2 phosphorylation was observed at an intracellular pH of 6.64. Inhibition of MEK1/2 [MAPK (mitogen-activated protein kinase)/ERK kinase 1/2) by pre-treatment of ARVM with U0126 or adenoviral expression of dominant-negative D208A-MEK1 protein prevented the phosphorylation of ERK1/2 by sustained intracellular acidosis, as did inhibition of Raf-1 with GW 5074 or ZM 336372. Interference with Ras signalling by the adenoviral expression of dominant-negative N17-Ras protein or with FPT III (farnesyl protein transferase inhibitor III) also prevented acidosis-induced ERK1/2 phosphorylation, whereas inhibiting G-protein signalling [by adenoviral expression of RGS4 or Lsc, the RGS domain of p115 RhoGEF (guanine nucleotide-exchange factor)] or protein kinase C (with bisindolylmaleimide I) had no effect. Our data show that, in ARVM, sustained intracellular acidosis activates ERK1/2 through proximal activation of the classical Ras/Raf/MEK pathway.

    The Biochemical journal 2006;399;3;493-501

  • Somatic mosaicism for an HRAS mutation causes Costello syndrome.

    Gripp KW, Stabley DL, Nicholson L, Hoffman JD and Sol-Church K

    Division of Medical Genetics, A. I. duPont Hospital for Children, Wilmington, Delaware 19899, USA. kgripp@nemours.org

    De novo heterozygous HRAS point mutations have been reported in more than 81 patients with Costello syndrome (CS), but genotype/phenotype correlation remains incomplete because the majority of patients share a common mutation, G12S, seen in 65/81 (80%). Somatic HRAS mutations have previously been identified in solid tumors, and mutation hot spots related to a gain-of-function effect of the gene product are known. The germline mutations causing CS occur at these hot spots and convey a gain-of-function effect, thus accounting for the greatly increased cancer risk. Diagnostic testing for HRAS mutations is now available and the identification of a mutation in a patient with consistent clinical findings confirms a diagnosis of CS. It is not clear yet if the absence of an HRAS mutation precludes a diagnosis of CS. Because there is a significant overlap in the clinical findings of Costello, cardio-facio-cutaneous, and Noonan syndromes, diagnostic uncertainty remains in patients lacking an HRAS mutation. We report here on a female with findings suggestive of CS in whom mutation analysis performed with standard techniques on white blood cell derived DNA did not show an HRAS mutation. However, analysis of DNA derived from three independently collected buccal swabs showed a sequence change qualitatively consistent with the G12S mutation. Allelic quantitation showed the presence of the mutation in approximately 25%-30% of the sampled buccal cells. In this patient, standard technology failed to identify the disease causing mutation on DNA derived from a blood sample, highlighting the potential pitfalls in the interpretation of negative mutation studies. This is the first reported CS patient mosaic for the common HRAS mutation, likely due to a somatic mutation occurring very early in fetal development.

    Funded by: NCRR NIH HHS: 1 P20 RR020173-01

    American journal of medical genetics. Part A 2006;140;20;2163-9

  • Ras-induced modulation of CXCL10 and its receptor splice variant CXCR3-B in MDA-MB-435 and MCF-7 cells: relevance for the development of human breast cancer.

    Datta D, Flaxenburg JA, Laxmanan S, Geehan C, Grimm M, Waaga-Gasser AM, Briscoe DM and Pal S

    Division of Nephrology, Children's Hospital Boston, Boston, MA 02115, USA.

    Interactions between chemokines and chemokine receptors have been proposed recently to be of importance in the development and progression of cancer. Human breast cancer cells express the chemokine CXCL10 (IP-10) and also its receptor CXCR3. In this study, we have investigated the role of Ras activation in the regulation of CXCL10 and its receptor splice variant CXCR3-B in two human breast cancer cell lines MDA-MB-435 and MCF-7. In cotransfection assays, using a full-length CXCL10 promoter-luciferase construct, we found that the activated form of Ras, Ha-Ras(12V), promoted CXCL10 transcriptional activation. Ras significantly increased CXCL10 mRNA and protein expression as observed by real-time PCR, fluorescence-activated cell sorting analysis, and ELISA. Selective inhibition of Ha-Ras by small interfering RNA (siRNA) decreased CXCL10 mRNA expression in a dose-dependent manner. Further, using effector domain mutants of Ras, we found that Ras-induced overexpression of CXCL10 is mediated primarily through the Raf and phosphatidylinositol 3-kinase signaling pathways. We also observed that the expression of the splice variant CXCR3-B, known to inhibit cell proliferation, was significantly down-regulated by Ras. Selective inhibition of CXCR3-B using siRNA resulted in an increase in CXCL10-mediated breast cancer cell proliferation through G(i) proteins and likely involving CXCR3-A. Finally, we observed intense expression of CXCL10 and CXCR3 in association with human breast cancer in situ, indicating that these observations may be of pathophysiologic significance. Together, these results suggest that activation of Ras plays a critical role in modulating the expression of both CXCL10 and CXCR3-B, which may have important consequences in the development of breast tumors through cancer cell proliferation.

    Funded by: NHLBI NIH HHS: HL74436, R01 HL074436; NIDDK NIH HHS: DK64182, T32 DK007726; PHS HHS: DBU16011

    Cancer research 2006;66;19;9509-18

  • Ras-dependent recruitment of c-Myc for transcriptional activation of nucleophosmin/B23 in highly malignant U1 bladder cancer cells.

    Yeh CW, Huang SS, Lee RP and Yung BY

    Cancer Biochemistry Laboratory, Department of Pharmacology, College of Medicine, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-San, Tao-Yuan 333, Taiwan, R.O.C.

    U1 bladder cancer cells of high malignancy exhibited higher proliferation capacity than U4 premalignant cells. Higher expression of Ras, c-Myc, and nucleophosmin/B23 and greater c-Myc transactivation and nucleophosmin/B23 promoter activities were detected in U1 cells compared with U4 cells. Moreover, c-Myc and nucleophosmin/B23 were increased in U1 but not in U4 cells upon serum stimulation from quiescence. Likewise, only in U1 cells could serum stimulate transcriptional activity of nucleophosmin/B23 promoter and c-Myc response element. The increase of nucleophosmin/B23 promoter activity could be abrogated by mitogen-activated protein kinase/extracellular signal-regulated kinase activating kinase inhibitor and was associated with recruitment of c-Myc to the promoter. U1 cells constitutively expressing dominant-negative Ras reduced the levels of Ras, nucleophosmin/B23, and p-ERK, and consequently abolished the serum-induced up-regulation of nucleophosmin/B23 promoter activity and c-Myc promoter recruitment. Our results indicate that Ras and c-Myc play important roles in the up-regulation of nucleophosmin/B23 during proliferation of cells associated with a high degree of malignancy, thus outlining a signaling cascade involving these factors in the cancer cells.

    Molecular pharmacology 2006;70;4;1443-53

  • Strain-stimulated hypertrophy in cardiac myocytes is mediated by reactive oxygen species-dependent Ras S-glutathiolation.

    Pimentel DR, Adachi T, Ido Y, Heibeck T, Jiang B, Lee Y, Melendez JA, Cohen RA and Colucci WS

    Cardiovascular Medicine Section, Department of Medicine, Boston University Medical Center, Boston, MA 02118, USA.

    Although reactive oxygen species (ROS) appear to play a central role in mediating myocardial hypertrophy in response to hemodynamic overload, little is known about the molecular targets by which ROS regulate growth signaling. In cardiac myocytes, we tested the hypothesis that mechanical strain causes cellular hypertrophy via ROS-dependent post-translational modification of Ras leading to activation of the Raf/Mek/Erk growth pathway. Cyclic mechanical strain increased Ras activity by 1.5 to 1.6-fold. Adenoviral overexpression of the N17 dominant negative mutant of Ras inhibited strain-stimulated Erk activation and protein synthesis. Strain-stimulated Ras activation was inhibited by overexpression of catalase, indicating that it is redox-dependent. Strain caused S-glutathiolation of Ras, which was inhibited by catalase overexpression and reversed by DTT. MALDI-TOF mass spectrometry demonstrated that in myocytes subjected to strain there was S-glutathiolation of Ras at Cys118. Adenoviral overexpression of a mutated Ras in which Cys118 was substituted with serine inhibited strain-stimulated S-glutathiolation of Ras, Erk activation and protein synthesis. Overexpression of glutaredoxin-1 likewise inhibited strain-stimulated Ras S-glutathiolation, Ras activation, Erk activation and protein synthesis. These findings indicate that mechanical strain causes ROS-dependent S-glutathiolation of Ras at Cys118, leading to myocyte hypertrophy via activation of the Raf/Mek/Erk pathway.

    Funded by: NCI NIH HHS: CA95191, CA96989; NHLBI NIH HHS: HL007969, HL07224, HL20612, HL55620, HL61639, N01-HV-28178; NIA NIH HHS: AG27080

    Journal of molecular and cellular cardiology 2006;41;4;613-22

  • Rheb inhibits C-raf activity and B-raf/C-raf heterodimerization.

    Karbowniczek M, Robertson GP and Henske EP

    Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.

    The Ras-Raf-MEK signaling cascade is critical for normal development and is activated in many forms of cancer. We have recently shown that B-Raf kinase interacts with and is inhibited by Rheb, the target of the GTPase-activating domain of the tuberous sclerosis complex 2 gene product tuberin. Here, we demonstrate for the first time that activation of Rheb is associated with decreased B-Raf and C-Raf phosphorylation at residues Ser-446 and Ser-338, respectively, concomitant with a decrease in the activities of both kinases and decreased heterodimerization of B-Raf and C-Raf. Importantly, the impact of Rheb on B-Raf/C-Raf heterodimerization and kinase activity are rapamycin-insensitive, indicating that they are independent of Rheb activation of the mammalian target of rapamycin-Raptor complex. In addition, we found that Rheb inhibits the association of B-Raf with H-Ras. Taken together, these results support a central role of Rheb in the regulation of the Ras/B-Raf/C-Raf/MEK signaling network.

    Funded by: NIDDK NIH HHS: DK 51052

    The Journal of biological chemistry 2006;281;35;25447-56

  • Recurring HRAS mutation G12S in Dutch patients with Costello syndrome.

    van Steensel MA, Vreeburg M, Peels C, van Ravenswaaij-Arts CM, Bijlsma E, Schrander-Stumpel CT and van Geel M

    Department of Dermatology, University Hospital Maastricht, Maastricht, The Netherlands. mvst@sder.azm.nl

    Costello syndrome (CS) is a rare multiple congenital anomaly/mental retardation syndrome characterized by coarse face, loose skin and cardiomyopathy. It is often associated with benign and malignant tumors. Several groups have now demonstrated that CS is caused by recurring mutations in the HRAS gene in different ethnic groups. Here, we describe three unrelated Dutch patients and show that they all have the same mutation, G12S, in HRAS. To our knowledge, our patients are the first Dutch to be analysed. The syndrome seems to be genetically homogeneous. We discuss the pertinent nosology of the syndrome.

    Experimental dermatology 2006;15;9;731-4

  • Ligand-independent regulation of ErbB4 receptor phosphorylation by activated Ras.

    Tal-Or P, Erlich S, Porat-Shliom N, Goldshmit Y, Ben-Baruch G, Shaharabani E, Kloog Y and Pinkas-Kramarski R

    Department of Neurobiochemistry, Tel Aviv University, Ramat Aviv, 69978, Israel.

    The ErbB family of receptor tyrosine kinases regulates cell growth, differentiation and survival. Activation of the receptors is induced by specific growth factors in an autocrine, paracrine or juxtacrine manner. The activated ErbB receptors turn on a large variety of signaling cascades, including the prominent Ras-dependent signaling pathways. The activated Ras can induce secretion of growth factors such as EGF and neuregulin, which activate their respective receptors. In the present study, we demonstrate for the first time that activated Ras can activate ErbB4 receptor in a ligand-independent manner. Expression of constitutively active H-Ras(12V), K-Ras(12V) or N-Ras(13V) in PC12-ErbB4 cells induced ErbB4-receptor phosphorylation, indicating that each of the most abundant Ras isoforms can induce receptor activation. NRG-induced phosphorylation of ErbB4 receptor was blocked by the soluble ErbB4 receptor, which had no effect on the Ras-induced receptor phosphorylation. Moreover, conditioned medium from H-Ras(12V)-transfected PC12-ErbB4 cells had no effect on receptor phosphorylation. It thus indicates that Ras induces ErbB4 phosphorylation in a ligand-independent manner. Each of the Ras effector domain mutants, H-Ras(12V)S35, H-Ras(12V)C40, and H-Ras(12V)G37, which respectively activate Raf1, PI3K, and RalGEF, induced a small but significant receptor phosphorylation. The PI3K inhibitor LY294002 and the MEK inhibitor PD98059 caused a partial inhibition of the Ras-induced ErbB4 receptor phosphorylation. Using a mutant ErbB4 receptor, which lacks kinase activity, we demonstrated that the Ras-mediated ErbB4 phosphorylation depends on the kinase activity of the receptor and facilitates ligand-independent neurite outgrowth in PC12-ErbB4 cells. These experiments demonstrate a novel mechanism controlling ErbB receptor activation. Ras induces ErbB4 receptor phosphorylation in a non-autocrine manner and this activation depends on multiple Ras effector pathways and on ErbB4 kinase activity.

    Journal of cellular biochemistry 2006;98;6;1482-94

  • H-RAS 81 polymorphism is significantly associated with aneuploidy in follicular tumors of the thyroid.

    Castro P, Soares P, Gusmão L, Seruca R and Sobrinho-Simões M

    Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Porto, Portugal.

    Follicular thyroid tumors are often aneuploid. It was advanced that chromosomal instability is closely associated to RAS mutations, but such association remains unproven. H-RAS can be alternatively spliced in two different proteins, p21 and p19, the former being the active protein. In order to investigate the relationship between RAS mutational status and ploidy in thyroid tumors, we analysed RAS genes in a series of 99 follicular lesions (14 nodular goiters, 70 follicular adenomas and 15 follicular carcinomas), eight thyroid carcinoma cell lines and a control group of 102 blood donors, correlating the presence of RAS mutations with the ploidy of the tumors and evaluating the two spliced forms of H-RAS. Overall, 20% of the follicular tumors harbored RAS mutations and 62% of the patients with follicular tumors (and 51% of blood donors) harbored the H-RAS 81T --> C polymorphism. The presence of RAS mutations was not associated with aneuploidy. The H-RAS polymorphism did not seem to confer a higher propensity for neoplastic transformation as it was also found in hyperplastic lesions, but was strongly associated with aneuploidy (P<0.0001). The presence of the H-RAS 81T --> C polymorphism was associated with significantly higher amounts of total H-RAS mRNA expression, higher amounts of p21 isoform and a higher fraction of neoplastic cells in S phase. Our results suggest that the H-RAS 81T --> C polymorphism may induce aneuploidy through overexpression of the active p21 isoform of H-RAS.

    Oncogene 2006;25;33;4620-7

  • Laterally spreading type of colorectal adenoma exhibits a unique methylation phenotype and K-ras mutations.

    Hiraoka S, Kato J, Tatsukawa M, Harada K, Fujita H, Morikawa T, Shiraha H and Shiratori Y

    Department of Gastroenterology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.

    Laterally spreading tumors (LST), characterized by superficial extension along the colonic lumen, have recently been detected by colonoscopy. However, genetic and epigenetic characteristics of these tumors were scarcely reported.

    Methods: A total of 205 sporadic colorectal adenoma tissues (157 protruded-type, 23 granular-type LST (G-LST), 12 flat-type LST (F-LST), and 13 flat-type smaller than 1 cm) were collected. CpG island methylator phenotype (CIMP) was determined by examination of methylation status at p16, methylated in tumor (MINT) 1, 2, 12, and 31 loci. K-ras codon 12 and 13 point mutations were also examined. The relationship between macroscopic appearance and CIMP status or K-ras mutations was analyzed.

    Results: Among adenomas larger than 1 cm, CpG island methylation involving 2 or more loci (CIMP-high) was more likely to be observed in G-LST (14/23, 61%) than in protruded-type adenomas (18/73, 25%) (P = .002). The prevalence of K-ras mutations in G-LST (18/23, 78%) was significantly higher than that in protruded-type adenomas (18/73, 25%) (P < .0001). Moreover, the prevalence of CIMP-high and K-ras mutations in G-LST located in the proximal colon was much higher (11/13, 85%; and 12/13, 92%, respectively). In contrast, F-LST exhibited low prevalence of CIMP-high (1/12, 8%) and K-ras mutations (2/12, 16%).

    Conclusions: High prevalence of CIMP-high and K-ras mutations in G-LST, especially in the proximal colon, could strongly suggest that G-LST appearance is associated with a unique carcinogenic pathway.

    Gastroenterology 2006;131;2;379-89

  • G1/S arrest induced by histone deacetylase inhibitor sodium butyrate in E1A + Ras-transformed cells is mediated through down-regulation of E2F activity and stabilization of beta-catenin.

    Abramova MV, Pospelova TV, Nikulenkov FP, Hollander CM, Fornace AJ and Pospelov VA

    Institute of Cytology, Russian Academy of Sciences, St. Petersburg.

    Tumor cells are often characterized by a high and growth factor-independent proliferation rate. We have previously shown that REF cells transformed with oncogenes E1A and c-Ha-Ras do not undergo G(1)/S arrest of the cell cycle after treatment with genotoxic factors. In this work, we used sodium butyrate, a histone deacetylase inhibitor, to show that E1A + Ras transformants were able to stop proliferation and undergo G(1)/S arrest. Apart from inducing G(1)/S arrest, sodium butyrate was shown to change expression of a number of cell cycle regulatory genes. It down-regulated cyclins D1, E, and A as well as c-myc and cdc25A and up-regulated the cyclin-kinase inhibitor p21(waf1). Accordingly, activities of cyclin E-Cdk2 and cyclin A-Cdk2 complexes in sodium butyrate-treated cells were decreased substantially. Strikingly, E2F1 expression was also down-modulated at the levels of gene transcription, the protein content, and the E2F transactivating capability. To further study the role of p21(waf1) in the sodium butyrate-induced G(1)/S arrest and the E2F1 down-modulation, we established E1A + Ras transformants from mouse embryo fibroblast cells with deletion of the cdkn1a (p21(waf1)) gene. Despite the absence of p21(waf1), sodium butyrate-treated mERas transformants reveal a slightly delayed G(1)/S arrest as well as down-modulation of E2F1 activity, implying that the observed effects are mediated through an alternative p21(waf1)-independent signaling pathway. Subsequent analysis showed that sodium butyrate induced accumulation of beta-catenin, a downstream component of the Wnt signaling. The results obtained indicate that the antiproliferative effect of histone deacetylase inhibitors on E1A + Ras-transformed cells can be mediated, alongside other mechanisms, through down-regulation of E2F activity and stabilization of beta-catenin.

    Funded by: PHS HHS: G-3-00-336

    The Journal of biological chemistry 2006;281;30;21040-51

  • Release of RASSF1C from the nucleus by Daxx degradation links DNA damage and SAPK/JNK activation.

    Kitagawa D, Kajiho H, Negishi T, Ura S, Watanabe T, Wada T, Ichijo H, Katada T and Nishina H

    The Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan.

    Stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) responds to a variety of stress stimuli and controls cell fates such as cell cycle entrance, apoptosis and senescence. Stimuli such as ultraviolet irradiation and chemical reagents that damage genomic DNA induce the activation of the SAPK/JNK signaling pathway. However, it is unclear how the signal arising in the nucleus owing to DNA damage is transmitted to SAPK/JNK in the cytoplasm. Here, we report that the nuclear components Daxx and Ras-association domain family 1C (RASSF1C) link DNA damage to SAPK/JNK activation in HeLa cells. In response to DNA damage, Daxx localized in promyelocytic leukaemia-nuclear bodies (PML-NBs) undergoes ubiquitination and degradation. RASSF1C, a tumor suppressor and newly identified binding partner of Daxx, is constitutively anchored by Daxx in PML-NBs but is released from the nucleus when Daxx is degraded. This released RASSF1C translocates to cytoplasmic microtubules and participates in the activation of SAPK/JNK. Our data define a novel mechanism by which the Daxx-RASSF1C complex in PML-NBs couples nuclear DNA damage to the cytoplasmic SAPK/JNK signaling pathway.

    The EMBO journal 2006;25;14;3286-97

  • Neuronal nitric oxide synthase-induced S-nitrosylation of H-Ras inhibits calcium ionophore-mediated extracellular-signal-regulated kinase activity.

    Raines KW, Cao GL, Lee EK, Rosen GM and Shapiro P

    Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201, USA.

    nNOS (neuronal nitric oxide synthase) is a constitutively expressed enzyme responsible for the production of NO* from L-arginine and O2. NO* acts as both an intra- and an inter-cellular messenger that mediates a variety of signalling pathways. Previous studies from our laboratory have demonstrated that nNOS production of NO* blocks Ca2+-ionophore-induced activation of ERK1/2 (extracellular-signal-regulated kinase 1/2) of the mitogen-activated protein kinases through a mechanism involving Ras G-proteins and Raf-1 kinase. Herein we describe a mechanism by which NO* blocks Ca2+-mediated ERK1/2 activity through direct modification of H-Ras. Ca2+-mediated ERK1/2 activation in NO*-producing cells could be restored by exogenous expression of constitutively active mitogen-activated protein kinase kinase 1. In contrast, exogenous expression of constitutively active mutants of Raf-1 and H-Ras only partially restored ERK1/2 activity, by 50% and 10% respectively. On the basis of these findings, we focused on NO*-mediated mechanisms of H-Ras inhibition. Assays for GTP loading and H-Ras interactions with the Ras-binding domain on Raf-1 demonstrated a decrease in H-Ras activity in the presence of NO*. We demonstrate that S-nitrosylation of H-Ras occurs in nNOS-expressing cells activated with Ca2+ ionophore. Mutation of a putative nitrosylation site at Cys118 inhibited S-nitrosylation and restored ERK1/2 activity by constitutively active H-Ras even in the presence of NO*. These findings indicate that intracellular generation of NO* by nNOS leads to S-nitrosylation of H-Ras, which interferes with Raf-1 activation and propagation of signalling through ERK1/2.

    Funded by: NCI NIH HHS: CA-10529; NIBIB NIH HHS: EB-2034, P41 EB002034; NIGMS NIH HHS: R25 GM055036, R25-GM55036

    The Biochemical journal 2006;397;2;329-36

  • Detection of N-RAS and K-RAS in their active GTP-bound form in acute myeloid leukemia without activating RAS mutations.

    Ehmann F, Horn S, Garcia-Palma L, Wegner W, Fiedler W, Giehl K, Mayr GW and Jücker M

    Center of Experimental Medicine, Institute of Biochemistry and Molecular Biology I, Cellular Signal Transduction, University Hospital Hamburg-Eppendorf, Hamburg, Germany.

    RAS genes, predominantly N-RAS and K-RAS, have been implicated in the pathogenesis of acute myeloid leukemia (AML), due to activating RAS mutations detectable in approximately 20% of AML patients. In the present study, RAS proteins were detected in their activated, GTP-bound form, in AML patients (n = 10) not expressing mutated forms of H-RAS, K-RAS and N-RAS. Further analysis revealed the simultaneous presence of N-RAS and K-RAS proteins in the GTP-bound state in seven out of 10 AML samples. In four out of 10 samples the levels of RAS-GTP were comparable to an AML cell line (TF-1) with an activating N-RAS mutation (Q61P). The detection of RAS-GTP in AML patients without RAS mutations further supports a functional role of RAS proteins in the pathogenesis of AML and may explain the observed effects of RAS inhibitors in some AML patients in the absence of activating RAS mutations.

    Leukemia & lymphoma 2006;47;7;1387-91

  • Frequency of RAS gene mutation and its cooperative genetic events in Southeast Asian adult acute myeloid leukemia.

    Auewarakul CU, Lauhakirti D and Tocharoentanaphol C

    Department of Medicine, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand. cicaw@mahidol.ac.th

    RAS gene as one of the most frequently mutated genes in acute myeloid leukemia (AML) has become an attractive target for molecular therapy. The role of oncogenic RAS and its associated genetic events in AML are not yet defined. We examined the frequency of RAS mutation in 239 Thai de novo adult AML patients using polymerase chain reaction-single-strand conformational polymorphism analysis. Thirty-five RAS mutations were found in 32 cases (13%) predominantly classified as M1/M2 (53%) followed by M4/M5 subtype (38%). Ten cases were positive for N-RAS codon 12, 11 cases for N-RAS codon 61, 13 cases for N-RAS codon 13, and one case for K-RAS codon 13. No mutation was found in K-RAS exon 2 or H-RAS. The most common base substitution was the G to A transition at codon 13. Most M1/M2 cases had mutations at codon 12 or 13, whereas M4/M5 cases preferentially affected codon 61. Half of the patients with RAS mutations had abnormal karyotypes with the majority involving chromosomes 21, 11 and 7. Four patients had core-binding factor leukemia and four additional patients had coexisting FLT3 or AML1 mutation. One patient had RAS, FLT3 and t(8;21) and the other had RAS, AML1 point mutation and del(9q). In conclusion, mutation of RAS gene was not as common in the Thais as in the western population. Several additional genetic abnormalities occurred in RAS-mutated patients. Future molecular-targeting approaches should take into account the multiple genetic events that coexist with RAS mutations in AML patients.

    European journal of haematology 2006;77;1;51-6

  • Influence of single nucleotide polymorphisms in H-Ras and cyclin D1 genes on oral cancer susceptibility.

    Sathyan KM, Nalinakumari KR, Abraham T and Kannan S

    Division of Cancer Research, Regional Cancer Centre, Thiruvananthapuram 695011, Kerala, India.

    Although tobacco usage and alcohol consumption are the major risk factors for oral cancer, there are individual variations in genetic susceptibility to oral cancer. The Ras pathway plays an important role in oral carcinogenesis. High percentage of Ras mutation in oral carcinoma was reported from India. Cyclin D1, a downstream member of the Ras pathway, was also shown to be overexpressed in the majority of oral cancers and the overexpression was shown to be associated with poor prognosis. In the present study, we have evaluated the association of the single nucleotide polymorphisms in the H-Ras (C81T) and cyclin D1 (A870G and C1722G) genes and oral cancer risk in 176 oral cancer cases and 142 hospital based controls matched by age and sex. All the polymorphisms studied conformed to Hardy-Weinberg equilibrium. The comparison of the CCND1 A870G and C1722G genotype frequencies in cases and controls did not show any significant association with oral cancer risk. In H-Ras C81T polymorphism, TC+CC genotype showed a one and half fold increased risk (OR=1.59) for oral cancer. On stratified analysis, the observed increased risk was more evident among men (OR=2), while such an increased risk was not seen among women. Thus, our data suggests that the variant 'C' allele of the H-Ras (C81T) is associated with a higher risk for oral carcinoma, particularly in male population and thus, this polymorphism could be a low penetrance gene predisposition factor for oral carcinoma.

    Oral oncology 2006;42;6;607-13

  • Neurofibromin is a novel regulator of RAS-induced signals in primary vascular smooth muscle cells.

    Li F, Munchhof AM, White HA, Mead LE, Krier TR, Fenoglio A, Chen S, Wu X, Cai S, Yang FC and Ingram DA

    Department of Pediatrics, Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, 1044 W. Walnut Street, Indianapolis, IN 46202, USA.

    Neurofibromatosis type I (NF1) is a genetic disorder caused by mutations in the NF1 tumor suppressor gene. Neurofibromin is encoded by NF1 and functions as a negative regulator of Ras activity. NF1 patients develop renal artery stenosis and arterial occlusions resulting in cerebral and visceral infarcts. Further, NF1 patients develop vascular neurofibromas where tumor vessels are invested in a dense pericyte sheath. Although it is well established that aberrations in Ras signaling lead to human malignancies, emerging data generated in genetically engineered mouse models now implicate perturbations in the Ras signaling axis in vascular smooth muscular cells (VSMCs) as central to the initiation and progression of neointimal hyperplasia and arterial stenosis. Despite these observations, the function of neurofibromin in regulating VSMC function and how Ras signals are terminated in VSMCs is virtually unknown. Utilizing VSMCs harvested from Nf1+/- mice and primary human neurofibromin-deficient VSMCs, we identify a discrete Ras effector pathway, which is tightly regulated by neurofibromin to limit VSMC proliferation and migration. Thus, these studies identify neurofibromin as a novel regulator of Ras activity in VSMCs and provide a framework for understanding cardiovascular disease in NF1 patients and a mechanism by which Ras signals are attenuated for maintaining VSMC homeostasis in blood vessel walls.

    Funded by: NCI NIH HHS: 1K08 CA 096579-01, P30 CA 82709; NINDS NIH HHS: P50 NS 052606

    Human molecular genetics 2006;15;11;1921-30

  • Acquisition of JAK2, PTPN11, and RAS mutations during disease progression in primary myelodysplastic syndrome.

    Chen CY, Lin LI, Tang JL, Tsay W, Chang HH, Yeh YC, Huang CF, Chiou RJ, Yao M, Ko BS, Chen YC, Lin KH, Lin DT and Tien HF

    Leukemia 2006;20;6;1155-8

  • Incidence and prognostic impact of c-Kit, FLT3, and Ras gene mutations in core binding factor acute myeloid leukemia (CBF-AML).

    Boissel N, Leroy H, Brethon B, Philippe N, de Botton S, Auvrignon A, Raffoux E, Leblanc T, Thomas X, Hermine O, Quesnel B, Baruchel A, Leverger G, Dombret H, Preudhomme C, Acute Leukemia French Association (ALFA) and Leucémies Aiguës Myéloblastiques de l'Enfant (LAME) Cooperative Groups

    Service d'Hématologie Adulte, Hôpital Saint-Louis, Paris, France.

    In core binding factors (CBF) acute myeloid leukemia (AML), the disruption of CBFalpha/beta genes impairs normal hematopoietic differentiation and is supposed to cooperate with additional mutations promoting proliferation. The incidence and the prognosis of receptor tyrosine kinase (RTK) c-Kit and FLT3 mutations and Ras mutations were evaluated in 103 pediatric and adult patients with CBF-AML. c-Kit mutations were present in 17% patients. c-Kit exon 8 mutations were more frequent in inv(16) than in t(8;21) subset (20 versus 6%). Only one patient had FLT3-ITD but FLT3-D835 was as frequent as reported in AML population (7%). Ras mutations were significantly more frequent in inv(16) than in t(8;21) subset (36 versus 8%, P=0.001). RTK mutations were associated with a higher white blood cell count (WBC) (36 versus 21 G/L, P=0.05). FLT3 mutations were significantly associated with a shorter EFS and survival (P<0.0001 and P=0.0002) owing to an excess of early events. c-Kit mutations were associated with a shorter EFS and RFS (P=0.002 and P=0.003) in t(8;21) but not inv(16) patients. As previously observed, Ras mutations did not affect prognosis. Screening for RTK mutations may help to identify patients with a more adverse outcome and thus susceptible to benefit from intensified protocols or RTK inhibitors.

    Leukemia 2006;20;6;965-70

  • Mutations of the PTPN11 and RAS genes in rhabdomyosarcoma and pediatric hematological malignancies.

    Chen Y, Takita J, Hiwatari M, Igarashi T, Hanada R, Kikuchi A, Hongo T, Taki T, Ogasawara M, Shimada A and Hayashi Y

    Department of Pediatrics, Graduate School of Medicine, University of Tokyo, Tokyo, Japan.

    PTPN11 has been identified as a causative gene in Noonan syndrome (NS), responsible for about 50% of cases of NS. Given the association between NS and an increased risk of some malignancies, notably leukemia and probably some solid tumors including neuroblastoma (NB) and rhabdomyosarcoma (RMS), recent studies have reported that gain-of-function somatic mutations in PTPN11 occur in some hematological malignancies, especially de novo juvenile myelomonocytic leukemia (JMML) and in some solid tumors such as NB, although at a low frequency. In a screen for mutations of PTPN11 in 7 cell lines and 30 fresh tumors of RMS and in 25 cell lines and 40 fresh tumors of NB, we identified a missense mutation (A72T) in an embryonal RMS patient. In the RMS samples, we also detected mutations of NRAS in 1 cell line and 1 patient; both mutations were in embryonal RMSs and had no PTPN11 mutations. No mutations of PTPN11 were detected in NB. In 95 leukemia cell lines and 261 fresh leukemia samples including 22 JMMLs, 9 kinds of missense mutations were detected in 17 leukemia samples, which included 11 (50.0%) mutations in JMML samples and lower frequencies in other hematological malignancies. Furthermore, we identified 4 (18.2%) NRAS mutations and 1 (4.5%) KRAS mutation in 5 JMML samples, 1 of which had a concomitant PTPN11 mutation. Our data suggest that mutations of PTPN11 as well as RAS play a role in the pathogenesis of not only myeloid hematological malignancies but also a subset of RMS malignancies.

    Genes, chromosomes & cancer 2006;45;6;583-91

  • Patterns of K-ras mutation in colorectal carcinomas from Iran and Italy (a Gruppo Oncologico dell'Italia Meridionale study): influence of microsatellite instability status and country of origin.

    Bishehsari F, Mahdavinia M, Malekzadeh R, Verginelli F, Catalano T, Sotoudeh M, Bazan V, Agnese V, Esposito DL, De Lellis L, Semeraro D, Colucci G, Hormazdi M, Rakhshani N, Cama A, Piantelli M, Iacobelli S, Russo A, Mariani-Costantini R and Gruppo Oncologico dell'Italia Meridionale

    Department of Oncology and Neurosciences, University G. d'Annunzio, and Center of Excellence on Aging (CeSI), G. d'Annunzio University Foundation, Chieti, Italy.

    Background: K-ras mutations are a key step in colorectal cancer progression. Such mutations have been widely studied in case series from Western countries but there are few data on the rate and spectrum of mutations in tumors from countries where the epidemiological features of the disease are different.

    Tumor samples from 182 Iranian colorectal cancer patients (170 sporadic cases and 12 HNPCC cases) were screened for K-ras mutations at codons 12, 13 and 61 by sequencing analysis. The cases were also characterized for microsatellite instability at mononucleotide repeats by PCR and fragment analysis, and classified according to microsatellite instability status. The frequency and the spectrum of K-ras mutations were compared with those observed in a series of colorectal cancer patients from Italy.

    Results: K-ras mutations were observed in 68/182 (37.4%) cases. Mutation frequencies were similar in HNPCC-associated, sporadic MSI-H and sporadic microsatellite-stable (MSS) tumors. However, the G13D substitution was more frequent in HNPCC (3/4, 75%) and sporadic MSI-H (7/11, 63.6%) tumors compared to sporadic MSS tumors (11/53, 20.4%) (P <0.01). Comparison of mutations in the two series from Iran and Italy showed a significantly higher frequency of G13D among Italian patients.

    Conclusions: While the frequency of K-ras mutations could be similar, the mutational spectrum could be differentially influenced by genetic and environmental factors.

    Annals of oncology : official journal of the European Society for Medical Oncology 2006;17 Suppl 7;vii91-6

  • p19ras brings a new twist to the regulation of p73 by Mdm2.

    Harms KL and Chen X

    Department of Cell Biology, University of Alabama, Birmingham, AL 35294, USA.

    p53 is the most commonly mutated gene in human cancer. It is now known that the p53 family proteins p63 and p73 play important roles in tumor suppression as well as in development. Because p63 and p73 are rarely mutated in human cancer, understanding the signaling pathways that activate p63 and p73 will not only shed light on the developmental processes regulated by p63 and p73 but may also yield insight into ways to harness p63 and p73 activity for cancer therapy. Recent research has shown that an alternative splice form of c-H-ras, called p19ras, is a positive regulator of p73beta through a mechanism that involves the E3 ubiquitin ligase Mdm2. Implications for this previously unidentified means of regulation are discussed in light of tumor suppression and are extended to p53 and p63.

    Science's STKE : signal transduction knowledge environment 2006;2006;337;pe24

  • Implications of NRAS mutations in AML: a study of 2502 patients.

    Bacher U, Haferlach T, Schoch C, Kern W and Schnittger S

    Munich Leukemia Laboratory, Department of Internal Medicine III, University Hospital Munich, Ludwig-Maximilians University, Max-Lebsche-Platz 31, 81377 Munich, Germany.

    We analyzed 2502 patients with acute myeloid leukemia at diagnosis for NRAS mutations around the hot spots at codons 12, 13, and 61 and correlated the results to cytomorphology, cytogenetics, other molecular markers, and prognostic relevance of these mutations. Two hundred fifty-seven (10.3%) of 2502 patients had NRAS mutations (NRAS(mut)). Most mutations (112 of 257; 43.6%) were found at codon 12, mostly resulting in changes from glycine to asparagine. The history of AML did not differ significantly in association with NRAS mutations. The subgroups with inv(16)/t(16;16) and inv(3)/t(3;3) showed a significantly higher frequency of NRAS(mut) (50 of 133, 37.6% [P < .001], and 11 of 41, 26.8% [P = .004], respectively) than the total cohort. In addition, in these 2 subgroups, mutations of codon 61 were significantly overrepresented (both P < .001). In contrast, NRAS mutations were significantly underrepresented in t(15;17) (2 of 102; 2%; P = .005) in the subgroup with MLL/11q23 rearrangements (3 of 77; 3.9%; P = .061) and in the complex aberrant karyotype (4 of 258; 1.6%; P < .001). Overall, we did not find a significant prognostic impact of NRAS(mut) for overall survival, event-free survival, and disease-free survival. However, there was a trend to better survival in most subgroups, especially when other molecular markers (FLT3-LM, MLL-PTD, and NPM) were taken into account.

    Blood 2006;107;10;3847-53

  • Expression of genes involved in the regulation of p16 in psoriatic involved skin.

    Mark EB, Jonsson M, Asp J, Wennberg AM, Mölne L and Lindahl A

    Department of Dermatology, Sahlgrenska University Hospital, Göteborg University, 413 45, Göteborg, Sweden. elisabeth.bjorntorp@medic.gu.se

    It has been suggested that the up-regulation of the tumour suppressor p16 gene and induction of senescence protect the phenotype of psoriatic involved skin from malignant transformation. On the other hand, Id1, which is inversely correlated with p16 has been shown to be up-regulated in psoriatic involved skin. To test the hypothesis that there may be an altered regulation of p16 in psoriatic involved skin, we have measured genes involved in the Igf-1 receptor signalling through the Ras/MAPK cascade. Igf-1R, IGFBP3, hRas, Ets2, JunB, Egr-1, Id1, MIDA1 and p16 gene expressions were measured using quantitative real-time PCR in total RNA isolated from punch biopsies from psoriatic involved (n = 9) and uninvolved skin (n = 9) and from cutaneous squamous cell cancer (SCC) involved (n = 8) and uninvolved skin (n = 8). The IGFBP3, hRas, JunB, Egr-1, Id1 and MIDA1 genes were up-regulated in psoriatic involved skin compared with uninvolved skin. The p16, JunB and MIDA1 genes were up-regulated in SCC involved skin compared with uninvolved skin. Our results indicate that there may be a balance between the proliferation and induction of senescence in psoriasis. This balance may vary and the psoriatic involved skin represented in this study appears to be in a proliferative state rather than senescence. Furthermore, we suggest that the noted up-regulation of JunB, which has been shown to up-regulate p16, in combination with the previously reported elevation of p16 expression in psoriatic involved skin, may indicate activation of a pathway by which JunB may protect the psoriatic plaque by inducing p16 in an event of malignant stress.

    Archives of dermatological research 2006;297;10;459-67

  • HIV-1 Tat regulates endothelial cell cycle progression via activation of the Ras/ERK MAPK signaling pathway.

    Toschi E, Bacigalupo I, Strippoli R, Chiozzini C, Cereseto A, Falchi M, Nappi F, Sgadari C, Barillari G, Mainiero F and Ensoli B

    AIDS National Center, Istituto Superiore di Sanità, 00161 Rome, Italy.

    Tat, the transactivator of HIV-1 gene expression, is released by acutely HIV-1-infected T-cells and promotes adhesion, migration, and growth of inflammatory cytokine-activated endothelial and Kaposi's sarcoma cells. It has been previously demonstrated that these effects of Tat are due to its ability to bind through its arginine-glycine-aspartic (RGD) region to the alpha5beta1 and alphavbeta3 integrins. However, the signaling pathways linking Tat to the regulation of cellular functions are incompletely understood. Here, we report that Tat ligation on human endothelial cells results in the activation of the small GTPases Ras and Rac and the mitogen-activated protein kinase ERK, specifically through its RGD region. In addition, we demonstrated that Tat activation of Ras, but not of Rac, induces ERK phosphorylation. We also found that the receptor proximal events accompanying Tat-induced Ras activation are mediated by tyrosine phosphorylation of Shc and recruitment of Grb2. Moreover, Tat enabled endothelial cells to progress through the G1 phase in response to bFGF, and the process is linked to ERK activation. Taken together, these data provide novel evidence about the ability of Tat to activate the Ras-ERK cascade which may be relevant for endothelial cell proliferation and for Kaposi's sarcoma progression.

    Molecular biology of the cell 2006;17;4;1985-94

  • p19ras interacts with and activates p73 by involving the MDM2 protein.

    Jeong MH, Bae J, Kim WH, Yoo SM, Kim JW, Song PI and Choi KH

    Laboratory of Molecular Biology, Department of Biological Science, College of Natural Sciences, Chung-Ang University, Seoul 156-756, Korea.

    p73beta is a structural and functional homologue of p53, a tumor suppressor gene. In this study, we identified a novel p73beta-binding protein, p19ras, by the yeast two-hybrid screening method. Alternative splicing of the proto-oncogene H-ras pre-mRNA has led to two distinct transcripts, p19ras and p21ras. In both endogenous and overexpressed systems, we confirmed that p19ras binds to full-length p73beta in vivo and in vitro. Coexpression of p19ras with p73beta stimulated the transcriptional activity of p73beta. Ras proteins are known to be small membrane-localized guanine nucleotide-binding proteins. However, unlike other Ras proteins, p19ras is localized in the nucleus and the cytosol and its interaction with p73beta occurred exclusively in the nucleus. Oncogenic MDM2 (mouse double minutes 2) is a known repressor of p73 transcriptional activity. In this study, when p19ras was bound to MDM2, it further inhibited the association of MDM2 to the p73beta protein. In addition, p19ras abolished MDM2-mediated transcriptional repression of p73beta. Therefore, this study presents a novel pathway of Ras signaling that occurs in the nucleus, involving p19ras and p73beta. Furthermore, a p19ras-mediated novel regulatory mechanism of p73 involving the MDM2 protein is described.

    The Journal of biological chemistry 2006;281;13;8707-15

  • Galphaq binds to p110alpha/p85alpha phosphoinositide 3-kinase and displaces Ras.

    Ballou LM, Chattopadhyay M, Li Y, Scarlata S and Lin RZ

    Department of Medicine, Division of Hematology, Stony Brook University, Stony Brook, NY 11794, USA.

    Several studies have reported that activation of G(q)-coupled receptors inhibits PI3K (phosphoinositide 3-kinase) signalling. In the present study, we used purified proteins to demonstrate that Galpha(q) directly inhibits p110alpha/p85alpha PI3K in a GTP-dependent manner. Activated Galpha(q) binds to the p110alpha/p85alpha PI3K with an apparent affinity that is seven times stronger than that for Galpha(q).GDP as measured by fluorescence spectroscopy. In contrast, Galpha(q) did not bind to the p110gamma PI3K. Fluorescence spectroscopy experiments also showed that Galpha(q) competes with Ras, a PI3K activator, for binding to p110alpha/p85alpha. Interestingly, co-precipitation studies using deletion mutants showed that Galpha(q) binds to the p85-binding domain of p110alpha and not to the Ras-binding domain. Expression of constitutively active Galpha(q)Q209L in cells inhibited Ras activation of the PI3K/Akt pathway but had no effect on Ras/Raf/MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] signalling. These results suggest that activation of G(q)-coupled receptors leads to increased binding of Galpha(q).GTP to some isoforms of PI3K, which might explain why these receptors inhibit this signalling pathway in certain cell types.

    Funded by: NIDDK NIH HHS: DK62722, R01 DK062722; NIGMS NIH HHS: GM53132, R01 GM053132

    The Biochemical journal 2006;394;Pt 3;557-62

  • Mutational analysis of K-ras and Ras protein expression in larynx squamous cell carcinoma.

    Ruíz-Godoy R LM, Garcia-Cuellar CM, Herrera González NE, Suchil BL, Pérez-Cárdenas E, Sácnchez-Pérez Y, Suárez-Roa ML and Meneses A

    Basic Research Subdirection, National Institute of Cancerology, Tlalpan, Mexico.

    The ras gene family (H, K and N-ras) encodes the Ras protein, a GTPase-activating protein that regulates several signal transduction pathways including cellular proliferation and differentiation. Mutations in codons 12, 13 and 61 of the ras genes constitute one of the most frequent alterations in human cancer. In the Western Hemisphere, a low frequency of mutations in these genes has been observed in head and neck carcinomas; a higher frequency has been found in countries such as India and Taiwan. Increased protein expression is a relatively frequent event in larynx carcinomas. This study was aimed to evaluate the participation of the k-ras gene and Ras expression in 20 Mexican patients with larynx squamous carcinoma, 2 with dysplasia and 4 with normal mucosa. Samples (of 26 patients) were embedded in paraffin and immunohistochemical analysis was performed for the Ras protein, as well as amplification of the k-ras gene exon 1 (108 bp) by laser capture microdissection. Then, DNA extraction, PCR and sequencing were performed looking for possible mutation in codons 12 and 13. All patients with larynx carcinoma were men, median age 62 years. Eighty-five percent of the patients had risk factors such as smoking and/or alcohol consumption, 25% were in clinical stages I and II, and 75% in stages III and IV; 45% of the patients presented tumor recurrence or persistence. In this study, no mutations were found in codons 12 or 13 of the k-ras gene; however, protein expression was observed in 95% of the samples and a higher expression of the protein was associated with tumor recurrence or persistence, although this was not statistically significant. Unexpectedly, well-differentiated carcinomas and dysplasias presented an increase in protein expression. These results suggest that ras may be involved in early stages of larynx carcinogenesis and may be activated by other mechanisms different from mutations, such as epigenetic events.

    Journal of experimental & clinical cancer research : CR 2006;25;1;73-8

  • RAS-mediated epigenetic inactivation of OPCML in oncogenic transformation of human ovarian surface epithelial cells.

    Mei FC, Young TW, Liu J and Cheng X

    Department of Pharmacology and Toxicology, Sealy Center for Cancer Cell Biology, School of Medicine, The University of Texas Medical Branch, Galveston, Texas 77555-1031, USA.

