G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
G00000876 (Mus musculus)

Databases (8)

Curated Gene
OTTHUMG00000004093 (Vega human gene)
ENSG00000175793 (Ensembl human gene)
2810 (Entrez Gene)
491 (G2Cdb plasticity & disease)
SFN (GeneCards)
601290 (OMIM)
Marker Symbol
HGNC:10773 (HGNC)
Protein Sequence
P31947 (UniProt)

Synonyms (1)


Literature (102)

Pubmed - other

  • Outcome prediction and risk assessment by quantitative pyrosequencing methylation analysis of the SFN gene in advanced stage, high-risk, neuroblastic tumor patients.

    Banelli B, Bonassi S, Casciano I, Mazzocco K, Di Vinci A, Scaruffi P, Brigati C, Allemanni G, Borzì L, Tonini GP and Romani M

    Laboratory of Tumor Genetics, Istituto Nazionale per la Ricerca sul Cancro, IST, Genova, Italy.

    The aim of our study was to identify threshold levels of DNA methylation predictive of the outcome to better define the risk group of stage 4 neuroblastic tumor patients. Quantitative pyrosequencing analysis was applied to a training set of 50 stage 4, high risk patients and to a validation cohort of 72 consecutive patients. Stage 4 patients at lower risk and ganglioneuroma patients were included as control groups. Predictive thresholds of methylation were identified by ROC curve analysis. The prognostic end points of the study were the overall and progression-free survival at 60 months. Data were analyzed with the Cox proportional hazard model. In a multivariate model the methylation threshold identified for the SFN gene (14.3.3sigma) distinguished the patients presenting favorable outcome from those with progressing disease, independently from all known predictors (Training set: Overall Survival HR 8.53, p = 0.001; Validation set: HR 4.07, p = 0.008). The level of methylation in the tumors of high-risk patients surviving more than 60 months was comparable to that of tumors derived from lower risk patients and to that of benign ganglioneuroma. Methylation above the threshold level was associated with reduced SFN expression in comparison with samples below the threshold. Quantitative methylation is a promising tool to predict survival in neuroblastic tumor patients. Our results lead to the hypothesis that a subset of patients considered at high risk-but displaying low levels of methylation-could be assigned at a lower risk group.

    International journal of cancer 2010;126;3;656-68

  • Up-regulation of 14-3-3sigma (Stratifin) is associated with high-grade CIN and high-risk human papillomavirus (HPV) at baseline but does not predict outcomes of HR-HPV infections or incident CIN in the LAMS study.

    Syrjänen S, Naud P, Sarian L, Derchain S, Roteli-Martins C, Longatto-Filho A, Tatti S, Branca M, Erzen M, Hammes LS, Costa S and Syrjänen K

    Department of Oncology & Radiotherapy, Turku University Hospital, Turku, Finland.

    To assess whether the potentially high-risk (HR) human papillomavirus (HPV)-related up-regulation of 14-3-3sigma (stratifin) has implications in the outcome of HPV infections or cervical intraepithelial neoplasia (CIN) lesions, cervical biopsy specimens from 225 women in the Latin American Screening Study were analyzed for 14-3-3sigma expression using immunohistochemical analysis. We assessed its associations with CIN grade and HR HPV at baseline and value in predicting outcomes of HR-HPV infections and the development of incident CIN 1+ and CIN 2+. Expression of 14-3-3sigma increased in parallel with the lesion grade. Up-regulation was also significantly related to HR-HPV detection (P = .004; odds ratio, 2.71; 95% confidence interval, 1.37-5.35) and showed a linear relationship to HR-HPV loads (P = .003). 14-3-3sigma expression was of no value in predicting the outcomes (incident, persistent, clearance) of HR-HPV infections or incident CIN 1+ and CIN 2+. 14-3-3sigma is not inactivated in cervical carcinoma and CIN but is up-regulated on transition from CIN 2 to CIN 3. Its normal functions in controlling G(1)/S and G(2)/M checkpoints are being bypassed by HR HPV.

    American journal of clinical pathology 2010;133;2;232-40

  • Targeted proteomic analysis of 14-3-3sigma in nasopharyngeal carcinoma.

    Huang WG, Cheng AL, Chen ZC, Peng F, Zhang PF, Li MY, Li F, Li JL, Li C, Yi H, Li XH, Yi B and Xiao ZQ

    Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, Changsha 410008, China.

    14-3-3sigma is a potential tumor suppressor, and loss of 14-3-3sigma expression plays an important role in carcinogenesis and metastasis. To explore the possible mechanism of 14-3-3sigma in nasopharyngeal carcinoma (NPC) invasion and metastasis, targeted proteomic analysis was performed on 14-3-3sigma-associated proteins from NPC cells. As the results, 112 proteins associated with 14-3-3sigma were identified, and four 14-3-3sigma-interacted proteins: keratin 8, epidermal growth factor receptor (EGFR), small GTP-binding protein RAB7, and p53 were confirmed by coimmunoprecipitation and Western blot analysis. The 14-3-3sigma-associated proteins could be grouped into eight clusters based on their molecule functions. Protein-protein interaction (PPI) analysis indicated that 14-3-3sigma/EGFR/keratin 8 interactions may be involved in the invasion and metastasis of NPC. 14-3-3sigma/EGFR/keratin 8 could form complexes in NPC cells. 14-3-3sigma downregulation in NPC may lead to the overexpression of EGFR and keratin 8, which increases the invasion ability of NPC cells possibly by activating the downstream signal molecules and reorganizing cytoskeleton. The data suggest that the biological functions of 14-3-3sigma in NPC are diversified, and 14-3-3sigma could inhibit the in vitro invasive ability of NPC cells possibly through 14-3-3sigma/EGFR/keratin 8 interaction.

    The international journal of biochemistry & cell biology 2010;42;1;137-47

  • 14-3-3sigma and p21 synergize to determine DNA damage response following Chk2 inhibition.

    Meng S, Arbit T, Veeriah S, Mellinghoff IK, Fang F, Vivanco I, Rohle D and Chan TA

    Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.

    DNA damage checkpoints are critical for preventing tumorigenesis and regulating the response of cells to genotoxic agents. It is believed that the coordinated actions of a number of effectors underlie proper checkpoint function. The kinase Chk2, p21 and 14-3-3sigma have each been shown to be independent effectors of the G(2) DNA damage checkpoint. However, the relative roles of these proteins remain unclear. To help elucidate this question, we have perturbed each of these 3 genes in combination in human cells. We show that Chk2 depletion causes markedly increased sensitivity to DNA damage in p21(-/-), 14-3-3sigma(-/-) cells but not in cells lacking only one or none of these genes. This greater sensitivity was due to an increase in apoptosis following DNA damage and not due to exacerbation of G(2) checkpoint defects. Pharmacologic inhibition of Chk2 in p21(-/-), 14-3-3sigma(-/-) cells also resulted in greater sensitivity to DNA damage. Our data indicates that p21 and 14-3-3sigma synergize as molecular determinants of sensitivity to DNA damage following Chk2 inhibition, and Chk2 modulates the biological rheostat that determines whether a cancer cell undergoes arrest versus death after treatment with a chemotherapeutic agent. These findings have implications for the targeting of Chk2 in human cancers.

    Cell cycle (Georgetown, Tex.) 2009;8;14;2238-46

  • Expression of 14-3-3sigma in cervical squamous cell carcinomas: relationship with clinical outcome.

    Holm R, Ali T, Svendsrud DH, Nesland JM, Kristensen GB and Lyng H

    Division of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway. ruth.holm@radiumhospitalet.no

    14-3-3 sigma (sigma) sequesters the cdc2-cyclin B1 complex in the cytoplasm resulting in G2 arrest. Inactivation and reduced expression of 14-3-3sigma have been reported in a varity of cancers. In the present study, we investigated the expression of 14-3-3sigma in a series of 297 cervical squamous cell carcinoma (SCC) to clarify the prognostic value. Using immunohistochemical methods we found high levels of 14-3-3sigma protein in cytoplasm of 143 (48.1%), in nucleus of 113 (38.0%) and in both cytoplasm and nucleus of 147 (49.5%) cases, whereas, low levels were present in cytoplasm of 154 (51.9%), in nucleus of 184 (62.0%) and in both cytoplasm and nucleus of 150 (50.5%) cases. Levels of 14-3-3sigma mRNA measured by reverse-transcription polymerase chain reaction (RT-PCR) and 14-3-3sigma protein were not significant associated. 14-3-3sigma expression in cytoplasm, nuclear and cytoplasm/nuclear were not significantly correlated to disease-specific survival or disease-free survival. In conclusion, reduced expression of 14-3-3sigma protein in the cytoplasm and shuttle of 14-3-3sigma protein into the nucleus in a relatively high number of cases indicate that 14-3-3sigma may be important in the carcinogenesis of cervical SCCs by two different mechanisms; reduction and nuclear translocation of 14-3-3sigma protein. Furthermore, the non-significant correlation between expression levels of 14-3-3sigma mRNA and protein support a post-transcriptional regulation in cervical SCCs. The protein has no prognostic value in cervical cancers.

    Oncology reports 2009;22;1;11-5

  • Identificating 14-3-3 sigma as a lymph node metastasis-related protein in human lung squamous carcinoma.

    Li DJ, Deng G, Xiao ZQ, Yao HX, Li C, Peng F, Li MY, Zhang PF, Chen YH and Chen ZC

    Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, 87# Xiangya Road, Changsha, Hunan 410008, China.

    In this study, we aim to screen metastasis-related proteins in human lung squamous carcinoma (LSC) using laser capture microdissection and a proteomic approach. Twenty two differential proteins were identified from pooled microdissected primary LSC and matched lymph node (LN) metastatic tissues. Expression of the differential protein 14-3-3 sigma was determined by Western blotting and immunohistochemistry. In cell invasion assay, down-regulated 14-3-3 sigma by siRNA increased in vitro invasive ability of HTB-182 and A549 cells, up-regulation of 14-3-3 sigma by pcDNA3.0/14-3-3 sigma decreased in vitro invasive ability of HTB-182 and A549 cells. The data suggest that 14-3-3 sigma is a potential LN metastasis-related protein in LSC, and its dysregulation might play an important role in the LN metastatic process of LSC.

    Cancer letters 2009;279;1;65-73

  • 14-3-3sigma-dependent resistance to cisplatin.

    Han Z, Dimas K, Tian X, Wang Y, Hemmi H, Yamada K, Kato N, Pantazis P, Ramanujam RJ, Anant S, Wyche JH and Houchen CW

    Department of Preclinical Sciences, New York College of Podiatric Medicine, New York, NY 10035, U.S.A. zhan@nycpm.edu

    Background: A major factor that impedes the clinical success of cisplatin-based chemotherapy for cancer is cisplatin resistance by cancer cells.

    The sensitivity of parental HCT116 human colon cancer cell line and its isogenic gene-knockout sub-lines to cisplatin was determined by clonogenicity assay; furthermore, p53 activation, p21 expression, cell cycle arrest and senescence in these cells after cisplatin treatment were investigated.

    Results: Parental cells were six times more resistant than 14-3-3sigma-knockout (sigma-KO) cells to cisplatin. Moreover, activation of p53, p53-dependent expression of p21 and p21-dependent senescence were observed in sigma-KO, but not parental cells after a treatment with a low cisplatin dose.

    Conclusion: A 14-3-3sigma-dependent mechanism inhibits p53 activation in parental cells treated with a low cisplatin dose, thereby blocking p21 expression that is essential for senescence and consequently conferring to the parental cells a significant degree of resistance to cisplatin.