    Opioid binding protein/cell adhesion molecule-like gene (OPCML), a recently identified tumor-suppressor, is frequently inactivated by allele loss and CpG island promoter methylation in epithelial ovarian cancer. Since elevated activation of the RAS signaling pathway, including overexpression of HER-2/neu and mutations of RAS and BRAF, is common in human ovarian carcinoma, we examined the cellular effect of oncogenic RAS on the expression status of OPCML in a genetically defined human ovarian cancer model. Our study revealed that RAS(V12)-mediated oncogenic transformation was accompanied by a concomitant loss of OPCML expression. Methylation-sensitive PCR analysis showed that the OPCML promoter was hypermethylated in RAS-transformed human ovarian epithelial cells (T29H) and that treatment with the DNA methyltransferase inhibitor 5'-aza-2'-deoxycytidine promoted demethylation of the OPCML promoter and restored OPCML expression in T29H cells. Furthermore, suppression of oncogenic RAS activity by stable siRNA specific for HRAS(V12) led to the demethylation and re-expression of OPCML in T29H cells, demonstrating that oncogenic RAS activity is directly responsible for the observed OPCML promoter hypermethylation and epigenetic gene silencing of OPCML. Taken together, our study suggests that elevation of the RAS signaling pathway may play an important role in epigenetic inactivation of OPCML in human epithelial ovarian cancer.

    Funded by: NIGMS NIH HHS: GM060170

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2006;20;3;497-9

  • RIG1 inhibits the Ras/mitogen-activated protein kinase pathway by suppressing the activation of Ras.

    Tsai FM, Shyu RY and Jiang SY

    Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan.

    The retinoid-inducible gene 1 (RIG1) protein is a retinoid-inducible growth regulator. Previous studies have shown that the RIG1 protein inhibits the signaling pathways of Ras/mitogen-activated protein kinases. However, neither the mode of action nor the site of inhibition of RIG1 is known. This study investigated the effects of RIG1, and the mechanisms responsible for these effects, on the activation of Ras proteins in HtTA cervical cancer cells. RIG1 reduced the levels of activated Ras (Ras-GTP) and total Ras protein in cells transfected with mutated H-, N-, or K-Ras(G12V), or in cells transfected with the wild type H- or N-Ras followed by stimulation with epidermal growth factor. The half-life of Ras protein decreased from more than 36 h in control cells to 18 h in RIG1-transfected cells. RIG1 immunoprecipitated with the Ras protein in co-transfected cellular lysates. In contrast to the predominant plasma membrane localization in control cells, the H-Ras fusion protein EGFP-H-Ras was localized within a discrete cytoplasmic compartment where it co-localized with RIG1. RIG1 inhibited more than 93% of the Elk- and CHOP-mediated transactivation induced by H- or K-Ras(G12V). However, RIG1 did not inhibit the transactivation induced by MEK1 or MEK3, and failed to suppress the phosphorylation of extracellular signal-regulated kinases 1 and 2 induced by the constitutively activated B-Raf(V599E). The RIG1 with carboxyl terminal truncation (RIG1DeltaC) did not immunoprecipitate with Ras and had no effect on Ras activation or transactivation of the downstream signal pathways. These data indicate that RIG1 exerts its inhibitory effect at the level of Ras activation, which is independent of Ras subtype but dependent on the membrane localization of the RIG1 protein. This inhibition of Ras activation may be mediated through downregulation of Ras levels and alteration of Ras subcellular distribution.

    Cellular signalling 2006;18;3;349-58

  • Structure of a transient intermediate for GTP hydrolysis by ras.

    Ford B, Hornak V, Kleinman H and Nassar N

    Department of Physiology and Biophysics, Basic Sciences Tower, Stony Brook University, Stony Brook, New York 11794, USA.

    The flexibility of the conserved 57DTAGQ61 motif is essential for Ras proper cycling in response to growth factors. Here, we increase the flexibility of the 57DTAGQ61 motif by mutating Gln61 to Gly. The crystal structure of the RasQ61G mutant reveals a new conformation of switch 2 that bears remarkable structural homology to an intermediate for GTP hydrolysis revealed by targeted molecular dynamics simulations. The mutation increased retention of GTP and inhibited Ras binding to the catalytic site, but not to the distal site of Sos. Most importantly, the thermodynamics of RafRBD binding to Ras are altered even though the structure of switch 1 is not affected by the mutation. Our results suggest that interplay and transmission of structural information between the switch regions are important factors for Ras function. They propose that initiation of GTP hydrolysis sets off the separation of the Ras/effector complex even before the GDP conformation is reached.

    Funded by: NIGMS NIH HHS: T32 GM008444

    Structure (London, England : 1993) 2006;14;3;427-36

  • The RAP1 guanine nucleotide exchange factor Epac2 couples cyclic AMP and Ras signals at the plasma membrane.

    Li Y, Asuri S, Rebhun JF, Castro AF, Paranavitana NC and Quilliam LA

    Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, and Walther Cancer Institute, Indianapolis, Indiana 46202, USA.

    Epac-1 and -2 (exchange proteins directly activated by cyclic AMP) are guanine-nucleotide exchange factors for the GTPases Rap1 and -2. Epac2 but not Epac1 was found to possess a RA (Ras association) domain similar to that found in the Ras effector Ral-GDS. This domain specifically bound Ras-GTP, enabling oncogenic Ras to translocate Epac2 from the cytosol to the plasma membrane. Consequently, a small pool of plasma membrane-bound Rap1 was activated at the expense of bulk Rap1 located on intracellular organelles. Whereas translocation of Epac2 was not mimicked by challenge with epidermal growth factor alone, costimulation with forskolin, prostaglandin E2, or an Epac-selective cyclic AMP analog-induced rapid relocation of GFP-Epac2 but not -Epac1 to the plasma membrane in a Ras-dependent manner. Deletion of the cyclic AMP-binding domain overcame the need for nucleotide, suggesting that this domain normally masked the RA domain in the resting GEF. Thus, Epac2 can respond to costimulation by agonists that jointly elevate Ras-GTP and cyclic AMP levels, activating a specific pool of Rap1 at the plasma membrane. Therefore, despite its previous description as a Ras antagonist or independently functioning GTPase, Rap1/Krev-1 may additionally act downstream of Ras in cells that express the cyclic AMP-regulated GEF, Epac2.

    Funded by: NCI NIH HHS: CA108647, R01 CA108647, R01 CA108647-02; PHS HHS: T32-H07774

    The Journal of biological chemistry 2006;281;5;2506-14

  • Prenylation is not necessary for endogenous Ras activation in non-malignant cells.

    Khwaja A, Dockrell ME, Hendry BM and Sharpe CC

    Department of Renal Medicine, King's College London, Bessemer Road, London SE5 9PJ, United Kingdom.

    Ras monomeric GTPases are pivotal to many core cellular processes such as proliferation and differentiation. The post-translational prenylation of Ras with a farnesyl or a geranylgeranyl moiety is thought to be critical for its membrane binding and consequent signaling activity. Inhibitors of Ras prenylation have an anti-proliferative effect in some Ras-transformed cells. We present a study of the effects of prenylation inhibitors on endogenous, wild-type Ras in three renal cell types, namely primary adult human renal fibroblasts, primary adult human mesangial cells, and a primate renal fibroblast cell line (Vero cells). We have previously demonstrated that Ras is necessary for normal proliferation in these cells. Here we show that Ras is farnesylated and not geranylgeranylated in all three cell types. Furthermore, inhibiting Ras farnesylation has no effect on cell proliferation or Ras activation. Although inhibiting geranylgeranylation in these cells does inhibit proliferation, this is through an Ras-independent mechanism. Non-prenylated Ras is able to localize to the plasma membrane, bind Raf when cells are stimulated by epidermal growth factor or platelet-derived growth factor, and activate the Ras downstream effectors mitogen-activated protein kinase and phosphotidylinositol 3-kinase. We conclude that in wild-type cells, endogenous Ras does not need to be prenylated to be active.

    Journal of cellular biochemistry 2006;97;2;412-22

  • Erbin inhibits RAF activation by disrupting the sur-8-Ras-Raf complex.

    Dai P, Xiong WC and Mei L

    Program of Developmental Neurobiology, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, 30912, USA.

    Erbin is a member of the LAP (leucine-rich repeat (LRR) and PDZ domain) family. It inhibits Ras-mediated activation of ERK in response to growth factors. In this study, we investigated the mechanisms by which Erbin regulates the Ras-Raf-MEK pathway. The N-terminal LRR domain was necessary and sufficient to inhibit neuregulin-activated expression of epsilon416-Luc, a reporter of ERK activation. On the other hand, Erbin had no effect on Ras activation, but it attenuated neuregulin-induced Raf activation, suggesting that Erbin may regulate Raf activation by Ras. Via the LRR domain, Erbin interacts with Sur-8, a scaffold protein necessary for the Ras-Raf complex. Expression of Erbin attenuated the interaction of Sur-8 with active Ras and Raf. Moreover, Erbin-shRNA, which suppressed Erbin expression at mRNA and protein levels, increased the interaction of Sur-8 with Ras and Raf, ERK activation, and neuregulin-induced expression of endogenous acetylcholine receptor epsilon-subunit mRNA. These results demonstrate a regulatory role of Erbin in the Ras-Raf-MEK pathway, suggesting that Erbin may inhibit ERK activation by disrupting the Sur-8-Ras/Raf interaction.

    The Journal of biological chemistry 2006;281;2;927-33

  • HRAS mutation analysis in Costello syndrome: genotype and phenotype correlation.

    Gripp KW, Lin AE, Stabley DL, Nicholson L, Scott CI, Doyle D, Aoki Y, Matsubara Y, Zackai EH, Lapunzina P, Gonzalez-Meneses A, Holbrook J, Agresta CA, Gonzalez IL and Sol-Church K

    Division of Medical Genetics, A. I. duPont Hospital for Children, Wilmington, DE 19899, USA. kgripp@nemours.org

    Costello syndrome is a rare condition comprising mental retardation, distinctive facial appearance, cardiovascular abnormalities (typically pulmonic stenosis, hypertrophic cardiomyopathy, and/or atrial tachycardia), tumor predisposition, and skin and musculoskeletal abnormalities. Recently mutations in HRAS were identified in 12 Japanese and Italian patients with clinical information available on 7 of the Japanese patients. To expand the molecular delineation of Costello syndrome, we performed mutation analysis in 34 North American and 6 European (total 40) patients with Costello syndrome, and detected missense mutations in HRAS in 33 (82.5%) patients. All mutations affected either codon 12 or 13 of the protein product, with G12S occurring in 30 (90.9%) patients of the mutation-positive cases. In two patients, we found a mutation resulting in an alanine substitution in position 12 (G12A), and in one patient, we detected a novel mutation (G13C). Five different HRAS mutations have now been reported in Costello syndrome, however genotype-phenotype correlation remains incomplete.

    Funded by: NCRR NIH HHS: 1P20 RR 020173-01

    American journal of medical genetics. Part A 2006;140;1;1-7

  • Identification by differential display of IL-24 autocrine loop activated by ras oncogenes.

    Tan Z, Wang M and Liang P

    Department of Cell Biology, Vanderbilt-Ingram Cancer Center, School of Medicine, Vanderbilt University, Nashville, TN, USA.

    Ras signaling pathway is thought to control the expression of a subset of yet-to-be-defined genes that are crucial for cell growth and differentiation. Here we have identified by differential display a novel oncogenic Ras target gene encoding a new cytokine. Biochemical studies reveal that this cytokine, which we named IL-24, is a member of IL-10 family of cytokines, and it signals through two hetorodimeric receptors, whose expression is also upregulated by ras oncogenes. Thus, IL-24 and its receptors may represent a novel autocrine loop coordinately activated by ras oncogenes.

    Methods in molecular biology (Clifton, N.J.) 2006;317;207-18

  • Mutations in APC, CTNNB1 and K-ras genes and expression of hMLH1 in sporadic colorectal carcinomas from the Netherlands Cohort Study.

    Lüchtenborg M, Weijenberg MP, Wark PA, Saritas AM, Roemen GM, van Muijen GN, de Bruïne AP, van den Brandt PA and de Goeij AF

    Nutrition and Toxicology Research Institute Maastricht, Department of Epidemiology, Maastricht University, Maastricht, The Netherlands. mluchten@crch.hawaii.edu

    Background: The early to intermediate stages of the majority of colorectal tumours are thought to be driven by aberrations in the Wnt (APC, CTNNB1) and Ras (K-ras) pathways. A smaller proportion of cancers shows mismatch repair deficiency. The aim of this study was to analyse the co-occurrence of these genetic alterations in relation to tumour and patient characteristics.

    Methods: In a group of 656 unselected sporadic colorectal cancer patients, aberrations in the APC, K-ras, CTNNB1 genes, and expression of hMLH1 were investigated. Additionally, tumours were divided in groups based on molecular features and compared with respect to patient's age at diagnosis, sex, family history of colorectal cancer, tumour sub-localisation, Dukes' stage and differentiation.

    Results: Mutations at the phosphorylation sites (codons 31, 33, 37, and 45) in the CTNNB1 gene were observed in tumours from only 5/464 patients. Tumours with truncating APC mutations and activating K-ras mutations in codons 12 and 13 occurred at similar frequencies (37% (245/656) and 36% (235/656), respectively). Seventeen percent of tumours harboured both an APC and a K-ras mutation (109/656). Nine percent of all tumours (58/656) lacked hMLH1 expression. Patients harbouring a tumour with absent hMLH1 expression were older, more often women, more often had proximal colon tumours that showed poorer differentiation when compared to patients harbouring tumours with an APC and/or K-ras mutation.

    Conclusion: CTNNB1 mutations seem to be of minor importance in sporadic colorectal cancer. The main differences in tumour and patient characteristics are found between groups of patients based on mismatch repair deficiency.

    BMC cancer 2005;5;160

  • Ras-induced spreading and wound closure in human epidermal keratinocytes.

    Tscharntke M, Pofahl R, Krieg T and Haase I

    Department Dermatology, University of Cologne and Centre for Molecular Medicine, University of Cologne (CMMC), Cologne, Germany.

    Although it is known that growth factor signaling cascades are active during epithelial wound healing, signals that regulate reepithelialization after wounding are not very well characterized. The small GTP binding protein Ras is a molecular switch involved in the regulation of signals originating from different growth factor receptors. We have investigated consequences of its activation in primary human keratinocytes. We provide evidence that activation of Ras can lead to shape changes of keratinocytes caused by rearrangements of the actin cytoskeleton that result in membrane protrusion and ruffling. Similar shape changes were found in the migrating tip of newly formed epithelium in mouse wounds. These cytoskeletal changes occur independently of keratinocyte terminal differentiation, and they can determine the speed of wound epithelialization in vitro. Using various mutant constructs and specific pharmacological inhibitors, we found that the effects of activated Ras on the cytoskeleton of keratinocytes are mediated by a phosphatidylinositol 3 kinase-independent activation of Rac. Our results suggest that growth factor-induced, Ras-mediated changes of keratinocyte shape may be an important mechanism that determines the speed of wound epithelialization.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2005;19;13;1836-8

  • Reduction in the requirement of oncogenic Ras signaling to activation of PI3K/AKT pathway during tumor maintenance.

    Lim KH and Counter CM

    Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.

    While tumors become addicted to oncogenes like Ras, the microenvironment in which tumor cells reside changes during tumorigenesis; the cells are surrounded initially by normal tissue and later by tumor tissue. Hence, we asked if Ras exerts its oncogenic effects through the same set of effectors during different stages of tumorigenesis. We now show in human cells that the Ras effector pathways MAPK, RalGEF, and PI3K are required to initiate tumor growth. Conversely, activation of the PI3K/AKT pathway replaced Ras once tumors formed, although other effectors were still activated independently of Ras, presumably by factors provided upon the establishment of a tumor microenvironment. Thus, as tumorigenesis progresses the addiction of cancers to their initiating oncogene is reduced to, at least in the case of Ras, the PI3K/AKT pathway.

    Funded by: NCI NIH HHS: R01-CA94184

    Cancer cell 2005;8;5;381-92

  • Platelet-derived growth factor and reactive oxygen species (ROS) regulate Ras protein levels in primary human fibroblasts via ERK1/2. Amplification of ROS and Ras in systemic sclerosis fibroblasts.

    Svegliati S, Cancello R, Sambo P, Luchetti M, Paroncini P, Orlandini G, Discepoli G, Paterno R, Santillo M, Cuozzo C, Cassano S, Avvedimento EV and Gabrielli A

    Istituto di Clinica Medica Generale, Ematologia ed Immunologia Clinica, Universita' di Ancona, 60020 Ancona, Italy.

    The levels of Ras proteins in human primary fibroblasts are regulated by PDGF (platelet-derived growth factor). PDGF induced post-transcriptionally Ha-Ras by stimulating reactive oxygen species (ROS) and ERK1/2. Activation of ERK1/2 and high ROS levels stabilize Ha-Ras protein, by inhibiting proteasomal degradation. We found a remarkable example in vivo of amplification of this circuitry in fibroblasts derived from systemic sclerosis (scleroderma) lesions, producing vast excess of ROS and undergoing rapid senescence. High ROS, Ha-Ras, and active ERK1/2 stimulated collagen synthesis, DNA damage, and accelerated senescence. Conversely ROS or Ras inhibition interrupted the signaling cascade and restored the normal phenotype. We conclude that in primary fibroblasts stabilization of Ras protein by ROS and ERK1/2 amplifies the response of the cells to growth factors and in systemic sclerosis represents a critical factor in the onset and progression of the disease.

    The Journal of biological chemistry 2005;280;43;36474-82

  • Analysis of K-ras mutations and expression of cyclooxygenase-2 and gastrin protein in laterally spreading tumors.

    Mukawa K, Fujii S, Takeda J, Kitajima K, Tominaga K, Chibana Y, Fujita M, Ichikawa K, Tomita S, Ono Y, Imura J, Kawamata H, Chiba T, Hiraishi H, Terano A and Fujimori T

    Department of Surgical and Molecular Pathology, Dokkyo University School of Medicine, Tochigi, Japan.

    Recent advances in colonoscopic techniques have led to the increased detection of, and interest in, superficial type colorectal tumors, and a new category, the 'laterally spreading tumor (LST)', has been proposed. However, the characteristics of the genetic alterations in these LSTs have not yet been fully determined. We therefore classified LSTs as LST-granular (LST-G) or LST-non-granular (LST-NG), according to their macroscopic appearance, and examined the genetic alterations in these two tumor groups compared with those in protruded type tumors.

    Methods: We obtained a total of 62 colorectal tumors, including 26 protruded type, 17 LST-G and 19 LST-NG, from specimens resected surgically or endoscopically. We examined K-ras codon 12 mutations by using the polymerase chain reaction-restriction fragment length polymorphism method and by fluorescence direct sequencing. We also performed immunohistochemistry to analyze cyclooxygenase (COX)-2 and gastrin abnormalities.

    Results: The incidence of K-ras mutation was 50.0% in protruded type tumors, 76.5% in LST-G, and 26.3% in LST-NG. The frequencies of COX-2 overexpression were 73.1, 88.2, and 31.6%, respectively, and those of gastrin overexpression were 61.5, 82.4, and 26.3%, respectively. Therefore, LST-G is similar to protruded type tumors in that the incidence of K-ras mutation and the frequencies of COX-2 and gastrin overexpression are high. LST-NG differs from both of these tumor types in that the values of these three indicators are all low.

    Conclusions: These results show that LST-G and LST-NG have different genetic alterations.

    Journal of gastroenterology and hepatology 2005;20;10;1584-90

  • Germline mutations in HRAS proto-oncogene cause Costello syndrome.

    Aoki Y, Niihori T, Kawame H, Kurosawa K, Ohashi H, Tanaka Y, Filocamo M, Kato K, Suzuki Y, Kure S and Matsubara Y

    Department of Medical Genetics, Tohoku University School of Medicine, 1-1 Seiryo-machi, Sendai 980-8574, Japan. aokiy@mail.tains.tohoku.ac.jp

    Costello syndrome is a multiple congenital anomaly and mental retardation syndrome characterized by coarse face, loose skin, cardiomyopathy and predisposition to tumors. We identified four heterozygous de novo mutations of HRAS in 12 of 13 affected individuals, all of which were previously reported as somatic and oncogenic mutations in various tumors. Our observations suggest that germline mutations in HRAS perturb human development and increase susceptibility to tumors.

    Funded by: Telethon: GTF04002

    Nature genetics 2005;37;10;1038-40

  • Proteomics analysis of H-RAS-mediated oncogenic transformation in a genetically defined human ovarian cancer model.

    Young T, Mei F, Liu J, Bast RC, Kurosky A and Cheng X

    Department of Pharmacology and Toxicology, The University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1031, USA.

    RAS is a small GTP binding protein mutated in approximately 30% human cancer. Despite its important role in the initiation and progression of human cancer, the underlying mechanism of RAS-induced human epithelial transformation remains elusive. In this study, we probe the cellular and molecular mechanisms of RAS-mediated transformation, by profiling two human ovarian epithelial cell lines. One cell line was immortalized with SV40 T/t antigens and the human catalytic subunit of telomerase (T29), while the second cell line was transformed with an additional oncogenic ras(V12) allele (T29H). In total, 32 proteins associated with RAS-mediated transformation have been identified using peptide mass fingerprinting. These protein targets are involved in several cellular pathways, including metabolism, redox balance, calcium signaling, apoptosis, and cellular methylation. One such target, the 40 kDa procaspase 4 is significantly upregulated at the protein level in RAS-transformed T29H cells, related directly to signaling through MEK, but not PI3 kinase. Cellular caspase 4 activity is, however, suppressed in the T29H cells, suggesting that the maturation process of caspase 4 is abrogated in RAS-transformed T29H cells. Consistent with this notion, transformed T29H cells were less susceptible to the toxic effects of anti-Fas antibody than were immortalized, nontransformed T29 cells, associated with less activation of caspase 4. This study demonstrates that functional proteomic analysis of a genetically defined cancer model provides a powerful approach toward systematically identifying cellular targets associated with oncogenic transformation.

    Funded by: NCI NIH HHS: R24 CA088317, R24CA88317; NHLBI NIH HHS: N01-HV-28184, N01HV28184; NIEHS NIH HHS: ES06676, P30 ES006676; NIGMS NIH HHS: GM060170, R01 GM060170, R01 GM066170

    Oncogene 2005;24;40;6174-84

  • DHHC9 and GCP16 constitute a human protein fatty acyltransferase with specificity for H- and N-Ras.

    Swarthout JT, Lobo S, Farh L, Croke MR, Greentree WK, Deschenes RJ and Linder ME

    Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

    Covalent lipid modifications mediate the membrane attachment and biological activity of Ras proteins. All Ras isoforms are farnesylated and carboxyl-methylated at the terminal cysteine; H-Ras and N-Ras are further modified by palmitoylation. Yeast Ras is palmitoylated by the DHHC cysteine-rich domain-containing protein Erf2 in a complex with Erf4. Here we report that H- and N-Ras are palmitoylated by a human protein palmitoyltransferase encoded by the ZDHHC9 and GCP16 genes. DHHC9 is an integral membrane protein that contains a DHHC cysteine-rich domain. GCP16 encodes a Golgi-localized membrane protein that has limited sequence similarity to yeast Erf4. DHHC9 and GCP16 co-distribute in the Golgi apparatus, a location consistent with the site of mammalian Ras palmitoylation in vivo. Like yeast Erf2.Erf4, DHHC9 and GCP16 form a protein complex, and DHHC9 requires GCP16 for protein fatty acyltransferase activity and protein stability. Purified DHHC9.GCP16 exhibits substrate specificity, palmitoylating H- and N-Ras but not myristoylated G (alphai1) or GAP-43, proteins with N-terminal palmitoylation motifs. Hence, DHHC9.GCP16 displays the properties of a functional human ortholog of the yeast Ras palmitoyltransferase.

    Funded by: NCI NIH HHS: CA050211, R01 CA050211, R01 CA050211-15A2; NIGMS NIH HHS: GM51466

    The Journal of biological chemistry 2005;280;35;31141-8

  • Human diploid fibroblasts are resistant to MEK/ERK-mediated disruption of the actin cytoskeleton and invasiveness stimulated by Ras.

    Sukezane T, Oneyama C, Kakumoto K, Shibutani K, Hanafusa H and Akagi T

    Laboratory of Molecular Oncology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan.

    Ras-induced transformation is characterized not only by uncontrolled proliferation but also by drastic morphological changes accompanied by the disruption of the actin cytoskeleton. Previously, we reported that human fibroblasts are more resistant than rodent fibroblasts to Ras-induced transformation. To explore the molecular basis for the difference in susceptibility to Ras-induced transformation, we investigated the effect of activated H-Ras on the actin cytoskeleton in human diploid fibroblasts and in rat embryo fibroblasts, both of which are immortalized by SV40 early region. We demonstrate here that Ras-induced morphological changes, decreased expression of tropomyosin isoforms, and suppression of the ROCK/LIMK/Cofilin pathway observed in the rat fibroblasts were not detected in the human fibroblasts even with high expression levels of Ras. We also show that activation of the MEK/ERK pathway sufficed to induce all of these alterations in the rat fibroblasts, whereas the human fibroblasts were refractory to these MEK/ERK-mediated changes. In addition to morphological changes, we demonstrated that the expression of activated Ras induced an invasive phenotype in the rat, but not in the human fibroblasts. These studies provide evidence for the existence of human-specific mechanisms that resist Ras/MEK/ERK-mediated transformation.

    Oncogene 2005;24;36;5648-55

  • Mutation analysis of p53, K-ras, and BRAF genes in colorectal cancer progression.

    Calistri D, Rengucci C, Seymour I, Lattuneddu A, Polifemo AM, Monti F, Saragoni L and Amadori D

    Division of Oncology and Diagnostics, Morgagni-Pierantoni Hospital, Forlì, Italy. biomolec@ausl.fo.it

    Gene mutations in APC, K-ras, and p53 are thought to be essential events for colorectal cancer development. Recent data seem to indicate that K-ras and p53 mutations rarely co-exist in the same tumor, indicating that these alterations do not represent a synergistic evolutionary pathway. Moreover, an inverse relation between K-ras gene activation and BRAF mutations has been demonstrated, suggesting alternative pathways for colorectal cancer transformation. To reconstruct the chronological modulation of these gene mutations during cell transformation and colorectal cancer progression, mutations of p53, K-ras, and BRAF genes were analyzed by Single Strand Conformation Polymorphism (SSCP) or sequencing analysis in 100 colorectal cancer samples, evenly distributed among different Dukes' stages. We found mutations in p53, K-ras, and BRAF genes in 35%, 30%, and 4% of tumors, respectively, and observed a minimal or no co-presence of these gene alterations. Moreover, the frequency of molecular p53 mutations increased as tumor stage increased, suggesting an important role for this gene in the progression of colorectal cancer. Conversely, K-ras or BRAF genes were not related to tumor stage or location. These data seem to indicate the absence of a co-presence of the genes, highlighting the possibility of multiple pathways for colorectal tumor progression. Moreover, mutations in p53, K-ras, and BRAF are not present in about one-third of colorectal cancers and therefore other gene mutations need to be investigated to better understand molecular mechanisms at the basis of cell transformation and the progression of colorectal cancer.

    Journal of cellular physiology 2005;204;2;484-8

  • Ras activity regulates cyclin E degradation by the Fbw7 pathway.

    Minella AC, Welcker M and Clurman BE

    Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

    The Skp1-Cullin1 F-box protein-Fbw7 ubiquitin ligase regulates phosphorylation-dependent cyclin E degradation, and disruption of this pathway is associated with genetic instability and tumorigenesis. Fbw7 is a human tumor suppressor that is targeted for mutation in primary cancers. However, mechanisms other than mutation of Fbw7 may also disrupt cyclin E proteolysis in cancers. We show that oncogenic Ha-Ras activity regulates cyclin E degradation by the Fbw7 pathway. Activated Ras impairs Fbw7-driven cyclin E degradation, and, conversely, inhibition of normal Ras activity decreases cyclin E abundance. Moreover, activation of the mitogen-activated protein kinase pathway is the essential Ras function that inhibits cyclin E turnover, and activated Ha-Ras expression inhibits both the binding of cyclin E to Fbw7 and cyclin E ubiquitination. Last, we found that oncogenic Ras activity potentiates cyclin E-induced genetic instability but only when cyclin E is susceptible to degradation by Fbw7. Thus, we conclude that Ras activity regulates Fbw7-mediated cyclin E proteolysis and suggest that impaired cyclin E proteolysis is a mechanism through which Ras mutations promote tumorigenesis.

    Funded by: NCI NIH HHS: K08 CA101800-02, K08CA101800, R01 CA102742-02, R01CA102742, R01CA84069

    Proceedings of the National Academy of Sciences of the United States of America 2005;102;27;9649-54

  • Effects of HRAS oncogene on cell cycle progression in a cervical cancer-derived cell line.

    Córdova-Alarcón E, Centeno F, Reyes-Esparza J, García-Carrancá A and Garrido E

    Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, México, D.F., México

    Background: Human papillomavirus (HPV) infection is the most prevalent factor in anogenital cancers. However, epidemiological surveys and molecular data indicate that viral presence is not enough to induce cervical cancer, suggesting that cellular factors could play a key role. One of the most important genes involved in cancer development is the RAS oncogene, and activating mutations in this gene have been associated with HPV infection and cervical neoplasia. Thus, we determined the effect of HRAS oncogene expression on cell proliferation in a cell line immortalized by E6 and E7 oncogenes.

    Methods: HPV positive human cervical carcinoma-derived cell lines (HeLa), previously transfected with the HRAS oncogene or the empty vector, were used. We first determined the proliferation rate and cell cycle profile of these cells by using flow cytometry and BrdU incorporation assays. In order to determine the signaling pathway regulated by HRAS and implicated in the alteration of proliferation of these cells, we used specific chemical inhibitors to inactivate the Raf and PI3K pathways.

    Results: We observed that HeLa cells stably transfected with oncogenic HRAS progressed faster than control cells on the cell cycle by reducing their G1 phase. Additionally, HRAS overexpression accelerated the G1/S transition. Specific chemical inhibitors for PI3K and MEK activities indicated that both PI3K/AKT and RAF/MEK/ERK pathways are involved in the HRAS oncogene-induced reduction of the G1 phase.

    Conclusions: Our results suggest that the HRAS oncogene could play an important role in the development of cervical cancer, in addition to the presence of HPV, by reducing the G1 phase and accelerating the G1/S transition of infected cells.

    Archives of medical research 2005;36;4;311-6

  • Microsatellite instability in sporadic and inherited colon adenocarcinomas from Greek patients: correlation with several clinicopathological characteristics.

    Michalopoulos NV, Saetta AA, Lazaris AC, Gigelou F, Koilakou S and Patsouris E

    Department of Pathology, Medical School, National and Kapodistrian University of Athens, Goudi, Athens, Greece.

    Microsatellite instability seems to play a significant role in colorectal carcinogenesis, as it is reported to occur in HNPCC patients as well as in a proportion of sporadic cases. The aim of this study was to examine the presence of microsatellite instability in relation to other commonly observed genetic abnormalities and clinicopathological characteristics of sporadic and inherited colorectal cancers.

    Methodology: One hundred and three sporadic colorectal adenocarcinomas and 9 adenocarcinomas from HNPCC patients were histologically evaluated. The presence of microsatellite instability was investigated at six loci. K-ras and p53 mutations, p53 LOH, hMLH1 expression and methylation status were examined as well. Statistical analysis was performed to define possible correlations of the observed genetic alterations with the clinicopathological characteristics of the analysed tumors.

    Results: High-grade microsatellite instability was found in 14% of sporadic adenocarcinomas and in 78% of adenocarcinomas from HNPCC patients. K-ras and p53 mutations were found in 29% and 28% of sporadic adenocarcinomas respectively and in 0% and 22% of the 9 HNPCC cases. A statistically significant correlation was noticed in sporadic tumors between the presence of MSI-H and tumor location at the proximal colon, as well as with the female gender.

    Conclusions: Sporadic MSI+ colon adenocarcinomas seem to represent a distinct entity with a unique profile of genetic changes, different from those observed in HNPCC or MSI negative sporadic tumors.

    Acta gastro-enterologica Belgica 2005;68;3;294-301

  • Oncogenic Ras blocks transforming growth factor-beta-induced cell-cycle arrest by degradation of p27 through a MEK/Erk/SKP2-dependent pathway.

    Schepers H, Wierenga AT, Eggen BJ and Vellenga E

    Division of Hematology, Department of Medicine, University Medical Center Groningen, The Netherlands.

    Objective: To examine whether oncogenic Ras affects transforming growth factor (TGF)-beta-mediated cell-cycle arrest in hematopoietic cells and the downstream signal transduction pathway involved in the interference with TGF-beta-induced cell-cycle arrest.

    Two leukemic cell lines bearing N-Ras(L61) mutations; HL-60 and TF-1, and the M1 cell line with wt Ras were investigated for their response to TGF-beta. Signal transduction inhibitors, overexpression and RNA interference studies were performed to investigate the involvement of the various proteins.

    Results: Although TGF-beta signal transduction was not affected, G0-G1 arrest was absent in HL-60 and TF-1 cells due to the absence of p27. Overexpression of p27 restored TGF-beta-induced cell-cycle arrest, as well as interfering in Ras-mediated signaling. The farnesyl transferase inhibitor L744832 and the MEK inhibitor U0126 both restored p27 levels and cell-cycle arrest in response to TGF-beta. The absence of p27 protein is due to elevated levels of the ubiquitin ligase SKP2, which complexes with and targets p27 for degradation. RNA interference for SKP2 and treatment of these cells with the proteasome inhibitor MG132 restored p27 levels, corresponding with decreasing SKP2 levels after interfering in N-Ras signal transduction. P27, phosphorylated at threonine 187, is nuclear localized in N-Ras-containing cells. Mutation of this residue to alanine rendered p27 insensitive to degradation.

    Conclusion: N-Ras(L61) transformed cells lack a G0-G1 arrest upon TGF-beta treatment due to absence of p27. p27 is degraded through a MapK-, and SKP2-dependent pathway. Overexpression of p27 results in restoration of cell-cycle arrest upon TGF-beta treatment.

    Experimental hematology 2005;33;7;747-57

  • Ras-mediated loss of the pro-apoptotic response protein Par-4 is mediated by DNA hypermethylation through Raf-independent and Raf-dependent signaling cascades in epithelial cells.

    Pruitt K, Ulkü AS, Frantz K, Rojas RJ, Muniz-Medina VM, Rangnekar VM, Der CJ and Shields JM

    Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, 27599-7295, USA.

    The apoptosis-promoting protein Par-4 has been shown to be down-regulated in Ras-transformed NIH 3T3 fibroblasts through the Raf/MEK/ERK MAPK pathway. Because mutations of the ras gene are most often found in tumors of epithelial origin, we explored the signaling pathways utilized by oncogenic Ras to down-regulate Par-4 in RIE-1 and rat ovarian surface epithelial (ROSE) cells. We determined that constitutive activation of the Raf, phosphatidylinositol 3-kinase, or Ral guanine nucleotide exchange factor effector pathway alone was not sufficient to down-regulate Par-4 in RIE-1 or ROSE cells. However, treatment of Ras-transformed RIE-1 or ROSE cells with the MEK inhibitors U0126 and PD98059 increased Par-4 protein expression. Thus, although oncogenic Ras utilizes the Raf/MEK/ERK pathway to down-regulate Par-4 in both fibroblasts and epithelial cells, Ras activation of an additional signaling pathway(s) is required to achieve the same outcome in epithelial cells. Methylation-specific PCR showed that the par-4 promoter is methylated in Ras-transformed cells through a MEK-dependent pathway and that treatment with the DNA methyltransferase inhibitor azadeoxycytidine restored Par-4 mRNA transcript and protein levels, suggesting that the mechanism for Ras-mediated down-regulation of Par-4 is by promoter methylation. Support for this possibility is provided by our observation that Ras transformation was associated with up-regulation of Dnmt1 and Dnmt3 DNA methyltransferase expression. Finally, ectopic Par-4 expression significantly reduced Ras-mediated growth in soft agar, but not morphological transformation, highlighting the importance of Par-4 down-regulation in specific aspects of Ras-mediated transformation of epithelial cells.

    Funded by: NCI NIH HHS: CA63071, CA84511

    The Journal of biological chemistry 2005;280;24;23363-70

  • Transformation by oncogenic RAS sensitizes human colon cells to TRAIL-induced apoptosis by up-regulating death receptor 4 and death receptor 5 through a MEK-dependent pathway.

    Drosopoulos KG, Roberts ML, Cermak L, Sasazuki T, Shirasawa S, Andera L and Pintzas A

    Laboratory of Signal Mediated Gene Expression, Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece.

    RAS oncogenes play a major role in cancer development by activating an array of signaling pathways, most notably mitogen-activated protein kinases, resulting in aberrant proliferation and inhibition of apoptotic signaling cascades, rendering transformed cells resistant to extrinsic death stimuli. However, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is able to kill specific tumor cells through the engagement of its receptors, death receptor 4 (DR4) and death receptor 5 (DR5), and the activation of apoptotic pathways, providing promising targets for anticancer therapies. In this study, we show that TRAIL induces cell death in human colon adenocarcinoma cells in a MEK-dependent manner. We also report a prolonged MEK-dependent activation of ERK1/2 and increased c-FOS expression induced by TRAIL in this system. Our study reveals that transformation of the colon cell line Caco-2 by Ki- and mainly by Ha-ras oncogenes sensitizes these cells to TRAIL-induced apoptosis by causing specific MEK-dependent up-regulation of DR4 and DR5. These observations taken together reveal that RAS-MEK-ERK1/2 signaling pathway can sensitize cells to TRAIL-induced apoptosis by up-regulating DR4 and DR5 and overall imply that TRAIL-based therapeutic strategies using TRAIL agonists could be used in cases of human colon cancers bearing RAS mutations.

    The Journal of biological chemistry 2005;280;24;22856-67

  • Role of Ras/PKCzeta/MEK/ERK1/2 signaling pathway in angiotensin II-induced vascular smooth muscle cell proliferation.

    Zhao Y, Liu J, Li L, Liu L and Wu L

    Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing, 100083, China.

    The role of protein kinase C (PKC) and its cross talk with extracellular signal-regulated kinase (ERK) cascade in angiotensin II (AngII)-elicited vascular smooth muscle cell (VSMC) proliferation are still unclear. In this study, the PKC pathway of AngII to activate ERK1/2 and induce cell proliferation was investigated in rat aortic smooth muscle cells. The proliferation of VSMCs was tested by [3H]-thymidine incorporation assay. Phosphorylated and non-phosphorylated PKCzeta, ERK1/2, Elk-1, and mitogen-activated ERK-activating kinase (MEK) were estimated by Western blot analysis. The interactions of signal molecules were examined by immunoprecipitation. AngII-induced VSMC proliferation and activation of ERK1/2 and nuclear transcription factor Elk-1 were all down-regulated by PKC non-specific inhibitor (staurosporine) and PKCzeta pseudosubstrate inhibitor (PS-PKCzeta). Dominant negative Ras transfection into VSMCs decreased AngII-induced PKCzeta and ERK1/2 phosphorylation. AngII stimulated the association of PKCzeta with Ras. AngII-induced MEK phosphorylation was inhibited by PKCzeta pseudosubstrate inhibitor and the PKCzeta-MEK complex was detected by immunoprecipitation. These results suggest that PKCzeta isoform is involved in VSMC proliferation and Elk-1 activation. AngII can activate ERK1/2 by Ras/PKCzeta/MEK pathway, which may be one of the important signal transduction pathways in AngII-induced VSMC proliferation.

    Regulatory peptides 2005;128;1;43-50

  • Rheb binds and regulates the mTOR kinase.

    Long X, Lin Y, Ortiz-Vega S, Yonezawa K and Avruch J

    Diabetes Unit and Medical Services and Department of Molecular Biology, Massachusetts General Hospital, Boston, 02114, USA.

    Background: The target of rapamycin (TOR), in complex with the proteins raptor and LST8 (TOR complex 1), phosphorylates the p70S6K and 4E-BP1 to promote mRNA translation. Genetic evidence establishes that TOR complex activity in vivo requires the small GTPase Rheb, and overexpression of Rheb can rescue TOR from inactivation in vivo by amino-acid withdrawal. The Tuberous Sclerosis heterodimer (TSC1/TSC2) functions as a Rheb GTPase activator and inhibits TOR signaling in vivo.

    Results: Here, we show that Rheb binds to the TOR complex specifically, independently of its ability to bind TSC2, through separate interactions with the mTOR catalytic domain and with LST8. Rheb binding to the TOR complex in vivo and in vitro does not require Rheb guanyl nucleotide charging but is modulated by GTP and impaired by certain mutations (Ile39Lys) in the switch 1 loop. Nucleotide-deficient Rheb mutants, although capable of binding mTOR in vivo and in vitro, are inhibitory in vivo, and the mTOR polypeptides that associate with nucleotide-deficient Rheb in vivo lack kinase activity in vitro. Reciprocally, mTOR polypeptides bound to Rheb(Gln64Leu), a mutant that is nearly 90% GTP charged, exhibit substantially higher protein kinase specific activity than mTOR bound to wild-type Rheb.

    Conclusions: The TOR complex 1 is a direct target of Rheb-GTP, whose binding enables activation of the TOR kinase.

    Funded by: NCI NIH HHS: CA-73818; NIDDK NIH HHS: DK-17776, DK007028

    Current biology : CB 2005;15;8;702-13

  • H-Ras-specific activation of Rac-MKK3/6-p38 pathway: its critical role in invasion and migration of breast epithelial cells.

    Shin I, Kim S, Song H, Kim HR and Moon A

    College of Pharmacy, Duksung Women's University, Seoul 132-714, Korea.

    Human tumors frequently exhibit constitutively activated Ras signaling, which contributes to the malignant phenotype. Mounting evidence suggests unique roles of the Ras family members, H-Ras, N-Ras and K-Ras, in normal and pathological conditions. In an effort to dissect distinct Ras isoform-specific functions in malignant phenotypic changes, we previously established H-Ras- and N-Ras-activated MCF10A human breast epithelial cell lines. Using these, we showed that p38 kinase is a key signaling molecule differentially regulated between H-Ras and N-Ras, leading to H-Ras-specific induction of invasive and migrative phenotypes. The present study is to further investigate H-Ras- and N-Ras-mediated signaling pathways and to unveil how these pathways are integrated for regulation of invasive/migrative phenotypic conversion of human breast epithelial cells. Here we report that the Rac-MAPK kinase (MKK)3/6-p38 pathway is a unique signaling pathway activated by H-Ras, leading to the invasive/migrative phenotype. In contrast, Raf-MEK-ERK and phosphatidylinositol 3-kinase-Akt pathways, which are fundamental to proliferation and differentiation, are activated by both H-Ras and N-Ras. A significant role for p38 in cell invasion is further supported by the observation that p38 activation by MKK6 transfection is sufficient to induce invasive and migrative phenotypes in MCF10A cells. Activation of the MKK6-p38 pathway results in a marked induction of matrix metalloproteinase (MMP)-2, whereas it had little effect on MMP-9, suggesting MMP-2 up-regulation by MKK6-p38 pathway as a key step for H-Ras-induced invasion and migration. We also provide evidence for cross-talk among the Rac, Raf, and phosphatidylinositol 3-kinase pathways critical for regulation of MMP-2 and MMP-9 expression and invasive phenotype. Taken together, the present study elucidated the role of the Rac-MKK3/6-p38 pathway leading to H-Ras-specific induction of malignant progression in breast epithelial cells, providing implications for developing therapeutic strategies for mammary carcinoma to target Ras downstream signaling molecules required for malignant cancer cell behavior but less critical for normal cell functions.

    The Journal of biological chemistry 2005;280;15;14675-83

  • Superoxide anion radical modulates the activity of Ras and Ras-related GTPases by a radical-based mechanism similar to that of nitric oxide.

    Heo J and Campbell SL

    Department of Biochemistry and Biophysics, University of North Carolina, 530 Mary Ellen Jones Building Chapel Hill, North Carolina 27599-7260, USA.

    Ras GTPases cycle between inactive GDP-bound and active GTP-bound states to modulate a diverse array of processes involved in cellular growth control. The activity of Ras is up-regulated by cellular agents, including both protein (guanine nucleotide exchange factors) and redox-active agents (nitric oxide (NO) and superoxide anion radical (O2*). We have recently elucidated the mechanism by which NO promotes guanine nucleotide dissociation of redox-active NKCD motif-containing Ras and Ras-related GTPases. In this study, we show that guanine nucleotide dissociation is enhanced upon exposure of the redox-active GTPases, Ras and Rap1A, to O2* and provide evidence for the efficient guanine nucleotide reassociation in the presence of the radical quenching agent ascorbate to complete guanine nucleotide exchange. In vivo, guanine nucleotide reassociation is necessary to populate Ras in its biologically active GTP-bound form after the dissociation of GDP. We further show that treatment of the redox-active GTPases with O2* releases GDP in form of an unstable the oxygenated GDP adduct, putatively assigned as 5-oxo-GDP. 5-Oxo-GDP was not produced from either the C118S or the F28L Ras variants upon the treatment of O2*, supporting the involvement of residues Cys118 and Phe28 in O2*-mediated Ras guanine nucleotide dissociation. These results indicate that the mechanism of O2*-mediated Ras guanine nucleotide dissociation is similar to that of NO/O2-mediated Ras guanine nucleotide dissociation.

    Funded by: NCI NIH HHS: R01CA89614-01A1; NHLBI NIH HHS: P01 HL45100

    The Journal of biological chemistry 2005;280;13;12438-45

  • The RET/PTC-RAS-BRAF linear signaling cascade mediates the motile and mitogenic phenotype of thyroid cancer cells.

    Melillo RM, Castellone MD, Guarino V, De Falco V, Cirafici AM, Salvatore G, Caiazzo F, Basolo F, Giannini R, Kruhoffer M, Orntoft T, Fusco A and Santoro M

    Istituto di Endocrinologia ed Oncologia Sperimentale del CNR G. Salvatore, Dipartimento di Biologia e Patologia Cellulare e Molecolare, University Federico II, Naples, Italy.

    In papillary thyroid carcinomas (PTCs), rearrangements of the RET receptor (RET/PTC) and activating mutations in the BRAF or RAS oncogenes are mutually exclusive. Here we show that the 3 proteins function along a linear oncogenic signaling cascade in which RET/PTC induces RAS-dependent BRAF activation and RAS- and BRAF-dependent ERK activation. Adoptive activation of the RET/PTC-RAS-BRAF axis induced cell proliferation and Matrigel invasion of thyroid follicular cells. Gene expression profiling revealed that the 3 oncogenes activate a common transcriptional program in thyroid cells that includes upregulation of the CXCL1 and CXCL10 chemokines, which in turn stimulate proliferation and invasion. Thus, motile and mitogenic properties are intrinsic to transformed thyroid cells and are governed by an epistatic oncogenic signaling cascade.

    The Journal of clinical investigation 2005;115;4;1068-81

  • Perturbation of epidermal growth factor receptor complex formation and Ras signalling in cells harbouring the hepatitis C virus subgenomic replicon.

    Macdonald A, Chan JK and Harris M

    School of Biochemistry and Microbiology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.

    Hepatitis C virus non-structural NS5A protein inhibits epidermal growth factor (EGF)-stimulated activation of the Ras-ERK mitogen-activated protein kinase pathway at a point upstream of Ras activation. To determine the mechanism of this inhibition, the events occurring between the EGF receptor and Ras in Huh-7 cells harbouring the HCV subgenomic replicon were investigated. It was shown that, following EGF stimulation, these cells exhibited decreased EGF receptor tyrosine phosphorylation, aberrant recruitment of the adaptor proteins ShcA and Grb2 to the EGF receptor, reduced phosphorylation of ShcA and reduced Ras activation in comparison with control cells. These data are consistent with effects of NS5A and/or other components of the replicon on multiple events occurring upstream of Ras.

    The Journal of general virology 2005;86;Pt 4;1027-33

  • Cooperation and selectivity of the two Grb2 binding sites of p52Shc in T-cell antigen receptor signaling to Ras family GTPases and Myc-dependent survival.

    Patrussi L, Savino MT, Pellegrini M, Paccani SR, Migliaccio E, Plyte S, Lanfrancone L, Pelicci PG and Baldari CT

    Department of Evolutionary Biology, University of Siena, Via Aldo Moro 2, Siena 53100, Italy.

    Shc proteins participate in a variety of processes regulating cell proliferation, survival and apoptosis. The two ubiquitously expressed isoforms, p52Shc/p46Shc, couple tyrosine kinase receptors to Ras by recruiting Grb2/Sos complexes to a membrane-proximal localization. Tyrosine residues 239/240 and 317 become phosphorylated following receptor engagement and, as such, form two Grb2 binding sites, which have been proposed to be differentially coupled to Myc-dependent survival and to fos-dependent proliferation, respectively. Here, we have addressed the individual function of YY239/240 and Y317 in T-cell antigen receptor (TCR) signaling. We show that p52Shc is phosphorylated on both YY239/240 and Y317 following TCR engagement. Mutation of either YY239/240 or Y317 results in impaired interaction with Grb2 and inhibition of Ras/MAP kinase activation and CD69 induction, supporting a role for both Grb2 binding sites in this function. Substitution of either YY239/240 or Y317 also results in a defective activation of Rac and the coupled stress kinases JNK and p38. Furthermore, mutation of Y317 or, to a larger extent, of YY239/240, results in increased activation-induced cell death, which in cells expressing the FF239/240 mutant is accompanied by impaired TCR-dependent c-myc transcription. The data underline a pleiotropic and nonredundant role of Shc, mediated by both YY239/240 and Y317, in T-cell activation and survival.

    Oncogene 2005;24;13;2218-28

  • An acylation cycle regulates localization and activity of palmitoylated Ras isoforms.

    Rocks O, Peyker A, Kahms M, Verveer PJ, Koerner C, Lumbierres M, Kuhlmann J, Waldmann H, Wittinghofer A and Bastiaens PI

    Department of Structural Biology, Max Planck Institute for Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.

    We show that the specific subcellular distribution of H- and Nras guanosine triphosphate-binding proteins is generated by a constitutive de/reacylation cycle that operates on palmitoylated proteins, driving their rapid exchange between the plasma membrane (PM) and the Golgi apparatus. Depalmitoylation redistributes farnesylated Ras in all membranes, followed by repalmitoylation and trapping of Ras at the Golgi, from where it is redirected to the PM via the secretory pathway. This continuous cycle prevents Ras from nonspecific residence on endomembranes, thereby maintaining the specific intracellular compartmentalization. The de/reacylation cycle also initiates Ras activation at the Golgi by transport of PM-localized Ras guanosine triphosphate. Different de/repalmitoylation kinetics account for isoform-specific activation responses to growth factors.

    Science (New York, N.Y.) 2005;307;5716;1746-52

  • Conformational states of Ras complexed with the GTP analogue GppNHp or GppCH2p: implications for the interaction with effector proteins.

    Spoerner M, Nuehs A, Ganser P, Herrmann C, Wittinghofer A and Kalbitzer HR

    Institut für Biophysik und physikalische Biochemie, Universität Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany.

    The guanine nucleotide-binding protein Ras occurs in solution in two different states, state 1 and state 2, when the GTP analogue GppNHp is bound to the active center as detected by (31)P NMR spectroscopy. Here we show that Ras(wt).Mg(2+).GppCH(2)p also exists in two conformational states in dynamic equilibrium. The activation enthalpy DeltaH(++)(12) and the activation entropy DeltaS(++)(12) for the transition from state 1 to state 2 are 70 kJ mol(-1) and 102 J mol(-1) K(-1), within the limits of error identical to those determined for the Ras(wt).Mg(2+).GppNHp complex. The same is true for the equilibrium constants K(12) = [2]/[1] of 2.0 and the corresponding DeltaG(12) of -1.7 kJ mol(-1) at 278 K. This excludes a suggested specific effect of the NH group of GppNHp on the equilibrium. The assignment of the phosphorus resonance lines of the bound analogues has been done by two-dimensional (31)P-(31)P NOESY experiments which lead to a correction of the already reported assignments of bound GppNHp. Mutation of Thr35 in Ras.Mg(2+).GppCH(2)p to serine leads to a shift of the conformational equilibrium toward state 1. Interaction of the Ras binding domain (RBD) of Raf kinase or RalGDS with Ras(wt) or Ras(T35S) shifts the equilibrium completely to state 2. The (31)P NMR experiments suggest that, besides the type of the side chain of residue 35, a main contribution to the conformational equilibrium in Ras complexes with GTP and GTP analogues is the effective acidity of the gamma-phosphate group of the bound nucleotide. A reaction scheme for the Ras-effector interaction is presented which includes the existence of two conformations of the effector loop and a weak binding state.

    Biochemistry 2005;44;6;2225-36

  • Regulation of Raf-1 by direct feedback phosphorylation.

    Dougherty MK, Müller J, Ritt DA, Zhou M, Zhou XZ, Copeland TD, Conrads TP, Veenstra TD, Lu KP and Morrison DK

    Laboratory of Protein Dynamics and Signaling, National Cancer Institute-Frederick, Frederick, MD 21702, USA.

    The Raf-1 kinase is an important signaling molecule, functioning in the Ras pathway to transmit mitogenic, differentiative, and oncogenic signals to the downstream kinases MEK and ERK. Because of its integral role in cell signaling, Raf-1 activity must be precisely controlled. Previous studies have shown that phosphorylation is required for Raf-1 activation, and here, we identify six phosphorylation sites that contribute to the downregulation of Raf-1 after mitogen stimulation. Five of the identified sites are proline-directed targets of activated ERK, and phosphorylation of all six sites requires MEK signaling, indicating a negative feedback mechanism. Hyperphosphorylation of these six sites inhibits the Ras/Raf-1 interaction and desensitizes Raf-1 to additional stimuli. The hyperphosphorylated/desensitized Raf-1 is subsequently dephosphorylated and returned to a signaling-competent state through interactions with the protein phosphatase PP2A and the prolyl isomerase Pin1. These findings elucidate a critical Raf-1 regulatory mechanism that contributes to the sensitive, temporal modulation of Ras signaling.

    Funded by: NIGMS NIH HHS: R01GM58556

    Molecular cell 2005;17;2;215-24

  • The role of RAS oncogene in survival of patients with lung cancer: a systematic review of the literature with meta-analysis.

    Mascaux C, Iannino N, Martin B, Paesmans M, Berghmans T, Dusart M, Haller A, Lothaire P, Meert AP, Noel S, Lafitte JJ and Sculier JP

    Department of Intensive Care and Thoracic Oncology, Institut Jules Bordet, Centre des Tumeurs de l'Université Libre de Bruxelles, Belgium. celine.mascaux@bordet.be

    The proto-oncogene RAS, coding for a 21 kDa protein (p21), is mutated in 20% of lung cancer. However, the literature remains controversial on its prognostic significance for survival in lung cancer. We performed a systematic review of the literature with meta-analysis to assess its possible prognostic value on survival. Published studies on lung cancer assessing prognostic value of RAS mutation or p21 overexpression on survival were identified by an electronic search. After a methodological assessment, we estimated individual hazard ratios (HR) estimating RAS protein alteration or RAS mutation effect on survival and combined them using meta-analytic methods. In total, 53 studies were found eligible, with 10 concerning the same cohorts of patients. Among the 43 remaining studies, the revelation method was immunohistochemistry (IHC) in nine and polymerase chain reaction (PCR) in 34. Results in terms of survival were significantly pejorative, significantly favourable, not significant and not conclusive in 9, 1, 31, 2, respectively. In total, 29 studies were evaluable for meta-analysis but we aggregated only the 28 dealing with non-small-cell lung cancer (NSCLC) and not the only one dealing with small-cell-lung cancer (SCLC). The quality scores were not statistically significantly different between studies with or without significant results in terms of survival, allowing us to perform a quantitative aggregation. The combined HR was 1.35 (95% CI: 1.16-1.56), showing a worse survival for NSCLC with KRAS2 mutations or p21 overexpression and, particularly, in adenocarcinomas (ADC) (HR 1.59; 95% CI 1.26-2.02) and in studies using PCR (HR 1.40; 95% CI 1.18-1.65) but not in studies using IHC (HR 1.08; 95% CI 0.86-1.34). RAS appears to be a pejorative prognostic factor in terms of survival in NSCLC globally, in ADC and when it is studied by PCR.

    British journal of cancer 2005;92;1;131-9

  • Myc antagonizes Ras-mediated growth arrest in leukemia cells through the inhibition of the Ras-ERK-p21Cip1 pathway.

    Vaqué JP, Navascues J, Shiio Y, Laiho M, Ajenjo N, Mauleon I, Matallanas D, Crespo P and León J

    Grupo de Biología Molecular del Cáncer, Departamento de Biología Molecular, Unidad de Biomedicina del Consejo Superior de Investigaciones Cientiíficas, Facultad de Medicina, Universidad de Cantabria, 39011 Santander, Spain.

    Even though RAS usually acts as a dominant transforming oncogene, in primary fibroblasts and some established cell lines Ras inhibits proliferation. This can explain the virtual absence of RAS mutations in some types of tumors, such as chronic myeloid leukemia (CML). We report that in the CML cell line K562 Ras induces p21Cip1 expression through the Raf-MEK-ERK pathway. Because K562 cells are deficient for p15INK4b, p16INK4a, p14ARF, and p53, this would be the main mechanism whereby Ras up-regulates p21 expression in these cells. Accordingly, we also found that Ras suppresses K562 growth by signaling through the Raf-ERK pathway. Because c-Myc and Ras cooperate in cell transformation and c-Myc is up-regulated in CML, we investigated the effect of c-Myc on Ras activity in K562 cells. c-Myc antagonized the induction of p21Cip1 mediated by oncogenic H-, K-, and N-Ras and by constitutively activated Raf and ERK2. Activation of the p21Cip1 promoter by Ras was dependent on Sp1/3 binding sites in K562. However, mutational analysis of the p21 promoter and the use of a Gal4-Sp1 chimeric protein strongly suggest that c-Myc affects Sp1 transcriptional activity but not the binding of Sp1 to the p21 promoter. c-Myc-mediated impairment of Ras activity on p21 expression required a transactivation domain, a DNA binding region, and a Max binding region. Moreover, the effect was independent of Miz1 binding to c-Myc. Consistent with its effect on p21Cip1 expression, c-Myc rescued cell growth inhibition induced by Ras. The data suggest that in particular tumor types, such as those associated with CML, c-Myc contributes to tumorigenesis by inhibiting Ras antiproliferative activity.

    The Journal of biological chemistry 2005;280;2;1112-22

  • Persistence of K-ras mutations in plasma after colorectal tumor resection.

    Lindforss U, Zetterquist H, Papadogiannakis N and Olivecrona H

    Karolinska University Hospital, Huddinge, Sweden. Ulrik.Lindforss@cfss.ki.se

    Free DNA in the circulation is increased five-to ten-fold in patients with solid tumours compared to healthy controls. A range of tumor-specific mutated DNA has been shown to be readily extractable and possible to analyse from plasma and serum in these patients. K-ras oncogene mutations are an early event in a subset of colorectal tumors and have been found in 30-60% of patients with colorectal carcinoma (CRC). The presence of tumor-derived k-ras gene mutations in the circulation has previously been described before surgery. The aim of this study was to characterize the presence of mutant k-ras in plasma in the short-term postoperative period after radical surgery of CRC patients, and further to characterize this in relation to relapse of the disease. Tumors and corresponding plasma pre- and postoperatively on day three after surgery were collected from 25 patients with CRC (Dukes' stage A-D). Biopsies for DNA extraction from the tumors were collected from the most invasive parts microscopically. After PCR amplification of the k-ras gene (codon 12 and 13), the presence of mutations was analysed by TGGE (temperature gradient gel electrophoresis). Twenty four/25 patients underwent putatively curative resections. Sixteen of the 25 patients (64%) expressed k-ras mutations in their tumor. Of these, 9 patients (56%) also had detectable k-ras mutations in preoperative plasma samples. On day three postoperatively, 8 of these patients persistently were found to have mutant k-ras in the plasma. This was not correlated with tumor stage. None of the 9 tumor mutation-negative cases expressed mutated k-ras in their plasma pre- or postoperatively. The results indicate that plasma mutant k-ras can be detected pre- and early postoperatively in all stages of colorectal neoplasia. No correlation between short-term postoperative persistence of mutant plasma-DNA and disease recurrence at follow-up was found. However, the use of k-ras as a marker during postoperative follow-up and as a possible tool for early detection of recurrent disease must be further characterized.

    Anticancer research 2005;25;1B;657-61

  • Expression of Ras GTPase isoforms in normal and diseased pancreas.

    Kocher HM, Senkus R, Moorhead J, Al-Nawab M, Patel AG, Benjamin IS and Hendry BM

    Department of Surgery, King's College Hospital, Guy's King's and St Thomas' School of Medicine, King's College London, London, UK.

    Background: Ki-Ras is well studied in its oncogenic form in relation to pancreatic pathologies. However, the individual contribution of each of the wild-type Ras isoforms (Ha-, Ki-, and N-) in pancreatic cells in health and disease is unknown.

    Methods: Archival formalin-fixed, paraffin-embedded specimens of normal (n = 6) and malignant pancreas (n = 35) were used for immuno-histochemical detection of Ras isoforms using a modified polymer system. In addition, immunogold labelling for Ras isoforms was done for subcellular localisation under electron microscopy.

    Results: Pancreatic ductal cells expressed Ha-Ras in the cytoplasm, with Ki-Ras in the apical region and N-Ras (50% of cases) in a supranuclear distribution. Pancreatic acinar cells express all three isoforms with some nuclear expression of Ki-Ras and supranuclear expression of N-Ras. Islets show Ki- and Ha-Ras mainly with differential expression of Ha-Ras (beta cells showing less Ha-Ras and more Ki-Ras than alpha cells). Electron microscopy shows that Ha-Ras is mainly localised in the endoplasmic reticulum and Golgi apparatus of the acinar cells with some plasma membrane localisation of Ki-Ras in the ductal cells. There was no change in any of the Ras isoform expression in the ductal or acinar cells in various malignancies studied (Mann-Whitney U test, p > 0.1).

    Conclusions: Ras isoforms have distinct and separate cellular and subcellular distribution that may persist even in the malignantly transformed state. Understanding this distinct functional distribution patterns in detail is an essential step if mutant Ki-Ras is to be targeted in the pancreas by genetic or molecular therapeutic tools.

    Pancreatology : official journal of the International Association of Pancreatology (IAP) ... [et al.] 2005;5;2-3;205-14

  • Toll-like receptor 2 and mitogen- and stress-activated kinase 1 are effectors of Mycobacterium avium-induced cyclooxygenase-2 expression in macrophages.

    Pathak SK, Bhattacharyya A, Pathak S, Basak C, Mandal D, Kundu M and Basu J

    Department of Chemistry, Bose Institute, 93/1 Acharya Prafulla Chandra Road, Calcutta 700009, India.

    Understanding how pathogenic mycobacteria subvert the protective immune response is crucial to the development of strategies aimed at controlling mycobacterial infections. Prostaglandin E(2) exerts an immunosuppressive function in the context of mycobacterial infection. Because cyclooxygenase-2 (COX-2) is a rate-limiting enzyme in prostaglandin biosynthesis, there is a need to delineate the mechanisms through which pathogenic mycobacteria regulate COX-2 expression in macrophages. Our studies demonstrate that the NF-kappaB and CRE elements of the COX-2 promoter are critical to Mycobacterium avium-induced COX-2 gene expression. M. avium-triggered signaling originates at the Toll-like receptor 2 (TLR2). Ras associates with TLR2 and activates the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK), whereas tumor necrosis factor receptor-associated factor 6 (TRAF6)/transforming growth factor beta-activated kinase 1 (TAK1)-dependent signaling activates p38 MAPK. Both ERK and p38 MAPK activation converge to regulate the activation of mitogen- and stress-activated kinase 1 (MSK1). MSK1 mediates the phosphorylation of the transcription factor CREB accounting for its stimulatory effect on CRE-dependent gene expression. M. avium-triggered cytoplasmic NF-kappaB activation following IkappaB phosphorylation is necessary but not sufficient for COX-2 promoter-driven gene expression. MSK1 activation is also essential for M. avium-triggered NF-kappaB-dependent gene expression, presumably mediating nucleosomal modifications. These studies demonstrate that the nuclear kinase MSK1 is necessary in regulating the pathogen-driven expression of a gene by controlling two transcription factors. The attenuation of MSK1 may therefore have potential benefit in restricting survival of pathogenic mycobacteria in macrophages.

    The Journal of biological chemistry 2004;279;53;55127-36

  • Crystallographic analysis of CaaX prenyltransferases complexed with substrates defines rules of protein substrate selectivity.

    Reid TS, Terry KL, Casey PJ and Beese LS

    Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.

    Post-translational modifications are essential for the proper function of many proteins in the cell. The attachment of an isoprenoid lipid (a process termed prenylation) by protein farnesyltransferase (FTase) or geranylgeranyltransferase type I (GGTase-I) is essential for the function of many signal transduction proteins involved in growth, differentiation, and oncogenesis. FTase and GGTase-I (also called the CaaX prenyltransferases) recognize protein substrates with a C-terminal tetrapeptide recognition motif called the Ca1a2X box. These enzymes possess distinct but overlapping protein substrate specificity that is determined primarily by the sequence identity of the Ca1a2X motif. To determine how the identity of the Ca1a2X motif residues and sequence upstream of this motif affect substrate binding, we have solved crystal structures of FTase and GGTase-I complexed with a total of eight cognate and cross-reactive substrate peptides, including those derived from the C termini of the oncoproteins K-Ras4B, H-Ras and TC21. These structures suggest that all peptide substrates adopt a common binding mode in the FTase and GGTase-I active site. Unexpectedly, while the X residue of the Ca1a2X motif binds in the same location for all GGTase-I substrates, the X residue of FTase substrates can bind in one of two different sites. Together, these structures outline a series of rules that govern substrate peptide selectivity; these rules were utilized to classify known protein substrates of CaaX prenyltransferases and to generate a list of hypothetical substrates within the human genome.

    Funded by: NCRR NIH HHS: RR07707; NIGMS NIH HHS: GM52382

    Journal of molecular biology 2004;343;2;417-33

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • RIAM, an Ena/VASP and Profilin ligand, interacts with Rap1-GTP and mediates Rap1-induced adhesion.

    Lafuente EM, van Puijenbroek AA, Krause M, Carman CV, Freeman GJ, Berezovskaya A, Constantine E, Springer TA, Gertler FB and Boussiotis VA

    Department of Medical Oncology, Dana-Farber Cancer Institute, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.

    The small GTPase Rap1 induces integrin-mediated adhesion and changes in the actin cytoskeleton. The mechanisms that mediate these effects of Rap1 are poorly understood. We have identified RIAM as a Rap1-GTP-interacting adaptor molecule. RIAM defines a family of adaptor molecules that contain a RA-like (Ras association) domain, a PH (pleckstrin homology) domain, and various proline-rich motifs. RIAM also interacts with Profilin and Ena/VASP proteins, molecules that regulate actin dynamics. Overexpression of RIAM induced cell spreading and lamellipodia formation, changes that require actin polymerization. In contrast, RIAM knockdown cells had reduced content of polymerized actin. RIAM overexpression also induced integrin activation and cell adhesion. RIAM knockdown displaced Rap1-GTP from the plasma membrane and abrogated Rap1-induced adhesion. Thus, RIAM links Rap1 to integrin activation and plays a role in regulating actin dynamics.

    Funded by: NIAID NIH HHS: AI 41584, AI 43552, AI 46584; NIGMS NIH HHS: GM68676

    Developmental cell 2004;7;4;585-95

  • Change in protein flexibility upon complex formation: analysis of Ras-Raf using molecular dynamics and a molecular framework approach.

    Gohlke H, Kuhn LA and Case DA

    Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

    Changes in flexibility upon protein-protein complex formation of H-Ras and the Ras-binding domain of C-Raf1 have been investigated using the molecular framework approach FIRST (Floppy Inclusion and Rigid Substructure Topology) and molecular dynamics simulations (MD) of in total approximately 35 ns length. In a computational time of about one second, FIRST identifies flexible and rigid regions in a single, static three-dimensional molecular framework, whose vertices represent protein atoms and whose edges represent covalent and non-covalent (hydrogen bond and hydrophobic) constraints and fixed bond angles within the protein. The two methods show a very good agreement with respect to the identification of changes in flexibility in both binding partners on a local scale. This implies that flexibility can be successfully predicted by identifying which bonds limit motion within a molecule and how they are coupled. In particular, as identified by MD, the beta-sheet in Raf shows considerably more pronounced orientational correlations in the bound state compared to the unbound state. Similarly, FIRST assigns the beta-sheet to the largest rigid cluster of the complex. Interestingly, FIRST allows us to identify that interactions across the interface (but not conformational changes upon complex formation) result in the observed rigidification. Since regions of the beta-sheet of Raf that do not interact directly with Ras become rigidified, this also demonstrates the long-range aspect to rigidity percolation. Possible implications of the change of flexibility of the Ras-binding domain of Raf on the activation of Raf upon complex formation are discussed. Finally, the sensitivity of FIRST results with respect to the representation of non-covalent interactions used as constraints is probed.

    Funded by: NCRR NIH HHS: RR12255; NIGMS NIH HHS: GM67249

    Proteins 2004;56;2;322-37

  • Selective occurrence of ras mutations in benign and malignant thyroid follicular neoplasms in Taiwan.

    Liu RT, Hou CY, You HL, Huang CC, Hock-Liew, Chou FF, Wang PW and Cheng JT

    Division of Metabolism, Chang Gung Memorial Hospital, Kaohsiung, Taiwan.

    Previous studies have demonstrated that point mutations in all three ras genes (H-ras, K-ras, and N-ras) may occur in thyroid neoplasia. However, the overall incidence of ras mutations in thyroid tumors and their frequency in specific histologic types varies widely in different series. Many earlier studies have chosen allele-specific oligonucleotide hybridization approaches to examine ras mutations without further confirmation of the positive samples by DNA sequencing. In this study, mutational hot spots in exon 1 (codons 12/13) and exon 2 (codon 61) of the H-ras, K-ras, and N-ras were polymerase chain reaction (PCR) amplified and sequenced with an automatic sequencer. ras mutations were detected in 4 of 89 (4.5%) benign and malignant thyroid tumors. Three of 8 follicular carcinomas exhibited mutations in codon 61 of H-ras, K-ras, and N-ras, respectively, and mutation at codon 61 of N-ras was found in 1 of 12 follicular adenomas. No mutations were observed in the other tumors, which included 20 nodular goiters, 5 Hürthle cell adenomas, 42 papillary carcinomas, and 2 undifferentiated carcinomas. Our results, obtained by the direct sequencing technique, indicate a lower overall prevalence of ras oncogenes in thyroid tumors than reports in earlier series. However, the frequency of ras mutations in specific histotype of thyroid tumors and their exclusive involvement of codon 61 in our series are similar to those studies utilizing DNA sequencing to detect or to confirm ras gene alterations. The selective occurrence of ras mutations in benign and malignant follicular neoplasms indicates that ras gene alterations have a specific and early role in the development of follicular type of thyroid tumors in Taiwan.

    Thyroid : official journal of the American Thyroid Association 2004;14;8;616-21

  • The CD40-induced signaling pathway in endothelial cells resulting in the overexpression of vascular endothelial growth factor involves Ras and phosphatidylinositol 3-kinase.

    Flaxenburg JA, Melter M, Lapchak PH, Briscoe DM and Pal S

    Division of Nephrology, Children's Hospital, 300 Longwood Avenue, Boston, MA 02115, USA.

    Ligation of endothelial cell (EC) CD40 induces the expression of several proinflammatory cytokines as well as angiogenesis factors, including vascular endothelial growth factor (VEGF). Moreover, despite the reported importance of CD40 in cell-mediated immunity, little is known of the CD40-induced signaling pathways in EC. In this study, we have investigated the function of the Ras signaling pathway(s) for CD40-induced overexpression of VEGF. EC were transiently transfected with a full-length VEGF promoter-luciferase construct and a dominant-inhibitory mutant of Ras (Ras17N). Following transfection, ligation of CD40 with soluble CD40 ligand resulted in a significant increase in VEGF transcriptional activation, and the inhibitory mutant of Ras blocked this CD40-induced VEGF overexpression. Using EMSA and Western blot analysis, we demonstrated that CD40-dependent binding of nuclear protein(s) to the VEGF promoter and CD40-induced VEGF protein expression in EC were also inhibited by the Ras mutant. Immunoprecipitation studies revealed that ligation of CD40 on EC promoted an increased association of Ras with its effector molecules Raf, Rho, and phosphatidylinositol 3-kinase (PI3K). But, cotransfection of effector-loop mutants of Ras determined that only PI3K was functional for Ras-induced VEGF transcription. Also, wortmanin and a dominant-inhibitory mutant of PI3K inhibited CD40-induced overexpression of VEGF. Together these findings demonstrate that both Ras and PI3K are intermediaries in CD40-induced regulation of VEGF in EC. We believe our findings are of importance in several chronic inflammatory diseases, including atherosclerosis and allograft rejection associated with both CD40-CD40 ligand signaling as well as VEGF expression and function.

    Funded by: NHLBI NIH HHS: F32 HL 73613; NIAID NIH HHS: AI 46756; NIDDK NIH HHS: DK 64182

    Journal of immunology (Baltimore, Md. : 1950) 2004;172;12;7503-9

  • Predictive factors for pancreatic cancer in patients with chronic pancreatitis in association with K-ras gene mutation.

    Arvanitakis M, Van Laethem JL, Parma J, De Maertelaer V, Delhaye M and Devière J

    Department of Hepatogastroenterology, Erasme Hospital, Université Libre de Bruxelles, Brussels, Belgium. maarvani@ulb.ac.be

    Chronic pancreatitis is considered to be a predisposing factor for pancreatic ductal adenocarcinoma (PAC). The purpose of this study was to examine the prognostic value of a finding of mutated (K- ras) gene in predicting the development of PAC in patients with chronic pancreatitis.

    The pancreatic duct brushings of 146 patients with chronic pancreatitis were examined in order to identify K- ras gene mutations. A total of 112 patients were followed up (median duration 42 months) using clinical evaluation, serum CA19 - 9 levels, and imaging studies.

    Results: One or more K- ras mutations were found in 57 of the 146 patients with chronic pancreatitis (39 %). Patients harboring K- ras mutations had a higher incidence of persistent alcohol consumption ( P = 0.041) and of prior rupture of the main pancreatic duct ( P = 0.040). A finding of nuclear atypia in brushing cytology was also more common in patients with K- ras mutation ( P = 0.048). Out of the 112 patients who were followed up, PAC occurred in four of the 44 patients who had a K- ras mutation, but in none of the 68 patients with the wild genotype ( P = 0.022). PAC occurred in three of the 25 patients who did not have pancreatic calcifications ( P = 0.034) and in four of the 54 patients who had demonstrated exocrine insufficiency, but in none of the 58 patients with preserved exocrine function ( P = 0.051). Using stepwise logistic regression, the absence of calcifications, the presence of exocrine insufficiency, and the presence of K- ras mutation were identified as independent predictive factors for cancer development in all patients with chronic pancreatitis.

    Conclusions: K- ras gene mutations occur in chronic pancreatitis and are associated with evolution towards PAC. The absence of pancreatic calcifications and the presence of exocrine insufficiency were identified as additional predictive factors for the development of PAC.

    Endoscopy 2004;36;6;535-42

  • Identification and characterization of rain, a novel Ras-interacting protein with a unique subcellular localization.

    Mitin NY, Ramocki MB, Zullo AJ, Der CJ, Konieczny SF and Taparowsky EJ

    Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-2054, USA.

    The Ras small GTPase functions as a signaling node and is activated by extracellular stimuli. Upon activation, Ras interacts with a spectrum of functionally diverse downstream effectors and stimulates multiple cytoplasmic signaling cascades that regulate cellular proliferation, differentiation, and apoptosis. In addition to the association of Ras with the plasma membrane, recent studies have established an association of Ras with Golgi membranes. Whereas the effectors of signal transduction by activated, plasma membrane-localized Ras are well characterized, very little is known about the effectors used by Golgi-localized Ras. In this study, we report the identification of a novel Ras-interacting protein, Rain, that may serve as an effector for endomembrane-associated Ras. Rain does not share significant sequence similarity with any known mammalian proteins, but contains a Ras-associating domain that is found in RalGDS, AF-6, and other characterized Ras effectors. Rain interacts with Ras in a GTP-dependent manner in vitro and in vivo, requires an intact Ras core effector-binding domain for this interaction, and thus fits the definition of a Ras effector. Unlike other Ras effectors, however, Rain is localized to perinuclear, juxta-Golgi vesicles in intact cells and is recruited to the Golgi by activated Ras. Finally, we found that Rain cooperates with activated Raf and causes synergistic transformation of NIH3T3 cells. Taken together, these observations support a role for Rain as a novel protein that can serve as an effector of endomembrane-localized Ras.

    Funded by: NCI NIH HHS: CA09634, CA42978; NIAMS NIH HHS: AR41115; NIGMS NIH HHS: GM08737

    The Journal of biological chemistry 2004;279;21;22353-61

  • Identification of a novel domain of Ras and Rap1 that directs their differential subcellular localizations.

    Nomura K, Kanemura H, Satoh T and Kataoka T

    Department of Molecular and Cellular Biology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.

    The small GTPase Ha-Ras and Rap1A exhibit high mutual sequence homology and share various target proteins. However, they exert distinct biological functions and exhibit differential subcellular localizations; Rap1A is predominantly localized in the perinuclear region including the Golgi apparatus and endosomes, whereas Ha-Ras is predominantly localized in the plasma membrane. Here, we have identified a small region in Rap1A that is crucial for its perinuclear localization. Analysis of a series of Ha-Ras-Rap1A chimeras shows that Ha-Ras carrying a replacement of amino acids 46-101 with that of Rap1 exhibits the perinuclear localization. Subsequent mutational studies indicate that Rap1A-type substitutions within five amino acids at positions 85-89 of Ha-Ras, such as NNTKS85-89TAQST, NN85-86TA, and TKS87-89QST, are sufficient to induce the perinuclear localization of Ha-Ras. In contrast, substitutions of residues surrounding this region, such as FAI82-84YSI and FEDI90-93FNDL, have no effect on the plasma membrane localization of Ha-Ras. A chimeric construct consisting of amino acids 1-134 of Rap1A and 134-189 of Ha-Ras, which harbors both the palmitoylation and farnesylation sites of Ha-Ras, exhibits the perinuclear localization like Rap1A. Introduction of a Ha-Ras-type substitution into amino acids 85-89 (TAQST85-89NNTKS) of this chimeric construct causes alteration of its predominant subcellular localization site from the perinuclear region to the plasma membrane. These results indicate that a previously uncharacterized domain spanning amino acids 85-89 of Rap1A plays a pivotal role in its perinuclear localization. Moreover, this domain acts dominantly over COOH-terminal lipid modification of Ha-Ras, which has been considered to be essential and sufficient for the plasma membrane localization.

    The Journal of biological chemistry 2004;279;21;22664-73

  • Polymorphisms in DNA repair and metabolic genes in bladder cancer.

    Sanyal S, Festa F, Sakano S, Zhang Z, Steineck G, Norming U, Wijkström H, Larsson P, Kumar R and Hemminki K

    Department of Biosciences at Novum, Karolinska Institute, S-14157 Huddinge, Sweden.

    We investigated the association of urinary bladder cancer with genetic polymorphisms in the xeroderma pigmentosum complementation group C (XPC), group D (XPD) and group G (XPG), X-ray repair cross-complementing group 1 (XRCC1) and group 3 (XRCC3), Nijmegen breakage syndrome 1 (NBS1), cyclin D1, methylene-tetrahydrofolate reductase (MTHFR), NAD(P)H dehydrogenase quinone 1 (NQO1), H-ras and glutathione S-transferase theta 1 (GSTT1) genes. Bladder cancer patients from the different hospitals in Stockholm County Council area and matching controls were genotyped for different polymorphisms. The frequency of the variant allele for A/C polymorphism in exon 15 of the XPC gene was significantly higher in the bladder cancer cases than in the controls (OR 1.49, 95% CI 1.16-1.92, P = 0.001). The variant allele homozygote genotype for the T/C polymorphism in exon 1 of the H-ras gene was associated with a decreased risk for bladder cancer (OR 0.12, 95% CI 0.02-0.67, P = 0.006). The variant allele genotypes for the single nucleotide polymorphisms (SNPs) in DNA repair genes, XPG and NBS1, showed a marginal association with the occurrence of bladder cancer (OR 0.38, 95% CI 0.15-0.94, P = 0.03 and OR 1.64, 95% CI 0.92-2.90, P = 0.09, respectively). We also report a positive correlation between the null homozygote of GSTT1 with the risk of bladder cancer (OR 2.54, 95% CI 1.32-4.98, P = 0.003). For other polymorphisms included in this study, NBS1 Glu185Gln, XPD Lys751Gln, XPG Asp1104His, XRCC1 Arg399Gln, XRCC3 Thr241Met, cyclin D1 Pro242Pro, MTHFR Ala222Val and Glu429Ala, NQO1 Arg139Trp and Pro187Ser, no significant differences for genotype distributions and allele frequencies between the bladder cancer cases and the controls were observed in the present study.

    Carcinogenesis 2004;25;5;729-34

  • H-ras mutation is an additional event of sarcomatous transformation in aerodigestive spindle cell carcinoma.

    Shiratsuchi H, Hirakawa N, Saito T, Oda Y, Yamamoto H, Tomita K, Yamamoto T and Tsuneyoshi M

    Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

    The conversion from a carcinomatous component to a sarcomatous one in spindle cell carcinoma (SPCC) of the upper aerodigestive tract is thought to occur via a series of molecular alterations; however the detailed mechanism is still unknown. We examined mutations at the H-ras and p53 genes in 16 SPCCs of upper aerodigestive tracts using PCR-RFLP, PCR-SSCP and direct sequencing analysis. The two distinct components, sarcomatous and carcinomatous components in SPCC, were analyzed independently. p53 mutations were detected in both components of SPCC (50.0%, 8/16), and those in the sarcomatous component were completely in accordance with those in the carcinomatous one. In contrast, H-ras mutations were detected only in the sarcomatous component (12.5%, 2/16), and not in the carcinomatous one (0%, 0/16). There was a statistically significant difference in prognosis between the patients with the H-ras mutation (n=2) and those without (n=14); the former had poorer prognosis (P=0.0049). Our results seem to suggest that the H-ras mutation is a relatively uncommon event in SPCC; however, the presence of H-ras mutations may be associated with a more malignant potential in SPCC, while actually occurring during the sarcomatous change itself.

    Oncology reports 2004;11;3;597-604

  • p53 deficiency provokes urothelial proliferation and synergizes with activated Ha-ras in promoting urothelial tumorigenesis.

    Gao J, Huang HY, Pak J, Cheng J, Zhang ZT, Shapiro E, Pellicer A, Sun TT and Wu XR

    Veterans Affairs Medical Center in New York, NY 10010, USA.

    Mutation and deletion of the p53 tumor suppressor gene are arguably the most prevalent among the multiple genetic alterations found in human bladder cancer, but these p53 defects are primarily associated with the advanced diseases, and their roles in bladder tumor initiation and in synergizing with oncogenes in tumor progression have yet to be defined. Using the mouse uroplakin II gene promoter, we have targeted into urothelium of transgenic mice a dominant-negative mutant of p53 that lacks the DNA-binding domain but retains the tetramerization domain. Urothelium-expressed p53 mutant binds to and stabilizes the endogenous wild-type p53, induces nuclear abnormality, hyperplasia and occasionally dysplasia, without eliciting frank carcinomas. Concurrent expression of the p53 mutant with an activated Ha-ras, the latter of which alone induces urothelial hyperplasia, fails to accelerate tumor formation. In contrast, the expression of the activated Ha-ras in the absence of p53, as accomplished by crossing the activated Ha-ras transgenic mice with the p53 knockout mice, results in early-onset bladder tumors that are either low-grade superficial papillary or high grade in nature. These results provide the first in vivo experimental evidence that p53 deficiency predisposes the urothelium to hyperproliferation, but is insufficient for bladder tumorigenesis; that the mere reduction of p53 dosage, as produced in transgenic mice expressing the dominant-negative p53 or in heterozygous p53 knockouts, is incapable of synergizing with Ha-ras to induce bladder tumors; and that the complete loss of p53 is a prerequisite for collaborating with activated Ha-ras to promote bladder tumorigenesis.

    Funded by: NIDDK NIH HHS: DK52206, DK56903

    Oncogene 2004;23;3;687-96

  • The small GTP-binding protein, Rhes, regulates signal transduction from G protein-coupled receptors.

    Vargiu P, De Abajo R, Garcia-Ranea JA, Valencia A, Santisteban P, Crespo P and Bernal J

    Instituto de Investigaciones Biomédicas Alberto Sols. Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, 28029 Madrid, Spain.

    The Ras homolog enriched in striatum, Rhes, is the product of a thyroid hormone-regulated gene during brain development. Rhes and the dexamethasone-induced Dexras1 define a novel distinct subfamily of proteins within the Ras family, characterized by an extended variable domain in the carboxyl terminal region. We have carried this study because there is a complete lack of knowledge on Rhes signaling. We show that in PC12 cells, Rhes is targeted to the plasma membrane by farnesylation. We demonstrate that about 30% of the native Rhes protein is bound to GTP and this proportion is unaltered by typical Ras family nucleotide exchange factors. However, Rhes is not transforming in murine fibroblasts. We have also examined the role of Rhes in cell signaling. Rhes does not stimulate the ERK pathway. By contrast, it binds to and activates PI3K. On the other hand, we demonstrate that Rhes impairs the activation of the cAMP/PKA pathway by thyroid-stimulating hormone, and by an activated beta2 adrenergic receptor by a mechanism that suggests uncoupling of the receptor to its cognate heterotrimeric complex. Overall, our results provide the initial insights into the role in signal transduction of this novel Ras family member.

    Oncogene 2004;23;2;559-68

  • Ras regulates assembly of mitogenic signalling complexes through the effector protein IMP.

    Matheny SA, Chen C, Kortum RL, Razidlo GL, Lewis RE and White MA

    Department of Cell Biology, UT Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9039, USA.

    The signal transduction cascade comprising Raf, mitogen-activated protein (MAP) kinase kinase (MEK) and MAP kinase is a Ras effector pathway that mediates diverse cellular responses to environmental cues and contributes to Ras-dependent oncogenic transformation. Here we report that the Ras effector protein Impedes Mitogenic signal Propagation (IMP) modulates sensitivity of the MAP kinase cascade to stimulus-dependent activation by limiting functional assembly of the core enzymatic components through the inactivation of KSR, a scaffold/adaptor protein that couples activated Raf to its substrate MEK. IMP is a Ras-responsive E3 ubiquitin ligase that, on activation of Ras, is modified by auto-polyubiquitination, which releases the inhibition of Raf-MEK complex formation. Thus, Ras activates the MAP kinase cascade through simultaneous dual effector interactions: induction of Raf kinase activity and derepression of Raf-MEK complex formation. IMP depletion results in increased stimulus-dependent MEK activation without alterations in the timing or duration of the response. These observations suggest that IMP functions as a threshold modulator, controlling sensitivity of the cascade to stimulus and providing a mechanism to allow adaptive behaviour of the cascade in chronic or complex signalling environments.

    Nature 2004;427;6971;256-60

  • Hepatocarcinogenesis in mice with beta-catenin and Ha-ras gene mutations.

    Harada N, Oshima H, Katoh M, Tamai Y, Oshima M and Taketo MM

    Banyu Tsukuba Research Institute (Merck), Tsukuba, Japan.

    We have established previously a mouse strain containing a mutant beta-catenin allele of which exon 3 was sandwiched by loxP sequences [Catnb(lox(ex3))]. In this mouse strain, a Wnt-activating beta-catenin mutation alone is insufficient for hepatocarcinogenesis, but additional mutations or epigenetic changes may be required. Here we report that hepatocellular carcinoma develops at the 100% incidence in mice with simultaneous mutations in the beta-catenin and H-ras genes that are introduced by adenovirus-mediated Cre expression. Although H-ras mutation alone rapidly causes large cell dysplasia in the hepatocytes, these cells show no autonomous growth within 1 week after infection of the Cre-adenovirus. However, simultaneous induction of an additional mutation in the beta-catenin gene causes a clonal expansion of such dysplastic cells, followed by nodular formation and development of hepatocellular carcinoma. These results indicate that beta-catenin mutations play a critical role in hepatocarcinogenesis in cooperation with another oncogene and that these mice provide a convenient model to investigate early steps of hepatocarcinogenesis.

    Cancer research 2004;64;1;48-54

  • [Study of HRas1 minisatellite frequencies in children with thyroid papillary cancer].

    Iarmolinskiĭ DG, Kartel NA, Ermak G, Demidchik IuE and Figge J

    The "rare" alleles of HRas1 gene minisatellite are well-known factor of predisposition to many kinds of cancer. We have studied HRas1 minisatellite frequencies among patients with papillary thyroid cancer which is related to consequences of Chernobyl accident. The HRas1 minisatellite was analysed in 32 patients who suffered from papillary carcinoma and underwent operation in 1996-2001 and in 75 Belorussian residents. Of 64 HRas1 alleles revealed in patients 14 were defined as "rare" (21.9%); in the control group we have detected 17 "rare" alleles (11.3%) of the examined 150 alleles. The higher frequency of "rare" HRas1 minisatellite alleles in patient group was statistically significant (p < 0.01). We can suppose that the "rare" alleles of HRas1 minisatellite are associated with increased risk of papillary thyroid cancer formation in children and adolescents after Chernobyl accident.

    TSitologiia i genetika 2004;38;1;9-14

  • Clinical usefulness of K-RAS mutation detection in colorectal cancer and in surgical margins of the colon.

    Okulczyk B, Kovalchuk O, Piotrowski Z, Myśliwiec P and Chyczewski L

    The 2nd Department of General and Gastroenterological Surgery, Medical University of Białystok, Poland.

    The incidence of K-RAS gene mutations in tumour and surgical margins was investigated in 63 patients with adenocarcinomas of varied clinical stage and histological grade. Point mutations of codon 12 K-RAS gene were detected, using the PCR-RFPL technique in cancer tissue in 23 patients (36.5%) and in colon margin mucosa in 1 patient (3.7%), out of 27 examined subjects. No significant correlations were found between the mutations and clinical features. Tumours, located in the left colon, and mucinous neoplasms displayed a higher incidence of mutations. No correlation was observed with either Dukes or TMN clinical advancement.

    Roczniki Akademii Medycznej w Białymstoku (1995) 2004;49 Suppl 1;52-4

  • SIAH-1 interacts with CtIP and promotes its degradation by the proteasome pathway.

    Germani A, Prabel A, Mourah S, Podgorniak MP, Di Carlo A, Ehrlich R, Gisselbrecht S, Varin-Blank N, Calvo F and Bruzzoni-Giovanelli H

    Laboratory of Vascular Biology and Gene Therapy, Centro Cardiologico Fondazione-IRCCS, Via Parea 4, 20138 Milano, Italy.

    SIAH-1 and SIAH-2 are the human members of an evolutionary highly conserved E3 ligase family. SIAH-1 is a p53 and p21(Waf-1/Cip-1) induced gene during apoptosis and tumor suppression. In stable-transfected clones of MCF-7 cells, SIAH-1 overexpression was associated with apoptosis, mitotic alterations and p21(Waf-1/Cip-1) induction of expression. Using a two-hybrid screening, we identified here the transcriptional corepressor CtBP-interacting protein (CtIP) as a SIAH-1-interacting protein. CtIP has been proposed as a regulator of p21(Waf-1/Cip-1) gene transcription through a protein complex involving BRCA1. We demonstrate that SIAH-1 associates with CtIP both in vitro and in vivo. This interaction led to CtIP degradation by the ubiquitin-proteasome pathway. As expected, SIAH-1 induced p21(Waf-1/Cip-1) transcription in Jurkat-T cell. Surprisingly, a SIAH protein deleted of its RING finger, SIAH-1DeltaN, which is able to interact with CtIP but does not promote its degradation, also induced transcription from the p21(Waf-1) promoter in a similar extent as did SIAH-1. Our results suggest that p21(Waf-1/Cip-1) induction by SIAH-1 could not be mediated by CtIP degradation.

    Oncogene 2003;22;55;8845-51

  • The HRAS1 variable number of tandem repeats and risk of breast cancer.