    Anticancer research 2009;29;6;2009-14

  • The 14-3-3sigma gene promoter is methylated in both human melanocytes and melanoma.

    Liu S, Howell P, Ren S, Fodstad O and Riker AI

    Ochsner Cancer Institute, Department of Surgery 1514 Jefferson Highway, BH334 New Orleans, LA 70121, USA. suhu_liu@dfci.harvard.edu

    Background: Recent evidence demonstrates that 14-3-3sigma acts as a tumor suppressor gene inactivated by methylation of its 5' CpG islands in epithelial tumor cells, while remaining un-methylated in normal human epithelia. The methylation analysis of 14-3-3sigma has been largely overlooked in melanoma.

    Methods: The methylation status of 14-3-3sigma CpG island in melanocytes and melanoma cells was analyzed by methylation-specific sequencing (MSS) and quantitative methylation-specific PCR (Q-MSP). 14-3-3sigma mRNA and protein expression in cell lines was detected by real-time RT-PCR and western blot. Melanoma cells were also treated by 5-aza-2'-deoxycytidine (DAC), a demethylating agent, and/or histone deacetylase inhibitor, Trichostatin A (TSA), to evaluate their effects on 14-3-3sigma gene expression.

    Results: 14-3-3sigma is hypermethylated in both human melanocytes and most melanoma cells in a lineage-specific manner, resulting in the silencing of 14-3-3sigma gene expression and the active induction of 14-3-3sigma mRNA and protein expression following treatment with DAC. We also observed a synergistic effect upon gene expression when DAC was combined with TSA. The promoter methylation status of 14-3-3sigma was analyzed utilizing Q-MSP in 20 melanoma tissue samples and 10 cell lines derived from these samples, showing that the majority of melanoma samples maintain their hypermethylation status of the 14-3-3sigma gene.

    Conclusion: 14-3-3sigma is hypermethylated in human melanoma in a cell-linage specific manner. Spontaneous demethylation and re-expression of 14-3-3sigma is a rare event in melanoma, indicating 14-3-3sigma might have a tentative role in the pathogenesis of melanoma.

    BMC cancer 2009;9;162

  • Identification and quantification of preterm birth biomarkers in human cervicovaginal fluid by liquid chromatography/tandem mass spectrometry.

    Shah SJ, Yu KH, Sangar V, Parry SI and Blair IA

    Center for Excellence in Environmental Toxicology, Department of Obstetrics and Gynecology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6160, USA.

    Spontaneous preterm birth (PTB) before 37 completed weeks of gestation resulting from preterm labor (PTL) is a leading contributor of perinatal morbidity and mortality. Early identification of at-risk women by reliable screening tests could alleviate this health issue; however, conventional methods such as obstetric history and clinical risk factors, uterine activity monitoring, biochemical markers, and cervical sonography for screening women at risk for PTB have proven unsuccessful in lowering the rate of PTB. Cervicovaginal fluid (CVF) might prove to be a useful, readily available biological fluid for identifying diagnostic PTB biomarkers. Human columnar epithelial endocervical-1 (End1) and vaginal (Vk2) cell secretomes were employed to generate a stable isotope labeled proteome (SILAP) standard to facilitate characterization and relative quantification of proteins present in CVF. The SILAP standard was prepared using stable isotope labeling by amino acids in cell culture (SILAC) of End1 and Vk2 through seven passages. The labeled secreted proteins from both cell lines were combined and characterized by liquid-chromatography-tandem mass spectrometry (LC-MS/MS). In total, 1211 proteins were identified in the End1-Vk2 SILAP standard, with 236 proteins being consistently identified in each of the replicates analyzed. Individual proteins were found to contain <0.5% of the endogenous unlabeled forms. Identified proteins were screened to provide a set of 15 candidates that have either previously been identified as potential PTB biomarkers or could be linked mechanistically to PTB. Stable isotope dilution LC-multiple reaction monitoring (MRM/MS) assays were then developed for conducting relative quantification of the 15 candidate biomarkers in human CVF samples from term and PTB cases. Three proteins were significantly elevated in PTB cases (desmoplakin isoform 1, stratifin, and thrombospondin 1 precursor), providing a foundation for further validation in larger patient cohorts.

    Funded by: NCRR NIH HHS: UL1 RR024134, UL1 RR024134-02, UL1RR024134; NICHD NIH HHS: U01 HD050088, U01HD050088; NIEHS NIH HHS: P30 ES013508, P30 ES013508-03, P30ES013508

    Journal of proteome research 2009;8;5;2407-17

  • Inactivation of 14-3-3 sigma by promoter methylation correlates with metastasis in nasopharyngeal carcinoma.

    Yi B, Tan SX, Tang CE, Huang WG, Cheng AL, Li C, Zhang PF, Li MY, Li JL, Yi H, Peng F, Chen ZC and Xiao ZQ

    Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China.

    14-3-3 sigma, the downstream target of p53, is a negative regulator of cell cycle G2-M phase checkpoint in response to DNA damage. Our previous comparative proteomics study showed that 14-3-3 sigma was downregulated or lost in nasopharyngeal carcinoma (NPC) tissue compared with non-cancerous nasopharyngeal epithelial tissue (NNET). In this study, we further investigated for the epigenetic mechanism of 14-3-3 sigma inactivation. Methylation-specific PCR showed 14-3-3 sigma promoter methylation in 100% of analyzed NPC cell lines (4/4) but not in immortalized human nasopharyngeal epithelial cell line NP69. Treatment of the four NPC cell lines with the methyltransferase inhibitor 5-aza-2'-dC resulted in the demethylation and upregulation of 14-3-3 sigma. In tissues, 14-3-3 sigma promoter methylation occurred at a higher frequency in NPC, 63/75 (84%), compared to adjacent NNET, 7/25 (28%), and fully methylated 14-3-3 sigma promoter was detected in NPC but not in any of adjacent NNET. RT-PCR, Western blotting, and immunohistochemistry showed that 14-3-3 sigma expression was downregulated or lost in NPC with methylation, and there was a negative correlation between the expression levels and methylation statuses of 14-3-3 sigma gene. In addition, the patients with methylated 14-3-3 sigma presented a higher frequency of lymph node and distant metastasis, and an advanced clinical stage, and overexpression of 14-3-3 sigma in NPC cell line 5-8F with high metastatic potential was able to inhibit its in vitro invasive ability. Our data are the first to show that 14-3-3 sigma is frequently inactivated by promoter methylation in NPC and this aberrant methylation correlates with lymph node and distant metastasis.

    Journal of cellular biochemistry 2009;106;5;858-66

  • 14-3-3sigma gene silencing during melanoma progression and its role in cell cycle control and cellular senescence.

    Schultz J, Ibrahim SM, Vera J and Kunz M

    Department of Cardiac Surgery, University of Rostock, Rostock, Germany. schultzj@gmx.de

    Background: The family of 14-3-3 proteins plays an important role in cancer biology by interfering with intracellular signalling pathways and cell cycle checkpoints. The 14-3-3sigma isoform acts as a tumor suppressor and is often inactivated during tumor development.

    Results: Here, we demonstrate enhanced CpG methylation of the 14-3-3sigma gene in lymph node and cutaneous melanoma metastases compared with primary tumors, associated with dramatically reduced mRNA expression. In line with this, treatment of different metastatic melanoma cell lines with 5-aza-2'-deoxycytidine (5-Aza-CdR), a potent inhibitor of cytosine methylation, significantly induces 14-3-3sigma protein expression. Additional treatment with histone deacetylase inhibitor 4-phenylbutyric acid (Pba) further enhances 14-3-3sigma expression. Induction of 14-3-3sigma expression by 5-Aza-CdR/Pba treatment leads to almost complete inhibition of cell proliferation, with cells predominantly arrested in G2-M. The antiproliferative effect of 5-Aza-CdR/Pba was reversed in 14-3-3sigma knockdown cells. Similarly, melanoma cell lines stably overexpressing 14-3-3sigma show dramatically reduced cell proliferation rates. Moreover, synchronous 14-3-3sigma stably overexpressing cells do not progress through cell cycle, but display a permanent increase in the population of 4n DNA containing cells. Interestingly, overexpression of 14-3-3sigma induces senescence of melanoma cells and is involved in melanoma cell senescence under genotoxic stress. Finally, 14-3-3sigma knockdown supports migratory capacity of melanoma cells in vitro, while 14-3-3sigma overexpression has opposing effects.

    Conclusion: Taken together, the present report indicates that epigenetic silencing of 14-3-3sigma might contribute to tumor progression in malignant melanoma via loss of cell cycle control, impaired cellular senescence program and support of migratory capacity.

    Molecular cancer 2009;8;53

  • Higher expression levels of 14-3-3sigma in ductal carcinoma in situ of the breast predict poorer outcome.

    Yoon NK, Seligson DB, Chia D, Elshimali Y, Sulur G, Li A, Horvath S, Maresh E, Mah V, Bose S, Bonavida B and Goodglick L

    Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095-1747, USA.

    The protein 14-3-3sigma is involved in the regulation of cellular processes such as apoptosis, cell cycle progression and proliferation. Disruption of protein expression has been implicated in a number of malignancies. Here we examine the expression pattern of 14-3-3sigma in breast cancer and specifically consider whether expression in ductal carcinoma in situ (DCIS) lesions is predictive of disease outcome. We examined 14-3-3sigma protein expression and localization using immunohistochemical staining on a high-density tissue microarray consisting of 157 invasive breast cancer patients. Statistical analyses were used to assess the correlation of 14-3-3sigma expression with clinico-pathological parameters and patient outcome. We observed a statistically significant increase in 14-3-3sigma protein expression in ductal hyperplasia, DCIS, and invasive ductal carcinoma (IDC) as compared normal glandular epithelium. In IDC, lower expression of 14-3-3sigma tended to predicted poorer survival time while in DCIS lesions, there was a stronger correlation between relatively higher levels of 14-3-3sigma predicting shorter survival time. Further, of patients who had concurrent DCIS and IDC lesions, those that exhibited a decrease of 14-3-3sigma expression from DCIS to IDC had significantly shorter survival time. Our findings indicate that 14-3-3sigma expression may be a useful prognostic indicator for survival in patients with breast cancer with an elevated 14-3-3sigma in earlier disease predicting a less favorable disease outcome. To our knowledge this is the first published study associating 14-3-3sigma protein expression with breast cancer survival.

    Funded by: NCI NIH HHS: 2 P30 CA16042-29, CA-86366, P30 CA016042, P30 CA016042-29, U24 CA086366, U24 CA086366-06

    Cancer biomarkers : section A of Disease markers 2009;5;4;215-24

  • Cooperative effect of p21Cip1/WAF-1 and 14-3-3sigma on cell cycle arrest and apoptosis induction by p14ARF.

    Hemmati PG, Normand G, Gillissen B, Wendt J, Dörken B and Daniel PT

    Department of Hematology, Oncology and Tumor Immunology, University Medical Center Charité, Campus Berlin-Buch, Germany.

    P14(ARF) (p19(ARF) in the mouse) plays a central role in the regulation of cellular proliferation. Although the capacity of p14(ARF) to induce a cell cycle arrest in G1 phase depends on a functional p53/p21-signaling axis, the G2 arrest triggered by p14(ARF) is p53/p21-independent. Using isogeneic HCT116 cells either wild-type or homozygously deleted for p21, 14-3-3sigma or both, we further investigated the cooperative effect of p21 and 14-3-3sigma on cell cycle regulation and apoptosis induction by p14(ARF). In contrast to DNA damage, which induces mitotic catastrophe in 14-3-3sigma-deficient cells, we show here that the expression of p14(ARF) triggers apoptotic cell death, as evidenced by nuclear DNA fragmentation and induction of pan-caspase activities, irrespective of the presence or absence of 14-3-3sigma. The activation of the intrinsic mitochondrial apoptosis pathway by p14(ARF) was confirmed by cytochrome c release from mitochondria and induction of caspase-9- (LEHDase) and caspase-3/7-like (DEVDase) activities. Moreover, 14-3-3sigma/p21 double-deficient cells were exceedingly sensitive to apoptosis induction by p14(ARF) as compared to wild-type cells or cells lacking either gene alone. Notably, p14(ARF)-induced apoptosis was preceded by an arrest in the G2 phase of cell cycle, which coincided with downregulation of cdc2 (cdk1) protein expression and lack of its nuclear localization. This indicates that p14(ARF) impairs mitotic entry by targeting the distal DNA damage-signaling pathway and induces apoptotic cell death, rather than mitotic catastrophe, out of a transient G2 arrest. Furthermore, our data delineate that the disruption of G2/M cell cycle checkpoint control critically determines the sensitivity of the cell toward p14(ARF)-induced mitochondrial apoptosis.

    Oncogene 2008;27;53;6707-19

  • 14-3-3sigma Modulates pancreatic cancer cell survival and invasiveness.

    Neupane D and Korc M

    Department of Medicine and Pharmacology and Toxicology, Dartmouth-Hitchcock Medical Center and Dartmouth Medical School, Hanover, New Hamsphire 03756, USA.

    Purpose: The purpose of the present study was to investigate the potential role of 14-3-3sigma in pancreatic ductal adenocarcinoma (PDAC).

    14-3-3 isoform expression was determined by real-time quantitative PCR in laser capture normal pancreatic ductal cells and pancreatic cancer cells and in 5 pancreatic cancer cell lines. PANC-1 cells, with low levels of 14-3-3sigma, were stably transfected with a human 14-3-3sigma cDNA. Conversely, high endogenous 14-3-3sigma levels in T3M4 cells were suppressed by specific short hairpin RNA. Apoptosis, motility, and invasiveness were studied.

    Results: The cancer cells in 7 PDAC samples expressed high levels of 14-3-3sigma mRNA by quantitative PCR when compared with normal pancreatic duct cells. 14-3-3sigma protein levels were high in BxPC3, COLO-357, and T3M4 cells, intermediate in ASPC-1 cells, and low in PANC-1 cells. Most cell lines released detectable amount of 14-3-3sigma into conditioned medium. Overexpression of 14-3-3sigma in PANC-1 cells led to resistance to cisplatinum-induced apoptosis, increased basal migration, and increased invasion in response to epidermal growth factor and insulin-like growth factor-I. By contrast, short hairpin RNA-mediated knockdown of endogenous 14-3-3sigma in T3M4 cells did not alter migration but led to enhanced cisplatinum sensitivity, increased invasiveness in response to epidermal growth factor, and decreased invasiveness in response to insulin-like growth factor-I.

    Conclusions: 14-3-3sigma contributes to the chemoresistance of pancreatic cancer cells and exerts cell type-dependent effects on cell migration and invasion. Therefore, strategies aimed at suppressing 14-3-3sigma expression and function may have a therapeutic benefit in subgroups of patients with PDAC.

    Funded by: NCI NIH HHS: CA-10130, R01 CA101306, R01 CA101306-05

    Clinical cancer research : an official journal of the American Association for Cancer Research 2008;14;23;7614-23

  • The clinicopathological and prognostic impact of 14-3-3 sigma expression on vulvar squamous cell carcinomas.

    Wang Z, Tropè CG, Suo Z, Trøen G, Yang G, Nesland JM and Holm R

    Division of Pathology, The Norwegian Radium Hospital, Rikshospitalet University Hospital, Oslo, Norway. zhihui.wang@radiumhospitalet.no

    Background: 14-3-3 sigma promotes G2/M cell cycle arrest by sequestering cyclin B1-CDC2 complex in cytoplasm. Down-regulation of 14-3-3sigma, which has been demonstrated in various carcinomas, may contribute to malignant transformation. However, the exact role of 14-3-3sigma in the pathogenesis of vulvar carcinoma is not fully characterized, and the prognostic impact of 14-3-3sigma protein expression is still unknown.

    Methods: We investigated the 14-3-3sigma expression in a series of 302 vulvar squamous cell carcinomas using immunohistochemistry and its associations with clinicopathological factors and clinical outcome.

    Results: In cytoplasm, nucleus and cytoplasm/nucleus of vulvar carcinomas high 14-3-3sigma protein expression was found in 72%, 59% and 75% of the carcinomas, respectively, and low levels in 28%, 41% and 25% of the cases, respectively. High level of 14-3-3sigma in cytoplasm, nucleus and cytoplasm/nucleus was significantly correlated to large tumor diameter (p = 0.001, p = 0.002 and p = 0.001, respectively) and deep invasion (p = 0.01, p = 0.001 and p = 0.007, respectively). Variations of 14-3-3sigma protein expression were not associated to disease-specific survival.

    Conclusion: Our results indicate that 14-3-3sigma may be involved in the development of a subset of vulvar squamous cell carcinomas by down-regulation of 14-3-3sigma protein. Neither cytoplasmic nor nuclear level of 14-3-3sigma expression was associated with prognosis.

    BMC cancer 2008;8;308

  • Loss of 14-3-3 sigma protein expression and presence of human papillomavirus type 16 E6 in oral squamous cell carcinoma.

    Bhawal UK, Sugiyama M, Nomura Y, Kuniyasu H and Tsukinoki K

    BDS, Department of Diagnostic Science, Division of Pathology & High Tech Research Center, Kanagawa Dental College, Yokosuka 238-8580, Japan. bhawal2002@yahoo.co.in

    Objective: To confirm the expression of 14-3-3 sigma in oral malignant lesions and in adjacent nonmalignant oral epithelium to provide a clue to the involvement in the cell cycle progression and note any association with human papillomavirus (HPV) status. 14-3-3 Sigma plays important roles in a wide range of vital regulatory processes, including signal transduction, apoptosis, cell cycle progression, and DNA replication. 14-3-3 Sigma is an exclusive epithelial marker, and data on its expression in different malignancies are very scarce.

    Design: Western blotting, immunohistochemical analysis, and polymerase chain reaction were performed.

    Setting: An academic university laboratory.

    Patients: Adults with known oral squamous cell carcinomas (SCCs) that were surgically resected.

    The DNA of HPV-16 E6 was detected by polymerase chain reaction, and protein expression of 14-3-3 sigma was evaluated by Western blot and immunohistochemical analysis.

    Results: The immunoreactive 14-3-3 sigma protein was detected mainly in the cytoplasm of differentiated squamous cells of oral SCC lesions as well as adjacent nonmalignant squamous mucosa. Immunoreactivity for 14-3-3 sigma was observed in 93% of SCC lesions (27 of 29), including HPV-negative cases. No significant association was observed between 14-3-3 sigma expression and clinicopathologic parameters. A statistically significant correlation was found between 14-3-3 sigma protein expression and the Ki-67 labeling index. 14-3-3 Sigma expression was correlated inversely with HPV-16 E6.

    Conclusion: These findings suggest that 14-3-3 sigma may act as a negative regulator of the cell cycle progression in oral SCC.

    Archives of otolaryngology--head & neck surgery 2008;134;10;1055-9

  • Phosphorylation-dependent binding of 14-3-3 terminates signalling by the Gab2 docking protein.

    Brummer T, Larance M, Herrera Abreu MT, Lyons RJ, Timpson P, Emmerich CH, Fleuren ED, Lehrbach GM, Schramek D, Guilhaus M, James DE and Daly RJ

    Cancer Research Program, Garvan Institute of Medical Research, Sydney, New South Wales, Australia.

    Grb2-associated binder (Gab)2 functions downstream of a variety of receptor and cytoplasmic tyrosine kinases as a docking platform for specific signal transducers and performs important functions in both normal physiology and oncogenesis. Gab2 signalling is promoted by its association with specific receptors through the adaptor Grb2. However, the molecular mechanisms that attenuate Gab2 signals have remained unclear. We now demonstrate that growth factor-induced phosphorylation of Gab2 on two residues, S210 and T391, leads to recruitment of 14-3-3 proteins. Together, these events mediate negative-feedback regulation, as Gab2(S210A/T391A) exhibits sustained receptor association and signalling and promotes cell proliferation and transformation. Importantly, introduction of constitutive 14-3-3-binding sites into Gab2 renders it refractory to receptor activation, demonstrating that site-selective binding of 14-3-3 proteins is sufficient to terminate Gab2 signalling. Furthermore, this is associated with reduced binding of Grb2. This leads to a model where signal attenuation occurs because 14-3-3 promotes dissociation of Gab2 from Grb2, and thereby uncouples Gab2 from the receptor complex. This represents a novel regulatory mechanism with implications for diverse tyrosine kinase signalling systems.

    The EMBO journal 2008;27;17;2305-16

  • Regulation of 14-3-3sigma expression in human thyroid carcinoma is epigenetically regulated by aberrant cytosine methylation.

    Lal G, Padmanabha L, Provenzano M, Fitzgerald M, Weydert J and Domann FE

    Department of Surgery, Division of Surgical Oncology, University of Iowa Hospitals and Clinics, 200 Hawkins Drive, 4641 JCP, Iowa city, IA 52242, USA. Geeta-lal@uiowa.edu

    Increased 14-3-3sigma expression has been observed by immunohistochemistry in papillary and anaplastic tumors, but not follicular thyroid cancers. 14-3-3sigma mRNA expression and methylation status was examined in tumor cell lines and primary thyroid tissues using real-time RT-PCR, bisulfite sequencing and methylation-specific PCR. Most of the 27 CpG's in the gene's CpG island were methylated in normal thyroid, TPC-1, NPA, FTC-238 and 2-7, which did not express 14-3-3sigma. In contrast, they were unmethylated in KAK-1 and anaplastic lines KAT4 and DRO-90. 14-3-3sigma expression was not increased in thyroid carcinomas, the majority of which had a methylated CpG island. In addition, 5-aza-dC treatment increased 14-3-3sigma expression in the FTC-238 and NPA cell lines, which had low baseline expression. We conclude 14-3-3sigma expression in thyroid carcinomas is regulated by CpG island hypermethylation.

    Funded by: NCI NIH HHS: R01 CA073612, R01 CA073612-07, R01 CA073612-08, R01 CA73612

    Cancer letters 2008;267;1;165-74

  • Primary human keratinocytes externalize stratifin protein via exosomes.

    Chavez-Muñoz C, Morse J, Kilani R and Ghahary A

    Department of Surgery, BC Professional Burn and Wound Healing Research Lab, The University of British Columbia, Vancouver, British Columbia, Canada.

    Although, stratifin (SFN) is externalized by keratinocytes and stimulates the expression of matrix metalloproteinase-1 (MMP-1) in fibroblasts, its mechanism of externalization is not known. Here, we hypothesize that keratinocytes have a capacity to release stratifin through externalization of exosomes. To test this hypothesis, exosomes were purified from human keratinocyte conditioned medium (KCM) and analyzed for the presence of SFN by Western blot analysis using lysosomal-associated membrane protein 2 (LAMP-2) and heat shock cognate 70 (hsc70) as exosomal markers. The results showed the presence of SFN in keratinocyte lysate, concentrated KCM and exosomes, but not in concentrated unconditioned medium. Transmission electron microscopic examination revealed the presence of unique "saucer-like" structures characteristic of exosomes whose diameters were <100 nm. Similar to the recombinant SFN, the exosomes associated proteins stimulated MMP-1 expression in fibroblasts. Depletion of the exosomes markedly reduced this MMP-1 stimulatory effect. To further statistically confirm these findings, fibroblasts were treated with three different exosome preparations and the finding showed more than 7.4-fold increase in the level of MMP-1 in the treated cells. Furthermore, we found that approximately 1% of the total proteins contained in exosomes correspond to SFN. In conclusion, this study is the first report showing that keratinocytes have the capacity to produce exosomes through which some intracellular proteins such as SFN, with MMP-1 stimulating activity for fibroblasts, is externalized into keratinocyte microenvironment.