    Tamimi RM, Hankinson SE, Ding S, Gagalang V, Larson GP, Spiegelman D, Colditz GA, Krontiris TG and Hunter DJ

    Department of Epidemiology, Harvard School of Public Health, and Channing Laboratory, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA. rulla.tamimi@channing.harvard.edu

    Rare alleles at the HRAS1 variable number of tandem repeats (VNTRs) locus have been implicated in breast cancer risk. We assessed the association of rare HRAS1 alleles and breast cancer in a case-control study nested within the Nurses' Health Study cohort. Using PCR-based methods, 717 incident breast cancer cases and 798 controls were genotyped for the HRAS1 VNTRs. The prevalence of the rare alleles in breast cancer cases was not different compared with controls (10.7 versus 12.0%, respectively; P = 0.45, two-sided Cochran-Mantel-Haenzel chi(2) test). There was no evidence that women heterozygous (multivariate odds ratio, 0.97; 95% confidence interval, 0.73-1.27) or homozygous (multivariate odds ratio, 0.83; 95% confidence interval, 0.32-2.14) for rare alleles were at an increased risk of breast cancer or that a positive gene-dose effect existed. The results did not vary by menopausal status. Although as a group the rare alleles were not associated with breast cancer, one class of rare alleles between the common alleles of a3 and a4 was associated with a significantly increased risk. These results suggest that there is no overall association between rare alleles of the HRAS1 VNTR and breast cancer.

    Funded by: NCI NIH HHS: CA65725, CA87969, T32-CA09001

    Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 2003;12;12;1528-30

  • Lack of effect of human c-Ha-ras proto-oncogene overexpression on prostate carcinogenesis in probasin/SV40 T antigen transgenic rats.

    Hokaiwado N, Asamoto M, Cho YM, Tsuda H and Shirai T

    Department of Experimental Pathology and Tumor Biology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601. naoiwado@med.nagoya-cu.ac.jp

    We have previously reported that transgenic (Tg) rats bearing the SV40 T antigen under probasin promoter control (PB/SV40T) develop prostate carcinomas at 100% incidence, showing their prostate carcinoma growth to be completely androgen-dependent. Transgenic rats carrying three copies of the human c-Ha-ras proto-oncogene (Hras128) are also highly susceptible to carcinogen induction of multiple mammary carcinomas, in this case estrogen-independent, since ovariectomy does not affect mammary tumor formation. A relationship between ras/mitogen-activated protein kinase signaling and androgen responsiveness of prostate cancer cells has been reported. Therefore it is of interest to investigate whether expression of human c-Ha-ras affects the androgen-dependence of prostate carcinomas developing in the PB/SV40T Tg rat. For this purpose, we established double transgenic (rasTag) rats bearing both PB/SV40T and Hras128. In prostate tissues of the rasTag rats, expression of both human c-Ha-ras and SV40T was confirmed, but the prostate tumor incidence and growth were not significantly affected. Castration at 15 weeks of age induced complete tumor involution in the rasTag rats. These results indicate that the human c-Ha-ras proto-oncogene product does not influence the androgen-dependence of prostate carcinogenesis due to the probasin-mediated SV40 T antigen, despite the estrogen-independence of mammary carcinogenesis in Hras128 rats.

    Cancer science 2003;94;12;1042-5

  • Intersectin activates Ras but stimulates transcription through an independent pathway involving JNK.

    Mohney RP, Das M, Bivona TG, Hanes R, Adams AG, Philips MR and O'Bryan JP

    Laboratory of Signal Transduction, National Institute of Environmental Health Services, NIH/DHHS, Building 101, Research Triangle Park, NC 27709, USA.

    Intersectin (ITSN) is a molecular scaffold involved in regulating endocytosis and mitogenic signaling. We previously demonstrated that ITSN transformed rodent fibroblasts, accelerated hormone-induced maturation of Xenopus oocytes, and activated the Elk-1 transcription factor through an MEK- and Erk-independent mechanism. We now demonstrate that ITSN complexes with the Ras guanine nucleotide exchange factor Sos1 leading to increased RasGTP levels. Using fluorescence resonant energy transfer analysis, we demonstrate that ITSN complexes with Ras in living cells leading to Ras activation on intracellular vesicles. These vesicles contain epidermal growth factor receptor but are distinct from transferrin-positive vesicles. However, Ras is not required for ITSN stimulation of transcription. Rather, we demonstrate that ITSN signals through JNK to activate Elk-1. Although ITSN activation of Elk-1 was Ras-independent, ITSN cooperates with Ras to synergistically activate JNK. These findings indicate that ITSN activates multiple intracellular signaling pathways and suggest that this adaptor protein may coordinately regulate the activity of these pathways in vivo.

    The Journal of biological chemistry 2003;278;47;47038-45

  • The NMDA receptor is coupled to the ERK pathway by a direct interaction between NR2B and RasGRF1.

    Krapivinsky G, Krapivinsky L, Manasian Y, Ivanov A, Tyzio R, Pellegrino C, Ben-Ari Y, Clapham DE and Medina I

    Howard Hughes Medical Institute, Children's Hospital, 1309 Enders Building, 320 Longwood Avenue, Boston, MA 02115, USA.

    The NMDA subtype of glutamate receptors (NMDAR) at excitatory neuronal synapses plays a key role in synaptic plasticity. The extracellular signal-regulated kinase (ERK1,2 or ERK) pathway is an essential component of NMDAR signal transduction controlling the neuroplasticity underlying memory processes, neuronal development, and refinement of synaptic connections. Here we show that NR2B, but not NR2A or NR1 subunits of the NMDAR, interacts in vivo and in vitro with RasGRF1, a Ca(2+)/calmodulin-dependent Ras-guanine-nucleotide-releasing factor. Specific disruption of this interaction in living neurons abrogates NMDAR-dependent ERK activation. Thus, RasGRF1 serves as NMDAR-dependent regulator of the ERK kinase pathway. The specific association of RasGRF1 with the NR2B subunit and study of ERK activation in neurons with varied content of NR2B suggests that NR2B-containing channels are the dominant activators of the NMDA-dependent ERK pathway.

    Neuron 2003;40;4;775-84

  • 8-Hydroxyguanine in a mutational hotspot of the c-Ha-ras gene causes misreplication, 'action-at-a-distance' mutagenesis and inhibition of replication.

    Jałoszyński P, Masutani C, Hanaoka F, Perez AB and Nishimura S

    Banyu Tsukuba Research Institute in Collaboration with Merck Research Laboratories, Okubo 3, Tsukuba, Ibaraki 300-2611, Japan.

    Mutations in particular codons of c-Ha-ras have a strong activating potential, and an activated ras oncogene has been found in a number of human cancers. Using fragments of the human c-Ha-ras gene containing 8-hydroxyguanine (8-OH-G) in codon 12, we provide evidence for highly complex biochemical events leading to activation of the oncogene. Replication with DNA polymerases alpha (Pol(alpha)) and beta (Pol(beta)) led to misincorporation of dAMP, while DNA polymerase eta (Pol(eta)) caused additional insertion of dGMP. For the first time we report an 'action-at-a-distance' mutagenic effect for Pol(eta). Replication catalyzed by this enzyme resulted in misincorporating dAMP, dTMP and dGMP opposite non-oxidized guanine 3'-flanked by 8-OH-G. Interestingly, two adjacent 8-OH-G residues greatly relaxed the specificity of Pol(eta), which in this system was able to incorporate all four nucleotides. Moreover, two adjacent 8-OH-G residues completely blocked Pol(alpha) and strongly inhibited Pol(beta), whereas Pol(eta) was entirely resistant to this inhibition. These results suggest an important role for Pol(eta) in inducing hypermutability in codon 12. Our observations are important for understanding the consequences of 8-OH-G being positioned within the mutational hot spots of oncogenes, the outcome of which appears to be relatively complex even in minimal in vitro systems.

    Nucleic acids research 2003;31;21;6085-95

  • Ras participates in CpG oligodeoxynucleotide signaling through association with toll-like receptor 9 and promotion of interleukin-1 receptor-associated kinase/tumor necrosis factor receptor-associated factor 6 complex formation in macrophages.

    Xu H, An H, Yu Y, Zhang M, Qi R and Cao X

    Institute of Immunology, Second Military Medical University, Shanghai 200433, People's Republic of China.

    CpG oligodeoxynucleotides (ODN) activate immune cells to produce immune mediators by Toll-like receptor 9 (TLR9)-mediated signal transduction, which activates mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB) through the MyD88/IRAK/TRAF6 kinases cascade. However, the precise mechanisms of CpG ODN activation of immune cells have not been fully elucidated. The small GTP-binding protein Ras mediates MAPK activation in response to a variety of stimuli. Up to now, it is not clear whether Ras plays a role in CpG ODN signaling. In the present study, we found that the dominant-negative version of Ras (RasN17) and specific Ras inhibitor, FTI-277, inhibited CpG ODN-induced nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production by murine macrophage cell line RAW264.7. While overexpression of wild-type Ras enhanced CpG ODN-induced extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and NF-kappaB activation, overexpression of RasN17 inhibited CpG ODN-induced ERK, JNK, and NF-kappaB activation. RasN17 overexpression also inhibited CpG ODN-induced IRAK1/TRAF6 complex formation. Further studies revealed that CpG ODN activated Ras in a time- and dose-dependent manner, and Ras associated with TLR9 in a CpG ODN-dependent manner. Most interestingly, activation of Ras preceded the association of Ras with TLR9, giving rise to a possibility that Ras activation might not be dependent on the interaction between Ras and TLR9. Our data demonstrate for the first time that Ras can be activated by CpG ODN in macrophages, and Ras is involved in CpG ODN signaling as an early event by associating with TLR9 and promoting IRAK1/TRAF6 complex formation, and MAPK and NF-kappaB activation.

    The Journal of biological chemistry 2003;278;38;36334-40

  • Alternative splicing of the human proto-oncogene c-H-ras renders a new Ras family protein that trafficks to cytoplasm and nucleus.

    Guil S, de La Iglesia N, Fernández-Larrea J, Cifuentes D, Ferrer JC, Guinovart JJ and Bach-Elias M

    Centre d'Investigació Cardiovascular-Consejo Superior de Investigaciones Científicas, Barcelona, Spain.

    We characterized a novel protein of the Ras family, p19 (H-RasIDX). The c-H-ras proto-oncogene undergoes alternative splicing of the exon termed IDX. We show that the alternative p19 mRNA is stable and as abundant as p21 (p21 H-Ras4A) mRNA in all of the human tissues and cell lines tested. IDX is spliced into stable mRNA in different mammalian species, which present a high degree of nucleotide conservation. Both the endogenous and the transiently expressed p19 protein are detected in COS-1 and HeLa cells and show nuclear diffuse and speckled patterns as well as cytoplasmic localization. In yeast two-hybrid assays, p19 did not interact with two known p21 effectors, Raf1 and Rin1, but was shown to interact with RACK1, a scaffolding protein that promotes multiprotein complexes in different signaling pathways. This observation suggests that p19 and p21 play differential and complementary roles in the cell.

    Cancer research 2003;63;17;5178-87

  • Ha-ras overexpression mediated cell apoptosis in the presence of 5-fluorouracil.

    Tseng YS, Tzeng CC, Chiu AW, Lin CH, Won SJ, Wu IC and Liu HS

    Department of Microbiology and Immunology, National Cheng Kung University, College of Medicine, Tainan, Taiwan.

    By using a mouse NIH3T3 derivate designed 7-4 harboring the inducible Ha-ras oncogene, we demonstrated the close relationship between Ha-ras expression level and sensitization of 5-flurouracil (5-FU)-treated cells. Further studies revealed that the cells susceptible to 5-FU treatment died of apoptosis, which was demonstrated by caspase-3 activation, loss of mitochondria membrane potential (MMP), and DNA fragmentation. The 7-4 cells coexpressing dominant negative Ras (Ras(Asn17)), dominant negative Raf-1 (Raf-1(CB4)), Bcl-2, or active form of phosphatidylinositol 3-kinase (PI3K) became resistant to 5-FU, and apoptosis was prevented. In contrast, the cells coexpressing dominant negative Rac 1 (Rac1(Asn17)) or dominant negative Rho A (RhoA(Asn19)) showed no change of sensitivity to 5-FU. These results indicate that Ras, Bcl-2, as well as Raf-1 and PI3K pathways play pivotal roles in 5-FU-induced apoptosis under Ha-ras-overexpressed condition. Aberrant levels of cyclin E and p21(Cip/WAF-1) expression as well as Cdc 2 phosphorylation at Tyrosine 15 suggest that perturbation of G1/S and G2/M transitions in cell cycle might be responsible for 5-FU triggered apoptosis. Sensitization of Ha-ras-related cells to 5-FU was also demonstrated in human bladder cancer cells. Through understanding the mechanism of 5-FU induced apoptosis in tumor cells, a new direction toward the treatment of Ha-ras oncogene-related cancers with 5-FU at more optimal dosages is possible and combinational therapy with other drugs that suppress PI3K and Bcl-2 activities can also be considered.

    Experimental cell research 2003;288;2;403-14

  • Ha-ras oncogene mutation associated to progression of papillomavirus induced lesions of uterine cervix.

    Alonio LV, Picconi MA, Dalbert D, Mural J, Bartt O, Bazán G, Dominguez M and Teyssié AR

    Instituto Nacional de Enfermedades Infecciosas ANLIS 'Carlos G. Malbrán', Departamento Virologia, (1281) Av. Vélez Sársfield 563, Buenos Aires, Argentina. alonio@anlis.gov.ar

    Background: Epidemiological and virological surveys suggest that the HPV presence is not enough condition to generate anogenital cancer, others factors (genetic, environmental, hormonal, etc) may have an important role. Mutations of ras genes were observed in several human neoplasias, including cervical cancer.

    Objective: The aim of the study was to assess the frequency of Ha-ras oncogene mutations in cervical intraepithelial neoplasia (CIN) grade III and invasive squamous cell carcinomas and to examine this genetic factor in relation to HPV infection and the clinical evolution of cervical lesions.

    They were selected for (a) evaluation of the frequency of Ha-ras mutations: 39 cases of invasive carcinomas (InCa), 47 CIN III and 12 normal tissues taken from areas adjacent to the tumor (NT). (b) Retrospective follow-up: 18 cases of lesion progression; 9 cases of persistence and 12 of regression to mature or immature metaplasia after specific treatment. All biopsies obtained from each patient during the follow-up done between 5 and 10 years were included. HPV typing and scanning of possible mutations in Ha-ras were made by single-strand conformation polymorphism analysis/polymerase chain reaction.

    Results: HPV-DNA was detected in 95% of InCa and 84% of CIN III; HPV 16/18 was found in 65% of patients, mainly associated with persistent infection and lesion progression. The undetermined HPV types (18%) could indicate the circulation in our country of types other than those screened (6, 11, 16, 18, 31 and 33). Twenty percent of CIN III and 41% of InCa had patterns compatible with Ha-ras mutations. Mutated Ha-ras was detected in 61 and 44% of progression and persistence cases, respectively, including early stages of progression.

    Conclusions: Ha-ras mutations were detected in CIN II-III lesions; in mutated cases, the progression took place in under 2 years, then this detection may be an early predictive marker of rapid progression.

    Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology 2003;27;3;263-9

  • RASSF2 is a novel K-Ras-specific effector and potential tumor suppressor.

    Vos MD, Ellis CA, Elam C, Ulku AS, Taylor BJ and Clark GJ

    Department of Cell and Cancer Biology, NCI, National Institutes of Health, Rockville, Maryland 20850-3300, USA.

    Ras proteins regulate a wide range of biological processes by interacting with a broad assortment of effector proteins. Although activated forms of Ras are frequently associated with oncogenesis, they may also provoke growth-antagonistic effects. These include senescence, cell cycle arrest, differentiation, and apoptosis. The mechanisms that underlie these growth-inhibitory activities are relatively poorly understood. Recently, two related novel Ras effectors, NORE1 and RASSF1, have been identified as mediators of apoptosis and cell cycle arrest. Both of these proteins exhibit many of the properties normally associated with tumor suppressors. We now identify a novel third member of this family, designated RASSF2. RASSF2 binds directly to K-Ras in a GTP-dependent manner via the Ras effector domain. However, RASSF2 only weakly interacts with H-Ras. Moreover, RASSF2 promotes apoptosis and cell cycle arrest and is frequently down-regulated in lung tumor cell lines. Thus, we identify RASSF2 as a new member of the RASSF1 family of Ras effectors/tumor suppressors that exhibits a specificity for interacting with K-Ras.

    The Journal of biological chemistry 2003;278;30;28045-51

  • rgr oncogene: activation by elimination of translational controls and mislocalization.

    Hernández-Muñoz I, Benet M, Calero M, Jiménez M, Díaz R and Pellicer A

    Department of Pathology, New York University School of Medicine, New York, New York 10016, USA.

    Previous studies have identified a novel oncogene, rgr, which has homology to the guanine nucleotide exchange factor (GEF) Ral guanine dissociation stimulator (RALGDS). To determine the mechanism of activation of rgr, the wild-type form was isolated. rgr is expressed physiologically at very low levels, due, at least in part, to a long 5'-untranslated region that contains eight AUGs, which inhibit translation of the main open reading frame. When these regulatory sequences are removed, the wild-type gene is expressed at high levels. An investigation of how this GEF could transform cells showed that RGR interacts with RAS, supporting its involvement as a RAS-GEF. Because RAL is localized mainly to the Golgi, the expression of the RGR protein was identified in RK13 cells, a cell line that expresses endogenous rgr. RGR localizes to endomembranes. To determine its location upon transformation, a green fluorescent protein-RGR fusion protein was used to track the movement of RGR. Increasing amounts of expression result in enhanced localization of RGR to the plasma membrane. These results indicate that rgr is activated when its tight translational controls are eliminated and increased expression allows its relocation to the plasma membrane, where efficient activation of RAS occurs.

    Funded by: NCI NIH HHS: CA50434

    Cancer research 2003;63;14;4188-95

  • H-Ras modulates N-methyl-D-aspartate receptor function via inhibition of Src tyrosine kinase activity.

    Thornton C, Yaka R, Dinh S and Ron D

    Ernest Gallo Clinic and Research Center, University of California San Francisco, San Francisco, California 94110-3518, USA.

    Tyrosine phosphorylation of the NR2A and NR2B subunits of the N-methyl-d-aspartate (NMDA) receptor by Src protein-tyrosine kinases modulates receptor channel activity and is necessary for the induction of long term potentiation (LTP). Deletion of H-Ras increases both NR2 tyrosine phosphorylation and NMDA receptor-mediated hippocampal LTP. Here we investigated whether H-Ras regulates phosphorylation and function of the NMDA receptor via Src family protein-tyrosine kinases. We identified Src as a novel H-Ras binding partner. H-Ras bound to Src but not Fyn both in vitro and in brain via the Src kinase domain. Cotransfection of H-Ras and Src inhibited Src activity and decreased NR2A tyrosine phosphorylation. Treatment of rat brain slices with Tat-H-Ras depleted NR2A from the synaptic membrane, decreased endogenous Src activity and NR2A phosphorylation, and decreased the magnitude of hippocampal LTP. No change was observed for NR2B. We suggest that H-Ras negatively regulates Src phosphorylation of NR2A and retention of NR2A into the synaptic membrane leading to inhibition of NMDA receptor function. This mechanism is specific for Src and NR2A and has implications for studies in which regulation of NMDA receptor-mediated LTP is important, such as synaptic plasticity, learning, and memory and addiction.

    Funded by: NIAAA NIH HHS: R01 AA013438, R01AA/MH13438-01A1

    The Journal of biological chemistry 2003;278;26;23823-9

  • Identification of tetratricopeptide repeat 1 as an adaptor protein that interacts with heterotrimeric G proteins and the small GTPase Ras.

    Marty C, Browning DD and Ye RD

    Department of Pharmacology, College of Medicine, University of Illinois, Chicago, Illinois 60612, USA.

    The biological functions of heterotrimeric G proteins and small GTPases are modulated by both extracellular stimuli and intracellular regulatory proteins. Using Saccharomyces cerevisiae two-hybrid screening, we identified tetratricopeptide repeat 1 (TPR1), a 292-amino-acid protein with three TPR motifs, as a Galpha16-binding protein. The interaction was confirmed both in vitro and in transfected mammalian cells, where TPR1 also binds to several other Galpha proteins. TPR1 was found to interact with Ha-Ras preferentially in its active form. Overexpression of TPR1 promotes accumulation of active Ras. TPR1 was found to compete with the Ras-binding domain (RBD) of Raf-1 for binding to the active Ras, suggesting that it may also compete with Ras GTPase-activating protein, thus contributing to the accumulation of GTP-bound Ras. Expression of Galpha16 strongly enhances the interaction between TPR1 and Ras. Removal of the TPR1 N-terminal 112 residues abolishes potentiation by Galpha16 while maintaining the interaction with Galpha16 and the ability to discriminate active Ras from wild-type Ras. We have also observed that LGN, a Galphai-interacting protein with seven TPR motifs, binds Ha-Ras. Thus, TPR1 is a novel adaptor protein for Ras and selected Galpha proteins that may be involved in protein-protein interaction relating to G-protein signaling.

    Funded by: NIAID NIH HHS: AI33503, AI40176, R01 AI033503, R01 AI040176, R56 AI033503, R56 AI040176

    Molecular and cellular biology 2003;23;11;3847-58

  • Structural and biochemical studies of p21Ras S-nitrosylation and nitric oxide-mediated guanine nucleotide exchange.

    Williams JG, Pappu K and Campbell SL

    Department of Biochemistry and Biophysics, 530 Mary Ellen Jones Building, University of North Carolina, Chapel Hill 27599, USA.

    Ras is a guanine nucleotide-binding protein that cycles between inactive GDP-bound and active GTP-bound states to regulate a diverse array of cellular processes, including cell growth, apoptosis, and differentiation. The guanine nucleotide-bound state of Ras is tightly maintained by regulatory factors to promote regulated growth control. A class of regulatory molecules that lead to Ras activation are guanine nucleotide exchange factors (GEFs). Ras GEFs bind to Ras and facilitate GDP release, followed by GTP incorporation and Ras activation. Nitric oxide (NO) has also been shown to promote guanine nucleotide exchange (GNE) on Ras and increase cellular Ras-GTP levels, but the process by which NO-mediated GNE occurs is not clear. We initiated NMR structural and biochemical studies to elucidate how nitrosylation of Ras might lead to enhanced GNE. Surprisingly, our studies show that stable S-nitrosylation of Ras at Cys-118, does not affect the structure of Ras, its association with the Ras-binding domain of Raf (a downstream effector of Ras), or GNE rates relative to non-nitrosylated Ras. We have found, however, that the actual chemical process of nitrosylation, rather than the end-product of Ras S-nitrosylation, accounts for the enhanced GNE that we have observed and that has been previously observed by others.

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;11;6376-81

  • Targeting Ras signaling through inhibition of carboxyl methylation: an unexpected property of methotrexate.

    Winter-Vann AM, Kamen BA, Bergo MO, Young SG, Melnyk S, James SJ and Casey PJ

    Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA.

    The antifolate methotrexate is one of the most successful drugs in cancer chemotherapy. Although its efficacy is widely attributed to a decrease in nucleotide biosynthesis (1), methotrexate is known to increase homocysteine (2), a compound associated with an elevated risk of heart disease, Alzheimer's disease (3), and neural tube defects (4). A potential mechanism for the detrimental effects of homocysteine is cellular hypomethylation from an increase in S-adenosylhomocysteine (5), an inhibitor of methyltransferases including isoprenylcysteine carboxyl methyltransferase (Icmt). Among the substrates of Icmt is the monomeric G protein Ras, a critical component of many signaling pathways that regulate cell growth and differentiation. Because carboxyl methylation of Ras is important for proper plasma membrane localization and function (6), we investigated the role of Icmt in the antiproliferative effect of methotrexate. After methotrexate treatment of DKOB8 cells, Ras methylation is decreased by almost 90%. This hypomethylation is accompanied by a mislocalization of Ras to the cytosol and a 4-fold decrease in the activation of p44 mitogen-activated protein kinase and Akt. Additionally, cells lacking Icmt are highly resistant to methotrexate. Whereas cells expressing wild-type levels of Icmt are inhibited by methotrexate, stable expression of myristoylated H-Ras, which does not require carboxyl methylation for membrane attachment (7), confers resistance to methotrexate. These results suggest that inhibition of Icmt is a critical component of the antiproliferative effect of methotrexate, expanding our understanding of this widely used drug and identifying Icmt as a target for drug discovery.

    Funded by: NHLBI NIH HHS: HL41633; NIA NIH HHS: AG15451; NIGMS NIH HHS: GM46372

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;11;6529-34

  • Conformational states of the switch I region of Ha-ras-p21 in hinge residue mutants studied by fluorescence lifetime and fluorescence anisotropy measurements.

    Kuppens S, Hellings M, Jordens J, Verheyden S and Engelborghs Y

    Laboratory of Biomolecular Dynamics, Katholieke Universiteit Leuven, Celestijnenlaan 200D, B-3001 Leuven, Belgium.

    The hinge residues (Val29 and Ile36) of the switch I region (also known as the effector loop) of the Ha-ras-p21 protein have been mutated to glycines to accelerate the conformational changes typical for the effector loop. In this work, we have studied the influence of the combined mutations on the steady-state structure of the switch I region of the protein in both the inactive GDP-bound conformation as in the active GTP-bound conformation. Here, we use the fluorescence properties of the single tryptophan residue in the Y32W mutant of Ha-ras-p21. This mutant has already been used extensively as a reference form of the protein. Reducing the size of the side chains of the hinge residues not only accelerates the conformational changes but also affects the steady-state structures of the effector loop as indicated by the changes in the fluorescence properties. A thorough analysis of the fluorescence changes (quantum yield, lifetimes, etc.) proves that these changes are from a reshuffling between the rotamer populations of Trp. The population reshuffling is caused by the overall structural rearrangement along the switch I region. The effects are clearly more pronounced in the inactive GDP-bound conformation than in the active GTP-bound conformation. The effect of both mutations seems to be additive in the GDP-bound state, but cooperative in the GTP-bound state.

    Protein science : a publication of the Protein Society 2003;12;5;930-8

  • [Development of chronic obstructive pulmonary disease correlates with mini- and microsatellite locus instability].

    Chizhikov VV, Chikina SIu, Tatosian AG, Chuchalin AG and Zborovskaia IB

    Research Institute of Carcinogenesis, Cancer Research Center, Russian Academy of Medical Sciences, Moscow, 115478 Russia.

    Allele distribution at a highly polymorphic minisatellite adjacent to the c-Hras1 gene as well as deletions of microsatellite markers, D3S966, D3S1298, D9S171, and a microsatellite within p53 gene, were examined in bronchial epithelium specimens obtained from 53 chronic obstructive pulmonary disease (COPD) patients and healthy donors. A higher frequency of rare Hras1 minisatellite alleles in COPD patients than in the individuals without pulmonary pathology (6.6% versus 2.2%; P < 0.05) was shown. This difference was most pronounced in the group of ten COPD patients with idiopathic pulmonary fibrosis. Three of these patients had rare Hras1 minisatellite allele (P < 0.02 in comparison with healthy controls). Alterations in at least one of the microsatellite markers (deletions or microsatellite instability) were detected in bronchial epithelium samples obtained from: 4 of 10 COPD patients with pneumofibrosis (40%); 15 of 43 COPD patients (34.9%) without pneumofibrosis; and 8 of 20 tobacco smokers (40%) without pulmonary pathology. These defects were not observed in the analogous samples obtained from healthy nonsmoking individuals. No statistically significant differences were revealed between COPD patients and healthy smokers upon comparison of both the total number of molecular defects and the number of defects in the individual chromosomal loci. The total number of molecular defects revealed in bronchial epithelium samples from the individuals of two groups examined correlated with the intensity of exposure to tobacco smoke carcinogens (r = 0.28; P < 0.05). These findings suggest that rare alleles at the Hras1 locus may be associated with hereditary predisposition to COPD and the development of pneumofibrosis, while mutations in microsatellite markers result from exposure to tobacco smoke carcinogens and are not associated with the appearance of these pathologies.

    Genetika 2003;39;5;694-701

  • RAS point mutations and PAX8-PPAR gamma rearrangement in thyroid tumors: evidence for distinct molecular pathways in thyroid follicular carcinoma.

    Nikiforova MN, Lynch RA, Biddinger PW, Alexander EK, Dorn GW, Tallini G, Kroll TG and Nikiforov YE

    Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0529, USA.

    A series of 88 conventional follicular and Hürthle cell thyroid tumors were analyzed for RAS mutations and PAX8-PPAR gamma rearrangements using molecular methods and for galectin-3 and HBME-1 expression by immunohistochemistry. A novel LightCycler technology-based method was developed to detect point mutations in codons 12/13 and 61 of the H-RAS, K-RAS, and N-RAS genes. Forty-nine percent of conventional follicular carcinomas had RAS mutations, 36% had PAX8-PPAR gamma rearrangement, and only one (3%) had both. In follicular adenomas, 48% had RAS mutations, 4% had PAX8-PPAR gamma rearrangement, and 48% had neither. Follicular carcinomas with PAX8-PPAR gamma typically showed immunoreactivity for galectin-3 but not for HBME-1, tended to present at a younger patient age and be smaller size, and were almost always overtly invasive. In contrast, follicular carcinomas with RAS mutations most often displayed an HBME-1-positive/galectin-3-negative immunophenotype and were either minimally or overtly invasive. Hürthle cell tumors infrequently had PAX8-PPAR gamma rearrangement or RAS mutations. These results suggest that conventional follicular thyroid carcinomas develop through at least two distinct and virtually nonoverlapping molecular pathways initiated by either RAS point mutation or PAX8-PPAR gamma rearrangement.

    Funded by: NCI NIH HHS: CA75425, R01-CA88041; NCRR NIH HHS: M01 RR08084

    The Journal of clinical endocrinology and metabolism 2003;88;5;2318-26

  • Biallelic expression of HRAS and MUCDHL in human and mouse.

    Goldberg M, Wei M, Yuan L, Murty VV and Tycko B

    Department of Pediatrics, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, New York, NY 10032, USA.

    At least eight genes clustered in 1 Mb of DNA on human chromosome (Chr) 11p15.5 are subject to parental imprinting, with monoallelic expression in one or more tissues. Orthologues of these genes show conserved linkage and imprinting on distal Chr 7 of mice. The extended imprinted region has a bipartite structure, with at least two differentially methylated DNA elements (DMRs) controlling the imprinting of two sub-domains. We previously described three biallelically expressed genes ( MRPL23, 2G7 and TNNT3) in 100 kb of DNA immediately downstream of the imprinted H19 gene, suggesting that H19 marks one border of the imprinted region. Here we extend this analysis to two additional downstream genes, HRAS and MUCDHL (mu-protocadherin). We find that these genes are biallelically expressed in multiple fetal and adult tissues, both in humans and in mice. The mouse orthologue of a third gene, DUSP8, located between H19 and MUCDHL, is also expressed biallelically. The DMR immediately upstream of H19 frequently shows a net gain of methylation in Wilms tumors, either via Chr 11p15.5 loss of heterozygosity (LOH) or loss of imprinting (LOI), but changes in methylation in CpG-rich sequences upstream and within the MUCDHL gene are rare in these tumors and do not correlate with LOH or LOI. These findings are further evidence for a border of the imprinted region immediately downstream of H19, and the data allow the construction of an imprinting map that includes more than 20 genes, distributed over 3 Mb of DNA on Chr 11p15.5.

    Funded by: NCI NIH HHS: R01 CA60765

    Human genetics 2003;112;4;334-42

  • Overexpression of the peroxisome proliferator activated receptor alpha or the human c-Ha-ras transgene is not involved in tumorigenesis induced by di(2-ethylhexyl)phthalate in rasH2 mice.

    Toyosawa K, Okugawa K, Teranishi Y, Tanaka K and Matsuoka N

    Drug Research Division, Dainippon Pharmaceutical Co., Ltd., 33-94 Enoki, Suita, 564-0053, Osaka, Japan. kaoru-toyozawa@dianippon-pharm.co.jp

    The mRNA profiles for peroxisome proliferator activated receptor (PPAR) alpha and human c-Ha-ras genes were determined by real-time semi-quantitative polymerase chain reaction analysis of hepatocellular adenomas induced by di(2-ethylhexyl)phthalate (DEHP) in transgenic mice carrying a human prototype c-Ha-ras gene (rasH2 mice). The mRNA levels were essentially equal in hepatocellular adenomas and adjacent non-neoplastic hepatocytes, in spite of a remarkable elevation in the cell proliferation index in tumors determined by anti-Ki-67 immunohistochemistry. From the results, it is concluded that overexpression of PPARalpha or the transgene is not associated with the liver tumorigenesis induced by DEHP in rasH2 mice.

    Cancer letters 2003;192;2;199-203

  • Structural evidence for feedback activation by Ras.GTP of the Ras-specific nucleotide exchange factor SOS.

    Margarit SM, Sondermann H, Hall BE, Nagar B, Hoelz A, Pirruccello M, Bar-Sagi D and Kuriyan J

    Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook, Stony Brook, NY 11794, USA.

    Growth factor receptors activate Ras by recruiting the nucleotide exchange factor son of sevenless (SOS) to the cell membrane, thereby triggering the production of GTP-loaded Ras. Crystallographic analyses of Ras bound to the catalytic module of SOS have led to the unexpected discovery of a highly conserved Ras binding site on SOS that is located distal to the active site and is specific for Ras.GTP. The crystal structures suggest that Ras.GTP stabilizes the active site of SOS allosterically, and we show that Ras.GTP forms ternary complexes with SOS(cat) in solution and increases significantly the rate of SOS(cat)-stimulated nucleotide release from Ras. These results demonstrate the existence of a positive feedback mechanism for the spatial and temporal regulation of Ras.

    Cell 2003;112;5;685-95

  • Characterisation of PDZ-GEFs, a family of guanine nucleotide exchange factors specific for Rap1 and Rap2.

    Kuiperij HB, de Rooij J, Rehmann H, van Triest M, Wittinghofer A, Bos JL and Zwartkruis FJ

    Department of Physiological Chemistry and Centre for Biomedical Genetics, University Medical Centre Utrecht, Universiteitsweg 100, 3584 CG, Utrecht, The Netherlands.

    PDZ-GEF1 (RA-GEF/nRapGEP/CNrasGEF) is a guanine nucleotide exchange factor (GEF) characterised by the presence of a PSD-95/DlgA/ZO-1 (PDZ) domain, a Ras-association (RA) domain and a region related to a cyclic nucleotide binding domain (RCBD). These domains are in addition to a Ras exchange motif (REM) and GEF domain characteristic for GEFs for Ras-like small GTPases. PDZ-GEF1 efficiently exchanges nucleotides of both Rap1 and Rap2, but has also been implicated in mediating cAMP-induced Ras activation through binding of cAMP to the RCBD. Here we describe a new family member, PDZ-GEF2, of which we isolated two splice variants (PDZ-GEF2A and 2B). PDZ-GEF2 contains, in addition to the domains characteristic for PDZ-GEF1, a second, less conserved RCBD at the N-terminus. PDZ-GEF2 is also specific for both Rap1 and Rap2. We further investigated the possibility that PDZ-GEF2, like PDZ-GEF1, is a cAMP-responsive GEF for Ras. However, in contrast to previous results, we did not find any effect of either PDZ-GEF1 or PDZ-GEF2 on Ras in the absence or presence of cAMP. Moreover, affinity measurements by isothermic calorimetry showed that the RCBD of PDZ-GEF1 does not bind cAMP with a physiologically relevant affinity. We conclude that both PDZ-GEF1 and 2 are specific for Rap1 and Rap2 and unresponsive to cAMP and various other nucleotides.

    Biochimica et biophysica acta 2003;1593;2-3;141-9

  • An unusual H-Ras mutant isolated from a human multiple myeloma line leads to transformation and factor-independent cell growth.

    Crowder C, Kopantzev E, Williams K, Lengel C, Miki T and Rudikoff S

    Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

    Multiple myeloma (MM) is an incurable plasma cell malignancy. To investigate biochemical lesions associated with MM, we constructed an expression cDNA library from the OPM-2 human myeloma line. A highly transforming H-Ras mutant was identified by transfection analysis using NIH 3T3 cells. DNA sequencing demonstrated a single-point mutation at position 117 located in the guanine nucleotide-binding site resulting in a lysine-to-glutamic acid substitution. This mutant, H-Ras (K117E), was found to be constitutively activated in terms of GTP binding. We compared the biological effects of H-Ras (K117E) and H-Ras (G12V) in 32D murine hematopoietic progenitor cells. Whereas both Ras proteins are constitutively activated, 32D cells expressing H-Ras (G12V) are still dependent on IL-3 for survival and proliferation while cells carrying H-Ras (K117E) become IL-3 independent. Similar experiments conducted with the B9 line, an IL-6-dependent hybridoma, also demonstrated that B9/H-Ras (K117E) became IL-6-independent. Expression of H-Ras (K117E) in the human IL-6-dependent ANBL-6 myeloma line resulted in enhanced proliferation at suboptimal concentrations of IL-6. These observations suggest that H-Ras mutations at the binding site for the GTP nucleotide ring structure may also represent activating lesions and have additional biological effects when compared to previously described Ras mutants.

    Oncogene 2003;22;5;649-59

  • Novel raf kinase protein-protein interactions found by an exhaustive yeast two-hybrid analysis.

    Yuryev A and Wennogle LP

    Novartis Institute for Biomedical Research, Summit, NJ 07901, USA.

    We have performed an exhaustive unbiased yeast two-hybrid analysis to identify interaction partners of two human Raf kinase isoforms, A-Raf and C-Raf, using their N-terminal regulatory domain as "bait." A total of 20 different human proteins were found to interact with Raf isoforms. Several of these interactions were novel and an extensive bioinformatics evaluation was performed for each. The novel putative interactions include a signalosome component, TOPK/PBK kinase, and two new putative protein phosphatases. The cysteine-rich zinc-binding domain (CRD) of Raf was found to interact with all 20 proteins and to achieve isoform-specific interactions. Since similar putative CRDs are present in a variety of protein serine-threonine kinases, the data suggest that the CRD may function as a major protein-protein interaction domain of these kinases. We propose possible functional consequences of these novel Raf interactions.

    Genomics 2003;81;2;112-25

  • Erbin suppresses the MAP kinase pathway.

    Huang YZ, Zang M, Xiong WC, Luo Z and Mei L

    Department of Neurobiology, Civitan International Research Center, University of Alabama at Birmingham, 35294-0021, USA.

    We present evidence here that Erbin is a negative regulator of the Ras-Raf-Erk signaling pathway. Expression of Erbin decreases transcription of the AChR epsilon-subunit gene, an event that is mediated by Erk activation. Although it interacts with the ErbB2 C terminus through the PDZ domain, Erbin has no effect on ErbB2 tyrosine phosphorylation or binding to the adaptor proteins Shc and Grb2. In contrast, expression of Erbin greatly impairs activation of Erk, but not Akt, by ligands that activate receptor tyrosine kinases. Moreover, Erbin inhibits the Erk activation by active Ras, while it fails to do so in the presence of active Raf-1. Erbin associates with active Ras, but not inactive Ras nor Raf. Consistently, Erbin interferes with the interaction between Ras and Raf both in vivo and in vitro. Finally, overexpression of Erbin leads to inhibition of NGF-induced neuronal differentiation of PC12 cells, whereas down-regulation of endogenous Erbin by specific siRNA exhibits an opposite effect. Collectively, our study has identified Erbin as a novel suppressor of the Ras signaling by disrupting the Ras-Raf interaction.

    Funded by: NINDS NIH HHS: NS40480

    The Journal of biological chemistry 2003;278;2;1108-14

  • A single nucleotide polymorphism in the human H-ras proto-oncogene determines the risk of urinary bladder cancer.

    Johne A, Roots I and Brockmöller J

    Institute of Clinical Pharmacology, University Medical Center Charité, Humboldt University of Berlin, D-10098 Berlin, Germany. andreas.johne@charite.de

    Acquired mutations and inherited polymorphisms in the H-ras gene may modulate the risk of urinary bladder cancer. In DNA isolated from bladder cancer tissue, we screened the coding sequence of H-ras, including the exon-intron-junctions, for exon 1 (n = 68 patients), exon 2 (n = 50), and exons 3 and 4 (n = 25). Acquired mutations at codons 12 and 13 (exon 1) and codon 61 (exon 2), which had been described earlier in bladder cancer tissue, were not found in any of the tumors, but we identified a frequent polymorphism at nucleotide 81T-->C (exon 1) in a wobble position. The clinical impact of this polymorphism was investigated in a case-control study in which 312 patients with histologically verified bladder cancer were compared with 254 hospital controls; 13.5% of the cases but only 7.1% of controls were homozygous for the 81C-variant of this polymorphism [odds ratio (OR), 2.04; 95% confidence interval (CI), 1.15-3.61; P = 0.014]. The homozygous 81C genotype was overrepresented, particularly in the patient groups with poorly differentiated tumors (n = 145, >or=G3; OR, 2.22; 95% CI, 1.15-4.27; P = 0.017), muscle-invasive tumors (n = 107, >or=T(2); OR, 2.65; 95% CI, 1.35-5.23; P = 0.005), and flat transitional cell carcinoma (n = 45; OR, 3.69; 95% CI, 1.60-8.51; P = 0.002). In general, 81CC occurred more frequently in advanced types of bladder cancer. We conclude that individuals harboring the homozygous 81C-genotype of the H-ras proto-oncogene are at an increased risk of bladder cancer.

    Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 2003;12;1;68-70

  • RasGRP4 is a novel Ras activator isolated from acute myeloid leukemia.

    Reuther GW, Lambert QT, Rebhun JF, Caligiuri MA, Quilliam LA and Der CJ

    Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295, USA. greuther@med.unc.edu

    Although a number of genetic defects are commonly associated with acute myeloid leukemia (AML), a large percentage of AML cases are cytogenetically normal. This suggests a functional screen for transforming genes is required to identify genetic mutations that are missed by cytogenetic analyses. We utilized a retrovirus-based cDNA expression system to identify transforming genes expressed in cytogenetically normal AML patients. We identified a new member of the Ras guanyl nucleotide-releasing protein (RasGRP) family of Ras guanine nucleotide exchange factors, designating it RasGRP4. Subsequently, cDNA sequences encoding rodent and human RasGRP4 proteins were deposited in GenBank. RasGRP4 contains the same protein domain structure as other members of the RasGRP family, including a Ras exchange motif, a CDC25 homology domain, a C1/diacyglycerol-binding domain, and putative calcium-binding EF hands. We show that expression of RasGRP4 induces anchorage-independent growth of Rat1 fibroblasts. RasGRP4 is a Ras-specific activator and, interestingly, is highly expressed in peripheral blood leukocytes and myeloid cell lines. Unlike other RasGRP proteins, RasGRP4 is not expressed in the brain or in lymphoid cells. We demonstrated that 32D myeloid cells expressing RasGRP4 have elevated levels of activated Ras compared with control cells, and phorbol 12-myristate 13-acetate (PMA) treatment greatly enhanced Ras activation. PMA induced membrane localization of RasGRP4 and 32D cells expressing RasGRP4 were capable of cytokine-independent proliferation in the presence of PMA. We conclude that RasGRP4 is a member of the RasGRP family of Ras guanine nucleotide exchange factors that may play a role in myeloid cell signaling growth regulation pathways that are responsive to diacylglycerol levels.

    Funded by: NCI NIH HHS: CA 42978; PHS HHS: T32 H 07774

    The Journal of biological chemistry 2002;277;34;30508-14

  • HRAS1 variable number of tandem repeats polymorphism and risk of bladder cancer.

    van Gils CH, Conway K, Li Y and Taylor JA

    Molecular and Genetic Epidemiology Section, Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.

    The HRAS1 variable number of tandem repeats (VNTR) polymorphism, 1 kb downstream from the HRAS1 gene, has been reported to be associated with risk of various cancers. To examine whether individuals with rare HRAS1 VNTR alleles are at increased risk of bladder cancer we carried out a case control study with 230 bladder cancer cases and 203 hospital-based controls frequency-matched on ethnicity, gender and age. For genotyping we used a PCR-based long-gel electrophoretic assay that provides precise allele size discrimination. We did not find evidence of a strong overall effect of the HRAS1 VNTR on bladder cancer risk. Genotype data for whites and blacks were analyzed separately, but the number of black subjects was too small to estimate meaningful odds ratios. Compared to white subjects with 2 common alleles, the odds ratio (OR) for white subjects with 1 rare allele was 0.9 (95% confidence interval (CI) = 0.5-1.4) and for those with 2 rare alleles OR = 1.7 (95% CI = 0.6-5.4). HRAS1 genotype may be related to the prognosis of bladder cancer, however, because incident cases, i.e., newly diagnosed cases had a higher frequency of rare alleles than did prevalent cases, i.e., cases already existing at the time of recruitment. Repeating the analyses with incident cases only (n = 53), the OR for subjects with 1 rare allele was 1.2 (95% CI = 0.6-2.4) and for those with 2 rare alleles 3.2 (95% CI = 0.8-13.7). The number of incident cases was too small to draw firm conclusions on a possible association with a subgroup of tumors with a poor prognosis. Published 2002 Wiley-Liss, Inc.

    International journal of cancer 2002;100;4;414-8

  • Tiam1 mediates Ras activation of Rac by a PI(3)K-independent mechanism.

    Lambert JM, Lambert QT, Reuther GW, Malliri A, Siderovski DP, Sondek J, Collard JG and Der CJ

    Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

    Rac is a member of the Ras superfamily of GTPases and functions as a GDP/GTP-regulated switch. Formation of active Rac-GTP is stimulated by Dbl family guanine nucleotide exchange factors (GEFs), such as Tiam1 (ref. 2). Once activated, Rac stimulates signalling pathways that regulate actin organization, gene expression and cellular proliferation. Rac also functions downstream of the Ras oncoprotein in pathways that stimulate membrane ruffling, growth transformation, activation of the c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase, activation of the NF-kappa B transcription factor and promotion of cell survival. Although recent studies support phosphatidylinositol 3-OH kinase (PI(3)K)-dependent mechanisms through which Ras might activate Rac (refs 9,10), the precise mechanism remains to be determined. Here we demonstrate that Tiam1, a Rac-specific GEF, preferentially associates with activated GTP-bound Ras through a Ras-binding domain. Furthermore, activated Ras and Tiam1 cooperate to cause synergistic formation of Rac-GTP in a PI(3)K-independent manner. Thus, Tiam1 can function as an effector that directly mediates Ras activation of Rac.

    Funded by: NCI NIH HHS: CA69577; NIGMS NIH HHS: GM62299, GM62338

    Nature cell biology 2002;4;8;621-5

  • ADP-ribosylation and functional effects of Pseudomonas exoenzyme S on cellular RalA.

    Fraylick JE, Riese MJ, Vincent TS, Barbieri JT and Olson JC

    Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 165 Ashley Avenue, Suite 309, Charleston, South Carolina 29425, USA.