    Journal of cellular biochemistry 2008;104;6;2165-73

  • Quantitative detection of methylated ESR1 and 14-3-3-sigma gene promoters in serum as candidate biomarkers for diagnosis of breast cancer and evaluation of treatment efficacy.

    Martínez-Galán J, Torres B, Del Moral R, Muñoz-Gámez JA, Martín-Oliva D, Villalobos M, Núñez MI, Luna Jde D, Oliver FJ and Ruiz de Almodóvar JM

    Servicio de Oncología Radioterápica, Hospital Virgen de las Nieves, Granada, Spain.

    The aim of the present study was to investigate the association between gene hypermethylation and main clinicopathological features of breast cancer, including diagnosis and treatment response. A sensitive SYBR green methylation-specific PCR technique was used to analyze the utility of circulating DNA with CpG island hypermethylation of ESR1, APC, RARB, 14-3-3-sigma and E-cad gene promoter regions as breast cancer biomarkers. Analyses were conducted of preoperative sera from 106 women with breast cancer, 34 with benign breast disease and 74 with no evidence of breast disease and of post-treatment sera from 60 of the breast cancer patients. Mean serum values of methylated ESR1 and 14-3-3-sigma gene promoters significantly differed between breast cancer patients and healthy controls (p = 0.0112 for ESR1 and p = 0.0047 for 14-3-3-sigma). When their results were combined, it was found that hypermethylation of these two genes differentiated between breast cancer patients and healthy controls (p < 0.0001) with a sensitivity of 81% (95% confidence interval: 72-88%) and specificity of 88% (95% CI: 78-94%). Presence of methylated ESR1 in serum of breast cancer patients was associated with the ER negative phenotype (p = 0.0179). Serum hypermethylation at ESR1 and 14-3-3-sigma loci was observed in cancer patients, in situ carcinoma and benign breast disease. No significant differences in methylated ERS1 or 14-3-3-sigma values were observed between pre-surgery and post-treatment measurements. Preliminary clinical applications of this approach have revealed several shortcomings, including a frequent presence of methylated 14-3-3-sigma in sera from women with breast benign disease. These findings cast some doubts on the utility for early cancer diagnosis of highly sensitive techniques to identify hypermethylation of specific gene promoters in DNA extracted from serum. Although numerous issues remain to be resolved, the quantitative measurement of circulating methylated DNA remains a promising tool for cancer risk assessment.

    Cancer biology & therapy 2008;7;6;958-65

  • CARPs enhance p53 turnover by degrading 14-3-3sigma and stabilizing MDM2.

    Yang W, Dicker DT, Chen J and El-Deiry WS

    Laboratory of Molecular Oncology and Cell Cycle Regulation, Department of Medicine (Hematology/Oncology), University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

    CARP1 and CARP2 proteins (CARPs) are E3 ligases that target p53 as well as phospho-p53 for degradation. Because MDM2 is a critical regulator of p53 turnover, we investigated and found that CARPs associate with MDM2. We provide evidence that CARPs stabilize MDM2 by inhibiting MDM2 self-ubiquitination. CARPs together with MDM2 enhance p53 degradation, thereby inhibiting p53-mediated cell death. CARP protein levels correlate with MDM2 levels including under hypoxia where both are reduced. CARP2 was found to target 14-3-3sigma for degradation, leading to MDM2 stabilization. MDMX, a homolog of MDM2, is not absolutely required for MDM2 stabilization by CARPs, although overexpression of CARP2 enhances MDM2/MDMX interaction. Taken together, our study identifies novel mechanisms by which CARP proteins regulate the p53 signaling pathway.

    Funded by: NCI NIH HHS: CA-105008, CA-75138, CA-97100, CA-98101

    Cell cycle (Georgetown, Tex.) 2008;7;5;670-82

  • Toward a confocal subcellular atlas of the human proteome.

    Barbe L, Lundberg E, Oksvold P, Stenius A, Lewin E, Björling E, Asplund A, Pontén F, Brismar H, Uhlén M and Andersson-Svahn H

    Department of Biotechnology, AlbaNova University Center, Royal Institute of Technology, SE-106 91 Stockholm, Sweden.

    Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.

    Molecular & cellular proteomics : MCP 2008;7;3;499-508

  • DNA methylation patterns of the CDH1, RARB, and SFN genes in choroid plexus tumors.

    Losi-Guembarovski R, Kuasne H, Guembarovski AL, Rainho CA and Cólus IM

    Department of General Biology, Biological Sciences Center, State University of Londrina-UEL, Londrina, PR, Brazil.

    Genetic and epigenetic alterations in choroid plexus tumors, a rare neuroepithelial neoplasm most frequently detected in children, are poorly characterized. Epigenetic silencing associated with aberrant CpG island methylation is one mechanism leading to the loss of tumor suppressor functions in cancer cells. Using methylation-specific polymerase chain reaction, the methylation patterns of the genes CDH1 (E-cadherin), RARB (retinoic acid receptor, beta), and SFN (stratifin; 14-3-3sigma) were retrospectively investigated in eight choroid plexus tumors (five papillomas, two atypical papillomas, and one carcinoma), as well as in two normal cortexes obtained after autopsy from male individuals aged 6 months and 64 years. Among the six pediatric tumors, the mean age at diagnosis was 1.8 years old (range, 0.2-6) and the two adult tumors were detected in a 66-year-old man and a 45-year-old woman. A high frequency of hypermethylation was detected in CDH1 and SFN genes in tumoral and normal cortex tissues. Tumor-specific RARB hypermethylation was observed in four papillomas. Further studies are required to evaluate the role of aberrant methylation in choroid plexus tumor progression.

    Cancer genetics and cytogenetics 2007;179;2;140-5

  • Over-expression of 14-3-3sigma in budding colorectal cancer cells modulates cell migration in the presence of tenascin-C.

    Ide M, Saito K, Tsutsumi S, Tsuboi K, Yamaguchi S, Asao T, Kuwano H and Nakajima T

    Department of General Surgical Science (Surgery I), Gunma University Graduate School of Medicine, Gunma 371-8511, Japan. muide@showa.gunma-u.ac.jp

    Epigenetic silencing of the 14-3-3sigma gene by CpG hypermethylation has been reported in many kinds of cancers, but has been considered inapplicable in the colorectal variety. The expression of 14-3-3sigma in colorectal cancer is located primarily in the invasive area. The interaction between tumor cells and the extracellular matrix (ECM) is involved in tumor invasion. In the current study, we investigated the correlation between 14-3-3sigma expression and the ECM, focusing especially on the presence of tenascin-C (TNC) at the invasive area of colorectal cancers. Correlations between the immunohistochemical expression of 14-3-3sigma and TNC, as well as other clinicopathological factors, were evaluated in 123 colorectal carcinoma tissues. 14-3-3sigma expression was frequently observed in budding tumor cells in the invasive area and expression was significantly correlated with budding formation (p=0.001), pTNM classification (p=0.001) and stromal TNC expression (p=0.004). Using colorectal cancer cell lines and ECMs, the up-regulation of 14-3-3sigma mRNA levels was investigated by semi-quantitative RT-PCR. TNC surrounding the tumor cells increased 14-3-3sigma mRNA expression 1.8- to 2.2-fold in HCT116 cells. The effect of 14-3-3sigma over-expression on tumor cell migration was investigated using an agarose-cell droplet migration assay. Over-expression of 14-3-3sigma up-regulated HCT116 cell migration on TNC (p<0.001). We concluded that the expression of 14-3-3sigma in the invasive area modulates tumor cell migration in certain types of colorectal cancer and thus facilitates tumor progression.

    Oncology reports 2007;18;6;1451-6

  • Aberrant overexpression of an epithelial marker, 14-3-3sigma, in a subset of hematological malignancies.

    Motokura T, Nakamura Y and Sato H

    Department of Hematology and Oncology, Faculty of Medicine, The University of Tokyo, Tokyo, Japan. motokura@grape.med.tottori-u.ac.jp

    Background: 14-3-3sigma is a p53-mediated cell-cycle inhibitor in epithelial cells. The expression of 14-3-3sigma is frequently altered in cancers of epithelial origin associated with altered DNA methylation. Since its involvement in a non-epithelial tumor is unknown, we examined 14-3-3sigma expression in patients with haematological malignancies.

    Methods: We analyzed 41 hematopoietic cell lines and 129 patients with a variety of hematological malignancies for 14-3-3sigma expression with real-time RT-PCR. We also examined protein levels by Western blot analysis and DNA methylation status of the 14-3-3sigma gene by methylation-specific PCR analysis of bisulfite-treated DNA. In addition, mutations of p53 gene were identified by RT-PCR-SSCP analysis and the expression levels of 14-3-3sigma were compared with those of other cell-cycle inhibitor genes, CDKN2A and ARF.

    Results: The expression levels of 14-3-3sigma mRNA in almost all cell lines were low and comparable to those in normal hematopoietic cells except for 2 B-cell lines. On the contrary, 14-3-3sigma mRNA was aberrantly overexpressed frequently in mature lymphoid malignancies (30 of 93, 32.3%) and rarely in acute leukemia (3 of 35, 8.6%). 14-3-3sigma protein was readily detectable and roughly reflected the mRNA level. In contrast to epithelial tumors, methylation status of the 14-3-3sigma gene was not associated with expression in hematological malignancies. Mutations of p53 were identified in 12 patients and associated with lower expression of 14-3-3sigma. The expression levels of 14-3-3sigma, CDKN2A and ARF were not correlated with but rather reciprocal to one another, suggesting that simultaneous overexpression of any two of them is incompatible with tumor growth.

    Conclusion: 14-3-3sigma, an epithelial cell marker, was overexpressed significantly in a subset of mature lymphoid malignancies. This is the first report of aberrant 14-3-3sigma expression in non-epithelial tumors in vivo. Since the significance of 14-3-3sigma overexpression is unknown even in epithelial tumors such as pancreatic cancers, further analysis of regulation and function of the 14-3-3sigma gene in non-epithelial as well as epithelial tumors is warranted.

    BMC cancer 2007;7;217

  • Roles for negative cell regulator 14-3-3sigma in control of MDM2 activities.

    Yang HY, Wen YY, Lin YI, Pham L, Su CH, Yang H, Chen J and Lee MH

    Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

    The 14-3-3sigma, upregulated by p53 in response to DNA damage, can have a positive-feedback impact driving p53 activities and is a human cancer epithelial marker downregulated in various tumors. However, the precise roles of 14-3-3sigma during tumorigenesis are not well characterized. Here, we show that 14-3-3sigma is a critical regulator of murine double minute oncogene (MDM2). 14-3-3sigma interacts with MDM2 at the RING domain. The C-terminal region of 14-3-3sigma binds to MDM2 very efficiently. Importantly, 14-3-3sigma overexpression leads to destabilization of MDM2 through enhancing MDM2 self-ubiquitination and accelerating turnover rate. Conversely, loss of 14-3-3sigma results in a significant increase in MDM2 protein. Moreover, live-cell images indicated that 14-3-3sigma can affect the location of MDM2 from the nucleus to the cytoplasm, and that MDM2-mediated cytoplasmic localization of p53 can be reversed by the presence of 14-3-3sigma. Significantly, we further showed that 14-3-3sigma causes MDM2 downregulation, thereby stabilizing p53 and inhibiting tumor growth in animal tumors. Also, 14-3-3sigma blocks MDM2-mediated retinoblastoma degradation and p53 NEDDylation. Our results provide evidence that 14-3-3sigma is a pivotal MDM2 regulator involved in blocking a variety of activities of MDM2.