    Exoenzyme S (ExoS) is a bifunctional virulence factor directly translocated into eukaryotic cells by the type III secretory process of Pseudomonas aeruginosa. Bacterial translocation of ExoS into epithelial cells is associated with diverse effects on cell function, including inhibition of growth, alterations in cell morphology, and effects on adherence processes. Preferred substrates of the ADP-ribosyltransferase (ADPRT) portion of ExoS include low molecular weight G-proteins (LMWG-proteins) in the Ras family. In examining the ADP-ribosylation and functional effects of ExoS on RalA, ExoS was found to ADP-ribosylate endogenous RalA and recombinant RalADeltaCAAX at multiple sites, with Arg52 identified as the preferred site of ADP-ribosylation. The binding of RalA to the Ral binding domain (RBD) of its downstream effector, RalBP1, was inhibited by bacterially translocated ExoS, indicating an effect of ExoS on cellular RalA function. In vitro analyses confirmed that ADP-ribosylation of RalA directly interfered with its ability to bind to the RBD of RalBP1. The studies support the fact that RalA is a cellular substrate of bacterially translocated ExoS and that ADP-ribosylation by ExoS affects RalA interaction with its downstream effector, RalBP1.

    Funded by: NIAID NIH HHS: AI 30162, AI 45569

    Biochemistry 2002;41;30;9680-7

  • H-Ras/mitogen-activated protein kinase pathway inhibits integrin-mediated adhesion and induces apoptosis in osteoblasts.

    Tanaka Y, Nakayamada S, Fujimoto H, Okada Y, Umehara H, Kataoka T and Minami Y

    First Department of Internal Medicine, University of Occupational and Environmental Health School of Medicine, Kitakyushu 807-8555, Japan. tanaka@med.uoeh-u.ac.jp

    We have studied the relevance of H-Ras and its downstream effectors to osteoblast functions. 1) Purified human osteoblasts highly expressed integrins beta1, alpha4, alpha5, alpha6 and the activation epitope of beta1. However, these molecules were markedly down-regulated on osteoblasts transfected with expression vector encoding fully activated H-Ras(V12), H-Ras(V12)T35S, activating Raf-1/mitogen-activated protein kinase (MAPK), or an active Raf-1 but not on cells having H-Ras(V12)Y40C, a phosphoinositide 3-kinase (PI3K)-binding mutant. 2) Although osteoblasts spontaneously adhered to fibronectin and laminin in beta1-dependent manner, the expression of H-Ras(V12) or H-Ras(V12)T35S, but not H-Ras(V12)Y40C, in osteoblasts reduced their adhesion. 3) Osteoblasts bearing H-Ras(V12), H-Ras(V12)T35S, or Raf-1 failed to proliferate, whereas those with H-Ras(V12)Y40C proliferated well. (4) The up-regulation of Fas and down-regulation of Bcl-2 were observed in osteoblasts expressing H-Ras(V12), H-Ras(V12)T35S, or Raf-1. (5) Most of the cells having H-Ras(V12), H-Ras(V12)T35S, or Raf-1 became annexin-V(high)/propidium iodide (PI)(high or low) and terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling (TUNEL)(high)/PI(low) after 24 and 72 h incubation, respectively. Thus, we propose that H-Ras signals followed by Raf-1/MAPK pathway but not PI3K not only reduces beta(1)-mediated adhesion of osteoblasts to matrix proteins but induces apoptosis presumably via the Fas up-regulation and Bcl-2 down-regulation.

    The Journal of biological chemistry 2002;277;24;21446-52

  • Detection of rare K-ras codon 12 mutations using allele-specific competitive blocker PCR.

    McKinzie PB and Parsons BL

    Division of Genetic and Reproductive Toxicology, HFT-120, National Center for Toxicological Research, Food and Drug Administration, 3900 NCTR Rd., Jefferson, AR 72079, USA. pmckenzie@nctr.fda.gov

    Allele-specific competitive blocker PCR (ACB-PCR) is a sensitive allele-specific amplification method in which preferential amplification of the mutant allele occurs by using a primer that has more mismatches to the wild-type allele than to the mutant allele (mutant-specific primer, MSP). Additionally, a non-extendable primer with more mismatches to the mutant allele than to the wild-type allele (blocker primer, BP) competes with the MSP for binding to the wild-type allele, thereby reducing background amplification from the wild-type allele. ACB-PCR primer design is largely dependent upon the basepair substitution being measured, making it unclear if this method is broadly applicable. In an earlier study, an H-ras codon 61 CAA-->AAA mutation had been detected by ACB-PCR at a sensitivity of 10(-5). In this study, ACB-PCR was applied to two human K-ras codon 12 mutations: GGT-->GTT and GGT-->GAT. The method was optimized by systematically altering the concentrations of Perfect Match PCR Enhancer, MSP, BP, and dNTPs. For each mutation, mutant fractions as low as 10(-5) were detected, indicating that this assay can be used on a variety of base substitution mutations. In addition, the results suggest that the 3'-terminal mismatches between the MSP and wild-type allele may be used to predict the ACB-PCR conditions that will be appropriate for the detection of other base substitution mutations. The range of concentrations for each of these components is narrow, making this method relatively easy to apply to additional mutational targets.

    Mutation research 2002;517;1-2;209-20

  • The complex of Arl2-GTP and PDE delta: from structure to function.

    Hanzal-Bayer M, Renault L, Roversi P, Wittinghofer A and Hillig RC

    Max-Planck-Institut für molekulare Physiologie, Abteilung Strukturelle Biologie, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany.

    Arf-like (Arl) proteins are close relatives of the Arf regulators of vesicular transport, but their function is unknown. Here, we present the crystal structure of full-length Arl2-GTP in complex with its effector PDE delta solved in two crystal forms (Protein Data Bank codes 1KSG, 1KSH and 1KSJ). Arl2 shows a dramatic conformational change from the GDP-bound form, which suggests that it is reversibly membrane associated. PDE delta is structurally closely related to RhoGDI and contains a deep empty hydrophobic pocket. Further experiments show that H-Ras, Rheb, Rho6 and G alpha(i1) interact with PDE delta and that, at least for H-Ras, the intact C-terminus is required. We suggest PDE delta to be a specific soluble transport factor for certain prenylated proteins and Arl2-GTP a regulator of PDE delta-mediated transport.

    The EMBO journal 2002;21;9;2095-106

  • Refinement and delineation of the breakpoint regions of a chromosome 1;22 translocation in a patient with Costello syndrome.

    Maróti Z, Kutsche K, Sutajova M, Gal A, Nothwang HG, Czeizel AE, Tímár L and Sólyom E

    American journal of medical genetics 2002;109;3;234-7

  • The delta subunit of retinal rod cGMP phosphodiesterase regulates the membrane association of Ras and Rap GTPases.

    Nancy V, Callebaut I, El Marjou A and de Gunzburg J

    INSERM U-528, Institut Curie-Section de Recherche, 75005 Paris, France.

    Post-translational modifications of GTPases from the Ras superfamily enable them to associate with membrane compartments where they exert their biological activities. However, no protein acting like Rho and Rab dissociation inhibitor (GDI) that regulate the membrane association of Rho and Rab GTPases has been described for Ras and closely related proteins. We report here that the delta subunit of retinal rod phosphodiesterase (PDEdelta) is able to interact with prenylated Ras and Rap proteins, and to solubilize them from membranes, independently of their nucleotide-bound (GDP or GTP) state. We show that PDEdelta exhibits striking structural similarities with RhoGDI, namely conservation of the Ig-like fold and presence of a series of hydrophobic residues which could act as in RhoGDI to sequester the prenyl group of its target proteins, thereby providing structural support for the biochemical activity of PDEdelta. We observe that the overexpression of PDEdelta interferes with Ras trafficking and propose that it may play a role in the process that delivers prenylated proteins from endomembranes, once they have undergone proteolysis and carboxymethylation, to the structures that ensure trafficking to their respective resident compartments.

    The Journal of biological chemistry 2002;277;17;15076-84

  • Extracellular Nef protein activates signal transduction pathway from Ras to mitogen-activated protein kinase cascades that leads to activation of human immunodeficiency virus from latency.

    Tobiume M, Fujinaga K, Suzuki S, Komoto S, Mukai T and Ikuta K

    Department of Microbiology and Immunology, School of Medicine, Vanderbilt University, Nashville, Tennessee, USA.

    We previously reported that viral antigen expression was markedly up-regulated by stimulation with extracellular Nef, similar to the effects of tumor necrosis factor (TNF)-alpha and phorbol myristate acetate, in model cells for HIV-1 latency. In this study, we examined the molecular mechanism of this novel Nef function. Flow cytometry revealed specific binding of Nef on the surface of latently infected cells. Furthermore, activation of Ras in the cells was detected after treatment with Nef, indicating the involvement of Ras in Nef-mediated activation of HIV-1 from latency. This was also confirmed by the observations that HIV-1 long-terminal repeat-luciferase (LTR-Luc) activity was significantly up-regulated by introduction of the active Ras into uninfected cells, and that LTR-Luc activity observed in Nef-treated cells was specifically inhibited by introduction of a dominant negative Ras. In addition, PD98059 inhibited the activation of HIV-1 by Nef, but not by TNF-alpha. Thus, Nef-mediated reactivation of HIV-1 in latent model cells occurs by signal transduction from Ras to mitogen-activated protein kinase cascades.

    AIDS research and human retroviruses 2002;18;6;461-7

  • SmgGDS displays differential binding and exchange activity towards different Ras isoforms.

    Vikis HG, Stewart S and Guan KL

    Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, MI 48109-0606, USA.

    Ras family GTPases play central roles in a wide variety of biological responses, including cell proliferation, differentiation, and oncogenic transformation. We searched for novel guanine nucleotide exchange factors of HRas and isolated small G-protein dissociation stimulator (smgGDS), a guanine nucleotide exchange factor known to act on numerous Ras and Rho family GTPases. SmgGDS specifically interacts with both dominant negative and nucleotide free forms of H and NRas, but not with the corresponding oncogenic forms. An effector domain mutant of HRas, HRasN17G37, selectively lost the ability to bind smgGDS. However, smgGDS does not catalyze guanine nucleotide exchange on either H or NRas in vitro. In contrast, substrates of smgGDS, such as KRas, Rac1, and RhoA, bind to smgGDS in both active and inactive forms which requires the presence of poly-basic residues in the C-termini of the GTPases. Our data suggest that the C-terminal poly-basic region of small GTPases is important for both binding and nucleotide exchange by smgGDS. Furthermore, these data underscore the idea that mammalian Ras isoforms are not functionally equivalent.

    Oncogene 2002;21;15;2425-32

  • Mechanical force generation by G proteins.

    Kosztin I, Bruinsma R, O'Lague P and Schulten K

    Department of Physics and Astronomy, University of Missouri, Columbia, MO 65211, USA. kosztini@missouri.edu

    GTP-hydrolyzing G proteins are molecular switches that play a critical role in cell signaling processes. Here we use molecular dynamics simulations to show that Ras, a monomeric G protein, can generate mechanical force upon hydrolysis. The generated force levels are comparable to those produced by ATP-hydrolyzing motor proteins, consistent with the structural similarities of the catalytic region of motor proteins and G proteins. The force transduction mechanism is based on an irreversible structural change, produced by the hydrolysis, which triggers thermal switching between force-generating substates through changes in the configurational space of the protein.

    Funded by: NCRR NIH HHS: 5 P41 RR05969-04, P41 RR005969

    Proceedings of the National Academy of Sciences of the United States of America 2002;99;6;3575-80

  • Ras participates in the activation of p38 MAPK by interleukin-1 by associating with IRAK, IRAK2, TRAF6, and TAK-1.

    McDermott EP and O'Neill LA

    Department of Biochemistry and Biotechnology Institute, Trinity College, Dublin 2, Ireland.

    Interleukin-1 (IL-1) activates p38 MAP kinase via the small G protein Ras, and this activity can be down-regulated by another small G protein Rap. Here we have further investigated the role of Ras and Rap in p38 MAPK activation by IL-1. Transient transfection of cells with constitutively active forms of the known IL-1 signaling components MyD88, IRAK, and TRAF-6, or the upstream kinases MKK6 and MKK3, activated p38 MAPK. Dominant negative forms of these were found to inhibit activation of p38 MAPK by IL-1. Dominant negative RasN17 blocked the effect of the active forms of all but MKK3 and MKK6, indicating that Ras lies downstream of TRAF-6 but upstream of MKK3 and MKK6 on the pathway. Furthermore, the activation of p38 MAPK caused by overexpressing active RasVHa could not be inhibited using dominant negative mutants of MyD88, IRAK, or IRAK-2, or TRAF6, but could be inhibited by dominant negative MKK3 or MKK6. In the same manner, the inhibitory effect of Rap on the activation of p38 by IL-1 occurred at a point downstream of MyD88, IRAK, and TRAF6, since the activation of p38 MAPK by these components was inhibited by overexpressing active Rap1AV12, while neither MKK3 nor MKK6 were affected. Active RasVHa associated with IRAK, IRAK2, and TRAF6, but not MyD88. In addition we found a role for TAK-1 in the activation of p38 MAPK by IL-1, with TAK-1 also associating with active Ras. Our study suggests that upon activation Ras becomes associated with IRAK, Traf-6, and TAK-1, possibly aiding the assembly of this multiprotein signaling complex required for p38 MAPK activation by IL-1.

    The Journal of biological chemistry 2002;277;10;7808-15

  • The putative tumor suppressor RASSF1A homodimerizes and heterodimerizes with the Ras-GTP binding protein Nore1.

    Ortiz-Vega S, Khokhlatchev A, Nedwidek M, Zhang XF, Dammann R, Pfeifer GP and Avruch J

    Diabetes Unit and Medical Services, Massachusetts General Hospital, Boston, Massachusetts, MA 02114, USA.

    Nore and RASSF1A are noncatalytic proteins that share 50% identity over their carboxyterminal 300 AA, a segment that encompasses a putative Ras-Rap association (RA) domain. RASSF1 is expressed as several splice variants, each of which contain an RA domain, however the 340 AA RASSF1A, but not the shorter RASSF1C variant, is a putative tumor suppressor. Nore binds to Ras and several Ras-like GTPases in a GTP dependent fashion however neither RASSF1 (A or C) or the C. elegans Nore/RASSF1 homolog, T24F1.3 exhibit any interaction with Ras or six other Ras-like GTPases in a yeast two-hybrid expression assay. A low recovery of RASSF1A (but not RASSF1C) in association with RasG12V is observed however on transient expression in COS cells. Nore and RASSF1A can each efficiently homodimerize and heterodimerize with each other through their nonhomologous aminoterminal segments. Recombinant RASSF1C exhibits a much weaker ability to homodimerize or heterodimerize; thus the binding of RASSF1C to Nore is very much less than the binding of RASSF1A to Nore. The association of RASSF1A with RasG12V in COS cells appears to reflect the heterodimerization of RASSF1A with Nore, inasmuch the recovery of RASSF1A with RasG12V is increased by concurrent expression of full length Nore, and abolished by expression of Nore deleted of its RA domain. The preferential ability of RASSF1A to heterodimerize with Nore and thereby associate with Ras-like GTPases may be relevant to its putative tumor suppressor function.

    Funded by: NIDDK NIH HHS: T32DK07028

    Oncogene 2002;21;9;1381-90

  • Immunohistochemical analysis reveals a protective effect of H-ras expression mediated via apoptosis in node-negative breast cancer patients.

    Schondorf T, Rutzel S, Andrack A, Becker M, Hoopmann M, Breidenbach M and Gohring UJ

    Department of Gynecology and Obstetrics, University of Cologne, D-50931 Cologne, Germany. thomas.schoendorf@medizin.uni-koeln.de

    Recently, we were able to demonstrate the favourable indication of H-ras expression in the clinically very important group of node-negative breast cancer patients. In order to further resolve a conceivable cellular effect mediated by the H-ras signal transduction pathway, we ascertained the apoptotic activity of the tumor cells of this overall 298 patients group. To this end, fragmented genomic DNA of breast cancer tissue was determined by specific DNA-end-labelling and subsequent visualization, thereby indicating early apoptosis induction. Tissues were considered apoptotic by means of the apoptotic index (quotient of apoptotic tumor cells and total tumor cells). One hundred and eighty-nine tumors (63.4%) were negative for apoptosis and 109 tissues (36.6%) were considered apoptotic (mean apoptotic index 11.9%, range, 0 to 90%). H-ras expression correlated significantly (chi2-test, p=0.004) to apoptosis. Furthermore, restricted to the node-negative subgroup, both H-ras expression and apoptosis were indicative of a better outcome (log-rank test p=0.0001, and p=0.012, respectively) during the observation time (median 87 months). These data suggest that H-ras expression effects the particular breast tumor cells by induction of apoptosis in breast cancer patients in an early stage without node involvement. The study indicates a possible mechanism, by which H-ras may act protectively.

    International journal of oncology 2002;20;2;273-7

  • Localizing a control region in the pathway to leukotriene C(4) secretion following stimulation of human basophils with anti-IgE antibody.

    Miura K, Lavens-Phillips S and MacGlashan DW

    Johns Hopkins Asthma and Allergy Center, Baltimore, MD 21224, USA.

    Mediator release from human basophils is a self-limited process, but down-regulation of the signaling cascades leading to secretion of leukotriene C(4) (LTC(4)) is controlled independently of the pathway leading to IL-4 secretion. In the current studies, we have explored the regulation of upstream signaling events leading to activation of extracellular signal-related kinases (ERKs; previously shown to be required for LTC(4) generation) in human basophils. IgE-, but not FMLP-mediated activation, induced sustained tyrosine phosphorylation of syk, of shc, and an association of shc to the Grb2/son of sevenless 2 complex. In contrast, IgE-mediated activation resulted in transient activation of p21(ras) and mitogen-activated protein/ERK kinase 1, which were kinetically associated with phosphorylation of ERKs. The canonical Shc/Grb2/son of sevenless pathway to activation of p21(ras) is therefore sustained, while p21(ras) activity is not. We have previously shown that phosphatidylinositol 3 kinase activity is required for p21(ras) activity and, in the current studies, we show that of the p85-sensitive forms of p110 possible, basophils express only p110 delta and that there are no changes in association between p21(ras) and p110 delta in stimulated basophils. We used the generation of phospho-Akt as a marker of the presence of phosphatidylinositol-3,4,5-trisphosphate and found that phospho-Akt is transient on a time scale consistent with p21(ras) activity. On the basis of information obtained in these and other studies, we localize down-regulation of IgE-mediated LTC(4) secretion to a region of the signaling cascade antecedent to p21(ras) activation, downstream of phosphatidylinositol 3 kinase activity and probably involving regulation of phosphatidylinositol-3,4,5-trisphosphate levels.

    Funded by: NIAID NIH HHS: AI07290, AI20253

    Journal of immunology (Baltimore, Md. : 1950) 2001;167;12;7027-37

  • Basis for signaling specificity difference between Sos and Ras-GRF guanine nucleotide exchange factors.

    Tian X and Feig LA

    Department of Biochemistry, Tufts University School of Medicine, Boston, MA 02111, USA.

    Sos and Ras-GRF are two families of guanine nucleotide exchange factors that activate Ras proteins in cells. Sos proteins are ubiquitously expressed and are activated in response to cell-surface tyrosine kinase stimulation. In contrast, Ras-GRF proteins are expressed primarily in central nervous system neurons and are activated by calcium/calmodulin binding and by phosphorylation. Although both Sos1 and Ras-GRF1 activate the Ras proteins Ha-Ras, N-Ras, and Ki-Ras, only Ras-GRF1 also activates the functionally distinct R-Ras GTPase. In this study, we determined which amino acid sequences in these exchange factors and their target GTPases are responsible for this signaling specificity difference. Analysis of chimeras and individual amino acid exchanges between Sos1 and Ras-GRF1 revealed that the critical amino acids reside within an 11-amino acid segment of their catalytic domains between the second and third structurally conserved regions (amino acids (aa) 828-838 in Sos1 and 1057-1067 in Ras-GRF1) of Ras guanine nucleotide exchange factors. In Sos1, this segment is in helix B, which is known to interact with the switch 2 region of Ha-Ras. Interestingly, a similar analysis of Ha-Ras and R-Ras chimeras did not identify the switch 2 region of Ha-Ras as encoding specificity. Instead, we found a more distal protein segment, helix 3 (aa 91-103 in Ha-Ras and 117-129 in R-Ras), which interacts instead primarily with helix K (aa 1002-1016) of Sos1. These findings suggest that specificity derives from the fact that R-Ras-specific amino acids in the region analogous to Ha-Ras helix 3 prevent a functional interaction with Sos1 indirectly, possibly by preventing an appropriate association of its switch 2 region with helix B of Sos1. Although previous studies have shown that helix B of Sos1 and helix 3 of Ha-Ras are involved in promoting nucleotide exchange on Ras proteins, this study highlights the importance of these regions in establishing signaling specificity.

    The Journal of biological chemistry 2001;276;50;47248-56

  • Galectin-1 binds oncogenic H-Ras to mediate Ras membrane anchorage and cell transformation.

    Paz A, Haklai R, Elad-Sfadia G, Ballan E and Kloog Y

    Department of Neurobiochemistry, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, 69978 Tel-Aviv, Israel.

    Ras genes, frequently mutated in human tumors, promote malignant transformation. Ras transformation requires membrane anchorage, which is promoted by Ras farnesylcysteine carboxymethylester and by a second signal. Previously we showed that the farnesylcysteine mimetic, farnesylthiosalicylic acid (FTS) disrupts Ras membrane anchorage. To understand how this disruption contributes to inhibition of cell transformation we searched for new Ras-interacting proteins and identified galectin-1, a lectin implicated in human tumors, as a selective binding partner of oncogenic H-Ras(12V). The observed size of H-Ras(12V)-galectin-1 complex, which is equal to the sum of the molecular weights of Ras and galectin-1 indicates a direct binding interaction between the two proteins. FTS disrupted H-Ras(12V)-galectin-1 interactions. Overexpression of galectin-1 increased membrane-associated Ras, Ras-GTP, and active ERK resulting in cell transformation, which was blocked by dominant negative Ras. Galectin-1 antisense RNA inhibited transformation by H-Ras(12V) and abolished membrane anchorage of green fluorescent protein (GFP)-H-Ras(12V) but not of GFP-H-Ras wild-type (wt), GFP-K-Ras(12V), or GFP-N-Ras(13V). H-Ras(12V)-galectin-1 interactions establish an essential link between two proteins associated with cell transformation and human malignancies that can be exploited to selectively target oncogenic Ras proteins.

    Oncogene 2001;20;51;7486-93

  • The Ras-Byr2RBD complex: structural basis for Ras effector recognition in yeast.

    Scheffzek K, Grünewald P, Wohlgemuth S, Kabsch W, Tu H, Wigler M, Wittinghofer A and Herrmann C

    Max-Planck-Institut für molekulare Physiologie, Abt. Strukturelle Biologie, Otto-Hahn-Str. 11, 44227, Dortmund, Germany.

    Background: The small GTP binding protein Ras has important roles in cellular growth and differentiation. Mutant Ras is permanently active and contributes to cancer development. In its activated form, Ras interacts with effector proteins, frequently initiating a kinase cascade. In the lower eukaryotic Schizosaccharomyces pombe, Byr2 kinase represents a Ras target that in terms of signal-transduction hierarchy can be considered a homolog of mammalian Raf-kinase. The activation mechanism of protein kinases by Ras is not understood, and there is no detailed structural information about Ras binding domains (RBDs) in nonmammalian organisms.

    Results: The crystal structure of the Ras-Byr2RBD complex at 3 A resolution shows a complex architecture similar to that observed in mammalian homologous systems, with an interprotein beta sheet stabilized by predominantly polar interactions between the interacting components. The C-terminal half of the Ras switch I region contains most of the contact anchors, while on the Byr2 side, a number of residues from topologically distinct regions are involved in complex stabilization. A C-terminal helical segment, which is not present in the known mammalian homologous systems and which is part of the auto-inhibitory region, has an additional binding site outside the switch I region.

    Conclusions: The structure of the Ras-Byr2 complex confirms the Ras binding module as a communication element mediating Ras-effector interactions; the Ras-Byr2 complex is also conserved in a lower eukaryotic system like yeast, which is in contrast to other small GTPase families. The extra helical segment might be involved in kinase activation.

    Structure (London, England : 1993) 2001;9;11;1043-50

  • Downregulation of the Ras-mitogen-activated protein kinase pathway by the EphB2 receptor tyrosine kinase is required for ephrin-induced neurite retraction.

    Elowe S, Holland SJ, Kulkarni S and Pawson T

    Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada.

    Activation of the EphB2 receptor tyrosine kinase by clustered ephrin-B1 induces growth cone collapse and neurite retraction in differentiated NG108 neuronal cells. We have investigated the cytoplasmic signaling events associated with EphB2-induced cytoskeletal reorganization in these neuronal cells. We find that unlike other receptor tyrosine kinases, EphB2 induces a pronounced downregulation of GTP-bound Ras and consequently of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. A similar inhibition of the Ras-MAPK pathway was observed on stimulation of endogenous EphB2 in COS-1 cells. Inactivation of Ras, induced by ephrin B1 stimulation of NG108 neuronal cells, requires EphB2 tyrosine kinase activity and is blocked by a truncated form of p120-Ras GTPase-activating protein (p120-RasGAP), suggesting that EphB2 signals through the SH2 domain protein p120-RasGAP to inhibit the Ras-MAPK pathway. Suppression of Ras activity appears functionally important, since expression of a constitutively active variant of Ras impaired the ability of EphB2 to induce neurite retraction. In addition, EphB2 attenuated the elevation in ERK activation induced by attachment of NG108 cells to fibronectin, indicating that the EphB2 receptor can modulate integrin signaling to the Ras GTPase. These results suggest that a primary function of EphB2, a member of the most populous family of receptor tyrosine kinases, is to inactivate the Ras-MAPK pathway in a fashion that contributes to cytoskeletal reorganization and adhesion responses in neuronal growth cones.

    Molecular and cellular biology 2001;21;21;7429-41

  • Differences in patterns of activation of MAP kinases induced by oncogenic ras-p21 and insulin in oocytes.

    Ranginwale M, Smith S, Flom J, Chie L, Kanovsky M, Chung D, Friedman FK, Robinson RC, Brandt-Rauf PW, Yamaizumi Z, Michl J and Pincus MR

    Department of Pathology and Laboratory Medicine, New York Harbor VA Health Care System, 800 Poly Place, Brooklyn, New York 11209, USA.

    Oncogenic ras (Val 12-containing)-p21 protein induces oocyte maturation by a pathway that is blocked by peptides from effector domains of ras-p21, i.e., residues 35-47 (that block Val 12-p21-activated raf) and 96-110 and 115-126, which do not affect the ability of insulin-activated cellular p21 to induce maturation. Oncogenic p21 binds directly to jun-N-terminal kinase (JNK), which is blocked by the p21 96-110 and 115-126 peptides. This finding predicts that oncogenic p21, but not insulin, induces maturation by early and sustained activation of JNK. We now directly confirm this prediction by showing that oncogenic p21 induces activating phosphorylation of JNK (JNK-P) and of ERK (MAP kinase) (MAPK-P), whose levels correlate with oocyte maturation. p21 peptides 35-47 and 96-110 block formation of JNK-P and MAPK-P, further confirming this correlation and suggesting, unexpectedly, that raf-MEK-MAPK and JNK-jun pathways strongly interact on the oncogenic p21 pathway. In contrast, insulin activates only low levels of JNK-P, and, surprisingly, we find that insulin induces only low levels of MAPK-P, indicating that insulin and activated normal p21 utilize MAP kinase-independent signal transduction pathways.

    Funded by: NCI NIH HHS: CA 42500

    Experimental cell research 2001;269;1;162-9

  • Polymorphisms and colorectal tumor risk.

    Houlston RS and Tomlinson IP

    Section of Cancer Genetics, Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, UK. r.houlston@ICR.AC.UK

    Increasingly, studies of the relationship between common genetic variants and colorectal tumor risk are being proposed. To assess the evidence that any of these confers a risk, a systematic review and meta-analysis of published studies was undertaken.

    Methods: Fifty studies of the effect of common alleles of 13 genes on risk were identified. To clarify the impact of individual polymorphisms on risk, pooled analyses were performed.

    Results: Of the 50 studies identified, significant associations were seen in 16, but only 3 were reported in more than one study. Pooling studies, significant associations were only seen for 3 of the polymorphisms: adenomatosis polyposis coli (APC)-I1307K (odds ratio [OR] = 1.58, 95% confidence interval [CI]: 1.21-2.07); Harvey ras-1 variable number tandem repeat polymorphism (HRAS1-VNTR; OR = 2.50, 95% CI: 1.54-4.05); and methylenetetrahydrofolate reductase (MTHFR)(Val/Val) (OR = 0.76, 95% CI: 0.62-0.92). For tumor protein 53 (TP53), N-acetyl transferase 1 (NAT1), NAT2, glutathione-S transferase Mu (GSTM1), glutathione-S transferase Theta (GSTT1), and glutathione-S transferase Pi (GSTP1) polymorphisms, the best estimates are sufficient to exclude a 1.7-fold increase in risk of colorectal cancer.

    Conclusions: APC-I1307K, HRAS1-VNTR, and MTHFR variants represent the strongest candidates for low penetrance susceptibility alleles identified to date. Although their genotypic risks are modest, their high frequency in the population implies that they may well have considerable impact on colorectal cancer incidence. Determining precise risk estimates associated with other variants and gene-gene and gene-environment interactions will be contingent on further studies with sample sizes larger than typically used to date.

    Gastroenterology 2001;121;2;282-301

  • Prenylated Rab acceptor protein is a receptor for prenylated small GTPases.

    Figueroa C, Taylor J and Vojtek AB

    Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.

    Localization of Ras and Ras-like proteins to the correct subcellular compartment is essential for these proteins to mediate their biological effects. Many members of the Ras superfamily (Ha-Ras, N-Ras, TC21, and RhoA) are prenylated in the cytoplasm and then transit through the endomembrane system on their way to the plasma membrane. The proteins that aid in the trafficking of the small GTPases have not been well characterized. We report here that prenylated Rab acceptor protein (PRA1), which others previously identified as a prenylation-dependent receptor for Rab proteins, also interacts with Ha-Ras, RhoA, TC21, and Rap1a. The interaction of these small GTPases with PRA1 requires their post-translational modification by prenylation. The prenylation-dependent association of PRA1 with multiple GTPases is conserved in evolution; the yeast PRA1 protein associates with both Ha-Ras and RhoA. Earlier studies reported the presence of PRA1 in the Golgi, and we show here that PRA1 co-localizes with Ha-Ras and RhoA in the Golgi compartment. We suggest that PRA1 acts as an escort protein for small GTPases by binding to the hydrophobic isoprenoid moieties of the small GTPases and facilitates their trafficking through the endomembrane system.

    The Journal of biological chemistry 2001;276;30;28219-25

  • Differential actions of p60c-Src and Lck kinases on the Ras regulators p120-GAP and GDP/GTP exchange factor CDC25Mm.

    Giglione C, Gonfloni S and Parmeggiani A

    Groupe de Biophysique-Equipe 2, Ecole Polytechnique, Palaiseau, France. giglione@isv.cnrs-gif.fr

    It is known that the human Ras GTPase activating protein (GAP) p120-GAP can be phosphorylated by different members of the Src kinase family and recently phosphorylation of the GDP/GTP exchange factor (GEF) CDC25Mm/GRF1 by proteins of the Src kinase family has been revealed in vivo [Kiyono, M., Kaziro, Y. & Satoh, T. (2000) J. Biol. Chem. 275, 5441-5446]. As it still remains unclear how these phosphorylations can influence the Ras pathway we have analyzed the ability of p60c-Src and Lck to phosphorylate these two Ras regulators and have compared the activity of the phosphorylated and unphosphorylated forms. Both kinases were found to phosphorylate full-length or truncated forms of GAP and GEF. The use of the catalytic domain of p60c-Src showed that its SH3/SH2 domains are not required for the interaction and the phosphorylation of both regulators. Remarkably, the phosphorylations by the two kinases were accompanied by different functional effects. The phosphorylation of p120-GAP by p60c-Src inhibited its ability to stimulate the Ha-Ras-GTPase activity, whereas phosphorylation by Lck did not display any effect. A different picture became evident with CDC25Mm; phosphorylation by Lck increased its capacity to stimulate the GDP/GTP exchange on Ha-Ras, whereas its phosphorylation by p60c-Src was ineffective. Our results suggest that phosphorylation by p60c-Src and Lck is a selective process that can modulate the activity of p120-GAP and CDC25Mm towards Ras proteins.

    European journal of biochemistry / FEBS 2001;268;11;3275-83

  • Electroconvulsive shock increases the phosphorylation of Pyk2 in the rat hippocampus.

    Jeon SH, Oh SW, Kang UG, Ahn YM, Bae CD, Park JB and Kim YS

    Department of Molecular Cell Biology and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Korea.

    Recently we reported the activation MAPKs, MEK, and Rafs by electroconvulsive shock (ECS) in the rat hippocampus. However, the upstream pathways for the activation of Raf-MEK-MAPK cascade after ECS have not been studied yet. Since the proline-rich tyrosine kinase 2 (Pyk2) and Src were reported to be involved in the activation of the MAPKs in neuronal cells, we examined tyrosine phosphorylation and activation of Pyk2 in the rat hippocampus after ECS. ECS transiently increased the phosphorylation of Pyk2 at multiple tyrosine residues (Tyr-402, Tyr-580, and Tyr-881). The phosphorylations reached the peak at 1 min and returned to basal level by 10 min after ECS. At 1 min after ECS, the binding of Pyk2 to Src and Grb2, and of Grb2 to Ras increased. These results suggested that ECS activates Pyk2, which then transmits the signal to MAPK cascade via Src, Grb2, and Ras in the rat hippocampus.

    Biochemical and biophysical research communications 2001;282;4;1026-30

  • Diacylglycerol kinase zeta regulates Ras activation by a novel mechanism.

    Topham MK and Prescott SM

    The Huntsman Cancer Institute and Department of Internal Medicine, University of Utah, Salt Lake City, Utah 84112, USA.

    Guanine nucleotide exchange factors (GEFs) activate Ras by facilitating its GTP binding. Ras guanyl nucleotide-releasing protein (GRP) was recently identified as a Ras GEF that has a diacylglycerol (DAG)-binding C1 domain. Its exchange factor activity is regulated by local availability of signaling DAG. DAG kinases (DGKs) metabolize DAG by converting it to phosphatidic acid. Because they can attenuate local accumulation of signaling DAG, DGKs may regulate RasGRP activity and, consequently, activation of Ras. DGK zeta, but not other DGKs, completely eliminated Ras activation induced by RasGRP, and DGK activity was required for this mechanism. DGK zeta also coimmunoprecipitated and colocalized with RasGRP, indicating that these proteins associate in a signaling complex. Coimmunoprecipitation of DGK zeta and RasGRP was enhanced in the presence of phorbol esters, which are DAG analogues that cannot be metabolized by DGKs, suggesting that DAG signaling can induce their interaction. Finally, overexpression of kinase-dead DGK zeta in Jurkat cells prolonged Ras activation after ligation of the T cell receptor. Thus, we have identified a novel way to regulate Ras activation: through DGK zeta, which controls local accumulation of DAG that would otherwise activate RasGRP.

    The Journal of cell biology 2001;152;6;1135-43

  • Regulation of a novel human phospholipase C, PLCepsilon, through membrane targeting by Ras.

    Song C, Hu CD, Masago M, Kariyai K, Yamawaki-Kataoka Y, Shibatohge M, Wu D, Satoh T and Kataoka T

    Department of Physiology II, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.

    Phosphoinositide-specific phospholipase C (PI-PLC) plays a pivotal role in regulation of intracellular signal transduction from various receptor molecules. More than 10 members of human PI-PLC isoforms have been identified and classified into three classes beta, gamma, and delta, which are regulated by distinct mechanisms. Here we report identification of a novel class of human PI-PLC, named PLCepsilon, which is characterized by the presence of a Ras-associating domain at its C terminus and a CDC25-like domain at its N terminus. The Ras-associating domain of PLCepsilon specifically binds to the GTP-bound forms of Ha-Ras and Rap1A. The dissociation constant for Ha-Ras is estimated to be approximately 40 nm, comparable with those of other Ras effectors. Co-expression of an activated Ha-Ras mutant with PLCepsilon induces its translocation from the cytosol to the plasma membrane. Upon stimulation with epidermal growth factor, similar translocation of ectopically expressed PLCepsilon is observed, which is inhibited by co-expression of dominant-negative Ha-Ras. Furthermore, using a liposome-based reconstitution assay, it is shown that the phosphatidylinositol 4,5-bisphosphate-hydrolyzing activity of PLCepsilon is stimulated in vitro by Ha-Ras in a GTP-dependent manner. These results indicate that Ras directly regulates phosphoinositide breakdown through membrane targeting of PLCepsilon.

    The Journal of biological chemistry 2001;276;4;2752-7

  • Human breast cancer cells generated by oncogenic transformation of primary mammary epithelial cells.

    Elenbaas B, Spirio L, Koerner F, Fleming MD, Zimonjic DB, Donaher JL, Popescu NC, Hahn WC and Weinberg RA

    Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA.

    A number of genetic mutations have been identified in human breast cancers, yet the specific combinations of mutations required in concert to form breast carcinoma cells remain unknown. One approach to identifying the genetic and biochemical alterations required for this process involves the transformation of primary human mammary epithelial cells (HMECs) to carcinoma cells through the introduction of specific genes. Here we show that introduction of three genes encoding the SV40 large-T antigen, the telomerase catalytic subunit, and an H-Ras oncoprotein into primary HMECs results in cells that form tumors when transplanted subcutaneously or into the mammary glands of immunocompromised mice. The tumorigenicity of these transformed cells was dependent on the level of ras oncogene expression. Interestingly, transformation of HMECs but not two other human cell types was associated with amplifications of the c-myc oncogene, which occurred during the in vitro growth of the cells. Tumors derived from the transformed HMECs were poorly differentiated carcinomas that infiltrated through adjacent tissue. When these cells were injected subcutaneously, tumors formed in only half of the injections and with an average latency of 7.5 weeks. Mixing the epithelial tumor cells with Matrigel or primary human mammary fibroblasts substantially increased the efficiency of tumor formation and decreased the latency of tumor formation, demonstrating a significant influence of the stromal microenvironment on tumorigenicity. Thus, these observations establish an experimental system for elucidating both the genetic and cell biological requirements for the development of breast cancer.

    Funded by: NCI NIH HHS: 5 P01 CA80111-02

    Genes & development 2001;15;1;50-65

  • The Tg.AC (v-Ha-ras) transgenic mouse: nature of the model.

    Tennant RW, Stasiewicz S, Eastin WC, Mennear JH and Spalding JW

    National Institute of Environmental Health Sciences, Laboratory of Environmental Carcinogenesis and Mutagenesis, Research Triangle Park, North Carolina 27709, USA. tennant@niehs.nih.gov

    The Tg.AC (v-Ha-ras) transgenic mouse model provides a reporter phenotype of skin papillomas in response to either genotoxic or nongenotoxic carcinogens. In common with the conventional bioassay, the Tg.AC model responds to known human carcinogens and does not respond to noncarcinogens. It also does not respond to most chemicals that are positive in conventional bioassays principally at sites of high spontaneous tumor incidence. The mechanism of response of the Tg.AC model is related to the structure and genomic position of the transgene and the induction of transgene expression through specific mediated interactions between the chemicals and target cells in the skin.

    Toxicologic pathology 2001;29 Suppl;51-9

  • Nerve growth factor signaling, neuroprotection, and neural repair.

    Sofroniew MV, Howe CL and Mobley WC

    Department of Neurobiology and Brain Research Institute, University of California Los Angeles, Los Angeles, California 90095-1763, USA. sofroniew@mednet.ucla.edu

    Nerve growth factor (NGF) was discovered 50 years ago as a molecule that promoted the survival and differentiation of sensory and sympathetic neurons. Its roles in neural development have been characterized extensively, but recent findings point to an unexpected diversity of NGF actions and indicate that developmental effects are only one aspect of the biology of NGF. This article considers expanded roles for NGF that are associated with the dynamically regulated production of NGF and its receptors that begins in development, extends throughout adult life and aging, and involves a surprising variety of neurons, glia, and nonneural cells. Particular attention is given to a growing body of evidence that suggests that among other roles, endogenous NGF signaling subserves neuroprotective and repair functions. The analysis points to many interesting unanswered questions and to the potential for continuing research on NGF to substantially enhance our understanding of the mechanisms and treatment of neurological disorders.

    Funded by: NINDS NIH HHS: NS24054

    Annual review of neuroscience 2001;24;1217-81

  • Tg.AC genetically altered mouse: assay working group overview of available data.

    Eastin WC, Mennear JH, Tennant RW, Stoll RE, Branstetter DG, Bucher JR, McCullough B, Binder RL, Spalding JW and Mahler JF

    National Institute of Environmental Health Sciences, National Toxicology Program, Research Triangle Park, North Carolina 27713-2233, USA. eastin@niehs.nih.gov

    In a Government/Industry/Academic partnership to evaluate alternative approaches to carcinogenicity testing, 21 pharmaceutical agents representing a variety of chemical and pharmacological classes and possessing known human and or rodent carcinogenic potential were selected for study in several rodent models. The studies from this partnership project, coordinated by the International Life Sciences Institute, provide additional data to better understand the models' limitations and sensitivity in identifying carcinogens. The results of these alternative model studies were reviewed by members of Assay Working Groups (AWG) composed of scientists from government and industry with expertise in toxicology, genetics, statistics, and pathology. The Tg.AC genetically manipulated mouse was one of the models selected for this project based on previous studies indicating that Tg.AC mice seem to respond to topical application of either mutagenic or nonmutagenic carcinogens with papilloma formation at the site of application. This communication describes the results and AWG interpretations of studies conducted on 14 chemicals administered by the topical and oral (gavage and/or diet) routes to Tg.AC genetically manipulated mice. Cyclosporin A, an immunosuppresant human carcinogen, ethinyl estradiol and diethylstilbestrol (human hormone carcinogens) and clofibrate, an hepatocarcinogenic peroxisome proliferator in rodents, were considered clearly positive in the topical studies. In the oral studies, ethinyl estradiol and diethylstilbestrol were negative, cyclosporin was considered equivocal, and results were not available for the clofibrate study. Of the 3 genotoxic human carcinogens (phenacetin, melphalan, and cyclophosphamide), phenacetin was negative by both the topical and oral routes. Melphalan and cyclophosphamide are, respectively, direct and indirect DNA alkylating agents and topical administration of both caused equivocal responses. With the exception of clofibrate, Tg.AC mice did not exhibit tumor responses to the rodent carcinogens that were putative human noncarcinogens, (di(2-ethylhexyl) phthalate, methapyraline HCl, phenobarbital Na, reserpine, sulfamethoxazole or WY-14643, or the nongenotoxic, noncarcinogen, sulfisoxazole) regardless of route of administration. Based on the observed responses in these studies, it was concluded by the AWG that the Tg.AC model was not overly sensitive and possesses utility as an adjunct to the battery of toxicity studies used to establish human carcinogenic risk.

    Toxicologic pathology 2001;29 Suppl;60-80

  • Crystal structure and functional analysis of Ras binding to its effector phosphoinositide 3-kinase gamma.

    Pacold ME, Suire S, Perisic O, Lara-Gonzalez S, Davis CT, Walker EH, Hawkins PT, Stephens L, Eccleston JF and Williams RL

    MRC Laboratory of Molecular Biology Hills Road CB2 2QH, Cambridge, United Kingdom.

    Ras activation of phosphoinositide 3-kinase (PI3K) is important for survival of transformed cells. We find that PI3Kgamma is strongly and directly activated by H-Ras G12V in vivo or by GTPgammaS-loaded H-Ras in vitro. We have determined a crystal structure of a PI3Kgamma/Ras.GMPPNP complex. A critical loop in the Ras binding domain positions Ras so that it uses its switch I and switch II regions to bind PI3Kgamma. Mutagenesis shows that interactions with both regions are essential for binding PI3Kgamma. Ras also forms a direct contact with the PI3Kgamma catalytic domain. These unique Ras/PI3Kgamma interactions are likely to be shared by PI3Kalpha. The complex with Ras shows a change in the PI3K conformation that may represent an allosteric component of Ras activation.

    Funded by: Medical Research Council: MC_U105184308

    Cell 2000;103;6;931-43

  • Ras uses the novel tumor suppressor RASSF1 as an effector to mediate apoptosis.

    Vos MD, Ellis CA, Bell A, Birrer MJ and Clark GJ

    Department of Cell and Cancer Biology, NCI, National Institutes of Health, Rockville, Maryland 20850-3300, USA.

    Although activated Ras proteins are usually associated with driving growth and transformation, they may also induce senescence, apoptosis, and terminal differentiation. The subversion of these anti-neoplastic effects during Ras-dependent tumor development may be as important as the acquisition of the pro-neoplastic effects. None of the currently identified potential Ras effector proteins can satisfactorily explain the apoptotic action of Ras. Consequently, we have sought to identify novel Ras effectors that may be responsible for apoptosis induction. By examining the EST data base, we identified a potential Ras association domain in the tumor suppressor RASSF1. We now show that RASSF1 binds Ras in a GTP-dependent manner, both in vivo and directly in vitro. Moreover, activated Ras enhances and dominant negative Ras inhibits the cell death induced by transient transfection of RASSF1 into 293-T cells. This cell death appears to be apoptotic in nature, as RASSF1-transfected 293-T cells exhibit membrane blebbing and can be rescued by the addition of a caspase inhibitor. Thus, the RASSF1 tumor suppressor may serve as a novel Ras effector that mediates the apoptotic effects of oncogenic Ras.

    The Journal of biological chemistry 2000;275;46;35669-72

  • Relationships of TP53 codon 72 and HRAS1 polymorphisms with lung cancer risk in an ethnically diverse population.

    Pierce LM, Sivaraman L, Chang W, Lum A, Donlon T, Seifried A, Wilkens LR, Lau AF and Le Marchand L

    Etiology Program, Cancer Research Center of Hawaii, University of Hawaii, Honolulu 96813, USA.

    Tobacco smoking is a strong cause of lung cancer. However, because only a small proportion of smokers develop the disease, other factors, including genetic susceptibility, may be important in determining lung cancer risk. Polymorphisms in the TP53 tumor suppressor gene and HRAS1 proto-oncogene have been associated in some studies with this cancer; we sought to replicate these associations in an ethnically diverse population in Hawaii. We conducted a population-based case-control study among 334 incident lung cancer cases and 446 controls of Caucasian, Japanese, or Native Hawaiian origin. In-person interviews collected detailed information on lifestyle risk factors. DNA was extracted from peripheral blood leukocytes, and genotyping was performed using a PCR-based assay for the TP53 codon 72 polymorphism and Southern blot analysis and PCR for allelic polymorphisms in the HRAS1 minisatellite. Logistic regression analyses were used to compute odds ratios (ORs) and 95% confidence intervals (CIs) adjusting for smoking and other risk factors. The presence of two rare HRAS1 alleles was associated with a 2.2-fold (95% CI, 1.0-5.0) increased lung cancer risk for all ethnic groups combined. The association was present in Native Hawaiians (OR, 5.2; 95% CI, 1.1-24.4) and was suggested for Japanese (OR, 2.8; 95% CI, 0.6-12.5); no association was observed in Caucasians (OR, 0.8; 95% CI, 0.2-3.6). This association was also observed for each lung cancer cell type. The presence of only one rare allele did not increase risk for any ethnic group or cell type. No significant association was found between the TP53 codon 72 polymorphism and lung cancer [OR, 1.4 (95% CI, 0.8-2.4) for the Pro/Pro genotype compared with the Arg/Arg genotype]. This study suggests that the presence of two rare HRAS1 alleles confers an increased lung cancer risk in Native Hawaiians and Japanese but possibly not in Caucasians. The amino acid replacement of arginine by proline at codon 72 of TP53 appears not to be important in determining lung cancer risk in this population.

    Funded by: NCI NIH HHS: N01-PC-67001, R01-CA-55874

    Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 2000;9;11;1199-204

  • Stimulation of Ras guanine nucleotide exchange activity of Ras-GRF1/CDC25(Mm) upon tyrosine phosphorylation by the Cdc42-regulated kinase ACK1.

    Kiyono M, Kato J, Kataoka T, Kaziro Y and Satoh T

    Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan.

    Ras-GRF1 is a brain-specific guanine nucleotide exchange factor (GEF) for Ras, whose activity is regulated in response to Ca(2+) influx and G protein-coupled receptor signals. In addition, Ras-GRF1 acts as a GEF for Rac when tyrosine-phosphorylated following G protein-coupled receptor stimulation. However, the mechanisms underlying the regulation of Ras-GRF1 functions remain incompletely understood. We show here that activated ACK1, a nonreceptor tyrosine kinase that belongs to the focal adhesion kinase family, causes tyrosine phosphorylation of Ras-GRF1. On the other hand, kinase-deficient ACK1 exerted no effect. GEF activity of Ras-GRF1 toward Ha-Ras, as defined by in vitro GDP binding and release assays, was augmented after tyrosine phosphorylation by ACK1. In contrast, GEF activity toward Rac1 remained latent, implying that ACK1 does not represent a tyrosine kinase that acts downstream of G protein-coupled receptors. Consistent with enhanced Ras-GEF activity, accumulation of the GTP-bound form of Ras within the cell was shown through the use of Ras-binding domain pull-down assays. Furthermore, Ras-dependent activation of ERK2 by Ras-GRF1 was enhanced following co-expression of activated ACK1. These results implicate ACK1 as an upstream modulator of Ras-GRF1 and suggest a signaling cascade consisting of Cdc42, ACK1, Ras-GRF1, and Ras in neuronal cells.

    The Journal of biological chemistry 2000;275;38;29788-93

  • The junctional multidomain protein AF-6 is a binding partner of the Rap1A GTPase and associates with the actin cytoskeletal regulator profilin.

    Boettner B, Govek EE, Cross J and Van Aelst L

    Cold Spring Harbor Laboratories, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA.

    The AF-6 protein is a multidomain protein that contains two potential Ras-binding domains within its N terminus. Because of this feature, AF-6 has been isolated in both two-hybrid and biochemical approaches and is postulated to be a potential Ras-effector protein. Herein, we show that it is specifically the first Ras-binding domain of AF-6 that mediates this interaction and that the Ras-related Rap1A protein can associate with this motif even more efficiently than the oncogenic Ha-, K-, and N-Ras GTPases. We further demonstrate that both Ras and Rap1 interact with full-length AF-6 in vivo in mammalian cells and that a fraction of Rap1 colocalizes with AF-6 at the membrane. Dominant active Rap1A, in contrast to Ras, when introduced into epithelial MDCK and MCF-7 cells, does not perturb AF-6-specific residency in cell-cell adhesion complexes. In a pursuit to gain further understanding of the role of AF-6 in junctions, we identified profilin as an AF-6-binding protein. Profilin activates monomeric actin units for subsequent polymerization steps at barbed ends of actin filaments and has been shown to participate in cortical actin assembly. To our knowledge, AF-6 is the only integral component in cell-cell junctions discovered thus far that interacts with profilin and thus could modulate actin modeling proximal to adhesion complexes.

    Proceedings of the National Academy of Sciences of the United States of America 2000;97;16;9064-9

  • Evidence for a telomere-independent "clock" limiting RAS oncogene-driven proliferation of human thyroid epithelial cells.

    Jones CJ, Kipling D, Morris M, Hepburn P, Skinner J, Bounacer A, Wyllie FS, Ivan M, Bartek J, Wynford-Thomas D and Bond JA

    Cancer Research Campaign Laboratories, Department of Pathology, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, United Kingdom.

    An initiating role for RAS oncogene mutation in several epithelial cancers is supported by its high incidence in early-stage tumors and its ability to induce proliferation in the corresponding normal cells in vitro. Using retroviral transduction of thyroid epithelial cells as a model we ask here: (i) how mutant RAS can induce long-term proliferation in an epithelial cell in contrast to the premature senescence observed in fibroblasts; and (ii) what is the "clock" which eventually triggers spontaneous growth arrest even in epithelial clones generated by mutant RAS. The early response to RAS activation in thyroid epithelial cells showed two features not seen in fibroblasts: (i) a marked decrease in expression of the cyclin-dependent kinase inhibitor (CDKI) p27(kip1) and (ii) the absence of any induction of p21(waf1). When proliferation eventually ceased (after up to 20 population doublings) this occurred despite undiminished expression of mutant RAS and was tightly correlated with a return to the initial high level of p27(kip1) expression, together with the de novo appearance of p16(ink4a). Importantly, neither the CDKI changes nor the proliferative life span of RAS-induced epithelial clones was altered by induction of telomerase activity through forced expression of the catalytic subunit, hTERT, at levels sufficient to immortalize human fibroblasts. These data provide a basis for cell-type differences in sensitivity to RAS-induced proliferation which may explain the corresponding tumor-type specificity of RAS mutation. They also show for the first time in a primary human cell model that a telomere-independent mechanism can limit not only physiological but also oncogene-driven proliferation, pointing therefore to a tumour suppressor mechanism additional, or alternative, to the telomere clock.

    Molecular and cellular biology 2000;20;15;5690-9

  • Involvement of Ras and Ral in chemotactic migration of skeletal myoblasts.

    Suzuki J, Yamazaki Y, Li G, Kaziro Y, Koide H and Guang L

    Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan.

    In skeletal myoblasts, Ras has been considered to be a strong inhibitor of myogenesis. Here, we demonstrate that Ras is involved also in the chemotactic response of skeletal myoblasts. Expression of a dominant-negative mutant of Ras inhibited chemotaxis of C2C12 myoblasts in response to basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and insulin-like growth factor 1 (IGF-1), key regulators of limb muscle development and skeletal muscle regeneration. A dominant-negative Ral also decreased chemotactic migration by these growth factors, while inhibitors for phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase (MEK) showed no effect. Activation of the Ras-Ral pathway by expression of an activated mutant of either Ras, the guanine-nucleotide dissociation stimulator for Ral, or Ral resulted in increased motility of myoblasts. The ability of Ral to stimulate motility was reduced by introduction of a mutation which prevents binding to Ral-binding protein 1 or phospholipase D. These results suggest that the Ras-Ral pathway is essential for the migration of myoblasts. Furthermore, we found that Ras and Ral are activated in C2C12 cells by bFGF, HGF and IGF-1 and that the Ral activation is regulated by the Ras- and the intracellular Ca(2+)-mediated pathways. Taken together, our data indicate that Ras and Ral regulate the chemotactic migration of skeletal muscle progenitors.

    Molecular and cellular biology 2000;20;13;4658-65

  • Mapping of atypical protein kinase C within the nerve growth factor signaling cascade: relationship to differentiation and survival of PC12 cells.

    Wooten MW, Seibenhener ML, Neidigh KB and Vandenplas ML

    Department of Biological Sciences, Program in Cell and Molecular Biosciences, Auburn University, AL 36849, USA. mwwooten@ag.auburn.edu

    The pathway by which atypical protein kinase C (aPKC) contributes to nerve growth factor (NGF) signaling is poorly understood. We previously reported that in PC12 cells NGF-induced activation of mitogen-activated protein kinase (MAPK) occurs independently of classical and nonclassical PKC isoforms, whereas aPKC isoforms were shown to be required for NGF-induced differentiation. NGF-induced activation of PKC-iota was observed to be dependent on phosphatidylinositol 3-kinase (PI3K) and led to coassociation of PKC-iota with Ras and Src. Expression of dominant negative mutants of either Src (DN2) or Ras (Asn-17) impaired activation of PKC-iota by NGF. At the level of Raf-1, neither PKC-iota nor PI3 kinase was required for activation; however, PKC-iota could weakly activate MEK. Inhibitors of PKC-iota activity and PI3K had no effect on NGF-induced MAPK or p38 activation but reduced NGF-stimulated c-Jun N-terminal kinase activity. Src, PI3K, and PKC-iota were likewise required for NGF-induced NF-kappaB activation and cell survival, whereas Ras was not required for either survival or NF-kappaB activation but was required for differentiation. IKK existed as a complex with PKC-iota, Src and IkappaB. Consistent with a role for Src in regulating NF-kappaB activation, an absence of Src activity impaired recruitment of PKC-iota into an IKK complex and markedly impaired NGF-induced translocation of p65/NF-kappaB to the nucleus. These findings reveal that in PC12 cells, aPKCs comprise a molecular switch to regulate differentiation and survival responses coupled downstream to NF-kappaB. On the basis of these findings, Src emerges as a critical upstream regulator of both PKC-iota and the NF-kappaB pathway.

    Funded by: NINDS NIH HHS: R01 NS033661, R02-NS33661

    Molecular and cellular biology 2000;20;13;4494-504

  • Proteomic analysis of NMDA receptor-adhesion protein signaling complexes.

    Husi H, Ward MA, Choudhary JS, Blackstock WP and Grant SG

    Centre for Genome Research, Centre for Neuroscience, University of Edinburgh, West Mains Road, Edinburgh EH9 3JQ, UK.

    N-methyl-d-aspartate receptors (NMDAR) mediate long-lasting changes in synapse strength via downstream signaling pathways. We report proteomic characterization with mass spectrometry and immunoblotting of NMDAR multiprotein complexes (NRC) isolated from mouse brain. The NRC comprised 77 proteins organized into receptor, adaptor, signaling, cytoskeletal and novel proteins, of which 30 are implicated from binding studies and another 19 participate in NMDAR signaling. NMDAR and metabotropic glutamate receptor subtypes were linked to cadherins and L1 cell-adhesion molecules in complexes lacking AMPA receptors. These neurotransmitter-adhesion receptor complexes were bound to kinases, phosphatases, GTPase-activating proteins and Ras with effectors including MAPK pathway components. Several proteins were encoded by activity-dependent genes. Genetic or pharmacological interference with 15 NRC proteins impairs learning and with 22 proteins alters synaptic plasticity in rodents. Mutations in three human genes (NF1, Rsk-2, L1) are associated with learning impairments, indicating the NRC also participates in human cognition.

    Nature neuroscience 2000;3;7;661-9

  • Increased oxidative stress with gene alteration in urinary bladder urothelium after the Chernobyl accident.

    Romanenko A, Morimura K, Wanibuchi H, Salim EI, Kinoshita A, Kaneko M, Vozianov A and Fukushima S

    Department of Pathology, Institute of Urology and Nephrology, Academy of Medical Science of Ukraine, Kiev, Ukraine.

    We have previously shown that bladder urothelium of people living in the cesium-137 ((137)Cs)-contaminated areas of Ukraine demonstrates accumulation of stable p53 and p53 mutational inactivation, preferentially through G:C to A:T transition mutations at CpG dinucleotides, with a codon 245 hot spot. In the present study, we analyzed immuno-histochemically the relationship between oxidative stress markers and over-expression of p53 and H-ras in urinary bladder urothelium from 42 men with benign prostatic hyperplasia. Bladder mapping biopsies were obtained from 15 patients from a highly radiocontaminated area (group I), 14 patients from the less contaminated city of Kiev (group II) and 13 patients as a control group from "clean" (without radiocontamination) areas of Ukraine (group III). Irradiation cystitis with multiple foci of severe dysplasia and carcinoma in situ were observed in 15 of 15 (100%, group I) and 9 of 14 (64%, group II) cases, with 4 small transitional-cell carcinomas incidentally detected in groups I and II. Markedly elevated levels of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2) and 8-hydroxy-2;-deoxyguanosine (8-OHdG) were noted in these bladder urothelial lesions from groups I and II, accompanied by strong over-expression of p53 and less H-ras expression. These findings support the hypothesis that iNOS, COX-2 and 8-OHdG in bladder urothelium are induced by long-term exposure to low-dose radiation with a close relationship to p53 over-expression that could predispose to bladder carcinogenesis.

    International journal of cancer 2000;86;6;790-8

  • Synergistic activation of NFAT by HIV-1 nef and the Ras/MAPK pathway.

    Manninen A, Renkema GH and Saksela K

    Institute of Medical Technology, University of Tampere, P. O. Box 607, FIN-33101 and the Department of Clinical Chemistry, Tampere University Hospital, P. O. Box 2000, FIN-33521, Tampere, Finland.

    Nef is a lentiviral protein involved in pathogenesis of AIDS, but its molecular mechanisms of action remain incompletely understood. Here we report a novel effect of Nef on lymphocyte signaling, which is mediated via a T cell receptor (TCR)-independent contribution of Nef to induction of nuclear factor of activated T cells (NFAT), a transcription factor that plays a central role in coordinating T cell activation. Expression of Nef did not significantly alter the basal level of NFAT activity in Jurkat cells nor the increased activity following T cell receptor stimulation by anti-CD3 or anti-CD3 + anti-CD28. We also mimicked NFAT induction by TCR triggering by simultaneous activation of the Ras and calcium signaling pathways with phorbol 12-myristate 13-acetate and ionomycin, respectively. Strikingly, whereas activation of either of these pathways individually did not induce NFAT activity in control cells, in Nef-expressing cells phorbol 12-myristate 13-acetate treatment alone resulted in a 100-fold increase in NFAT-directed gene expression. Experiments with different dominant negative mutant signaling proteins, inhibitory chemicals, and Lck-deficient Jurkat cells revealed that this effect was mediated via activation of calcineurin by Nef-induced changes in calcium metabolism, but was independent of TCR-associated signaling events. This ability of Nef to substitute for triggering of the calcium pathway in induction of NFAT could promote activation of human immunodeficiency virus (HIV)-infected T cells in response to stimuli mediated via TCR or other cell surface receptors under conditions when activation of Ras rather than calcium signaling would otherwise predominate.

    The Journal of biological chemistry 2000;275;22;16513-7

  • The leucine-rich repeat protein SUR-8 enhances MAP kinase activation and forms a complex with Ras and Raf.

    Li W, Han M and Guan KL

    Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109 USA.

    Caenorhabditis elegans sur-8 encodes a positive regulator of Ras signaling. We investigated the mechanism by which the human Sur-8 homolog can positively regulate Ras-MAP kinase signaling in mammalian cells. Sur-8 expression enhances Ras- or EGF-induced Raf and ERK activation but has no effect on ERK activation induced by active Raf or MEK. Furthermore, Sur-8 expression does not increase AKT or JNK activation. Sur-8 interacts with Ras and Raf and is able to form a ternary complex with the two proteins. Thus, Sur-8 may function as a scaffold that enhances Ras-MAP kinase signal transduction by facilitating the interaction between Ras and Raf.

    Genes & development 2000;14;8;895-900

  • The Ras branch of small GTPases: Ras family members don't fall far from the tree.

    Reuther GW and Der CJ

    Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, North Carolina 27599-7295, USA. greuther@med.unc.edu

    The Ras branch of the Ras superfamily consists of small GTPases most closely related to Ras and include the R-Ras, Rap, Ral, Rheb, Rin and Rit proteins. Although our understanding of Ras signaling and biology is now considerable, recent observations suggest that Ras function is more complex than previously believed. First, the three Ras proteins may not be functionally identical. Second, Ras function involves functional cross-talk with their close relatives.

    Funded by: NCI NIH HHS: CA42978, CA55008, CA63071

    Current opinion in cell biology 2000;12;2;157-65

  • Lack of association of rare alleles in the HRAS variable number of tandem repeats (VNTR) region with adult glioma.

    Chen P, Wiencke JK, Conway K, Edmiston SN, Miike R and Wrensch M

    Department of Epidemiology and Biostatistics, School of Medicine, University of California, San Francisco 94143-0560, USA.

    HRAS rare alleles have been associated with the increased susceptibility to a variety of cancers. In the present study we examined the hypothesis that HRAS rare alleles are a risk factor for adult glioma in a population-based case-control study of adult glioma in six San Francisco Bay Area counties. We compared the prevalence of rare alleles in the variable number of tandem repeats region of HRAS in the germline DNA from 73 white adults who had gliomas with that of 65 controls. Overall, the prevalence of rare alleles in cases was not different from the prevalence of those in controls according to two definitions of rare alleles. We found that 25 of 73 (34%) of cases versus 25 of 65 (38%) of controls had at least one allele that was not 30, 46, 69, or 87 repeats; 4 of 73 (5%) of cases versus 6 of 65 (9%) of controls carried one or more alleles with 33, 39, 42, 53, 59, 63, 68, 105, or 114 repeats. The proportion of rare alleles was somewhat higher among subjects with anaplastic astrocytoma. Among women, cases were less likely than controls to have HRAS rare alleles, whereas among men, cases were slightly more likely to have HRAS rare alleles, but none of these results approach statistical significance. Our data do not suggest an excess of HRAS rare alleles among adult glioma cases.

    Funded by: NCI NIH HHS: CA52689, CA57220; NIEHS NIH HHS: ES04705

    Neuro-oncology 2000;2;2;120-4

  • Ras-induced cellular events (review).

    Ayllón V and Rebollo A

    Department of Immunology and Oncology, UAM, Madrid, Spain.

    Ras is a crucial regulator of cell growth in eukaryotic cells. Activated Ras can stimulate signal transduction cascades, leading to cell proliferation, differentiation or apoptosis. It is also one of the most commonly mutated genes in both solid tumours and haematologic neoplasias. In leukaemia and tumours, aberrant Ras signalling can be induced directly by Ras mutation or indirectly by altering genes that associate with Ras or its signalling pathways. A requisite for Ras function is localization to the plasma membrane, which is induced by the post-translational modification farnesylation. Molecules that interfere with this Ras modification have been used as antitumour agents. Ras is emerging as a dual regulator of cell functions, playing either positive or negative roles in the control of proliferation or apoptosis. The diversity of Ras-mediated effects may be related in part to the differential involvement of Ras homologues in distinct cellular processes or to the expanding array of Ras effectors.

    Molecular membrane biology 2000;17;2;65-73

  • Survival function of ERK1/2 as IL-3-activated, staurosporine-resistant Bcl2 kinases.

    Deng X, Ruvolo P, Carr B and May WS

    University of Florida Shands Cancer Center, Gainesville, FL 32610-0232, USA.

    Bcl2 phosphorylation at Ser-70 may be required for the full and potent suppression of apoptosis in IL-3-dependent myeloid cells and can result from agonist activation of mitochondrial protein kinase C (PKC). Paradoxically, expression of exogenous Bcl2 can protect parental cells from apoptosis induced by the potent PKC inhibitor, staurosporine (stauro). High concentrations of stauro of up to 1 microM only partially inhibit IL-3-stimulated Bcl2 phosphorylation but completely block PKC-mediated Bcl2 phosphorylation in vitro. These data indicate a role for a stauro-resistant Bcl2 kinase (SRK). We show that aurintricarboxylic acid (ATA), a nonpeptide activator of cellular MEK/mitogen-activated protein kinase (MAPK) kinase, can induce Ser-70 phosphorylation of Bcl2 and support survival of cells expressing wild-type but not the phosphorylation-incompetent S70A mutant Bcl2. A role for a MEK/MAPK as a responsible SRK was implicated because the highly specific MEK/MAPK inhibitor, PD98059, also can only partially inhibit IL-3-induced Bcl2 phosphorylation, whereas the combination of PD98059 and stauro completely blocks phosphorylation and synergistically enhances apoptosis. p44MAPK/extracellular signal-regulated kinase 1 (ERK1) and p42 MAPK/ERK2 are activated by IL-3, colocalize with mitochondrial Bcl2, and can directly phosphorylate Bcl2 on Ser-70 in a stauro-resistant manner both in vitro and in vivo. These findings suggest a role for the ERK1/2 kinases as SRKs. Thus, the SRKs can serve to functionally link the IL-3-stimulated proliferative and survival signaling pathways and, in a novel capacity, may explain how Bcl2 can suppress stauro-induced apoptosis. In addition, although the mechanism of regulation of Bcl2 by phosphorylation is not yet clear, our results indicate that phosphorylation may functionally stabilize the Bcl2-Bax heterodimerization.

    Funded by: NCI NIH HHS: CA44649, CA47993, R01 CA044649

    Proceedings of the National Academy of Sciences of the United States of America 2000;97;4;1578-83

  • H RAS mutations in haematologically normal individuals.

    Taylor C, Larghero J, Thomas C, Warren N, Carter G, Hughes D, Culligan D, Al-Sabah A, Whittaker JA, Chomienne C and Padua RA

    Hematology Department, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK.

    Introduction: Point mutations in N, K and H RAS have been found in adverse haematological malignancies. The background frequency of RAS mutations in the normal population has yet to be determined. Here we report the results of a screen for RAS mutations from normal individuals.

    DNA from peripheral blood or bone marrow from 115 haematologically normal individuals was screened for point mutations in N, K and H RAS, at amino acid positions 12, 13 and 61. The screening was done using polymerase chain reaction and oligonucleotide hybridisation and candidate mutations were subsequently confirmed by cloning and sequencing.

    Point mutations were identified in DNA from two of the 115 individuals. Both mutations resulted in an amino acid substitution at position 12 in H RAS. Both individuals with detectable H RAS mutations remain haematologically normal.

    The hematology journal : the official journal of the European Haematology Association 2000;1;6;399-402

  • The pre-hydrolysis state of p21(ras) in complex with GTP: new insights into the role of water molecules in the GTP hydrolysis reaction of ras-like proteins.

    Scheidig AJ, Burmester C and Goody RS

    Abteilung für Physikalische Biochemie, Max-Planck Institute for Molecular Physiology, Dortmund, 44227, Germany. scheidig@mpi-dortmund.mpg.de

    Background: In numerous biological events the hydrolysis of guanine triphosphate (GTP) is a trigger to switch from the active to the inactive protein form. In spite of the availability of several high-resolution crystal structures, the details of the mechanism of nucleotide hydrolysis by GTPases are still unclear. This is partly because the structures of the proteins in their active states had to be determined in the presence of non-hydrolyzable GTP analogues (e.g. GppNHp). Knowledge of the structure of the true Michaelis complex might provide additional insights into the intrinsic protein hydrolysis mechanism of GTP and related nucleotides.

    Results: The structure of the complex formed between p21(ras) and GTP has been determined by X-ray diffraction at 1.6 A using a combination of photolysis of an inactive GTP precursor (caged GTP) and rapid freezing (100K). The structure of this complex differs from that of p21(ras)-GppNHp (determined at 277K) with respect to the degree of order and conformation of the catalytic loop (loop 4 of the switch II region) and the positioning of water molecules around the gamma-phosphate group. The changes in the arrangement of water molecules were induced by the cryo-temperature technique.

    Conclusions: The results shed light on the function of Gln61 in the intrinsic GTP hydrolysis reaction. Furthermore, the possibility of a proton shuffling mechanism between two attacking water molecules and an oxygen of the gamma-phosphate group can be proposed for the basal GTPase mechanism, but arguments are presented that render this protonation mechanism unlikely for the GTPase activating protein (GAP)-activated GTPase.

    Structure (London, England : 1993) 1999;7;11;1311-24

  • Biochemical characterization of the Ras-related GTPases Rit and Rin.

    Shao H, Kadono-Okuda K, Finlin BS and Andres DA

    Department of Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 40536-0084, USA.

    We report the biochemical characterization of Rit and Rin, two members of the Ras superfamily identified by expression cloning. Recombinant Rit and Rin bind GTP and exhibit intrinsic GTPase activity. Conversion of Gln to Leu at position 79 (for Rit) or 78 (for Rin) (equivalent to position 61 in Ras) resulted in a complete loss of GTPase activity. Surprisingly, significant differences were found when the guanine nucleotide dissociation constants of Rit and Rin were compared with the majority of Ras-related GTPases. Both proteins display higher k(off) values for GTP than GDP in the presence of 10 mM Mg(2+). These GTP dissociation rates are 5- to 10-fold faster than most Ras-like GTPases. Despite these unique biochemical properties, our data support the notion that both Rit and Rin function as nucleotide-dependent molecular switches. To begin to address whether these proteins act as regulators of distinct signaling pathways, we examined their interaction with a series of known Ras-binding proteins by yeast two-hybrid analysis. Although Rit, Rin, and Ras have highly related effector domain sequences, Rit and Rin were found to interact with the known Ras binding proteins RalGDS, Rlf, and AF-6/Canoe but not with the Raf kinases, RIN1, or the p110 subunit of phosphatidylinositol 3-kinase. These interactions were GTP and effector domain dependent and suggest that RalGDS, Rlf, and AF-6 are Rit and Rin effectors. Their biochemical properties and interaction with a subset of known Ras effector proteins suggest that Rit and Rin may play important roles in the regulation of signaling pathways and cellular processes distinct from those controlled by Ras.

    Funded by: NEI NIH HHS: EY11231

    Archives of biochemistry and biophysics 1999;371;2;207-19

  • CrkL mediates Ras-dependent activation of the Raf/ERK pathway through the guanine nucleotide exchange factor C3G in hematopoietic cells stimulated with erythropoietin or interleukin-3.

    Nosaka Y, Arai A, Miyasaka N and Miura O

    First Department of Internal Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan.

    CrkL is an SH2 and SH3 domain-containing adaptor protein implicated in pathogenesis of chronic myelogenous leukemia. Here, we demonstrate that overexpression of CrkL enhances the erythropoietin (Epo)- or interleukin (IL)-3-induced activation of Elk-1 and the c-fos gene promoter activity in 32D/EpoR-Wt cells. Moreover, the Epo-induced activation of ERK1 and ERK2 was augmented and prolonged in cells inducibly overexpressing CrkL. A moderate increase in Epo-induced activation of JNK was also observed in cells overexpressing CrkL. Overexpression of C3G enhanced the Elk-1 activation synergistically with CrkL, while a C3G mutant lacking the guanine nucleotide exchange domain showed an inhibitory effect. Studies using a dominant negative Ha-Ras mutant demonstrated that the Elk-1 and ERK2 activation enhanced by CrkL and C3G was dependent on Ras. Consistent with this, the Epo-induced activation of Ras was augmented in cells inducibly overexpressing CrkL. Most importantly, a CrkL mutant defective in the SH2 or N-terminal SH3 domain showed an inhibitory effect on the Epo-induced activation of ERK2. These data indicate that the CrkL-C3G complex plays a role in Epo- or IL-3-induced, Ras-dependent activation of the Raf/ERK pathway leading to the activation of Elk-1 and the c-fos gene transcription.

    The Journal of biological chemistry 1999;274;42;30154-62

  • Transient palmitoylation supports H-Ras membrane binding but only partial biological activity.

    Coats SG, Booden MA and Buss JE

    Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa 50011, USA.

    H-Ras is >95% membrane-bound when modified by farnesyl and palmitate, but <10% membrane-bound if only farnesyl is present, implying that palmitate provides major support for membrane interaction. However the direct contribution of palmitate to H-Ras membrane interaction or the extent of its cooperation with farnesyl is unknown, because in the native protein the isoprenoid must be present before palmitate can be attached. To examine if palmitates can maintain H-Ras membrane association despite multiple cycles of turnover, a nonfarnesylated H-Ras(Cys186Ser) was constructed, with an N-terminal palmitoylation signal, derived from the GAP-43 protein. Although 40% of the GAP43:Ras(61Leu,186Ser) protein (G43:Ras61L) partitioned with membranes, the chimera had less than 10% of the transforming activity of fully lipidated H-Ras(61Leu) in NIH 3T3 cells. Poor focus formation was not due to incorrect targeting or gross structural changes, because G43:Ras61L localized specifically to plasma membranes and triggered differentiation of PC12 cells as potently as native H-Ras61L. Proteolytic digestion indicated that in G43:Ras61L both the N-terminal and the two remaining C-terminal cysteines of G43:Ras61L were palmitoylated. A mutant lacking all three C-terminal Cys residues had decreased membrane binding and differentiating activity. Therefore, even with correct targeting and palmitates at the C-terminus, G43:Ras61L was only partially active. These results indicate that although farnesyl and palmitate share responsibility for H-Ras membrane binding, each lipid also has distinct functions. Farnesyl may be important for signaling, especially transformation, while palmitates may provide potentially dynamic regulation of membrane binding.

    Funded by: NCI NIH HHS: CA51890

    Biochemistry 1999;38;39;12926-34

  • Bcl-2 differentially targets K-, N-, and H-Ras to mitochondria in IL-2 supplemented or deprived cells: implications in prevention of apoptosis.

    Rebollo A, Pérez-Sala D and Martínez-A C

    Department of Immunology and Oncology, Centro Nacional de Biotecnología, Universidad Autónoma, Campus de Cantoblanco, E-28049 Madrid, Spain.

    IL-2 deprivation triggers apoptosis in the murine T cell line TS1alphabeta, a process that can be blocked by overexpression of Bcl-2. Here we show that Bcl-2 and Ras proteins interact in mitochondria from TS1alphabeta cells in the presence or absence of IL-2, as evidenced by co-immunoprecipitation. All three Ras proteins, K-, N- and H-Ras, interact with Bcl-2; however, their mitochondrial localization is differentially regulated in IL-2-supplemented or -deprived cells. K-Ras is found in mitochondria only in IL-2-supplemented cells, whereas H-Ras is observed in mitochondria only after IL-2 withdrawal. N-Ras is detected in mitochondria under both experimental conditions. Bcl-2 transfection partially restored K- and N-Ras association with mitochondria in IL-2-deprived cells and rendered H-Ras association independent of IL-2 withdrawal. Inhibitors of Ras posttranslational processing did not alter the IL-2-induced differential pattern of mitochondrial localization. The processed forms of K- and N-Ras associated with mitochondria, although unprocessed H-Ras was also detected in mitochondria from mevastatin-treated cells. These results evidence a distinct behavior among the three Ras proteins in TS1alphabeta cells, depending on IL-2 supply, and suggest homologue-specific roles for Ras proteins in IL-2-dependent events.

    Oncogene 1999;18;35;4930-9

  • NF-kappaB is a target of AKT in anti-apoptotic PDGF signalling.

    Romashkova JA and Makarov SS

    Thurston Arthritis Research Center, University of North Carolina at Chapel Hill, 27599-7280, USA.

    The mechanisms of cell proliferation and transformation are intrinsically linked to the process of apoptosis: the default of proliferating cells is to die unless specific survival signals are provided. Platelet-derived growth factor (PDGF) is a principal survival factor that inhibits apoptosis and promotes proliferation, but the mechanisms mediating its anti-apoptotic properties are not completely understood. Here we show that the transcription factor NF-kappaB is important in PDGF signalling. NF-kappaB transmits two signals: one is required for the induction of proto-oncogene c-myc and proliferation, and the second, an anti-apoptotic signal, counterbalances c-Myc cytotoxicity. We have traced a putative pathway whereby PDGF activates NF-kappaB through Ras and phospatidylinositol-3-kinase (PI(3)K) to the PKB/Akt protein kinase and the IkappaB kinase (IKK); NF-kappaB thus appears to be a target of the anti-apoptotic Ras/PI(3)K/Akt pathway. We show that, upon PDGF stimulation, Akt transiently associates in vivo with IKK and induces IKK activation. These findings establish a role for NF-kappaB in growth factor signalling and define an anti-apoptotic Ras/PI(3)K/Akt/IKK/NF-kappaB pathway, thus linking anti-apoptotic signalling with transcription machinery.

    Nature 1999;401;6748;86-90

  • Myosin light chain kinase functions downstream of Ras/ERK to promote migration of urokinase-type plasminogen activator-stimulated cells in an integrin-selective manner.

    Nguyen DH, Catling AD, Webb DJ, Sankovic M, Walker LA, Somlyo AV, Weber MJ and Gonias SL

    Department of Biochemistry and Molecular Genetics, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.

    Urokinase-type plasminogen activator (uPA) activates the mitogen activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK) 1 and 2, in diverse cell types. In this study, we demonstrate that uPA stimulates migration of MCF-7 breast cancer cells, HT 1080 fibrosarcoma cells, and uPAR-overexpressing MCF-7 cells by a mechanism that depends on uPA receptor (uPAR)-ligation and ERK activation. Ras and MAP kinase kinase (MEK) were necessary and sufficient for uPA-induced ERK activation and stimulation of cellular migration, as demonstrated in experiments with dominant-negative and constitutively active mutants of these signaling proteins. Myosin light chain kinase (MLCK) was also required for uPA-stimulated cellular migration, as determined in experiments with three separate MLCK inhibitors. When MCF-7 cells were treated with uPA, MLCK was phosphorylated by a MEK-dependent pathway and apparently activated, since serine-phosphorylation of myosin II regulatory light chain (RLC) was also increased. Despite the transient nature of ERK phosphorylation, MLCK remained phosphorylated for at least 6 h. The uPA-induced increase in MCF-7 cell migration was observed selectively on vitronectin-coated surfaces and was mediated by a beta1-integrin (probably alphaVbeta1) and alphaVbeta5. When MCF-7 cells were transfected to express alphaVbeta3 and treated with uPA, ERK was still phosphorylated; however, the cells did not demonstrate increased migration. Neutralizing the function of alphaVbeta3, with blocking antibody, restored the ability of uPA to promote cellular migration. Thus, we have demonstrated that uPA promotes cellular migration, in an integrin-selective manner, by initiating a uPAR-dependent signaling cascade in which Ras, MEK, ERK, and MLCK serve as essential downstream effectors.

    Funded by: NCI NIH HHS: CA-53462

    The Journal of cell biology 1999;146;1;149-64

  • Aiolos transcription factor controls cell death in T cells by regulating Bcl-2 expression and its cellular localization.

    Romero F, Martínez-A C, Camonis J and Rebollo A

    Laboratoire d'Oncologie Cellulaire et Moleculaire, INSERM U363, Hôpital Cochin, 27 rue du Faubourg Saint-Jacques, F-75014 Paris, France.

    We searched for proteins that interact with Ras in interleukin (IL)-2-stimulated or IL-2-deprived cells, and found that the transcription factor Aiolos interacts with Ras. The Ras-Aiolos interaction was confirmed in vitro and in vivo by co-immunoprecipitation. Indirect immunofluorescence shows that IL-2 controls the cellular distribution of Aiolos and induces its tyrosine phosphorylation, required for dissociation from Ras. We also identified functional Aiolos-binding sites in the Bcl-2 promoter, which are able to activate the luciferase reporter gene. Mutation of Aiolos-binding sites within the Bcl-2 promoter inhibits transactivation of the reporter gene luciferase, suggesting direct control of Bcl-2 expression by Aiolos. Co-transfection experiments confirm that Aiolos induces Bcl-2 expression and prevents apoptosis in IL-2-deprived cells. We propose a model for the regulation of Bcl-2 expression via Aiolos.

    The EMBO journal 1999;18;12;3419-30

  • In vivo interaction of AF-6 with activated Ras and ZO-1.

    Yamamoto T, Harada N, Kawano Y, Taya S and Kaibuchi K

    Division of Signal Transduction, Nara Institute of Science and Technology, Ikoma, Nara, 630-0101, Japan.

    AF-6 contains two putative Ras-associating domains (RA domains) which are seen in several Ras effectors such as RalGDS and RIN1. We previously showed that an AF-6 fragment containing the amino-terminal (N-terminal) RA domain directly binds to activated Ras and ZO-1 in vitro. In this study, we showed that a single amino acid mutation in the N-terminal RA domain of AF-6 abolished the interaction of AF-6 with activated Ras and that the sites of this critical amino acid residue were similar to those for Raf-1 and RalGDS. The overexpression of the N-terminal RA domain of AF-6 inhibited the Ras-dependent c-fos promoter/enhancer stimulation in NIH3T3 cells. Endogenous AF-6 was coimmunoprecipitated with activated Ras from Rat1 cells expressing activated Ras. Moreover, we showed that AF-6 was coimmunoprecipitated with ZO-1 from Rat1 cells. Taken together, these results indicate that the Ras-interacting region on AF-6 is structurally similar to that on Raf-1 and on RalGDS and that AF-6 interacts with activated Ras and ZO-1 in vivo.

    Biochemical and biophysical research communications 1999;259;1;103-7

  • A novel pathway for tumor necrosis factor-alpha and ceramide signaling involving sequential activation of tyrosine kinase, p21(ras), and phosphatidylinositol 3-kinase.

    Hanna AN, Chan EY, Xu J, Stone JC and Brindley DN

    Signal Transduction Laboratories, and the Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2S2, Canada.

    Treatment of confluent rat2 fibroblasts with C2-ceramide (N-acetylsphingosine), sphingomyelinase, or tumor necrosis factor-alpha (TNFalpha) increased phosphatidylinositol (PI) 3-kinase activity by 3-6-fold after 10 min. This effect of C2-ceramide depended on tyrosine kinase activity and an increase in Ras-GTP levels. Increased PI 3-kinase activity was also accompanied by its translocation to the membrane fraction, increases in tyrosine phosphorylation of the p85 subunit, and physical association with Ras. Activation of PI 3-kinase by TNFalpha, sphingomyelinase, and C2-ceramide was inhibited by tyrosine kinase inhibitors (genistein and PP1). The stimulation of PI 3-kinase by sphingomyelinase and C2-ceramide was not observed in fibroblasts expressing dominant-negative Ras (N17) and the stimulation by TNFalpha was decreased by 70%. PI 3-kinase activation by C2-ceramide was not modified by inhibitors of acidic and neutral ceramidases, and it was not observed with the relatively inactive analog, dihydro-C2-ceramide. It is proposed that activation of Ras and PI 3-kinase by ceramide can contribute to signaling effects of TNFalpha that occur downstream of sphingomyelinase activation and result in increased fibroblasts proliferation.

    The Journal of biological chemistry 1999;274;18;12722-9

  • A non-farnesylated Ha-Ras protein can be palmitoylated and trigger potent differentiation and transformation.

    Booden MA, Baker TL, Solski PA, Der CJ, Punke SG and Buss JE

    Department of Biochemistry, Iowa State University, Ames, Iowa 50011, USA.

    Ha-Ras undergoes post-translational modifications (including attachment of farnesyl and palmitate) that culminate in localization of the protein to the plasma membrane. Because palmitate is not attached without prior farnesyl addition, the distinct contributions of the two lipid modifications to membrane attachment or biological activity have been difficult to examine. To test if palmitate is able to support these crucial functions on its own, novel C-terminal mutants of Ha-Ras were constructed, retaining the natural sites for palmitoylation, but replacing the C-terminal residue of the CAAX signal for prenylation with six lysines. Both the Ext61L and ExtWT proteins were modified in a dynamic fashion by palmitate, without being farnesylated; bound to membranes modestly (40% as well as native Ha-Ras); and retained appropriate GTP binding properties. Ext61L caused potent transformation of NIH 3T3 cells and, unexpectedly, an exaggerated differentiation of PC12 cells. Ext61L with the six lysines but lacking palmitates was inactive. Thus, farnesyl is not needed as a signal for palmitate attachment or removal, and a combination of transient palmitate modification and basic residues can support Ha-Ras membrane binding and two quite different biological functions. The roles of palmitate can therefore be independent of and distinct from those of farnesyl. Reciprocally, if membrane association can be sustained largely through palmitates, farnesyl is freed to interact with other proteins.

    Funded by: NCI NIH HHS: CA51890

    The Journal of biological chemistry 1999;274;3;1423-31

  • Premature senescence involving p53 and p16 is activated in response to constitutive MEK/MAPK mitogenic signaling.

    Lin AW, Barradas M, Stone JC, van Aelst L, Serrano M and Lowe SW

    Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.

    Oncogenic Ras transforms immortal rodent cells to a tumorigenic state, in part, by constitutively transmitting mitogenic signals through the mitogen-activated protein kinase (MAPK) cascade. In primary cells, Ras is initially mitogenic but eventually induces premature senescence involving the p53 and p16(INK4a) tumor suppressors. Constitutive activation of MEK (a component of the MAPK cascade) induces both p53 and p16, and is required for Ras-induced senescence of normal human fibroblasts. Furthermore, activated MEK permanently arrests primary murine fibroblasts but forces uncontrolled mitogenesis and transformation in cells lacking either p53 or INK4a. The precisely opposite response of normal and immortalized cells to constitutive activation of the MAPK cascade implies that premature senescence acts as a fail-safe mechanism to limit the transforming potential of excessive Ras mitogenic signaling. Consequently, constitutive MAPK signaling activates p53 and p16 as tumor suppressors.

    Funded by: NCI NIH HHS: CA13106, CA72982, P01 CA013106

    Genes & development 1998;12;19;3008-19

  • Presence of activated ras correlates with increased cysteine proteinase activities in human colorectal carcinomas.

    Kim K, Cai J, Shuja S, Kuo T and Murnane MJ

    Department of Pathology, Boston University School of Medicine, MA 02118, USA.

    The metastatic potential of ras-transfected cells has been attributed in part to significant ras induction of proteinase expression. To determine whether primary cancers also demonstrate higher cysteine proteinase activities in the presence of activated ras genes or altered ras protein expression, we have analyzed 60 primary human colorectal carcinomas for ras gene or protein changes together with the expression of cathepsins B and L. Cancers containing K-ras mutations (47% of 60 carcinomas) demonstrated greater increases in cathepsin L activity than cancers without K-ras mutations (p = 0.029), with particularly significant correlations for earlier stage cancers (Dukes' A and B carcinomas, p = 0.006). Western blots used to characterize ras protein patterns in the same cancer/normal pairs have demonstrated that N-ras protein is more highly expressed in colon tissues than H- or K-ras proteins and that N-ras overexpression occurs in almost 70% of colorectal cancers, with or without a concurrent change in electrophoretic mobility of N-ras protein. Our current study has now shown that N-ras protein overexpression alone does not significantly induce cathepsin B or L activity levels in colon cancers. However, carcinomas demonstrating altered N-ras protein forms, in the absence of any K- or N-ras mutations, expressed significantly higher levels of cathepsin B and L activities compared with carcinomas with normal N-ras protein banding patterns. Our data suggest that colorectal carcinomas with either K-ras mutations or altered forms of N-ras protein may increase their tumorigenic potential via the induction of cathepsin L or B expression levels. Our results also confirm that ras oncogene up-regulation of cathepsin B and L activities, previously reported in cultured cells, is a frequent event in primary human colorectal carcinomas.

    Funded by: NCI NIH HHS: CA 51865

    International journal of cancer 1998;79;4;324-33

  • The structural basis of the activation of Ras by Sos.

    Boriack-Sjodin PA, Margarit SM, Bar-Sagi D and Kuriyan J

    Laboratory of Molecular Biophysics, The Rockefeller University, New York, New York 10021, USA.

    The crystal structure of human H-Ras complexed with the Ras guanine-nucleotide-exchange-factor region of the Son of sevenless (Sos) protein has been determined at 2.8 A resolution. The normally tight interaction of nucleotides with Ras is disrupted by Sos in two ways. First, the insertion into Ras of an alpha-helix from Sos results in the displacement of the Switch 1 region of Ras, opening up the nucleotide-binding site. Second, side chains presented by this helix and by a distorted conformation of the Switch 2 region of Ras alter the chemical environment of the binding site for the phosphate groups of the nucleotide and the associated magnesium ion, so that their binding is no longer favoured. Sos does not impede the binding sites for the base and the ribose of GTP or GDP, so the Ras-Sos complex adopts a structure that allows nucleotide release and rebinding.

    Nature 1998;394;6691;337-43

  • SUR-8, a conserved Ras-binding protein with leucine-rich repeats, positively regulates Ras-mediated signaling in C. elegans.

    Sieburth DS, Sun Q and Han M

    Howard Hughes Medical Institute, Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, 80309-0347, USA.

    We describe the identification and characterization of a novel gene, sur-8, that positively regulates Ras-mediated signal transduction during C. elegans vulval development. Reduction of sur-8 function suppresses an activated ras mutation and dramatically enhances phenotypes of mpk-1 MAP kinase and ksr-1 mutations, while increase of sur-8 dosage enhances an activated ras mutation. sur-8 appears to act downstream of or in parallel to ras but upstream of raf. sur-8 encodes a conserved protein that is composed predominantly of leucine-rich repeats. The SUR-8 protein interacts directly with Ras but not with the Ras(P34G) mutant protein, suggesting that SUR-8 may mediate its effects through Ras binding. A structural and functional SUR-8 homolog in humans specifically binds K-Ras and N-Ras but not H-Ras in vitro.

    Funded by: NIGMS NIH HHS: GM47869

    Cell 1998;94;1;119-30

  • RasGRP, a Ras guanyl nucleotide- releasing protein with calcium- and diacylglycerol-binding motifs.

    Ebinu JO, Bottorff DA, Chan EY, Stang SL, Dunn RJ and Stone JC

    Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.

    RasGRP, a guanyl nucleotide-releasing protein for the small guanosine triphosphatase Ras, was characterized. Besides the catalytic domain, RasGRP has an atypical pair of "EF hands" that bind calcium and a diacylglycerol (DAG)-binding domain. RasGRP activated Ras and caused transformation in fibroblasts. A DAG analog caused sustained activation of Ras-Erk signaling and changes in cell morphology. Signaling was associated with partitioning of RasGRP protein into the membrane fraction. Sustained ligand-induced signaling and membrane partitioning were absent when the DAG-binding domain was deleted. RasGRP is expressed in the nervous system, where it may couple changes in DAG and possibly calcium concentrations to Ras activation.