    Funded by: NCI NIH HHS: CA16672, R01 CA089266, R01CA089266, R56 CA089266

    Oncogene 2007;26;52;7355-62

  • High frequency of promoter methylation of the 14-3-3 sigma and CAGE-1 genes, but lack of hypermethylation of the caveolin-1 gene, in primary adenocarcinomas and signet ring cell carcinomas of the urinary bladder.

    Kunze E and Schlott T

    Department of Osteopathology and Haematopathology, University of Göttingen, Göttingen, Germany. ekunze@med.uni-goettingen.de

    The molecular mechanisms underlying the histogenesis of nonurothelial carcinomas of the urinary bladder are not yet clearly understood. There is a growing body of evidence that, generally, epigenetic regulation mediated by methylation of normally unmethylated CpG islands at the 5' promoter regions of genes is involved in the modification of tumorigenesis. This prompted the current study to explore the methylation status of a broad panel of various genes implicated in cell differentiation and tumor suppression in 10 adenocarcinomas and 6 signet ring cell carcinomas of the bladder. Using methylation-specific PCR, we were able to detect a high frequency of promoter methylation of the 14-3-3 sigma (100%) and CAGE-1 (80%) genes in adenocarcinomas, and in nearly all signet ring cell carcinomas. The SYK and hDAB2IP genes proved to be hypermethylated in only single cases, whereas the caveolin-1 gene failed to be hypermethylated in all cases. The present data suggest that promoter methylation of the 14-3-3 sigma and CAGE-1 genes plays a crucial role during the phenotypical morphogenesis of vesical adenocarcinomas including signet ring cell carcinomas by an epigenetic mechanism.

    International journal of molecular medicine 2007;20;4;557-63

  • Methylation and intratumoural heterogeneity of 14-3-3 sigma in oral cancer.

    Bhawal UK, Tsukinoki K, Sasahira T, Sato F, Mori Y, Muto N, Sugiyama M and Kuniyasu H

    Department of Oral Maxillofacial Diagnostic Science, Division of Pathology, Kanagawa Dental College, Yokosuka 238-8580, Japan. bhawal2002@yahoo.co.in

    14-3-3 sigma has been a major G2/M checkpoint control gene and has demonstrated that its inactivation in various cancers occurs mostly by epigenetic hypermethylation, not by genetic change. This study investigated the methylation status and expression of the 14-3-3 sigma gene in 46 oral squamous cell carcinomas by methylation-specific polymerase chain reaction, reverse transcriptase-polymerase chain reaction, Western blotting and immunohistochemistry. Exons of the p53 gene were examined for mutations by sequencing analysis and CyclinD1 by immunohistochemistry. Methylation of the 14-3-3 sigma gene was detected in 13% (6/46) of the oral tumours, but not in corresponding adjacent non-malignant and normal gingival tissues. Intratumoural heterogeneity was found in the tumour tissues including three 14-3-3 sigma-methylated samples. Methylation of 14-3-3 sigma was detected in 3 SCC with p53 mutations and 3 with wild-type p53. Our major findings are: (a) methylation of 14-3-3 gene promoter is a rare event in oral cancer; (b) it is not always associated with 14-3-3 protein levels and there is no clear relationship between its methylation and p53 mutation; (c) loss of 14-3-3 sigma expression is associated with reduced CyclinD1 gene expression.

    Oncology reports 2007;18;4;817-24

  • 14-3-3 sigma isoform interacts with the cytoplasmic domain of the transmembrane BP180 in keratinocytes.

    Li Y, Lin X, Kilani RT, Jones JC and Ghahary A

    Department of Surgery, University of Alberta, Edmonton, Alberta, Canada.

    The protein bullous pemphigoid antigen-2 (BPAG2/BP180/collagen type XVII) plays a key role in attachment of basal keratinocytes to epidermal basement membrane. The binding of BP180 with either integrin alpha6, integrin beta4, or bullous pemphigoid antigen-1 (BPAG1/BP230) is critical for this attachment in skin. The protein 14-3-3 sigma, also known as stratifin and a marker for epithelial cells, is a member of a highly conserved small acidic 14-3-3 protein family naturally found in all eukaryotic cells. Here, we have used a 14-3-3sigma GST pull-down screening assay and showed that sigma (sigma) isoform of the 14-3-3 protein family interacts with the cytoplasmic N-terminal domain of BP180. Analysis of a series of truncated or deleted 14-3-3sigma revealed that only intact 14-3-3sigma molecule, but not any of its fragments can interact with BP180. This finding suggests that conformation and possible dimerization of 14-3-3 sigma is essential for this interaction. Further, a BP180 co-immunoprecipitation (IP) and its reverse IP assays were conducted and the results confirmed that 14-3-3 sigma interacts with cytoplasmic domain, but not ecto-domain of the BP180. In conclusion, the finding of this study provides evidence that 14-3-3sigma isoform interacts with BP180 which is a major component of hemidesmosome involved in the attachment of epidermis to the basement membrane in skin. However, the significance of this interaction in hemidesmosome formation and/or attachment needs to be explored.

    Funded by: NIAMS NIH HHS: R01 AR054184, R01 AR054184-18, R01 AR054184-20

    Journal of cellular physiology 2007;212;3;675-81

  • 14-3-3sigma negatively regulates the cell cycle, and its down-regulation is associated with poor outcome in intrahepatic cholangiocarcinoma.

    Kuroda Y, Aishima S, Taketomi A, Nishihara Y, Iguchi T, Taguchi K, Maehara Y and Tsuneyoshi M

    Department of Anatomic Pathology, Pathological Sciences, Graduate School of Medical Sciences, Kyushu University, Higashiku, Fukuoka, Japan.

    The 14-3-3sigma gene has been implicated in G2/M cell cycle arrest by p53, and the loss of 14-3-3sigma protein expression has been reported in diverse human cancers. However, the role of 14-3-3sigma in the signaling pathway of the cell cycle in the progression of intrahepatic cholangiocarcinoma has not been well understood. To clarify the role of 14-3-3sigma, we examined the protein expressions of 14-3-3sigma, cyclin B1, and p53 in 93 cases of intrahepatic cholangiocarcinoma by immunohistochemical staining. We also examined the correlation between these expressions and survival rate and clinicopathologic factors such as sex, age, tumor grade (ie, pathologic differentiation, tumor size, lymphatic permeation, vascular invasion, perineural invasion, lymph node metastasis), and tumor stage. Positive 14-3-3sigma protein expression (>30% of tumor cells) was observed in 67.7% (63/93) of cases of intrahepatic cholangiocarcinoma and was inversely correlated with cyclin B1 expression. No correlation was found between 14-3-3sigma expression and p53 expression or clinicopathologic factors; however, decreased 14-3-3sigma expression was an independent prognostic factor by multivariate survival analysis (P = .0282). Extensive methylation of 14-3-3sigma was found by methylation-specific polymerase chain reaction and sequence; however, no significant correlation was detected between methylation states and protein expression. These results indicate that depressed 14-3-3sigma protein is involved in the uncontrolled cell cycle in intrahepatic cholangiocarcinoma and that the decreased expression of 14-3-3sigma protein is a significant indicator of poor prognosis for patients with intrahepatic cholangiocarcinoma.

    Human pathology 2007;38;7;1014-22

  • [Effect of 14-3-3sigma on transcriptional activity of p73 gene].

    Sang MX, Geng CZ and Shan BE

    Department of The First General Surgery, The Fourth Affiliated Hospital, Hebei Medical University, Shijiazhuang, Hebei, 050011, PR China.

    p53 gene is the main regulator of 14-3-3sigma. Activated p53 could induce the expression of 14-3-3sigma, while 14-3-3sigma stabilizes the expression of p53 and enhances its transcriptional activity. p63 and p73, the members of p53 family, also have some functions similar to p53. This study was to investigate the effect of 14-3-3sigma on the transcriptional activity of p73.

    Methods: Luciferase reporter assay, reverse transcription-polymerase chain reaction (RT-PCR), and Western blot were used to evaluate the effect of 14-3-3sigma on the transcriptional activity of p73 in p53-deficient human lung carcinoma cell line H1299. Colony formation test was used to evaluate the effect of 14-3-3sigma on the transcriptional activity of p73 in p53-mutant human breast cancer cell line MDA-MB-436.

    Results: The luciferase activities induced by bax and p21WAF1 promotors were significantly higher in p73-transfected H1299 cells than in control H1299 cells (P<0.01), and were further increased by the transfection of p73 (25 ng) and 14-3-3sigma (100, 200, and 400 ng) in a dose-dependent manner (P<0.01). The expression of bax and p21WAF1 were higher in p73-transfected H1299 cells than in control H1299 cells, and were significantly higher in p73-and 14-3-3sigma-transfected H1299 cells than in p73-transfected H1299 cells (P<0.01). The number of colonies was fewer in p73-transfected MDA-MB-436 cells than in control MDA-MB-436 cells, and the colonies were significantly smaller in p73-and 14-3-3sigma-transfected H1299 cells than in p73-transfected H1299 cells (P<0.01).

    Conclusion: 14-3-3sigma can enhance the transcriptional activity of p73 in a dose-dependent manner.

    Ai zheng = Aizheng = Chinese journal of cancer 2007;26;5;489-93

  • Nuclear cyclin B1 in human breast carcinoma as a potent prognostic factor.

    Suzuki T, Urano T, Miki Y, Moriya T, Akahira J, Ishida T, Horie K, Inoue S and Sasano H

    Department of Pathology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi-ken, 980-8575, Japan. suzuki@patholo2.med.tohoku.ac.jp

    Cyclin B1 is translocated to the nucleus from the cytoplasm, and plays an essential role in cell proliferation through promotion of mitosis. Although overexpression of cyclin B1 was previously reported in breast carcinomas, the biological significance of the intracellular localization of cyclin B1 remains unclear. Therefore, in this study, we examined cyclin B1 immunoreactivity in 109 breast carcinomas, according to the intracellular localization, that is, nucleus, cytoplasm or total (nucleus or cytoplasm). Total cyclin B1 was detected in carcinoma cells in 42% of breast carcinomas examined, whereas nuclear and cytoplasmic cyclin B1 were positive in 17 and 35% of the cases, respectively. Total or cytoplasmic cyclin B1 were positively associated with histological grade, mitosis, Ki-67, p53, c-myc or 14-3-3sigma, and inversely correlated with estrogen or progesterone receptor. Nuclear cyclin B1 was significantly associated with tumor size, lymph node metastasis, histological grade, mitosis, Ki-67 or polo-like kinase 1. Only nuclear cyclin B1 was significantly associated with adverse clinical outcome of the patients, and multivariate analyses of disease-free and overall survival demonstrated nuclear cyclin B1 as the independent marker. A similar tendency was detected in the patients receiving adjuvant therapy after surgery. These results suggest that an onocogenic role of overexpressed cyclin B1 is mainly mediated in nuclei of breast carcinoma cells, and the nuclear translocation is regulated by polo-like kinase 1 and 14-3-3sigma. Nuclear cyclin B1-positive breast carcinoma is resistant to adjuvant therapy, and nuclear cyclin B1 immunoreactivity is a potent prognostic factor in breast carcinoma patients.