    Science (New York, N.Y.) 1998;280;5366;1082-6

  • Interactions of the amino acid residue at position 31 of the c-Ha-Ras protein with Raf-1 and RalGDS.

    Shirouzu M, Morinaka K, Koyama S, Hu CD, Hori-Tamura N, Okada T, Kariya K, Kataoka T, Kikuchi A and Yokoyama S

    Cellular Signaling Laboratory, Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi, Saitama 351-01, Japan.

    The Ras and Rap1A proteins can bind to the Raf and RalGDS families. Ras and Rap1A have Glu and Lys, respectively, at position 31. In the present study, we analyzed the effects of mutating the Glu at position 31 of the c-Ha-Ras protein to Asp, Ala, Arg, and Lys on the interactions with Raf-1 and RalGDS. The Ras-binding domain (RBD) of Raf-1 binds the E31R and E31K Ras mutants less tightly than the wild-type, E31A, and E31D Ras proteins; the introduction of the positively charged Lys or Arg residue at position 31 specifically impairs the binding of Ras with the Raf-1 RBD. On the other hand, the ability of the oncogenic RasG12V protein to activate Raf-1 in HEK293 cells was only partially reduced by the E31R mutation but was drastically impaired by the E31K mutation. Correspondingly, RasG12V(E31K) as well as Rap1A, but not RasG12V(E31R), exhibited abnormally tight binding with the cysteine-rich domain of Raf-1. On the other hand, the E31A, E31R, and E31K mutations, but not the E31D mutation, enhanced the RalGDS RBD-binding activity of Ras, indicating that the negative charge at position 31 of Ras is particularly unfavorable to the interaction with the RalGDS RBD. RasG12V(E31K), RasG12V(E31A), and Rap1A stimulate the RalGDS action more efficiently than the wild-type Ras in the liposome reconstitution assay. All of these results clearly show that the sharp contrast between the characteristics of Ras and Rap1A, with respect to the interactions with Raf-1 and RalGDS, depends on their residues at position 31.

    The Journal of biological chemistry 1998;273;13;7737-42

  • Identification of Nore1 as a potential Ras effector.

    Vavvas D, Li X, Avruch J and Zhang XF

    The Diabetes Unit and Medical Services and the Department of Medicine, Harvard Medical School, Massachusetts General Hospital East, Charlestown, Massachusetts 02129, USA.

    The small GTP-binding protein Ras is pivotal in transmitting growth and differentiation signals downstream of cell surface receptors. Many observations have indicated that Ras transmits signals from cell surface receptors into multiple pathways via direct interaction with different effectors in mammalian cells. We have identified a novel potential Ras effector or target named Nore1. Nore1 has no significant sequence similarity to known mammalian proteins and lacks an identifiable catalytic domain, but contains sequence motifs that predict DAG_PE binding and SH3 domain binding. We show that Nore1 directly interacts with Ras in vitro in a GTP-dependent manner, and the interaction requires an intact Ras effector domain. Nore1 becomes associated with Ras in situ following activation of epidermal growth factor receptor in COS-7 and in KB cells.

    Funded by: NIGMS NIH HHS: GM51281

    The Journal of biological chemistry 1998;273;10;5439-42

  • Identification of a specific effector of the small GTP-binding protein Rap2.

    Janoueix-Lerosey I, Pasheva E, de Tand MF, Tavitian A and de Gunzburg J

    Unité INSERM 248, Institut Curie, Section de Recherche, Paris, France.

    Rap2 is a small GTP-binding protein that belongs to the Ras superfamily and whose function is still unknown. To elucidate Rap2 function, we searched for potential effectors by screening a mouse brain cDNA library in a yeast two-hybrid system using as a bait a Rap2A protein bearing a mutation of Gly to Val at position 12. This strategy lead to the identification of a protein that interacts specifically with Rap2A complexed with GTP, and requires an intact effector domain of Rap2A for interaction; we designated this protein Rap2-interacting protein 8 (RPIP8). Biochemical data obtained from in vitro studies with purified proteins confirmed the genetic results. Mouse RPIP8 consists of 446 amino acids, bears a coiled-coil domain between residues 265 and 313, and is expressed principally in brain. Its human counterpart, of 400 amino acids, exhibits 93.7% identity in their common region. A search for similar sequences in expressed-sequence-tags databanks revealed the presence in human and rodents of mRNAs encoding the 400-residue and 446-residue forms of RPIP8. Furthermore a doublet of 45-50 kDa, corresponding to the 400-residue and 446-residue forms of the protein, was detected by western blotting of mouse brain extracts and lysates from pheochromocytoma PC12 cells and the pancreatic beta-cell lines HIT-T15 and RIN-m5F. Using transient transfections of HIT-T15 cells it was possible to demonstrate that [Val12]Rap2 and wild-type Rap2 could be immunoprecipitated with RPIP8. These data therefore argue for RPIP8 being a specific effector of the Rap2 protein in cells exhibiting neuronal properties.

    European journal of biochemistry 1998;252;2;290-8

  • Both farnesyltransferase and geranylgeranyltransferase I inhibitors are required for inhibition of oncogenic K-Ras prenylation but each alone is sufficient to suppress human tumor growth in nude mouse xenografts.

    Sun J, Qian Y, Hamilton AD and Sebti SM

    H Lee Moffitt Cancer Center, Drug Discovery Program and Department of Biochemistry and Molecular Biology at University of South Florida, Tampa 33612, USA.

    The ability of Ras oncoproteins to cause malignant transformation requires their post-translational modifications by prenyl groups. Because K-Ras can be both farnesylated and geranylgeranylated it is not known whether both farnesyltransferase and geranylgeranyltransferase I inhibitors are required for suppressing human tumor growth in whole animals. In this paper we report that oncogenic Ras processing, MAP kinase activation and growth in nude mice are inhibited by the farnesyltransferase inhibitor FTI-276 in H- and N-Ras transformed NIH3T3 cells; whereas in KB-Ras transformed NIH3T3 cells both FTI-276 and the geranylgeranyltransferase I inhibitor GGTI-297 are required for inhibition. Furthermore, human lung A-549 and Calu-1 carcinoma cell lines were found to co-express H-, N- and K-Ras. In Calu-1 cells, the processing of H- and N-Ras is inhibited greatly by FTI-276 but only partially by GGTI-297 whereas K-Ras processing inhibition requires both FTI-276 and GGTI-297. In contrast, in A-549 cells the processing of H- and N-Ras is inhibited only by FTI-276 and K-Ras processing is resistant to co-treatment with FTI-276 and GGTI-297. Yet, the growth in nude mice of A-549 and Calu-1 xenografts, both of which express K-Ras mutations, is inhibited by FTI-276 (80% inhibition) and GGTI-297 (60%). Furthermore, FTI-276 inhibits tumor growth of NIH3T3 cells transformed by a form of oncogenic H-Ras that is exclusively geranylgeranylated and whose processing is resistant to this inhibitor. Taken together, the results demonstrate that both FTase and GGTase I inhibitors are required for inhibition of K-Ras processing but that each alone is sufficient to suppress human tumor growth in nude mice.

    Funded by: NCI NIH HHS: CA-67771

    Oncogene 1998;16;11;1467-73

  • Signals transduced through the CD4 molecule interfere with TCR/CD3-mediated ras activation leading to T cell anergy/apoptosis.

    Tamma SM, Chirmule N, McCloskey TW, Oyaizu N, Kalyanaraman VS and Pahwa S

    Department of Pediatrics, North Shore University Hospital-New York University School of Medicine, Manhasset, New York 11030, USA.

    It has been previously demonstrated that the occupancy of CD4 molecules by the HIV-1 envelope glycoprotein gp120 results in marked inhibition of T cell receptor-CD3 complex (TCR/CD3) activation-induced IL-2 secretion. To elucidate the mechanism of inhibitory effects of gp160 on T cell signaling, we have investigated the intracellular biochemical events and biological output in response to anti-CD3 mAb activation of purified peripheral blood CD4+ T cells from healthy donors with and without prior exposure to HIV-1 gp160. Pretreatment with gp160 resulted in marked inhibition of tyrosine phosphorylation of p59(fyn), PLC-gamma1, ras activation, and TNF-alpha secretion in anti-CD3 mAb activated CD4+ T cells, and a subset of CD4+ cells underwent activation-induced cell death. The data presented here provide insight into the mechanism by which the interaction of HIV-1 envelope glycoproteins with CD4 molecules may alter TCR/CD3-activation-induced signal transduction resulting in anergy and apoptosis with consequent functional deficiency of CD4+ T cells.

    Funded by: NIAID NIH HHS: AI28281; NIDA NIH HHS: DA 05161

    Clinical immunology and immunopathology 1997;85;2;195-201

  • 14-3-3 zeta negatively regulates raf-1 activity by interactions with the Raf-1 cysteine-rich domain.

    Clark GJ, Drugan JK, Rossman KL, Carpenter JW, Rogers-Graham K, Fu H, Der CJ and Campbell SL

    Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA.

    Although Raf-1 is a critical effector of Ras signaling and transformation, the mechanism by which Ras promotes Raf-1 activation is complex and remains poorly understood. We recently reported that Ras interaction with the Raf-1 cysteine-rich domain (Raf-CRD, residues 139-184) may be required for Raf-1 activation. The Raf-CRD is located in the NH2-terminal negative regulatory domain of Raf-1 and is highly homologous to cysteine-rich domains found in protein kinase C family members. Recent studies indicate that the structural integrity of the Raf-CRD is also critical for Raf-1 interaction with 14-3-3 proteins. However, whether 14-3-3 proteins interact directly with the Raf-CRD and how this interaction may mediate Raf-1 function has not been determined. In the present study, we demonstrate that 14-3-3 zeta binds directly to the isolated Raf-CRD. Moreover, mutation of Raf-1 residues 143-145 impairs binding of 14-3-3, but not Ras, to the Raf-CRD. Introduction of mutations that impair 14-3-3 binding resulted in full-length Raf-1 mutants with enhanced transforming activity. Thus, 14-3-3 interaction with the Raf-CRD may serve in negative regulation of Raf-1 function by facilitating dissociation of 14-3-3 from the NH2 terminus of Raf-1 to promote subsequent events necessary for full activation of Raf-1.

    Funded by: NCI NIH HHS: CA42978, CA64569, CA70308; ...

    The Journal of biological chemistry 1997;272;34;20990-3

  • CD4 cross-linking (CD4XL) induces RAS activation and tumor necrosis factor-alpha secretion in CD4+ T cells.

    Tamma SM, Chirmule N, Yagura H, Oyaizu N, Kalyanaraman V and Pahwa S

    Department of Pediatrics, North Shore University Hospital, New York University School of Medicine, Manhasset, NY 11030, USA.

    CD4 molecules are the primary receptors for human immunodeficiency virus (HIV) and bind the envelope glycoprotein gp120 of HIV with high-affinity. We have previously shown that cross-linking of CD4 molecules (CD4XL) in normal peripheral blood mononuclear cells (PBMC) results in secretion of cytokines tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), but not of interleukin-2 (IL-2) or IL-4. To investigate the intracellular signaling events associated with CD4-gp120 interaction, we incubated CD4+ T cells from peripheral blood of HIV-negative healthy donors with HIV envelope protein gp160 alone or performed CD4XL with gp160 and anti-gp160 antibody. This procedure resulted in tyrosine phosphorylation of intracellular substrates p59fyn, zap 70, and p95vav and also led to ras activation, as assessed by conversion of rasGDP to rasGTP. The role of ras in CD4 signaling was further investigated using CD4+ Jurkat cells transfected with a dominant negative ras mutant. CD4+ T cells expressing dn-ras secreted significantly reduced levels of TNF-alpha in response to CD4XL. These studies indicate that interaction of HIV gp160 with CD4 molecules activates the ras pathway in T cells, which may result in the cells becoming unresponsive to subsequent stimulation.

    Funded by: NIAID NIH HHS: AI 128281; NIDA NIH HHS: DA 05161

    Blood 1997;90;4;1588-93

  • p110delta, a novel phosphatidylinositol 3-kinase catalytic subunit that associates with p85 and is expressed predominantly in leukocytes.

    Chantry D, Vojtek A, Kashishian A, Holtzman DA, Wood C, Gray PW, Cooper JA and Hoekstra MF

    ICOS Corporation, Bothell, Washington 98021, USA.

    We have identified a novel p110 isoform of phosphatidylinositol 3-kinase from human leukocytes that we have termed p110delta. In addition, we have independently isolated p110delta from a mouse embryo library on the basis of its ability to interact with Ha-RasV12 in the yeast two-hybrid system. This unique isoform contains all of the conserved structural features characteristic of the p110 family. Recombinant p110delta phosphorylates phosphatidylinositol and coimmunoprecipitates with p85. However, in contrast to previously described p110 subunits, p110delta is expressed in a tissue-restricted fashion; it is expressed at high levels in lymphocytes and lymphoid tissues and may therefore play a role in phosphatidylinositol 3-kinase-mediated signaling in the immune system.

    Funded by: NCI NIH HHS: CA54786

    The Journal of biological chemistry 1997;272;31;19236-41

  • Ras effectors and their role in mitogenesis and oncogenesis.

    Joneson T and Bar-Sagi D

    Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook 11794, USA.

    Ras proteins are membrane-bound GTP-binding proteins that play a critical role in the control of cell growth. Through a large number of genetic and biochemical studies it is becoming increasingly evident that the biological activity of Ras proteins is mediated by multiple signaling pathways. This review provides an account of the target proteins that interact with Ras and the functional consequences of these interactions. The relative contribution of the different Ras effector pathways to the mitogenic and oncogenic effects of Ras are discussed.

    Journal of molecular medicine (Berlin, Germany) 1997;75;8;587-93

  • The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants.

    Scheffzek K, Ahmadian MR, Kabsch W, Wiesmüller L, Lautwein A, Schmitz F and Wittinghofer A

    Max-Planck-Institut für molekulare Physiologie, Abteilung Strukturelle Biologie, Rheinlanddamm 201, 44139 Dortmund, Germany.

    The three-dimensional structure of the complex between human H-Ras bound to guanosine diphosphate and the guanosine triphosphatase (GTPase)-activating domain of the human GTPase-activating protein p120GAP (GAP-334) in the presence of aluminum fluoride was solved at a resolution of 2.5 angstroms. The structure shows the partly hydrophilic and partly hydrophobic nature of the communication between the two molecules, which explains the sensitivity of the interaction toward both salts and lipids. An arginine side chain (arginine-789) of GAP-334 is supplied into the active site of Ras to neutralize developing charges in the transition state. The switch II region of Ras is stabilized by GAP-334, thus allowing glutamine-61 of Ras, mutation of which activates the oncogenic potential, to participate in catalysis. The structural arrangement in the active site is consistent with a mostly associative mechanism of phosphoryl transfer and provides an explanation for the activation of Ras by glycine-12 and glutamine-61 mutations. Glycine-12 in the transition state mimic is within van der Waals distance of both arginine-789 of GAP-334 and glutamine-61 of Ras, and even its mutation to alanine would disturb the arrangements of residues in the transition state.

    Science (New York, N.Y.) 1997;277;5324;333-8

  • Interaction of activated Ras with Raf-1 alone may be sufficient for transformation of rat2 cells.

    Stang S, Bottorff D and Stone JC

    Department of Biochemistry, University of Alberta, Edmonton, Canada.

    v-H-ras effector mutants have been assessed for transforming activity and for the ability of the encoded proteins to interact with Raf-1-, B-Raf-, byr2-, ralGDS-, and CDC25-encoded proteins in the yeast two-hybrid system. Transformation was assessed in rat2 cells as well as in a mutant cell line, rv68BUR, that affords a more sensitive transformation assay. Selected mutant Ras proteins were also examined for their ability to interact with an amino-terminal fragment of Raf-1 in vitro. Finally, possible cooperation between different v-H-ras effector mutants and between effector mutants and overexpressed Raf-1 was assessed. Ras transforming activity was shown to correlate best with the ability of the encoded protein to interact with Raf-1. No evidence for cooperation between v-H-ras effector mutants was found. Signaling through the Raf1-MEK-mitogen-activated protein kinase cascade may be the only effector pathway contributing to RAS transformation in these cells.

    Molecular and cellular biology 1997;17;6;3047-55

  • Protein binding and signaling properties of RIN1 suggest a unique effector function.

    Han L, Wong D, Dhaka A, Afar D, White M, Xie W, Herschman H, Witte O and Colicelli J

    Department of Biological Chemistry, Molecular Genetics, and Immunology, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90095, USA.

    Human RIN1 was first characterized as a RAS binding protein based on the properties of its carboxyl-terminal domain. We now show that full-length RIN1 interacts with activated RAS in mammalian cells and defines a minimum region of 434 aa required for efficient RAS binding. RIN1 interacts with the "effector domain" of RAS and employs some RAS determinants that are common to, and others that are distinct from, those required for the binding of RAF1, a known RAS effector. The same domain of RIN1 that binds RAS also interacts with 14-3-3 proteins, extending the similarity between RIN1 and other RAS effectors. When expressed in mammalian cells, the RAS binding domain of RIN1 can act as a dominant negative signal transduction blocker. The amino-terminal domain of RIN1 contains a proline-rich sequence similar to consensus Src homology 3 (SH3) binding regions. This RIN1 sequence shows preferential binding to the ABL-SH3 domain in vitro. Moreover, the amino-terminal domain of RIN1 directly associates with, and is tyrosine phosphorylated by, c-ABL. In addition, RIN1 encodes a functional SH2 domain that has the potential to activate downstream signals. These data suggest that RIN1 is able to mediate multiple signals. A differential pattern of expression and alternate splicing indicate several levels of RIN1 regulation.

    Funded by: NCI NIH HHS: CA53867, CA56301, R01 CA056301, R01 CA071443; NIGMS NIH HHS: GM24787

    Proceedings of the National Academy of Sciences of the United States of America 1997;94;10;4954-9

  • Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin cytoskeleton by Ras.

    Rodriguez-Viciana P, Warne PH, Khwaja A, Marte BM, Pappin D, Das P, Waterfield MD, Ridley A and Downward J

    Imperial Cancer Research Fund, London, United Kingdom.

    The pathways by which mammalian Ras proteins induce cortical actin rearrangement and cause cellular transformation are investigated using partial loss of function mutants of Ras and activated and inhibitory forms of various postulated target enzymes for Ras. Efficient transformation by Ras requires activation of other direct effectors in addition to the MAP kinase kinase kinase Raf and is inhibited by inactivation of the PI 3-kinase pathway. Actin rearrangement correlates with the ability of Ras mutants to activate PI 3-kinase. Inhibition of PI 3-kinase activity blocks Ras induction of membrane ruffling, while activated PI 3-kinase is sufficient to induce membrane ruffling, acting through Rac. The ability of activated Ras to stimulate PI 3-kinase in addition to Raf is therefore important in Ras transformation of mammalian cells and essential in Ras-induced cytoskeletal reorganization.

    Cell 1997;89;3;457-67

  • P110delta, a novel phosphoinositide 3-kinase in leukocytes.

    Vanhaesebroeck B, Welham MJ, Kotani K, Stein R, Warne PH, Zvelebil MJ, Higashi K, Volinia S, Downward J and Waterfield MD

    Ludwig Institute for Cancer Research, 91 Riding House Street, London W1P 8BT, United Kingdom.

    Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that have been implicated in signal transduction through tyrosine kinase- and heterotrimeric G-protein-linked receptors. We report herein the cloning and characterization of p110delta, a novel class I PI3K. Like p110alpha and p110beta, other class I PI3Ks, p110delta displays a broad phosphoinositide lipid substrate specificity and interacts with SH2/SH3 domain-containing p85 adaptor proteins and with GTP-bound Ras. In contrast to the widely distributed p110alpha and beta, p110delta is exclusively found in leukocytes. In these cells, p110alpha and delta both associate with the p85alpha and beta adaptor subunits and are similarly recruited to activated signaling complexes after treatment with the cytokines interleukin 3 and 4 and stem cell factor. Thus, these class I PI3Ks appear not to be distinguishable at the level of p85 adaptor selection or recruitment to activated receptor complexes. However, distinct biochemical and structural features of p110delta suggest divergent functional/regulatory capacities for this PI3K. Unlike p110alpha, p110delta does not phosphorylate p85 but instead harbors an intrinsic autophosphorylation capacity. In addition, the p110delta catalytic domain contains unique potential protein-protein interaction modules such as a Pro-rich region and a basic-region leucine-zipper (bZIP)-like domain. Possible selective functions of p110delta in white blood cells are discussed.

    Proceedings of the National Academy of Sciences of the United States of America 1997;94;9;4330-5

  • Physical association with ras enhances activation of membrane-bound raf (RafCAAX).

    Mineo C, Anderson RG and White MA

    Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9039, USA.

    The transforming activity of artificially membrane-targeted Raf1 suggests that Ras-mediated recruitment of Raf1 to the plasma membrane is an important step in Raf1 activation. Cellular Ras is concentrated in the caveolae, a microdomain of the plasma membrane that is highly enriched in caveolin, glycosylphosphatidylinositol-anchored proteins, and signal transduction molecules. Growth factor stimulation recruits Raf1 to this membrane domain. Whether Ras simply promotes Raf1 association with caveolae membranes or also modulates subsequent activation events is presently unclear. We have identified a ras variant, ras12V,37G, that does not interact with Raf1 but does interact with a mutant raf1, raf1(257L). To examine the role of Ras in the activation of membrane-bound Raf1, raf1CAAX, and raf1(257L)CAAX, membrane-targeted variants of Raf1 and raf1(257L), respectively, were expressed in fibroblasts with or without coexpression of ras12V, 37G. Cell fractionation localized both raf1CAAX and raf1(257L)CAAX to caveolae membranes independent of ras12V,37G expression; however, coexpression of ras12V,37G enhanced the activation of raf(257L)CAAX, but not raf1CAAX, as monitored by induction of cellular transformation, increased Raf kinase activity, and induction of activated MAP kinase. These results suggest that the Ras/Raf1 interaction plays a role in Raf1 activation that is distinct from membrane recruitment.

    Funded by: NCI NIH HHS: CA71443; NHLBI NIH HHS: HL20948; NIGMS NIH HHS: GM43169

    The Journal of biological chemistry 1997;272;16;10345-8

  • The complexity of Raf-1 regulation.

    Morrison DK and Cutler RE

    Molecular Basis of Carcinogenesis Laboratory, ABL-Basic Research Program, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD 21702, USA.

    The activation of the serine/threonine kinase Raf-1 is proving to be an intricate multistep process. Recent advances in elucidating how Raf-1 becomes activated in response to signaling events have emphasized the role of phosphorylation and protein interactions in Raf-1 regulation. The picture clearly emerging is that Raf-1 activity can be regulated by multiple mechanisms.

    Current opinion in cell biology 1997;9;2;174-9

  • Signal transduction by the neurotrophin receptors.

    Kaplan DR and Miller FD

    Brain Tumor Research Centre, Montreal Neurological Institute, 3801 University Street, Montreal, PQ, Canada, H3A 2B4. mcdv@musica.mcgill.ca

    The neurotrophins signal cell survival, differentiation, growth cessation, and apoptosis through two cell surface receptors, the Trks and p75NTR (p75 neurotrophin receptor). Recent advances indicate that the particular events that are mediated by neurotrophins are dependent upon the cell type and the expression pattern of each neurotrophin receptor. For example, TrkA activation induces cell death of neural tumor cells, and survival and differentiation of neurons. Likewise, p75NTR, when activated in the absence of a strong Trk signal, induces apoptosis of neurons, while in the presence of Trk it enhances responses to neurotrophin. These differing responses point to a complex interplay between neurotrophin-stimulated survival, differentiation, and apoptosis pathways.

    Current opinion in cell biology 1997;9;2;213-21

  • Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a.

    Serrano M, Lin AW, McCurrach ME, Beach D and Lowe SW

    Cold Spring Harbor Laboratory, New York 11724, USA.

    Oncogenic ras can transform most immortal rodent cells to a tumorigenic state. However, transformation of primary cells by ras requires either a cooperating oncogene or the inactivation of tumor suppressors such as p53 or p16. Here we show that expression of oncogenic ras in primary human or rodent cells results in a permanent G1 arrest. The arrest induced by ras is accompanied by accumulation of p53 and p16, and is phenotypically indistinguishable from cellular senescence. Inactivation of either p53 or p16 prevents ras-induced arrest in rodent cells, and E1A achieves a similar effect in human cells. These observations suggest that the onset of cellular senescence does not simply reflect the accumulation of cell divisions, but can be prematurely activated in response to an oncogenic stimulus. Negation of ras-induced senescence may be relevant during multistep tumorigenesis.

    Funded by: NCI NIH HHS: CA13106, CA68040

    Cell 1997;88;5;593-602

  • RalGDS functions in Ras- and cAMP-mediated growth stimulation.

    Miller MJ, Prigent S, Kupperman E, Rioux L, Park SH, Feramisco JR, White MA, Rutkowski JL and Meinkoth JL

    Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6084, USA.

    Thyroid-stimulating hormone stimulates proliferation through both the cAMP-dependent protein kinase and Ras but not through Raf-1 and mitogen-activated and extracellular signal-related kinase kinase. We now report that thyroid-stimulating hormone represses mitogen-activated protein kinase activity and that microinjection of an effector domain mutant Ha-Ras protein, Ras(12V,37G), defective in Raf-1 binding and mitogen-activated protein kinase activation, stimulates DNA synthesis in quiescent and thyroid-stimulating hormone-treated thyrocytes. A yeast two-hybrid screen identified RalGDS as a Ras(12V,37G) binding protein and therefore a potential effector of Ras in these cells. Associations between Ras and RalGDS were observed in extracts prepared from thyroid cells. Microinjection of a mutant RalA(28N) protein thought to sequester RalGDS family members reduced DNA synthesis stimulated by Ras as well as cAMP-mediated DNA synthesis in two cell lines which respond to cAMP with mitogenesis. These results support the idea that RalGDS may be an effector of Ras in cAMP-mediated growth stimulation.

    Funded by: NCI NIH HHS: CA 50528, CA 58689; NIDDK NIH HHS: DK45696; ...

    The Journal of biological chemistry 1997;272;9;5600-5

  • Ras activation in platelets after stimulation of the thrombin receptor, thromboxane A2 receptor or protein kinase C.

    Shock DD, He K, Wencel-Drake JD and Parise LV

    Department of Pharmacology, University of North Carolina at Chapel Hill 27599-7365, USA.

    Several reports have indicated that the small G-protein Ras is not present immunologically in platelets. However, here we report the identification of Ras in platelets by immunoprecipitation with the Ras-specific monoclonal antibodies Y13-259 or Y13-238, followed by Western blotting. The presence of Ras was not due to contamination of samples with erythrocytes or leucocytes. Immunofluorescence studies indicated that Ras was present in a peripheral rim pattern in fixed, permeabilized platelets, suggesting an intracellular, plasma membrane location. Activation of platelets with the thrombin receptor peptide42-50, the prostaglandin H2/thromboxane A2 mimetic U46619 or phorbol 12-myristate 13-acetate induced a rapid increase in GTP-bound, activated Ras. In each case, this increase was inhibited by the protein kinase C (PKC) inhibitor bisindolylmaleimide GF 109203X, suggesting that Ras is activated downstream of PKC in platelets. Thus the activation of Ras in platelets by agonists will now allow consideration of multiple potential Ras-dependent signal transduction pathways in platelet activation processes.

    Funded by: NHLBI NIH HHS: 1-PO1-HL45100

    The Biochemical journal 1997;321 ( Pt 2);525-30

  • Identification of a novel RalGDS-related protein as a candidate effector for Ras and Rap1.

    Peterson SN, Trabalzini L, Brtva TR, Fischer T, Altschuler DL, Martelli P, Lapetina EG, Der CJ and White GC

    Division of Cell Biology, Glaxo Wellcome Inc., Research Triangle Park, North Carolina 27709, USA.

    Although Ras and Rap1 share interaction with common candidate effector proteins, Rap1 lacks the transforming activity exhibited by Ras proteins. It has been speculated that Rap antagonizes Ras transformation through the formation of nonproductive complexes with critical Ras effector targets. To understand further the distinct biological functions of these two closely related proteins, we searched for Rap1b-binding proteins by yeast two-hybrid screening. We identified multiple clones that encode the COOH-terminal sequences of a protein that shares sequence identity with RalGDS and RGL, which we have designated RGL2. A 158-amino acid COOH-terminal fragment of RGL2 (RGL2 C-158) bound to Ras superfamily proteins which shared identical effector domain sequences with Rap1 (Ha-Ras, R-Ras, and TC21). RGL2 C-158 binding was impaired by effector domain mutations in Rap1b and Ha-Ras. Furthermore, RGL2 C-158 bound exclusively to the GTP-, but not the GDP-bound form of Ha-Ras. Finally, coexpression of RGL2 C-158 impaired oncogenic Ras activation of transcription from a Ras-responsive promoter element and focus-forming activity in NIH 3T3 cells. We conclude that RGL2 may be an effector for Ras and/or Rap proteins.

    Funded by: NCI NIH HHS: CA42978, CA55008; NHLBI NIH HHS: HL26309; ...

    The Journal of biological chemistry 1996;271;47;29903-8

  • Loss of Raf-1-binding activity of v-Ha-Ras by the deletion of amino acid residues 64-72 and 143-151.

    Hiwasa T, Kasama M, Nakadai T, Sawada T and Sakiyama S

    Division of Biochemistry, Chiba Cancer Center Research Institute, Japan.

    In order to elucidate the molecular events in signal transduction, examination of the interaction between Ras and Raf-1 seems crucial. Many Raf-1 mutants have been investigated in terms of their binding activities to Ras, where only a few Ras mutants have been examined thus far. We have investigated the Raf-1-binding activities of v-Ha-Ras and 21 insertion/deletion mutants of this protein. The results show that the mutants have varying levels of Raf-1-binding activity that are related neither to their transforming activity nor to their guanine nucleotide-binding activity. Deletion in the effector domain of Ras did not completely abolish Raf-1-binding, whereas the deletion in amino acid residues 64-72 or 143-151 resulted in complete loss of Raf-1-binding activity.

    Cellular signalling 1996;8;5;393-6

  • Regulation of neuromodulatory actions of angiotensin II in the brain neurons by the Ras-dependent mitogen-activated protein kinase pathway.

    Yang H, Lu D, Yu K and Raizada MK

    Department of Physiology, University of Florida, College of Medicine, Gainesville, Florida 32610, USA.

    Angiotensin II (Ang II) stimulates norepinephrine transporter (NET) and tyrosine hydroxylase (TH) in the neurons, but the signal transduction mechanism of this neuromodulation is not understood. Treatment of neuronal cultures of hypothalamus-brainstem with Ang II resulted in a time- and dose-dependent activation of Ras, Raf-1, and mitogen-activated protein kinase. This activation was mediated by the interaction of Ang II with the AT1, receptor subtype and was associated with the redistribution of AT1 receptor with Ras and Raf-1 on the neuronal membrane. Treatment with antisense oligonucleotide (AON) to mitogen-activated protein kinase decreased mitogen-activated protein kinase immunoreactivity by 70% and attenuated Ang II stimulation of c-fos, NET, and TH mRNA levels. This demonstrates that induction of these genes requires mitogen-activated protein kinase activation by Ang II. In contrast, AON to mitogen-activated protein kinase failed to inhibit Ang II stimulation of plasminogen activator inhibitor-1 mRNA levels. These results suggest that AT1 receptors are coupled to a Ras-Raf-1 mitogen-activated protein kinase signal transduction pathway that is responsible for stimulation of NET and TH, two neuro-modulatory actions of Ang II in the brain.

    Funded by: PHS HHS: 33610 (NIH)

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1996;16;13;4047-58

  • p120 Ras GTPase-activating protein interacts with Ras-GTP through specific conserved residues.

    Miao W, Eichelberger L, Baker L and Marshall MS

    Department of Medicine, Division of Hematology/Oncology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.

    Previous structural studies of RasGAP have failed to clearly localize sites of Ras interaction to individual amino acids. Hypothesizing that sites of interaction with Ras-GTP would be conserved, 11 of the most highly conserved amino acid residues of RasGAP were changed by mutation. Each mutant protein was purified as a glutathione S-transferase catalytic domain fusion and analyzed for protein stability, Ras GTPase stimulating activity, affinity for Ras-GTP, and when possible, secondary structure. The majority of conserved positions were found to be important structurally but with no direct role in Ras interactions. However, Arg786, Lys831, and Arg925 were observed to be essential for binding to Ras-GTP but not for protein structure. RasGAP residues 890-902 (block 3A) were observed to be homologous to residues 1540-1552 of the yeast adenylyl cyclase with amino acid substitutions in both regions resulting in increased affinity for Ras. This is the first example of a conserved Ras interaction motif in distinct Ras effector proteins. Our data are supportive of a model for GAP/Ras-GTP association in which the conserved, positively charged Arg786, Lys831, and Arg925 residues form salt bridges with the conserved, negatively charged residues in the Ras effector loop.

    The Journal of biological chemistry 1996;271;26;15322-9

  • Activation of phosphoinositide 3-kinase by interaction with Ras and by point mutation.

    Rodriguez-Viciana P, Warne PH, Vanhaesebroeck B, Waterfield MD and Downward J

    Imperial Cancer Research Fund, London.

    We have reported previously that Ras interacts with the catalytic subunit of phosphoinositide 3-kinase (PI 3-kinase) in a GTP-dependent manner. The affinity of the interaction of Ras-GTP with p85alpha/p110alpha is shown here to be approximately 150 nM. The site of interaction on the p110alpha and beta isoforms of PI 3-kinase lies between amino acid residues 133 and 314. A point mutation in this region, K227E, blocks the GTP-dependent interaction of PI 3-kinase p110alpha with Ras in vitro and the ability of Ras to activate PI 3-kinase in intact cells. In addition, this mutation elevates the basal activity of PI 3-kinase in intact cells, suggesting a direct influence of the Ras binding site on the catalytic activity of PI 3-kinase. Using an in vitro reconstitution assay, it is shown that the interaction of Ras-GTP, but not Ras-GDP, with PI 3-kinase leads to an increase in its enzymatic activity. This stimulation is synergistic with the effect of tyrosine phosphopeptide binding to p85, particularly at suboptimal peptide concentrations. These data show that PI 3-kinase is regulated by a number of mechanisms, and that Ras contributes to the activation of this lipid kinase synergistically with tyrosine kinases.

    The EMBO journal 1996;15;10;2442-51

  • Palmitoylation of Ha-Ras facilitates membrane binding, activation of downstream effectors, and meiotic maturation in Xenopus oocytes.

    Dudler T and Gelb MH

    Department of Chemistry, University of Washington, Seattle 98195-1700, USA.

    Ras proteins serve as critical relays in signal transduction pathways that control growth and differentiation and must undergo posttranslational modifications before they become functional. While it is established that farnesylation is necessary for membrane binding and cellular functions of all Ras proteins, the significance of palmitoylation is unclear. We have studied the contribution of Ha-Ras palmitoylation for biological activity in Xenopus oocytes. In contrast to wild-type Ha-Ras, which binds to membranes and induces meiosis when microinjected into oocytes, a nonpalmitoylated but farnesylated and methylated mutant mislocalizes to the cytosol and fails to promote maturation. This lack of responsiveness correlates with the inability of the mutant to induce phosphorylation and activation of mitogen-activated protein kinase and maturation promoting factor, which are both strongly activated by wild-type Ha-Ras. Costimulation of oocytes with insulin increases their responsiveness to Ras and partially rescues the biological activity of the palmitoylation-resistant mutant. However, 25-50 times higher doses of mutant were required to elicit responses equivalent to wild-type Ha-Ras. These results suggest that palmitoylation and membrane association of Ha-Ras is necessary for efficient activation of the mitogen-activated protein kinase cascade in vivo and are consistent with a biochemical function for Ras as a membrane targeting signal for downstream effectors in this pathway.

    Funded by: NCI NIH HHS: CA52874; NIGMS NIH HHS: GM562

    The Journal of biological chemistry 1996;271;19;11541-7

  • Identification of neurofibromin mutants that exhibit allele specificity or increased Ras affinity resulting in suppression of activated ras alleles.

    Morcos P, Thapar N, Tusneem N, Stacey D and Tamanoi F

    Department of Microbiology and Molecular Genetics, University of California, Los Angeles 90095-1489, USA.

    Neurofibromin plays a critical role in the downregulation of Ras proteins in neurons and Schwann cells. Thus, the ability of neurofibromin to interact with Ras is crucial for its function, as mutations in NF1 that abolish this interaction fail to maintain function. To investigate the neurofibromin-Ras interaction in a systematic manner, we have carried out a yeast two-hybrid screen using a mutant of H-ras, H-rasD92K, defective for interaction with the GTPase-activated protein-related domain (GRD) of NF1. Two screens of a randomly mutagenized NF1-GRD library led to the identification of seven novel NF1 mutants. Characterization of the NF1-GRD mutants revealed that one class of mutants are allele specific for H-raSD92K. These mutants exhibit increased affinity for H-raSD92K and significantly reduced affinity for wild-type H-ras protein. Furthermore, they do not interact with another H-ras mutant defective for interaction with GTPase-activating proteins. Another class of mutants are high-affinity mutants which exhibit dramatically increased affinity for both wild-type and mutant forms of Ras. They also exhibit a striking ability to suppress the heat shock sensitive traits of activated RAS2G19v in yeast cells. Five mutations cluster within a region encompassing residues 1391 to 1436 (region II). Three NF1 patient mutations have previously been identified in this region. Two mutations that we identified occur in a region encompassing residues 1262 to 1276 (region I). Combining high-affinity mutations from both regions results in even greater affinity for Ras. These results demonstrate that two distinct regions of NF1-GRD are involved in the Ras interaction and that single amino acid changes can affect NF1's affinity for Ras.

    Funded by: NIGMS NIH HHS: GM52271; NINDS NIH HHS: NS30054

    Molecular and cellular biology 1996;16;5;2496-503

  • Co-purification and direct interaction of Ras with caveolin, an integral membrane protein of caveolae microdomains. Detergent-free purification of caveolae microdomains.

    Song KS, Li Shengwen, Okamoto T, Quilliam LA, Sargiacomo M and Lisanti MP

    Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142-1479, USA.

    Caveolae are plasma membrane specializations that have been implicated in signal transduction. Caveolin, a 21-24-kDa integral membrane protein, is a principal structural component of caveolae membranes in vivo. G protein alpha subunits are concentrated in purified preparations of caveolae membranes, and caveolin interacts directly with multiple G protein alpha subunits, including G(s), G(o), and G(i2). Mutational or pharmacologic activation of G alpha subunits prevents the interaction of caveolin with G proteins, indicating that inactive G alpha subunits preferentially interact with caveolin. Here, we show that caveolin interacts with another well characterized signal transducer, Ras. Using a detergent-free procedure for purification of caveolin-rich membrane domains and a polyhistidine tagged form of caveolin, we find that Ras and other classes of lipid-modified signaling molecules co-fractionate and co-elute with caveolin. The association of Ras with caveolin was further evaluated using two distinct in vitro binding assays. Wild-type H-Ras interacted with glutathione S-transferase (GST)-caveolin fusion proteins but not with GST alone. Using a battery of GST fusion proteins encoding distinct regions of caveolin, Ras binding activity was localized to a 41-amino acid membrane proximal region of the cytosolic N-terminal domain of caveolin. In addition, reconstituted caveolin-rich membranes (prepared with purified recombinant caveolin and purified lipids) interacted with a soluble form of wild-type H-Ras but failed to interact with mutationally activated soluble H-Ras (G12V). Thus, a single amino acid change (G12V) that constitutively activates Ras prevents or destabilizes this interaction. These results clearly indicate that (i) caveolin is sufficient to recruit soluble Ras onto lipid membranes and (ii) membrane-bound caveolin preferentially interacts with inactive Ras proteins. In direct support of these in vitro studies, we also show that recombinant overexpression of caveolin in intact cells is sufficient to functionally recruit a nonfarnesylated mutant of Ras (C186S) onto membranes, overcoming the normal requirement for lipid modification of Ras. Taken together, these observations suggest that caveolin may function as a scaffolding protein to localize or sequester certain caveolin-interacting proteins, such as wild-type Ras, within caveolin-rich microdomains of the plasma membrane.

    Funded by: NCI NIH HHS: CA-63139; NIGMS NIH HHS: GM-50443

    The Journal of biological chemistry 1996;271;16;9690-7

  • Ras-GTP levels are elevated in human NF1 peripheral nerve tumors.

    Guha A, Lau N, Huvar I, Gutmann D, Provias J, Pawson T and Boss G

    Programme in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, Toronto, Ontario, Canada.

    Neurofibromin, the gene product of NF1, is a Ras GTPase Activating Protein. The absence of neurofibromin leads to increased levels of Ras-GTP, which contributes to the proliferation of NF1 neurogenic sarcoma cell lines. Whether this pathogenic mechanism is applicable to benign and malignant peripheral nerve tumours from NF1 and non NF1 patients is not known, due to lack of a tissue based assay. We have adapted a colorimetric enzymatic assay for determining levels of Ras bound guanine nucleotides in tissues. Ras-GTP levels were increased in NF1 neurogenic sarcomas (15 times) and benign NF1 neurofibromas (four times), compared to non NF1 schwannomas. Neurofibromin was not expressed in NF1 sarcomas, in support of its important negative Ras regulatory role in the pathogenesis of NF1 peripheral nerve tumors.

    Funded by: NIGMS NIH HHS: GM49360; NINDS NIH HHS: NS-01590-05

    Oncogene 1996;12;3;507-13

  • Molecular analysis of Ras activation by tyrosine phosphorylated Vav.

    Gulbins E, Schlottmann K, Brenner B, Lang F and Coggeshall KM

    I. Institute of Physiology, University of Tuebingen, Germany.

    Vav has been shown to activate Ras (1-3) and is regulated by tyrosine phosphorylation (1) or binding of diglycerides (3) to the cysteine rich domain. In the present study employing different Ras activation assay techniques using [3H]GDP release or [32P]alpha GTP-binding from membrane-bound or soluble recombinant Ras, we demonstrate that Ras activity can be increased by tyrosine phosphorylated Vav upon cellular stimulation via the IL-2 receptor or the TCR/CD3-complex. Increase of [32P]alpha GTP-binding to Ras catalyzed by phosphorylated Vav is similar to the activity of immunoprecipitated Sos. The activity of Vav measured by binding of [32P]alpha GTP to Ras was linear with respect to the concentration of Vav protein used. To study molecular characteristics of this Vav-Ras interaction, we used several Ras mutants and demonstrate that Vav activity towards Ras depends on the integrity of the same or similar domains as Ras activation by SDC 25 or CDC 25.

    Funded by: NCI NIH HHS: CA64268

    Biochemical and biophysical research communications 1995;217;3;876-85

  • Cysteine-rich region of Raf-1 interacts with activator domain of post-translationally modified Ha-Ras.

    Hu CD, Kariya K, Tamada M, Akasaka K, Shirouzu M, Yokoyama S and Kataoka T

    Department of Physiology II, Kobe University School of Medicine, Japan.

    The interaction between "switch I/effector domain" of Ha-Ras and the Ras-binding domain (RBD, amino acid 51-131) of Raf-1 is essential for signal transduction. However, the importance of the "activator domain" (approximately corresponding to amino acids 26-28 and 40-49) of Ha-Ras and of the "cysteine-rich region" (CRR, amino acids 152-184) of Raf-1 have also been proposed. Here, we found that Raf-1 CRR interacts directly with Ha-Ras independently of RBD and that participation of CRR is necessary for efficient Ras-Raf binding. Furthermore, Ha-Ras carrying mutations (N26G and V45E) in the activator domain failed to bind CRR, whereas they bound RBD normally. On the contrary, Ha-Ras carrying mutations in the switch I/effector domain exhibited severely reduced ability to bind RBD, whereas their ability to bind CRR was unaffected. Mutants that bound to either RBD or CRR alone failed to activate Raf-1. Ha-Ras without post-translational modifications, which lacks the ability to activate Raf-1, selectively lost the ability to bind CRR. These results suggest that the activator domain of Ha-Ras participates in activation of Raf-1 through interaction with CRR and that post-translational modifications of Ha-Ras are required for this interaction.

    The Journal of biological chemistry 1995;270;51;30274-7

  • Ras-related proteins in signal transduction and growth control.

    McCormick F

    Onyx Pharmaceuticals, Richmond, California 94806, USA.

    Ras proteins are members of a superfamily of small GTPases that are involved in many aspects of cell growth control. The ras p21 protooncogene products, H-ras, K-ras, and N-ras, transmit signals from growth factor receptors to a cascade of protein kinases that begins with the Raf protooncogene product, and leads to alterations in transcription factors and cell cycle proteins in the nucleus. This cascade is controlled at several points: Ras p21 proteins are regulated by GAPs and by exchange factors, whose activities are altered by growth factor receptor activation (Boguski and McCormick, 1993: Nature 366:643-654). Transmission of signals from Ras to Raf is regulated by the Ras-related protein Rap1 (a protein capable of reverting cell transformation) and by cAMP. Other aspects of Ras p21 regulation will be discussed, including the existence of RasGDl proteins that inhibit GDP dissociation from Ras, and may thus regulate the level of active Ras in the cell. The role of Ras in activation of Raf kinase appears to be limited to the recruitment of Raf to the plasma membrane, at which time Raf becomes stably modified to render it active (Leevers et al., 1994: Nature 369:411-414; Stokoe et al., 1994: Science 264:1463-1467). The nature of these modifications is unclear. Raf in the plasma membrane becomes associated with insoluble structural cell components that may be part of the activation. Furthermore, Raf is associated with proteins of the 14-3-3 family that appear necessary for kinase activation. The 14-3-3 proteins interact with all three conserved regions of Raf, including the kinase domain. In addition to Raf, Ras proteins interact with two known classes of proteins in a manner consistent with effector functions: these are the GAPs and regulators of the Ras-related protein Ral referred to as RalGDS. These biochemical data suggest that other functional pathways are regulated by Ras, including, perhaps, pathways involved in regulating cell shape and motility. The protein R-Ras p21 is about 50% identical to the Ras p21 protooncogene product. This protein is incapable of transforming cells, even though it interacts with Raf and other putative Ras effectors (Fernandez-Sarabia and Bischoff, 1993: Nature 366:274-275). On the other hand, it has recently been shown that R-Ras binds to the protooncogene product Bcl-2, a protein that transforms B cells by blocking apoptosis. R-Ras is regulated by the same GAP molecules as H-Ras and the other Ras protooncogene products, and may therefore be activated in a manner co-ordinate with these growth-promoting proteins. The possible connection between R-Ras and apoptosis will be discussed.