    Cancer science 2007;98;5;644-51

  • 14-3-3sigma controls mitotic translation to facilitate cytokinesis.

    Wilker EW, van Vugt MA, Artim SA, Huang PH, Petersen CP, Reinhardt HC, Feng Y, Sharp PA, Sonenberg N, White FM and Yaffe MB

    Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

    14-3-3 proteins are crucial in a wide variety of cellular responses including cell cycle progression, DNA damage checkpoints and apoptosis. One particular 14-3-3 isoform, sigma, is a p53-responsive gene, the function of which is frequently lost in human tumours, including breast and prostate cancers as a result of either hypermethylation of the 14-3-3sigma promoter or induction of an oestrogen-responsive ubiquitin ligase that specifically targets 14-3-3sigma for proteasomal degradation. Loss of 14-3-3sigma protein occurs not only within the tumours themselves but also in the surrounding pre-dysplastic tissue (so-called field cancerization), indicating that 14-3-3sigma might have an important tumour suppressor function that becomes lost early in the process of tumour evolution. The molecular basis for the tumour suppressor function of 14-3-3sigma is unknown. Here we report a previously unknown function for 14-3-3sigma as a regulator of mitotic translation through its direct mitosis-specific binding to a variety of translation/initiation factors, including eukaryotic initiation factor 4B in a stoichiometric manner. Cells lacking 14-3-3sigma, in marked contrast to normal cells, cannot suppress cap-dependent translation and do not stimulate cap-independent translation during and immediately after mitosis. This defective switch in the mechanism of translation results in reduced mitotic-specific expression of the endogenous internal ribosomal entry site (IRES)-dependent form of the cyclin-dependent kinase Cdk11 (p58 PITSLRE), leading to impaired cytokinesis, loss of Polo-like kinase-1 at the midbody, and the accumulation of binucleate cells. The aberrant mitotic phenotype of 14-3-3sigma-depleted cells can be rescued by forced expression of p58 PITSLRE or by extinguishing cap-dependent translation and increasing cap-independent translation during mitosis by using rapamycin. Our findings show how aberrant mitotic translation in the absence of 14-3-3sigma impairs mitotic exit to generate binucleate cells and provides a potential explanation of how 14-3-3sigma-deficient cells may progress on the path to aneuploidy and tumorigenesis.

    Nature 2007;446;7133;329-32

  • Exquisite sensitivity of TP53 mutant and basal breast cancers to a dose-dense epirubicin-cyclophosphamide regimen.

    Bertheau P, Turpin E, Rickman DS, Espié M, de Reyniès A, Feugeas JP, Plassa LF, Soliman H, Varna M, de Roquancourt A, Lehmann-Che J, Beuzard Y, Marty M, Misset JL, Janin A and de Thé H

    Laboratoire de Pathologie, Assistance Publique/Hôpitaux de Paris, Hôpital Saint Louis, Paris, France.

    Background: In breast cancers, only a minority of patients fully benefit from the different chemotherapy regimens currently in use. Identification of markers that could predict the response to a particular regimen would thus be critically important for patient care. In cell lines or animal models, tumor protein p53 (TP53) plays a critical role in modulating the response to genotoxic drugs. TP53 is activated in response to DNA damage and triggers either apoptosis or cell-cycle arrest, which have opposite effects on cell fate. Yet, studies linking TP53 status and chemotherapy response have so far failed to unambiguously establish this paradigm in patients. Breast cancers with a TP53 mutation were repeatedly shown to have a poor outcome, but whether this reflects poor response to treatment or greater intrinsic aggressiveness of the tumor is unknown.

    In this study we analyzed 80 noninflammatory breast cancers treated by frontline (neoadjuvant) chemotherapy. Tumor diagnoses were performed on pretreatment biopsies, and the patients then received six cycles of a dose-dense regimen of 75 mg/m(2) epirubicin and 1,200 mg/m(2) cyclophosphamide, given every 14 days. After completion of chemotherapy, all patients underwent mastectomies, thus allowing for a reliable assessment of chemotherapy response. The pretreatment biopsy samples were used to determine the TP53 status through a highly efficient yeast functional assay and to perform RNA profiling. All 15 complete responses occurred among the 28 TP53-mutant tumors. Furthermore, among the TP53-mutant tumors, nine out of ten of the highly aggressive basal subtypes (defined by basal cytokeratin [KRT] immunohistochemical staining) experienced complete pathological responses, and only TP53 status and basal subtype were independent predictors of a complete response. Expression analysis identified many mutant TP53-associated genes, including CDC20, TTK, CDKN2A, and the stem cell gene PROM1, but failed to identify a transcriptional profile associated with complete responses among TP53 mutant tumors. In patients with unresponsive tumors, mutant TP53 status predicted significantly shorter overall survival. The 15 patients with responsive TP53-mutant tumors, however, had a favorable outcome, suggesting that this chemotherapy regimen can overcome the poor prognosis generally associated with mutant TP53 status.

    Conclusions: This study demonstrates that, in noninflammatory breast cancers, TP53 status is a key predictive factor for response to this dose-dense epirubicin-cyclophosphamide regimen and further suggests that the basal subtype is exquisitely sensitive to this association. Given the well-established predictive value of complete responses for long-term survival and the poor prognosis of basal and TP53-mutant tumors treated with other regimens, this chemotherapy could be particularly suited for breast cancer patients with a mutant TP53, particularly those with basal features.

    PLoS medicine 2007;4;3;e90

  • A probability-based approach for high-throughput protein phosphorylation analysis and site localization.

    Beausoleil SA, Villén J, Gerber SA, Rush J and Gygi SP

    Department of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, Massachusetts 02115, USA.

    Data analysis and interpretation remain major logistical challenges when attempting to identify large numbers of protein phosphorylation sites by nanoscale reverse-phase liquid chromatography/tandem mass spectrometry (LC-MS/MS) (Supplementary Figure 1 online). In this report we address challenges that are often only addressable by laborious manual validation, including data set error, data set sensitivity and phosphorylation site localization. We provide a large-scale phosphorylation data set with a measured error rate as determined by the target-decoy approach, we demonstrate an approach to maximize data set sensitivity by efficiently distracting incorrect peptide spectral matches (PSMs), and we present a probability-based score, the Ascore, that measures the probability of correct phosphorylation site localization based on the presence and intensity of site-determining ions in MS/MS spectra. We applied our methods in a fully automated fashion to nocodazole-arrested HeLa cell lysate where we identified 1,761 nonredundant phosphorylation sites from 491 proteins with a peptide false-positive rate of 1.3%.

    Funded by: NHGRI NIH HHS: HG03456; NIGMS NIH HHS: GM67945

    Nature biotechnology 2006;24;10;1285-92

  • p21WAF1/CIP1 and 14-3-3 sigma gene expression in degenerated aortic valves: a link between cell cycle checkpoints and calcification.

    Golubnitschaja O, Yeghiazaryan K, Skowasch D, Schild H and Bauriedel G

    Department of Radiology, Rheinische Friedrich-Wilhelms-University of Bonn, Bonn, Germany.

    The mechanisms underlying aortic valve degeneration are largely unknown. Cardiac tissue responds to a variety of stimuli by hypertrophic growth. Molecular mechanisms resulting in the hypertrophic response indicate similarity and overlap with those involved in both cell growth and death. We hypothesized cell cycle control to be the key event in progression regulation of heart valve degeneration followed by tissue mineralization. Human post-operative tissue samples of native non-rheumatic stenosed aortic valves were categorized according to absence (group 1) or presence of calcification (group 2). The samples were ex vivo examined for cell density and presence of macrophage (CD68), as well as expression of two checkpoint genes, p21WAF1/CIP1 and 14-3-3 sigma, arresting the G1 and G2 cell cycle phases, respectively. Expression rates were measured by "Real-Time"-PCR on transcriptional level. Target protein expression was measured and their co-localization in different kinds of valvular cells was tested using immunohistochemical analysis. Whereas macrophages were localized predominantly in sub-endothelial layer of valvular fibrosis, p21WAF1/CIP1 and 14-3-3 sigma expression was observed also in the valvular spongiosa co-localized with alpha-actin positive cells. Significantly higher cell density and inflammation grade were observed in group 2 versus group 1. Accordingly, p21WAF1/CIP1 and 14-3-3 sigma expression was several fold higher in group 1 versus group 2 on both transcription and translation levels. The present findings on degenerated aortic valves show that increased cell density accompanied with consequent calcification might be attributed to the down-regulation of both G1 and G2 checkpoint genes.

    Amino acids 2006;31;3;309-16

  • Promoter hypermethylation of the 14-3-3 sigma, SYK and CAGE-1 genes is related to the various phenotypes of urinary bladder carcinomas and associated with progression of transitional cell carcinomas.

    Kunze E, Wendt M and Schlott T

    Department of Osteopathology and Haematopathology, University of Göttingen, D-37099 Göttingen, Germany. ekunze@med.uni-goettingen.de

    To explore the significance of epigenetic mechanisms in urinary bladder carcinogenesis mediated by methylation of cytosine in CpG dinucleotides at 5' promoter regions, we analysed the methylation status of a broad panel of different genes in transitional cell carcinomas (TCC) and nonurothelial cancers, among which the 14-3-3 sigma, SYK and CAGE-1 genes were recognised as promising target genes. Using methylation-specific PCR, the rate of DNA hypermethylation proved to be related to the various histopathological cancer subtypes. The higher frequency of promoter methylation of the 14-3-3 sigma (57.1%) and SYK (64.3%) genes in high-grade, high-stage TCC in association with a reduced or even lacking immunohistochemical protein expression than in low-grade, low-stage TCC (28.6% and 42.9%, respectively), indicates that aberrant methylation of these genes plays an essential role in the progression of TCC. The importance of DNA hypermethylation in the conversion of TCC from a low to a high malignant potential was strongly supported by the finding that, unlike superficial low-grade TCC, advanced muscle invasive TCC showed a concurrent promoter methylation of the 14-3-3 sigma, SYK and CAGE-1 genes. Squamous cell carcinomas revealed a peak incidence of hypermethylation of the 14-3-3 sigma gene (80%), and conversely, the lowest methylation frequency of the SYK gene (13.3%). Undifferentiated small cell carcinomas disclosed a promoter methylation of the 14-3-3 sigma, SYK and CAGE-1 genes in only a quarter each for the cases. Although a correlation between the methylation status and gene activity in squamous cell and undifferentiated small cell carcinomas was not observed, the underexpression of the SYK protein products in both cancer types and additionally of the 14-3-3 sigma protein in small cell carcinomas appeared to be related to the aggressive clinical behaviour of both these nonurothelial bladder carcinomas. The relevance of the high frequency of DNA hypermethylation of the CAGE-1 antigen in TCC and squamous cell carcinomas merits further study, particularly in relation to anticancer immunotherapy. The methylation status of the PTEN, COX-2, RUNX-3 and HIC-1 genes was found to be unaltered. In conclusion, the different patterns of aberrant methylation of the 14-3-3 sigma, SYK and CAGE-1 genes in the various histopathological cancer types of the urinary bladder point to a role in tumor cell differentiation, resulting in the phenotypical conversion of TCC into nonurothelial carcinomas and in the progression of TCC to a more malignant potential.

    International journal of molecular medicine 2006;18;4;547-57

  • Dynamic profiling of the post-translational modifications and interaction partners of epidermal growth factor receptor signaling after stimulation by epidermal growth factor using Extended Range Proteomic Analysis (ERPA).

    Wu SL, Kim J, Bandle RW, Liotta L, Petricoin E and Karger BL

    Barnett Institute, Northeastern University, Boston, Massachusetts 01225, USA.

    In a recent report, we introduced Extended Range Proteomic Analysis (ERPA), an intermediate approach between top-down and bottom-up proteomics, for the comprehensive characterization at the trace level (fmol level) of large and complex proteins. In this study, we extended ERPA to determine quantitatively the temporal changes that occur in the tyrosine kinase receptor, epidermal growth factor receptor (EGFR), upon stimulation. Specifically A 431 cells were stimulated with epidermal growth factor after which EGFR was immunoprecipitated at stimulation times of 0, 0.5, 2, and 10 min as well as 4 h. High sequence coverage was obtained (96%), and methods were developed for label-free quantitation of phosphorylation and glycosylation. A total of 13 phosphorylation sites were identified, and the estimated stoichiometry was determined over the stimulation time points, including Thr(P) and Ser(P) sites in addition to Tyr(P) sites. A total of 10 extracellular domain N-glycan sites were also identified, and major glycoforms at each site were quantitated. No change in the extent of glycosylation with stimulation was observed as expected. Finally potential binding partners to EGFR were identified based on changes in the amount of protein pulled down with EGFR as a function of time of stimulation. Many of the 19 proteins identified are known binding partners of EGFR. This work demonstrates that comprehensive characterization provides a powerful tool to aid in the study of important therapeutic targets. The detailed molecular information will prove useful in future studies in tissue.

    Funded by: Intramural NIH HHS; NIGMS NIH HHS: GM 15847

    Molecular & cellular proteomics : MCP 2006;5;9;1610-27

  • Human astrocytes express 14-3-3 sigma in response to oxidative and DNA-damaging stresses.

    Satoh J, Tabunoki H, Nanri Y, Arima K and Yamamura T

    Department of Bioinformatics and Neuroinformatics, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo, Japan. satoj@my-pharm.ac.jp

    The 14-3-3 protein family consists of seven isoforms, most of which are expressed abundantly in neurons and glial cells, although the sigma isoform, a p53 target gene originally identified as an epithelium-specific marker, has not been identified in the human central nervous system. Here, we show that human astrocytes in culture expressed 14-3-3sigma under stress conditions. By Western blot, the expression of 14-3-3sigma, p53 and p21 was coordinately upregulated in astrocytes following exposure to hydrogen peroxide, 4-hydroxy-2-nonenal (4-HNE) or etoposide, a topoisomerase II inhibitor. 14-3-3sigma was induced by treatment with 5-aza-2'-deoxycytidine, suggesting a hypermethylated status of the gene promoter in astrocytes. In vivo, a small subset of hypertrophic reactive astrocytes, often showing a multinucleated morphology, expressed 14-3-3sigma in active demyelinating lesions of multiple sclerosis (MS) and ischemic lesions of cerebral infarction, where the expression of 4-HNE and 8-hydroxy-2'-deoxyguanosine was enhanced in reactive astrocytes. Microarray analysis of etoposide-treated astrocytes verified upregulation of p53-responsive genes and concurrent downregulation of mitotic checkpoint-regulatory genes. These observations suggest that 14-3-3sigma might serve as a marker of oxidative and DNA-damaging stresses inducing the mitotic checkpoint dysfunction in reactive astrocytes under pathological conditions.

    Neuroscience research 2006;56;1;61-72

  • 14-3-3sigma is a p37 AUF1-binding protein that facilitates AUF1 transport and AU-rich mRNA decay.

    He C and Schneider R

    Department of Microbiology, New York University School of Medicine, New York, NY, USA.

    Short-lived cytokine mRNAs contain an AU-rich destabilizing element (ARE). AUF1 proteins bind the ARE, undergo shuttling, and promote cytoplasmic ARE-mRNA decay through a poorly understood mechanism. We therefore identified AUF1-interacting proteins that may play a role in ARE-mRNA decay. We used mass-spectrometry to identify 14-3-3sigma protein as an AUF1-interacting protein. 14-3-3sigma binds selectively and strongly to p37 AUF1 and to a lesser extent to the p40 isoform, the two isoforms most strongly associated with ARE-mRNA decay, but not to the two larger isoforms, p42 and p45. The 14-3-3sigma interaction site on p37 was mapped to a region found only in the two smaller AUF1 isoforms and which overlaps a putative nuclear localization signal (NLS). Stable overexpression of 14-3-3sigma significantly increased cytoplasmic accumulation of p37 AUF1 and reduced the steady-state level and half-life of a reporter ARE-mRNA. siRNA silencing of AUF1 eliminated the effect of 14-3-3sigma overexpression. 14-3-3sigma therefore binds to p37 AUF1, retains it in the cytoplasm probably by masking its NLS, and enhances rapid turnover of ARE-mRNAs.

    Funded by: NIGMS NIH HHS: GM 60428, R01 GM060428

    The EMBO journal 2006;25;16;3823-31

  • p73-dependent induction of 14-3-3sigma increases the chemo-sensitivity of drug-resistant human breast cancers.

    Sang M, Li Y, Ozaki T, Ono S, Ando K, Yamamoto H, Koda T, Geng C and Nakagawara A

    Division of Biochemistry, Chiba Cancer Center Research Institute, Japan.

    It has been well documented that tumor suppressor p53 is mutated in about 50% of all human tumors. p53 status might be one of the critical determinants for the chemo-sensitivity of human tumors. In the present study, we have found that p53 family member p73 as well as 14-3-3sigma is down-regulated in response to adriamycin (ADR) in ADR-resistant human breast cancer-derived MBA-MD-436 cells which carry p53 mutation. Like p53, 14-3-3sigma was transactivated by p73 and, in turn, stabilized p73. Luciferase reporter analysis and colony formation assays demonstrated that 14-3-3sigma has an ability to enhance the p73-mediated transcriptional activity as well as its pro-apoptotic function. Furthermore, enforced expression of 14-3-3sigma increased the ADR sensitivity of MBA-MD-436 cells. Taken together, our present results strongly suggest that p73-dependent induction of 14-3-3sigma plays an important role in the regulation of chemo-sensitivity of breast cancers bearing p53 mutation.

    Biochemical and biophysical research communications 2006;347;1;327-33

  • CpG island promoter methylation and silencing of 14-3-3sigma gene expression in LNCaP and Tramp-C1 prostate cancer cell lines is associated with methyl-CpG-binding protein MBD2.

    Pulukuri SM and Rao JS

    Program of Cancer Biology, Department of Biomedical and Therapeutic Sciences, University of Illinois College of Medicine, Peoria, IL 61656, USA.

    14-3-3sigma proteins regulate numerous cellular processes that are important to cancer development. One of its biological roles involves G2 cell-cycle arrest following DNA damage. It has also been reported that the loss of 14-3-3sigma expression via CpG methylation may contribute to malignant transformation by impairing the G2 cell-cycle checkpoint function, thereby allowing an accumulation of genetic defects. However, how the CpG methylation-dependent silencing mechanism works in relation to promoter methylation associated with methyl-CpG-binding proteins (MeCPs) is still unclear. To better understand the mechanism, we first examined the methylation status of the 14-3-3sigma promoter-associated CpG islands and 14-3-3sigma gene expression in a subset of prostate cancer cell lines using methylation-specific PCR (MSP), an HhaI-based DNA methylation assay, and reverse transcription-PCR (RT-PCR). We found that the 14-3-3sigma expression is lost in LNCaP and Tramp-C1 prostate cancer cell lines and that this expression is restored after treatment with epigenetic silencing modifiers 5-aza-2'-deoxycytidine (5-aza) and trichostatin A (TSA). These results imply transcriptional silencing via promoter-associated CpG methylation. Chromatin immunoprecipitation analysis revealed that methyl-CpG-binding protein 2 (MBD2) is associated preferentially to the methylated CpG island in the 14-3-3sigma promoter in LNCaP and Tramp-C1 cells but not in 14-3-3sigma-expressing PC3 and DU145 cells, which contain an unmethylated CpG island in the 14-3-3sigma promoter region. The 14-3-3sigma gene silencing because of CpG methylation correlates with binding of MBD2. In addition, the activation of 14-3-3sigma gene expression by a combination of 5-aza and TSA also involves the release of the MBD2 from the 14-3-3sigma promoter-methylated CpG island in LNCaP and Tramp-C1 cells. Furthermore, MBD2 knockdown by siRNA stimulated 14-3-3sigma expression in LNCaP cells. We also investigated whether the loss of 14-3-3sigma expression in LNCaP and Tramp-C1 cells affects cell proliferation by MTT assays. Interestingly, we observed that 14-3-3sigma-inactivated LNCaP and Tramp-C1 cells had markedly decreased cell proliferation and protein expression of proliferation cell nuclear antigen (PCNA) after restoration of 14-3-3sigma expression with 5-aza and TSA treatment. On the other hand, the same treatment did not significantly affect 14-3-3sigma-active PC3 and DU145 cells, which normally express 14-3-3sigma. Finally, 14-3-3sigma knockdown by siRNA resulted in increased proliferation in PC3 and DU145 cells. These findings suggest that the transcriptional silencing of the 14-3-3sigma gene is caused by promoter CpG island methylation associated with MBD2, and that this may play an important role in prostate cancer progression during the invasive and metastatic stages of the disease.

    Funded by: NCI NIH HHS: CA 116708, CA 75557, CA 92393, CA 95058, R01 CA075557, R01 CA092393, R01 CA095058, R01 CA116708; NINDS NIH HHS: NS 47699, R01 NS047699

    Oncogene 2006;25;33;4559-72

  • Negative cell cycle regulator 14-3-3sigma stabilizes p27 Kip1 by inhibiting the activity of PKB/Akt.

    Yang H, Zhang Y, Zhao R, Wen YY, Fournier K, Wu HB, Yang HY, Diaz J, Laronga C and Lee MH

    Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

    The 14-3-3sigma (sigma) protein is a human cancer marker downregulated in various tumors, but its function has not been fully established. 14-3-3sigma is a negative regulator of cell cycle when overexpressed, but it is not clear whether 14-3-3sigma regulates cyclin-dependent kinase inhibitor p27(Kip1) to negatively affect cell cycle progression. Protein kinase B/Akt is a crucial regulator of oncogenic signal and can phosphorylate p27(Kip1) to enhance p27(Kip1)degradation, thereby promoting cell growth. Here, we show that 14-3-3sigma-mediated cell cycle arrest concurred with p27(Kip1) upregulation and Akt inactivation. We show that 14-3-3sigma blocks Akt-mediated acceleration of p27(Kip1) turnover rate. 14-3-3sigma inhibits Akt-mediated p27(Kip1) phosphorylation that targets p27(Kip1) for nuclear export and degradation. 14-3-3sigma inhibits cell survival and tumorigenicity of Akt-activating breast cancer cell. Low expression of 14-3-3sigma in human primary breast cancers correlates with cytoplasmic location of p27(Kip1). These data provide an insight into 14-3-3sigma activity and rational cancer gene therapy by identifying 14-3-3sigma as a positive regulator of p27 and as a potential anticancer agent.

    Funded by: NCI NIH HHS: CA16672, R01 CA089266, R01CA089266, R56 CA089266

    Oncogene 2006;25;33;4585-94

  • The DNA sequence and biological annotation of human chromosome 1.