    Molecular reproduction and development 1995;42;4;500-6

  • Ras binding to a C-terminal region of GAP.

    Molloy DP, Owen D and Grand RJ

    CRC Institute for Cancer Studies, Medical School, University of Birmingham, Edgbaston, UK.

    Using fluorescence spectroscopy we have identified a binding region for Ras on the GTPase activating protein (GAP) lying within residues 715-753. A synthetic peptide Y922, corresponding to residues 716-753 of GAP binds to wild type Ras showing 3.3-fold higher affinity for the GTP- over the GDP-bound forms of Ras. Binding is stabilised by Mg2+, although Y922 does not stimulate the GTPase activity of Ras. Peptide binding to the Y32A and Y40F Ras mutants showed equal affinity for both GDP- and GTP-bound forms, with binding to Y32A.GDP abolished in the absence of Mg2+. These results suggest that Y922 mimics the in vivo interactions shown by the intact p120GAP protein and provide the first direct demonstration of Ras interaction with GAP in the region 715-753.

    FEBS letters 1995;368;2;297-303

  • Raf1 interaction with Cdc25 phosphatase ties mitogenic signal transduction to cell cycle activation.

    Galaktionov K, Jessus C and Beach D

    Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, New York 11724, USA.

    The Ras and Raf1 proto-oncogenes transduce extracellular signals that promote cell growth. Cdc25 phosphatases activate the cell division cycle by dephosphorylation of critical threonine and tyrosine residues within the cyclin-dependent kinases. We show here that Cdc25 phosphatase associates with raf1 in somatic mammalian cells and in meiotic frog oocytes. Furthermore, Cdc25 phosphatase can be activated in vitro in a Raf1-dependent manner. We suggest that activation of the cell cycle by the Ras/Raf1 pathways might be mediated in part by Cdc25.

    Genes & development 1995;9;9;1046-58

  • Two distinct Raf domains mediate interaction with Ras.

    Brtva TR, Drugan JK, Ghosh S, Terrell RS, Campbell-Burk S, Bell RM and Der CJ

    Cirruculum in Genetics and Molecular Biology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill 27599, USA.

    A key event for Ras transformation involves the direct physical association between Ras and the Raf-1 kinase. This interaction promotes both Raf translocation to the plasma membrane and activation of Raf kinase activity. Although substantial experimental evidence has demonstrated that Raf residues 51-131 alone are sufficient for Ras binding, conflicting observations have suggested that the Raf cysteine-rich domain (residues 139-184) may also be important for interaction with Ras. To clarify the role of the Raf cysteine-rich domain in Ras-Raf binding, we have compared the ability of two distinct Raf fragments to interact with Ras using both in vitro Ras binding and in vivo Ras inhibition assays. First, we determined that both Raf sequences 2-140 and 139-186 (designated Raf-Cys) showed preferential binding to active, GTP-bound Ras in vitro. Second, we observed that Raf-Cys antagonized oncogenic Ras(Q61L)-mediated transactivation of Ras-responsive elements and focus-forming activity in NIH 3T3 cells and insulin-induced germinal vesicle breakdown in Xenopus laevis oocytes in vivo. This inhibitory activity suggests that Raf-Cys can interact with Ras in vivo. Taken together, these results suggest that Ras interaction with two distinct domains of Raf-1 may be important in Ras-mediated activation of Raf kinase activity.

    Funded by: NCI NIH HHS: CA42978, CA52072, CA55008; ...

    The Journal of biological chemistry 1995;270;17;9809-12

  • Biochemical analysis of MEK activation in NIH3T3 fibroblasts. Identification of B-Raf and other activators.

    Reuter CW, Catling AD, Jelinek T and Weber MJ

    Department of Microbiology, University of Virginia Health Sciences Center, School of Medicine, Charlottesville 22908, USA.

    Numerous potential activators of MEK have been identified, including c-Raf-1, B-Raf, c-Mos, and a family of MEK kinases. However, little information gives insight into the activators actually utilized in vivo. To address this, we have used column chromatography and a coupled MEK activation assay to identify in NIH3T3 cells, two major MEK activators, and a third insulin-specific activator. The first MEK activator has an apparent M(r) of 40,000-50,000, was immunologically distinct from A-Raf, B-Raf, c-Raf-1, c-MEKK, c-Mos, MEK1, and MEK2, and was rapidly activated by serum, platelet-derived growth factor (PDGF), insulin, thrombin, and phorbol ester. The second MEK activator was identified as B-Raf. Activation of 93-95 kDa B-Raf was observed in column fractions and B-Raf immunoprecipitates from cytosolic and particulate fractions after stimulation with serum or PDGF, but not insulin. c-Raf-1 from cytosol did not exhibit MEK activator activity; however, c-Raf-1 immunoprecipitates from the particulate fraction revealed MEK activator activity that was enhanced after stimulation with PDGF or phorbol ester, but not serum or insulin. Both c-Mos and c-MEKK were present in NIH3T3 fibroblasts but did not show MEK activator activity. These data provide direct evidence that 93-95-kDa B-Raf isozymes and an unidentified 40-50-kDa MEK activator are major agonist-specific MEK activators in NIH3T3 fibroblasts.

    Funded by: NCI NIH HHS: CA39076; NIGMS NIH HHS: GM47332

    The Journal of biological chemistry 1995;270;13;7644-55

  • A human protein selected for interference with Ras function interacts directly with Ras and competes with Raf1.

    Han L and Colicelli J

    Department of Biological Chemistry, UCLA School of Medicine 90024.

    The overexpression of some human proteins can cause interference with the Ras signal transduction pathway in the yeast Saccharomyces cerevisiae. The functional block is located at the level of the effector itself, since these proteins do not suppress activating mutations further downstream in the same pathway. We now demonstrate, with in vivo and in vitro experiments, that the protein encoded by one human cDNA (clone 99) can interact directly with yeast Ras2p and with human H-Ras protein, and we have named this gene rin1 (Ras interaction/interference). The interaction between Ras and Rin1 is enhanced when Ras is bound to GTP. Rin1 is not able to interact with either an effector mutant or a dominant negative mutant of H-Ras. Thus, Rin1 displays a human H-Ras interaction profile that is the same as that seen for Raf1 and yeast adenylyl cyclase, two known effectors of Ras. Moreover, Raf1 directly competes with Rin1 for binding to H-Ras in vitro. Unlike Raf1, however, the Rin1 protein resides primarily at the plasma membrane, where H-Ras is localized. These data are consistent with Rin1 functioning in mammalian cells as an effector or regulator of H-Ras.

    Funded by: NCI NIH HHS: CA-56301

    Molecular and cellular biology 1995;15;3;1318-23

  • Identification of the guanine nucleotide dissociation stimulator for Ral as a putative effector molecule of R-ras, H-ras, K-ras, and Rap.

    Spaargaren M and Bischoff JR

    ONYX Pharmaceuticals, Richmond, CA 94806.

    To identify proteins that bind to the Ras-related protein R-ras we performed a yeast two-hybrid cDNA library screen. Several clones were obtained encoding the C-terminal region of the guanine nucleotide dissociation stimulator for Ral (RalGDS). The R-ras-binding domain of RalGDS (RalGDS-RBD) is distinct from the conserved catalytic exchange factor regions. Using the two-hybrid system, we show that RalGDS-RBD interacts with H-ras, K-ras, and Rap, and with active but not with inactive point mutants of these Ras-like GTPases. Moreover, using purified proteins, we demonstrate the direct GTP-dependent interaction of the Ras-like GTPases with RalGDS-RBD and full-length RalGDS in vitro. Furthermore, we show that RalGDS-RBD and the Ras-binding domain of Raf-1 compete for binding to the Ras-like GTPases. These data indicate that RalGDS is a putative effector molecule for R-ras, H-ras, K-ras, and Rap.

    Proceedings of the National Academy of Sciences of the United States of America 1994;91;26;12609-13

  • RAS and RAF-1 form a signalling complex with MEK-1 but not MEK-2.

    Jelinek T, Catling AD, Reuter CW, Moodie SA, Wolfman A and Weber MJ

    Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville 22908.

    Recent studies have demonstrated the existence of a physical complex containing p21ras (RAS), p74raf-1 (RAF-1), and MEK-1. Although it is clear that formation of this complex depends on the activation state of RAS, it is not known whether this complex is regulated by the activation state of the cell and whether MEK-2 is also present in the complex. To analyze the regulation and specificity of this complex, we utilized immobilized RAS to probe lysates of cultured NIH 3T3 fibroblasts and analyzed the proteins complexing with RAS following serum starvation or stimulation. Complex formation among RAS, RAF-1, and MEK-1 was dependent only on RAS:GMP-PNP and not on cell stimulation. Incubations of lysates with immobilized RAS depleted all RAF-1 from the lysate but bound only a small fraction of cytosolic MEK-1, and further MEK-1 could bind immobilized RAS only if exogenous RAF-1 was added to the lysate. This indicates that binding of MEK-1 to RAS depends on the presence of RAF-1 or an equivalent protein. In contrast to MEK-1, MEK-2 was not detected in the RAS signalling complex. A proline-rich region of MEK-1 containing a phosphorylation site appears to be essential for signalling complex formation. Consistent with the preferential binding of MEK-1 to RAS:RAF-1, the basal activity of MEK-1 in v-ras-transformed cells was found to be elevated sixfold, whereas MEK-2 was elevated only twofold, suggesting that the RAS signalling pathway favors MEK-1 activation.

    Funded by: NCI NIH HHS: CA39076; NIGMS NIH HHS: GM 41220, GM 47332

    Molecular and cellular biology 1994;14;12;8212-8

  • Activated Ras interacts with the Ral guanine nucleotide dissociation stimulator.

    Hofer F, Fields S, Schneider C and Martin GS

    Department of Molecular and Cell Biology, University of California, Berkeley 94720-3204.

    The yeast two-hybrid system was used to identify proteins that interact with Ras. The H-Ras protein was found to interact with a guanine nucleotide dissociation stimulator (GDS) that has been previously shown to regulate guanine nucleotide exchange on another member of the Ras protein family, Ral. The interaction is mediated by the C-terminal, noncatalytic segment of the RalGDS and can be detected both in vivo, using the two-hybrid system, and in vitro, with purified recombinant proteins. The interaction of the RalGDS C-terminal segment with Ras is specific, dependent on activation of Ras by GTP, and blocked by a mutation that affects Ras effector function. These characteristics are similar to those previously demonstrated for the interaction between Ras and its putative effector, Raf, suggesting that the RalGDS may also be a Ras effector. Consistent with this idea, the RalGDS was found to inhibit the binding of Raf to Ras.

    Funded by: NCI NIH HHS: CA17542, CA28146

    Proceedings of the National Academy of Sciences of the United States of America 1994;91;23;11089-93

  • The ubiquitously expressed Syp phosphatase interacts with c-kit and Grb2 in hematopoietic cells.

    Tauchi T, Feng GS, Marshall MS, Shen R, Mantel C, Pawson T and Broxmeyer HE

    Department of Medicine (Hematology/Oncology), Indiana University School of Medicine, Indianapolis 46202.

    The c-kit proto-oncogene encodes a transmembrane tyrosine kinase receptor, which is important for the normal development of hematopoietic cells, melanoblasts, and germ cells. Autophosphorylation of c-kit receptor on tyrosine creates binding sites for cellular src homology 2 (SH2)-containing signaling molecules. The discovery of phosphotyrosine phosphatases that contain SH2 domains suggests roles for these molecules in growth factor signaling pathways. We found that Syp, a phosphotyrosine phosphatase widely expressed in all the tissues in mammals, associates with c-kit receptor after activation with its ligand, steel factor, in the factor-dependent cell line, M07e. Both NH2-terminal and COOH-terminal SH2 domains of Syp, made as glutathione S-transferase fusion proteins, were able to bind to the activated c-kit receptor in vitro. Furthermore, Syp became marginally phosphorylated on tyrosine upon c-kit receptor activation, and tyrosine-phosphorylated Syp was found to be complexed with Grb2 in steel factor-stimulated M07e cells. Direct binding between Syp and Grb2 was also observed in vitro. Last, Ras and Raf interacts in vitro as a result of steel factor-stimulated Ras activation. These results suggest that Syp may be an important signaling component downstream of the c-kit receptor and involved in activation of the Ras signaling pathway in hematopoietic cells.

    Funded by: NCI NIH HHS: R37 CA36464; NHLBI NIH HHS: R01 HL46549, R01 HL49202

    The Journal of biological chemistry 1994;269;40;25206-11

  • Critical binding and regulatory interactions between Ras and Raf occur through a small, stable N-terminal domain of Raf and specific Ras effector residues.

    Chuang E, Barnard D, Hettich L, Zhang XF, Avruch J and Marshall MS

    Department of Medicine, Indiana University, Indianapolis 46202.

    Genetic and biochemical evidence suggests that the Ras protooncogene product regulates the activation of the Raf kinase pathway, leading to the proposal that Raf is a direct mitogenic effector of activated Ras. Here we report the use of a novel competition assay to measure in vitro the relative affinity of the c-Raf-1 regulatory region for Ras-GTP, Ras-GDP, and 10 oncogenic and effector mutant Ras proteins. c-Raf-1 associates with normal Ras and the oncogenic V12 and L61 forms of Ras with equal affinity. The moderately transforming mutant Ras[E30K31] also bound to the c-Raf-1 regulatory region with normal affinity. Transformation-defective Ras effector mutants Ras[N33], Ras[S35], and Ras[N38] bound poorly. In contrast, the transformation defective Ras[G26I27] and Ras[E45] mutants bound to the c-Raf-1 regulatory region with nearly wild-type affinity. A stable, high-affinity Ras-binding region of c-Raf-1 was mapped to a 99-amino-acid subfragment of the first 257 residues. The smallest Ras-binding region identified consisted of N-terminal residues 51 to 131, although stable expression of the domain and high-affinity binding were improved by the presence of residues 132 to 149. Deletion of the Raf zinc finger region did not reduce Ras-binding affinity, while removal of the first 50 amino acids greatly increased affinity. Phosphorylation of Raf[1-149] by protein kinase A on serine 43 resulted in significant inhibiton of Ras binding. demonstrating that the mechanism of cyclic AMP downregulation results through structural changes occurring exclusively in this small Ras-binding domain.

    Funded by: NIDDK NIH HHS: DK07519

    Molecular and cellular biology 1994;14;8;5318-25

  • Solution structure and dynamics of ras p21.GDP determined by heteronuclear three- and four-dimensional NMR spectroscopy.

    Kraulis PJ, Domaille PJ, Campbell-Burk SL, Van Aken T and Laue ED

    Department of Biochemistry, University of Cambridge, U.K.

    A high-resolution solution structure of the GDP form of a truncated version of the ras p21 protein (residues 1-166) has been determined using NMR spectroscopy. Ras p21 is the product of the human ras protooncogene and a member of a ubiquitous eukaryotic gene family which is highly conserved in evolution. A virtually complete assignment (13C, 15N, and 1H), including stereospecific assignments of 54 C beta methylene protons and 10 C gamma methyl protons of valine residues, was obtained by analysis of three- and four-dimensional (3D and 4D) heteronuclear NMR spectra using a newly developed 3D/4D version of the ANSIG software. A total of 40 converged structures were computed from 3369 experimental restraints consisting of 3,167 nuclear Overhauser effect (NOE) derived distances, 14 phi and 54 chi 1 torsion angle restraints, 109 hydrogen bond distance restraints, and an additional 25 restraints derived from literature data defining interactions between the GDP ligand, the magnesium ion, and the protein. The structure in the region of residues 58-66 (loop L4), and to a lesser degree residues 30-38 (loop L2), is ill-defined. Analysis of the dynamics of the backbone 15N nuclei in the protein showed that residues within the regions 58-66, 107-109, and, to a lesser degree, 30-38 are dynamically mobile on the nanosecond time scale. The root mean square (rms) deviations between the 40 solution structures and the mean atomic coordinates are 0.78 A for the backbone heavy atoms and 1.29 A for all non-hydrogen atoms if all residues (1-166) are included in the analysis. If residues 30-38 and residues 58-66 are excluded from the analysis, the rms deviations are reduced to 0.55 and 1.00 A, respectively. The structure was compared to the most highly refined X-ray crystal structure of ras p21.GDP (1-189) [Milburn, M. V., Tong, L., de Vos, A. M., Brünger, A. T., Yamaizumi, Z., Nishimura, S., & Kim, S.-H. (1990) Science 24, 939-945]. The structures are very similar except in the regions found to be mobile by NMR spectroscopy. In addition, the second alpha-helix (helix-2) has a slightly different orientation. The rms deviation between the average of the solution structures and the X-ray crystal structure is 0.94 A for the backbone heavy atoms if residues 31-37 and residues 59-73 are excluded from the analysis.

    Biochemistry 1994;33;12;3515-31

  • JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain.

    Dérijard B, Hibi M, Wu IH, Barrett T, Su B, Deng T, Karin M and Davis RJ

    Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester 01605.

    The ultraviolet (UV) response of mammalian cells is characterized by a rapid and selective increase in gene expression mediated by AP-1 and NF-kappa B. The effect on AP-1 transcriptional activity results, in part, from enhanced phosphorylation of the c-Jun NH2-terminal activation domain. Here, we describe the molecular cloning and characterization of JNK1, a distant relative of the MAP kinase group that is activated by dual phosphorylation at Thr and Tyr during the UV response. Significantly, Ha-Ras partially activates JNK1 and potentiates the activation caused by UV. JNK1 binds to the c-Jun transactivation domain and phosphorylates it on Ser-63 and Ser-73. Thus, JNK1 is a component of a novel signal transduction pathway that is activated by oncoproteins and UV irradiation. These properties indicate that JNK1 activation may play an important role in tumor promotion.

    Funded by: NCI NIH HHS: CA50528, CA58396; NIGMS NIH HHS: GM37845

    Cell 1994;76;6;1025-37

  • Interaction of Ras and Raf in intact mammalian cells upon extracellular stimulation.

    Hallberg B, Rayter SI and Downward J

    Imperial Cancer Research Fund, London, United Kingdom.

    It has recently been shown that Ras proteins interact directly with Raf serine/threonine kinases in vitro and in the yeast two-hybrid system, leading to speculation that Raf proteins function as effectors for Ras. Here it is demonstrated that the endogenous Raf-1 protein co-immunoprecipitates with Ras from mammalian cells when the non-neutralizing anti-Ras monoclonal antibody Y13-238 is used. The formation of a Ras-Raf complex is absolutely dependent on prior treatment of the cells with a stimulus that activates Ras: phorbol ester or anti-T cell receptor antibody in the case of human peripheral blood T lymphoblasts, or epidermal growth factor in the case of Rat-1 fibroblasts. Up to 3% of cellular Raf-1 can be found in association with Ras. The association is not competed by addition of exogenous GST-Raf to the cell lysates and is therefore unlikely to be due to Ras-Raf binding after cell lysis. Specific interaction of Ras and Raf therefore occurs in intact mammalian cells in response to stimuli that cause Ras to become GTP-bound.

    The Journal of biological chemistry 1994;269;6;3913-6

  • Molecular cloning of the mouse grb2 gene: differential interaction of the Grb2 adaptor protein with epidermal growth factor and nerve growth factor receptors.

    Suen KL, Bustelo XR, Pawson T and Barbacid M

    Department of Molecular Biology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000.

    We report the isolation and molecular characterization of the mouse grb2 gene. The product of this gene, the Grb2 protein, is highly related to the Caenorhabditis elegans sem-5 gene product and the human GRB2 protein and displays the same SH3-SH2-SH3 structural motifs. In situ hybridization studies revealed that the mouse grb2 gene is widely expressed throughout embryonic development (E9.5 to P0). However, grb2 transcripts are not uniformly distributed, and in certain tissues (e.g., thymus) they appear to be regulated during development. Recent genetic and biochemical evidence has implicated the Grb2 protein in the signaling pathways that link cell surface tyrosine kinase receptors with Ras. We have investigated the association of the Grb2 protein with epidermal growth factor (EGF) and nerve growth factor (NGF) receptors in PC12 pheochromocytoma cells. EGF treatment of PC12 cells results in the rapid association of Grb2 with the activated EGF receptors, an interaction mediated by the Grb2 SH2 domain. However, Grb2 does not bind to NGF-activated Trk receptors. Mitogenic signaling of NGF in NIH 3T3 cells ectopically expressing Trk receptors also takes place without detectable association between Grb2 and Trk. These results suggest that whereas EGF and NGF can activate the Ras signaling pathway in PC12 cells, only the EGF receptor is likely to do so through a direct interaction with Grb2. Finally, binding studies with glutathione S-transferase fusion proteins indicate that Grb2 binds two distinct subsets of proteins which are individually recognized by its SH2 and SH3 domains. These observations add further support to the concept that Grb2 is a modular adaptor protein.

    Molecular and cellular biology 1993;13;9;5500-12

  • Direct interaction of Ras and the amino-terminal region of Raf-1 in vitro.

    Warne PH, Viciana PR and Downward J

    Imperial Cancer Research Fund, London, UK.

    The Ras proteins are key regulators of the growth of eukaryotic cells, but their direct target enzymes, or 'effectors', are unknown. The protein encoded by the c-raf-1 proto-oncogene is thought to function downstream of p21ras because disruption of Raf blocks signalling by Ras in a number of systems. Here we report that the amino-terminal cysteine-rich regulatory region of p74c-raf-1 expressed as a glutathione-S-transferase (GST) fusion protein binds directly to Ras with relatively high affinity (50 nM). The binding is strictly dependent on the Ras protein being in the active GTP-bound conformation rather than the inactive GDP-bound state. Raf-GST interacts with wild-type and oncogenic Ras (Val 12) but fails to interact with a biologically inert effector mutant of Ras (Ala 38) and a dominant negative mutant (Asn 17). A peptide based on the effector region of Ras inhibits the interaction. Raf-GST acts as a potent competitive inhibitor of the GTPase-activating proteins p120GAP and neurofibromin. In addition, Raf itself displays weak GTPase-stimulating activity towards Ras. It is therefore likely that Raf is a direct effector of Ras.

    Nature 1993;364;6435;352-5

  • Normal and oncogenic p21ras proteins bind to the amino-terminal regulatory domain of c-Raf-1.

    Zhang XF, Settleman J, Kyriakis JM, Takeuchi-Suzuki E, Elledge SJ, Marshall MS, Bruder JT, Rapp UR and Avruch J

    Diabetes Unit, Harvard Medical School, Charlestown, Massachusetts.

    In higher eukaryotes, the Ras and Raf-1 proto-oncoproteins transduce growth and differentiation signals initiated by tyrosine kinases. The Ras polypeptide and the amino-terminal regulatory domain of Raf-1 (residues 1-257) are shown to interact, directly in vitro and in a yeast expression system. Raf-1 (1-257) binds GTP-Ras in preference to GDP-Ras, and inhibits Ras-GAP activity. Mutations in and around the Ras effector domain impair Ras binding to Raf-1 (1-257) and Ras transforming activity in parallel.

    Nature 1993;364;6435;308-13

  • Detection of high incidence of H-RAS oncogene point mutations in acute myelogenous leukemia.

    Imamura N, Kuramoto A, Ishihara H and Shimizu S

    Department of Internal Medicine, Hiroshima University, Japan.

    We have been analyzing RAS p21 proteins and the DNA sequence of leukemic cells. We report here that these cells have high expression of H-RAS p21, which originates from point mutations of RAS oncogenes. The leukemic cells from six patients with acute myelogenous leukemia were separated from heparinized whole blood and bone marrow by a density gradient technique. The expression of RAS oncogenes was analyzed by a fluorescence-activated cell sorting with a panel of monoclonal antibodies. The high expression of DWP, which was reported to recognized activated RAS oncogene, was found in two patients and was associated with high levels of H-RAS expression. These facts prompted us to analyze the DNA sequence of RAS genes with an automated DNA sequencer. Unexpectedly, various kinds of H-RAS point mutations were found in all six cases, including two cases of hot-spot point mutation at codon 12, whereas K-RAS point mutation (no hot-spot point mutations) was found in six cases. The same H-RAS point mutations, at codons 10, 11, and 15, were found in all six cases. To our knowledge, there is no report on H-RAS point mutation in human leukemias. On the basis of these findings, we suggest that H-RAS point mutation together with p53 gene mutation may play an important role in leukemogenesis.

    American journal of hematology 1993;43;2;151-3

  • Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2.

    Chardin P, Camonis JH, Gale NW, van Aelst L, Schlessinger J, Wigler MH and Bar-Sagi D

    Institut de Pharmacologie Moleculaire et Cellulaire, CNRS, Valbonne, France.

    A human complementary DNA was isolated that encodes a widely expressed protein, hSos1, that is closely related to Sos, the product of the Drosophila son of sevenless gene. The hSos1 protein contains a region of significant sequence similarity to CDC25, a guanine nucleotide exchange factor for Ras from yeast. A fragment of hSos1 encoding the CDC25-related domain complemented loss of CDC25 function in yeast. This hSos1 domain specifically stimulated guanine nucleotide exchange on mammalian Ras proteins in vitro. Mammalian cells overexpressing full-length hSos1 had increased guanine nucleotide exchange activity. Thus hSos1 is a guanine nucleotide exchange factor for Ras. The hSos1 interacted with growth factor receptor-bound protein 2 (GRB2) in vivo and in vitro. This interaction was mediated by the carboxyl-terminal domain of hSos1 and the Src homology 3 (SH3) domains of GRB2. These results suggest that the coupling of receptor tyrosine kinases to Ras signaling is mediated by a molecular complex consisting of GRB2 and hSos1.

    Funded by: NCI NIH HHS: CA46370, CA55360

    Science (New York, N.Y.) 1993;260;5112;1338-43

  • Mutations of the p53 tumor suppressor gene and the ras gene family in intrahepatic cholangiocellular carcinomas in Japan and Thailand.

    Kiba T, Tsuda H, Pairojkul C, Inoue S, Sugimura T and Hirohashi S

    Pathology Division, National Cancer Center Research Institute, Tokyo, Japan.

    The incidence and pattern of mutations of the ras oncogenes and the p53 tumor suppressor gene have been shown to differ among different cancer types and even among the same cancer types with different etiological backgrounds. For example, in a previous study we showed that not only the etiology but also the incidence of point mutation of the c-Ki-ras oncogene in cholangiocellular carcinomas (CCCs) differ between Japanese and Thai patients. In the study presented here, we examined the incidence of mutations in the ras gene family and the p53 gene in CCCs of both Japanese and Thai patients by single-strand conformation polymorphism and direct sequencing analyses and compared the pattern of p53 mutation between these two CCC groups. Although the incidence of ras mutation differed markedly between Japanese (seven of 12, 58%) and Thai (two of 26, 8%) cases, the incidence of p53 mutation was similar: four of 12 (33%) and nine of 26 (35%), respectively. Except for one case in which deletion-insertion was detected in the second exon of the N-ras gene, all ras mutations occurred at codon 12 or 13 of the c-Ki-ras gene. All p53 mutations but one were detected in a highly conserved region, and the predominant form of the mutations was G:C-->A:T transition at CpG sites in both Japanese and Thai cases, similar to that reported for colorectal cancers. Therefore, in contrast to the ras oncogenes, mutation of the p53 gene was frequently involved in the development of CCCs in both Japanese and Thai patients, irrespective of any difference in etiology.

    Molecular carcinogenesis 1993;8;4;312-8

  • The p53 tumor-suppressor gene and ras oncogene mutations in oral squamous-cell carcinoma.

    Sakai E, Rikimaru K, Ueda M, Matsumoto Y, Ishii N, Enomoto S, Yamamoto H and Tsuchida N

    Department of Molecular Cellular Oncology and Microbiology, Faculty of Dentistry, Tokyo Medical and Dental University, Japan.

    The frequencies of mutations in the p53 tumor-suppressor gene and ras proto-oncogenes were investigated systematically in surgically resected oral squamous-cell carcinomas (SCCs) using single-strand conformation polymorphism (SSCP) and/or dot-blot hybridization analysis of DNA fragments which had been amplified by the polymerase chain reaction (PCR). p53 gene mutations, within the region of exons 5 to 8, were detected in 17 out of 27 (63%) tumor specimens. The role of p53 mutations in cell-line establishment was investigated. p53 gene mutations were detected in 5 out of 6 tissue samples from which cell lines were established and in 4 out of 5 specimens from which cell lines could not be established, suggesting that the presence of p53 gene mutations is not by itself sufficient for cell-line establishment. Tumor samples were also analyzed for point mutational activation of the ras proto-oncogenes. One out of 30 (3%) tumors showed an activating point mutation in codon 12 of H-ras, this being consistent with reports from Europe and USA but not with any from India. Compared to frequencies of the other genetic changes so far reported for oral SCC, the p53 mutations have been observed most often to undergo genetic change. p53 gene mutation is thus intimately involved in the genesis of oral SCC and consequently should be useful as a marker for the diagnosis of this neoplasm.

    International journal of cancer 1992;52;6;867-72

  • PCR detection of an insertion/deletion polymorphism in intron 1 of the HRAS1 locus.

    Tanci P, Genuardi M, Santini SA and Neri G

    Istituto di Genetica Umana, Università Cattolica del S.Cuore, Rome, Italy.

    Nucleic acids research 1992;20;5;1157

  • Posttranslational modification of Ha-ras p21 by farnesyl versus geranylgeranyl isoprenoids is determined by the COOH-terminal amino acid.

    Kinsella BT, Erdman RA and Maltese WA

    Weis Center for Research, Geisinger Clinic, Danville, PA 17822.

    ras proteins undergo posttranslational modification by a 15-carbon farnesyl isoprenoid at a cysteine within a defined COOH-terminal amino acid motif; i.e., Cys-Ali-Ali-Ser/Met (where Ali represents an aliphatic residue). In other low molecular mass GTP-binding proteins, cysteines are modified by 20-carbon geranylgeranyl groups within a Cys-Ali-Ali-Leu motif. We changed the terminal Ser-189 of Ha-ras p21 to Leu-189 by site-directed mutagenesis and found that the protein was modified by [3H]geranylgeranyl instead of [3H]farnesyl in an in vitro assay. Gel-permeation chromatography of [3H]mevalonate-labeled hydrocarbons released from immunoprecipitated ras proteins overexpressed in COS cells indicated that Ha-ras p21(Leu-189) was also a substrate for 20-carbon isoprenyl modification in vivo. Additional steps in Ha-ras p21 processing, normally initiated by farnesylation, appear to be supported by geranylgeranylation, based on metabolic labeling of Ha-ras p21(Leu-189) with [3H]palmitate and its subcellular localization in a particulate fraction from COS cells. These observations indicate that the amino acid occupying the terminal position (Xaa) in the Cys-Ali-Ali-Xaa motif constitutes a key structural feature by which Ha-ras p21 and other proteins with ras-like COOH-terminal isoprenylation sites are distinguished as substrates for farnesyl- or geranylgeranyltransferases.

    Funded by: NCI NIH HHS: CA-34569

    Proceedings of the National Academy of Sciences of the United States of America 1991;88;20;8934-8

  • Crystal structures at 2.2 A resolution of the catalytic domains of normal ras protein and an oncogenic mutant complexed with GDP.

    Tong LA, de Vos AM, Milburn MV and Kim SH

    Department of Chemistry, University of California, Berkeley 94720.

    The biological functions of ras proteins are controlled by the bound guanine nucleotide GDP or GTP. The GTP-bound conformation is biologically active, and is rapidly deactivated to the GDP-bound conformation through interaction with GAP (GTPase Activating Protein). Most transforming mutants of ras proteins have drastically reduced GTP hydrolysis rates even in the presence of GAP. The crystal structures of the GDP complexes of ras proteins at 2.2 A resolution reveal the detailed interaction between the ras proteins and the GDP molecule. All the currently known transforming mutation positions are clustered around the bound guanine nucleotide molecule. The presumed "effector" region and the GAP recognition region are both highly exposed. No significant structural differences were found between the GDP complexes of normal ras protein and the oncogenic mutant with valine at position 12, except the side-chain of the valine residue. However, comparison with GTP-analog complexes of ras proteins suggests that the valine side-chain may inhibit GTP hydrolysis in two possible ways: (1) interacting directly with the gamma-phosphate and altering its orientation or the conformation of protein residues around the phosphates; and/or (2) preventing either the departure of gamma-phosphate on GTP hydrolysis or the entrance of a nucleophilic group to attack the gamma-phosphate. The structural similarity between ras protein and the bacterial elongation factor Tu suggests that their common structural motif might be conserved for other guanine nucleotide binding proteins.

    Funded by: NCI NIH HHS: CA 45593

    Journal of molecular biology 1991;217;3;503-16

  • Modulation of guanine nucleotides bound to Ras in NIH3T3 cells by oncogenes, growth factors, and the GTPase activating protein (GAP).

    Gibbs JB, Marshall MS, Scolnick EM, Dixon RA and Vogel US

    Department of Molecular Biology, Merck, Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.

    The mitogenic activity of membrane-associated tyrosine kinases such as Src and the PDGF receptor appear to depend on Ras function. Ras biochemical activity involves regulation of a GTP/GDP cycle and the GTPase activating protein (GAP). Recently, PDGF and v-Src have been shown to stimulate tyrosine phosphorylation of GAP, linking these pathways at the biochemical level. To test whether PDGF and v-Src affect the Ras GTP/GDP cycle, we have measured the guanine nucleotides complexed to Ras in NIH3T3 cells and compared the ratio of GTP to total GTP + GDP detected (percent GTP). In normal quiescent NIH3T3 cells, PDGF stimulated the basal amount of GTP complexed to Ras (7%) by 2.1-fold to 15%. The effect was dependent on PDGF concentration and was observed maximally within 10 min following PDGF challenge. Ras was complexed to 22% GTP in NIH3T3 cells transformed by v-src or v-abl. Overexpression of GAP by 110-fold in NIH3T3 cells reduced the basal level of GTP complexed to Ras to 2.4%; upon challenge with PDGF, Ras was complexed to 6.6% GTP. These results indicate that PDGF receptor activation and tyrosine kinase-encoding oncogene products can stimulate Ras into the GTP complex and that GAP in intact mammalian cells can decrease the amount of GTP complexed to Ras.

    The Journal of biological chemistry 1990;265;33;20437-42

  • Identification and preliminary characterization of protein-cysteine farnesyltransferase.

    Manne V, Roberts D, Tobin A, O'Rourke E, De Virgilio M, Meyers C, Ahmed N, Kurz B, Resh M, Kung HF et al.

    Department of Molecular Biology, Squibb Institute for Medical Research, Princeton, NJ 08543-4000.

    Ras proteins must be isoprenylated at a conserved cysteine residue near the carboxyl terminus (Cys-186 in mammalian Ras p21 proteins) in order to exert their biological activity. Previous studies indicate that an intermediate in the mevalonate pathway, most likely farnesyl pyrophosphate, is the donor of this isoprenyl group. Inhibition of mevalonate synthesis reverts the abnormal phenotypes induced by the mutant RAS2Val-19 gene in Saccharomyces cerevisiae and blocks the maturation of Xenopus oocytes induced by an oncogenic Ras p21 protein of human origin. These results have raised the possibility of using inhibitors of the mevalonate pathway to block the transforming properties of ras oncogenes. Unfortunately, mevalonate is a precursor of various end products essential to mammalian cells, such as dolichols, ubiquinones, heme A, and cholesterol. In this study, we describe an enzymatic activity(ies) capable of catalyzing the farnesylation of unprocessed Ras p21 proteins in vitro at the correct (Cys-186) residue. This farnesylating activity is heat-labile, requires Mg2+ or Mn2+ ions, is linear with time and with enzyme concentration, and is present in all mammalian cell lines and tissues tested. Gel filtration analysis of a partially purified preparation of protein farnesyltransferase revealed two peaks of activity at 250-350 kDa and 80-130 kDa. Availability of an in vitro protein farnesyltransferase assay should be useful in screening for potential inhibitors of ras oncogene function that will not interfere with other aspects of the mevalonate pathway.

    Proceedings of the National Academy of Sciences of the United States of America 1990;87;19;7541-5

  • Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 A resolution: implications for the mechanism of GTP hydrolysis.

    Pai EF, Krengel U, Petsko GA, Goody RS, Kabsch W and Wittinghofer A

    Max-Planck-Institut für medizinische Forschung, Abteilung Biophysik, Heidelberg, FRG.

    The crystal structure of the H-ras oncogene protein p21 complexed to the slowly hydrolysing GTP analogue GppNp has been determined at 1.35 A resolution. 211 water molecules have been built into the electron density. The structure has been refined to a final R-factor of 19.8% for all data between 6 A and 1.35 A. The binding sites of the nucleotide and the magnesium ion are revealed in high detail. For the stretch of amino acid residues 61-65, the temperature factors of backbone atoms are four times the average value of 16.1 A2 due to the multiple conformations. In one of these conformations, the side chain of Gln61 makes contact with a water molecule, which is perfectly placed to be the nucleophile attacking the gamma-phosphate of GTP. Based on this observation, we propose a mechanism for GTP hydrolysis involving mainly Gln61 and Glu63 as activating species for in-line attack of water. Nucleophilic displacement is facilitated by hydrogen bonds from residues Thr35, Gly60 and Lys16. A mechanism for rate enhancement by GAP is also proposed.

    The EMBO journal 1990;9;8;2351-9

  • Unique guanine nucleotide binding properties of the human placental GTP-binding protein Gp.

    Tamir A, Fawzi AB and Northup JK

    Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510.

    Gp is a major GTP-binding protein of human placenta and platelets [Evans, T., Brown, M. L., Fraser, E. D., & Northup, J. K. (1986) J. Biol. Chem. 261, 7052-7059]. High-affinity guanine nucleotide binding is associated with a polypeptide migrating identically with H-ras on SDS-PAGE. We have characterized the interactions of preparations of purified human placental Gp with guanine nucleotides in detergent solution. Equilibrium binding studies with [35S]GTP gamma S, [3H]Gpp(NH)p, and [3H]GTP identified a single class of sites with a dissociation constant of 10 +/- 1, 153 +/- 61, and 125 +/- 77 nM for the ligands, respectively. These three ligands were mutually competitive with Ki values consistent with the Kd values from direct binding experiments. Competition for the binding of [3H]Gpp(NH)p was used to determine the specificity of the site. Ki values determined from this assay were 14 nM for GTP gamma S, 143 nM for Gpp(NH)p, 3.3 microM for GDP beta S, 69 nM for GTP, and 64 nM for GDP. ATP, ADP, cAMP, cGMP, and NAD+ had no detectable affinity for this site. While the equilibrium binding data fit well to a single class of sites, association kinetics of these ligands were better fit to two rate constants. Dissociation kinetics, however, were not clearly resolved into two rates.(ABSTRACT TRUNCATED AT 250 WORDS)

    Funded by: NIGMS NIH HHS: GM40154; OCPHP CDC HHS: PR 05358

    Biochemistry 1990;29;30;6947-54

  • Transforming genes from familial adenomatous polyposis patient cells detected by a tumorigenicity assay.

    Yuasa Y, Kamiyama T, Kato M, Iwama T, Ikeuchi T and Tonomura A

    Department of Hygiene and Oncology, School of Medicine, Tokyo Medical and Dental University, Japan.

    We tried to detect oncogenes associated with familial adenomatous polyposis by a tumorigenicity assay in nude mice. One polyp and two peripheral blood lymphocyte DNAs out of 12 samples from patients induced Alu-positive tumors. Lymphocyte DNAs from one of 5 healthy people also showed tumorigenic activity. The transforming genes of polyps from a patient and lymphocytes from a normal person were found to be the human N-ras gene. Since these N-ras genes were amplified in nude mouse tumors and did not show any alterations in the nucleotide sequences around codons 12 and 61, it is likely that the tumors were induced by the amplified normal N-ras genes. The transforming sequences from two patients' lymphocytes did not hybridize with 12 known oncogene probes, suggesting that these two genes are novel oncogenes or genes for which we have not yet examined the homology. One oncogene derived from a patient's lymphocytes was partially cloned and shown to be located on human chromosome 7. This gene did not hybridize with the met and erbB1 genes, which are potential oncogenes located on chromosome 7. These data indicate that this gene is a new oncogene.

    Oncogene 1990;5;4;589-96

  • Structure of the guanine-nucleotide-binding domain of the Ha-ras oncogene product p21 in the triphosphate conformation.

    Pai EF, Kabsch W, Krengel U, Holmes KC, John J and Wittinghofer A

    Max-Planck-Institut für medizinische Forschung, Abteilung Biophysik, Heidelberg, FRG.

    The crystal structure of the guanine-nucleotide-binding domain of p21 (amino acids 1-166) complexed to the guanosine triphosphate analogue guanosine-5'-(beta, gamma-imido)triphosphate (GppNp) has been determined at a resolution of 2.6 A. The topological order of secondary structure elements is the same as that of the guanine-nucleotide-binding domain of bacterial elongation factor EF-Tu. Many interactions between nucleotide and protein have been identified. The effects of point mutations and the conservation of amino-acid sequence in the guanine-nucleotide-binding proteins are discussed.

    Nature 1989;341;6239;209-14

  • All ras proteins are polyisoprenylated but only some are palmitoylated.

    Hancock JF, Magee AI, Childs JE and Marshall CJ

    Section of Cell and Molecular Biology, Royal Cancer Hospital, Chester Beatty Laboratories, London, England.

    The C-terminal CAAX motif of the yeast mating factors is modified by proteolysis to remove the three terminal amino acids (-AAX) leaving a C-terminal cysteine residue that is polyisoprenylated and carboxyl-methylated. Here we show that all ras proteins are polyisoprenylated on their C-terminal cysteine (Cys186). Mutational analysis shows palmitoylation does not take place on Cys186 as previously thought but on cysteine residues contained in the hypervariable domain of some ras proteins. The major expressed form of c-K-ras (exon 4B) does not have a cysteine residue immediately upstream of Cys186 and is not palmitoylated. Polyisoprenylated but nonpalmitoylated H-ras proteins are biologically active and associate weakly with cell membranes. Palmitoylation increases the avidity of this binding and enhances their transforming activity. Polyisoprenylation is essential for biological activity as inhibiting the biosynthesis of polyisoprenoids abolishes membrane association of p21ras.

    Cell 1989;57;7;1167-77

  • Activation of oncogenes in human oral cancer cells: a novel codon 13 mutation of c-H-ras-1 and concurrent amplifications of c-erbB-1 and c-myc.

    Tadokoro K, Ueda M, Ohshima T, Fujita K, Rikimaru K, Takahashi N, Enomoto S and Tsuchida N

    Department of Oral Microbiology, Faculty of Dentistry, Tokyo Medical and Dental University, Japan.

    By NIH3T3 transfection assay in conjunction with in vitro transient neomycin selection, activated c-H-ras-1 oncogenes were detected in two squamous cell carcinoma cell lines, ZA and HOC-313, newly established from human oral cancer patients. ZA had a point mutational activation at the 13th codon, this activation of c-H-ras-1 being novel in human cancer cells, while HOC-313 appeared to have an activation at the 12th codon. In ZA, 16- to 32-fold amplification of the EGF receptor gene, c-erbB-1 and a few-fold amplification of c-myc were detected. The significance of these findings is discussed in relation to multistep carcinogenesis in human cells.

    Oncogene 1989;4;4;499-505

  • Characterization of mutagen-activated cellular oncogenes that confer anchorage independence to human fibroblasts and tumorigenicity to NIH 3T3 cells: sequence analysis of an enzymatically amplified mutant HRAS allele.

    Stevens CW, Manoharan TH and Fahl WE

    McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.

    Treatment of diploid human fibroblasts with an alkylating mutagen has been shown to induce stable, anchorage-independent cell populations at frequencies (11 X 10(-4) consistent with an activating mutation. After treatment of human foreskin fibroblasts with the mutagen benzo[a]pyrene (+/-)anti- 7,8-dihydrodiol 9,10-epoxide and selection in soft agar, 17 anchorage-independent clones were isolated and expanded, and their cellular DNA was used to cotransfect NIH 3T3 cells along with pSV2neo. DNA from 11 of the 17 clones induced multiple NIH 3T3 cell tumors in recipient nude mice. Southern blot analyses showed the presence of human Alu repetitive sequences in all of the NIH 3T3 tumor cell DNAs. Intact, human HRAS sequences were observed in 2 of the 11 tumor groups, whereas no hybridization was detected when human KRAS or NRAS probes were used. Slow-migrating ras p21 proteins, consistent with codon 12 mutations, were observed i in the same two NIH 3T3 tumor cell groups that contained the human HRAS bands. Genomic DNA from one of these two human anchorage-independent cell populations (clone 21A) was used to enzymatically amplify a portion of exon 1 of the HRAS gene. Direct sequence analysis of the amplified DNA indicated equal presence of a wild-type (GGC) and mutant (GTC) allele of the HRAS gene. The results demonstrate that exposure of normal human cells to a common environmental mutagen yields HRAS GC----TA codon 12 transversions that have been commonly observed in human tumors. This oncogene as well as yet to be identified oncogene are also shown to stably confer anchorage-independence to human cells.

    Funded by: NCI NIH HHS: P30-CA07175, R37-CA42024

    Proceedings of the National Academy of Sciences of the United States of America 1988;85;11;3875-9

  • Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain.

    Adari H, Lowy DR, Willumsen BM, Der CJ and McCormick F

    Department of Molecular Biology, Cetus Corporation, Emeryville, CA 94608.

    A cytoplasmic protein that greatly enhances the guanosine triphosphatase (GTPase) activity of N-ras protein but does not affect the activity of oncogenic ras mutants has been recently described. This protein (GAP) is shown here to be ubiquitous in higher eukaryotes and to interact with H-ras as well as with N-ras proteins. To identify the region of ras p21 with which GAP interacts, 21 H-ras mutant proteins were purified and tested for their ability to undergo stimulation of GTPase activity by GAP. Mutations in nonessential regions of H-ras p21 as well as mutations in its carboxyl-terminal domain (residues 165-185) and purine binding region (residues 117 and 119) did not decrease the ability of the protein to respond to GAP. In addition, an antibody against the carboxyl-terminal domain did not block GAP activity, supporting the conclusion that GAP does not interact with this region. Transforming mutations at positions 12, 59, and 61 (the phosphoryl binding region) abolished GTPase stimulation by GAP. Point mutations in the putative effector region of ras p21 (amino acids 35, 36, and 38) were also insensitive to GAP. However, a point mutation at position 39, shown previously not to impair effector function, did not alter GAP-p21 interaction. These results indicate that GAP