    Gregory SG, Barlow KF, McLay KE, Kaul R, Swarbreck D, Dunham A, Scott CE, Howe KL, Woodfine K, Spencer CC, Jones MC, Gillson C, Searle S, Zhou Y, Kokocinski F, McDonald L, Evans R, Phillips K, Atkinson A, Cooper R, Jones C, Hall RE, Andrews TD, Lloyd C, Ainscough R, Almeida JP, Ambrose KD, Anderson F, Andrew RW, Ashwell RI, Aubin K, Babbage AK, Bagguley CL, Bailey J, Beasley H, Bethel G, Bird CP, Bray-Allen S, Brown JY, Brown AJ, Buckley D, Burton J, Bye J, Carder C, Chapman JC, Clark SY, Clarke G, Clee C, Cobley V, Collier RE, Corby N, Coville GJ, Davies J, Deadman R, Dunn M, Earthrowl M, Ellington AG, Errington H, Frankish A, Frankland J, French L, Garner P, Garnett J, Gay L, Ghori MR, Gibson R, Gilby LM, Gillett W, Glithero RJ, Grafham DV, Griffiths C, Griffiths-Jones S, Grocock R, Hammond S, Harrison ES, Hart E, Haugen E, Heath PD, Holmes S, Holt K, Howden PJ, Hunt AR, Hunt SE, Hunter G, Isherwood J, James R, Johnson C, Johnson D, Joy A, Kay M, Kershaw JK, Kibukawa M, Kimberley AM, King A, Knights AJ, Lad H, Laird G, Lawlor S, Leongamornlert DA, Lloyd DM, Loveland J, Lovell J, Lush MJ, Lyne R, Martin S, Mashreghi-Mohammadi M, Matthews L, Matthews NS, McLaren S, Milne S, Mistry S, Moore MJ, Nickerson T, O'Dell CN, Oliver K, Palmeiri A, Palmer SA, Parker A, Patel D, Pearce AV, Peck AI, Pelan S, Phelps K, Phillimore BJ, Plumb R, Rajan J, Raymond C, Rouse G, Saenphimmachak C, Sehra HK, Sheridan E, Shownkeen R, Sims S, Skuce CD, Smith M, Steward C, Subramanian S, Sycamore N, Tracey A, Tromans A, Van Helmond Z, Wall M, Wallis JM, White S, Whitehead SL, Wilkinson JE, Willey DL, Williams H, Wilming L, Wray PW, Wu Z, Coulson A, Vaudin M, Sulston JE, Durbin R, Hubbard T, Wooster R, Dunham I, Carter NP, McVean G, Ross MT, Harrow J, Olson MV, Beck S, Rogers J, Bentley DR, Banerjee R, Bryant SP, Burford DC, Burrill WD, Clegg SM, Dhami P, Dovey O, Faulkner LM, Gribble SM, Langford CF, Pandian RD, Porter KM and Prigmore E

    The Wellcome Trust Sanger Institute, The Wellcome Trust Genome Campus, Hinxton, Cambridgeshire CB10 1SA, UK. sgregory@chg.duhs.duke.edu

    The reference sequence for each human chromosome provides the framework for understanding genome function, variation and evolution. Here we report the finished sequence and biological annotation of human chromosome 1. Chromosome 1 is gene-dense, with 3,141 genes and 991 pseudogenes, and many coding sequences overlap. Rearrangements and mutations of chromosome 1 are prevalent in cancer and many other diseases. Patterns of sequence variation reveal signals of recent selection in specific genes that may contribute to human fitness, and also in regions where no function is evident. Fine-scale recombination occurs in hotspots of varying intensity along the sequence, and is enriched near genes. These and other studies of human biology and disease encoded within chromosome 1 are made possible with the highly accurate annotated sequence, as part of the completed set of chromosome sequences that comprise the reference human genome.

    Funded by: Medical Research Council: G0000107; Wellcome Trust

    Nature 2006;441;7091;315-21

  • Sensitizing hormone-refractory prostate cancer cells to drug treatment by targeting 14-3-3sigma.

    Han B, Xie H, Chen Q and Zhang JT

    Department of Pharmacology and Toxicology, Indiana University Cancer Center, Indiana University School of Medicine, Indianapolis, IN 46202, USA.

    Advanced and hormone-refractory prostate cancer has long been considered as a chemoresistant disease. Recently, it was found that 14-3-3sigma expression increases as prostate tumor progresses, and that 14-3-3sigma contributes significantly to drug resistance in breast cancers. We, thus, hypothesized that advanced and hormone-refractory prostate cancers may have an increased level of 14-3-3sigma, which in turn may contribute to drug resistance in advanced and hormone-refractory prostate cancers. In this study, we tested this hypothesis and found that, indeed, the expression level of 14-3-3sigma in androgen-independent prostate cancer cell lines DU145, PC3, and CWR22RV are much higher than that in the androgen-dependent cell line LNCaP, and that the androgen-independent cells are more resistant to mitoxantrone and Adriamycin than the androgen-dependent cells. Depleting 14-3-3sigma expression in DU145 and CWR22RV by RNA interference significantly sensitized these cells to mitoxantrone and Adriamycin by abrogating G2-M checkpoint and increasing apoptosis, whereas restoring 14-3-3sigma expression in LNCaP cells enhanced drug resistance. We also showed that 14-3-3sigma deficiency caused nuclear localization of Cdc2 and dephosphorylation of the Tyr15 residue upon DNA damage. Based on these studies, we propose that therapeutic intervention targeting 14-3-3sigma may be useful for sensitizing hormone-refractory prostate cancers to chemotherapy by both G2-M checkpoint abrogation and apoptosis enhancement.

    Funded by: NCI NIH HHS: CA94961

    Molecular cancer therapeutics 2006;5;4;903-12

  • Subcellular targeting of p33ING1b by phosphorylation-dependent 14-3-3 binding regulates p21WAF1 expression.

    Gong W, Russell M, Suzuki K and Riabowol K

    Southern Alberta Cancer Research Institute, Dept. of Biochemistry, University of Calgary, #370 Heritage Medical Research Building, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada.

    ING1 is a type II tumor suppressor that affects cell growth, stress signaling, apoptosis, and DNA repair by altering chromatin structure and regulating transcription. Decreased ING1 expression is seen in several human cancers, and mislocalization has been noted in diverse types of cancer cells. Aberrant targeting may, therefore, functionally inactivate ING1. Bioinformatics analysis identified a sequence between the nuclear localization sequence and plant homeodomain domains of ING1 that closely matched the binding motif of 14-3-3 proteins that target cargo proteins to specific subcellular locales. We find that the widely expressed p33(ING1b) splicing isoform of ING1 interacts with members of the 14-3-3 family of proteins and that this interaction is regulated by the phosphorylation status of ING1. 14-3-3 binding resulted in significant amounts of p33(ING1b) protein being tethered in the cytoplasm. As shown previously, ectopic expression of p33(ING1b) increased levels of the p21(Waf1) cyclin-dependent kinase inhibitor upon UV-induced DNA damage. Overexpression of 14-3-3 inhibited the up-regulation of p21(Waf1) by p33(ING1b), consistent with the idea that mislocalization blocks at least one of ING1's biological activities. These data support the idea that the 14-3-3 proteins play a crucial role in regulating the activity of p33(ING1b) by directing its subcellular localization.

    Molecular and cellular biology 2006;26;8;2947-54

  • 14-3-3gamma binds to MDMX that is phosphorylated by UV-activated Chk1, resulting in p53 activation.

    Jin Y, Dai MS, Lu SZ, Xu Y, Luo Z, Zhao Y and Lu H

    Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, OR 97239, USA.

    It has been shown that MDMX inhibits the activity of the tumor suppressor p53 by primarily cooperating with the p53 feedback regulator MDM2. Here, our study shows that this inhibition can be overcome by 14-3-3gamma and Chk1. 14-3-3gamma was identified as an MDMX-associated protein via an immuno-affinity purification-coupled mass spectrometry. Consistently, 14-3-3gamma directly interacted with MDMX in vitro, and this interaction was stimulated by MDMX phosphorylation in vitro and in cells. Interestingly, in response to UV irradiation, the wild-type, but not the kinase-dead mutant, Chk1 phosphorylated MDMX at serine 367, enhanced the 14-3-3gamma-MDMX binding and the cytoplasmic retaining of MDMX. The Chk1 specific inhibitor UCN-01 repressed all of these effects. Moreover, overexpression of 14-3-3gamma, but not its mutant K50E, which did not bind to MDMX, suppressed MDMX-enhanced p53 ubiquitination, leading to p53 stabilization and activation. Finally, ablation of 14-3-3gamma by siRNA reduced UV-induced p53 level and G1 arrest. Thus, these results demonstrate 14-3-3gamma and Chk1 as two novel regulators of MDMX in response to UV irradiation.

    Funded by: NCI NIH HHS: CA079721, CA095441, CA93614, R01 CA079721, R01 CA093614, R01 CA095441

    The EMBO journal 2006;25;6;1207-18

  • 14-3-3sigma, a p53 regulator, suppresses tumor growth of nasopharyngeal carcinoma.

    Yang H, Zhao R and Lee MH

    The University of Texas M.D. Anderson Cancer Center, Box 79, 1515 Holcombe Boulevard, Houston, TX 77030, USA.

    The 14-3-3sigma gene product, up-regulated by p53 in response to DNA damage, is involved in cell-cycle checkpoint control and is a human cancer epithelial marker down-regulated in various tumors. However, its role and function have not been established in nasopharyngeal carcinoma (NPC), a tumor of epithelial origin. Recently, we found that 14-3-3sigma interacts with p53 in response to DNA damage and stabilizes the expression of p53. In addition, we also showed that overexpression of 14-3-3sigma inhibits oncogene-activated tumorigenicity. In the present study, we investigated the tumor-suppressive role of 14-3-3sigma in NPC cells. We found that there is a failure to up-regulate 14-3-3sigma in response to DNA damage in two NPC cell lines that have p53 mutation. We also found that 14-3-3sigma interacted with protein kinase B/Akt and negatively regulated the activity of Akt. Overexpression of 14-3-3sigma inhibited NPC cell growth and blocks DNA synthesis. Overexpression of 14-3-3sigma also led to inhibition of anchorage-independent growth of NPC cells. In addition, we found that 14-3-3sigma sensitized NPC cells to apoptosis induced by the chemotherapeutic agent 2-methoxyestradiol. Overexpression of 14-3-3sigma in both NPC cell lines reduced the tumor volume in nude mice, which could have significance for clinical application. These findings provide an insight into the roles of 14-3-3sigma in NPC and suggest that approaches that modulate 14-3-3sigma activity may be useful in the treatment of NPC.

    Funded by: NCI NIH HHS: CA16672, R01CA 089266

    Molecular cancer therapeutics 2006;5;2;253-60

  • Increasing 14-3-3 sigma expression with declining estrogen receptor alpha and estrogen-responsive finger protein expression defines malignant progression of endometrial carcinoma.

    Nakayama H, Sano T, Motegi A, Oyama T and Nakajima T

    Department of Tumor Pathology, Gunma University, Graduate School of Medicine, Faculty of Medicine, Maebashi, Japan. hnakayam@med.gunma-u.ac.jp

    14-3-3 sigma (sigma) is a negative regulator of the cell cycle and contributes to G2 arrest. Lack of its expression due to hypermethylation of CpG islands has been reported in some carcinomas. A recent study showed that 14-3-3 sigma was down-regulated through proteolysis by estrogen-responsive finger protein (Efp). Here, we investigated the expression of 14-3-3 sigma, hormone receptors, Efp and p53 in 86 cases of endometrial adenocarcinoma and 46 cases of normal or non-neoplastic endometria by means of immunohistochemistry and methylation-specific polymerase chain reaction. In normal endometrium, 14-3-3 sigma was overexpressed in the mid- to late-secretory phase due to hypomethylation. In endometrial adenocarcinoma, 14-3-3 sigma expression was low in low grade endometrioid adenocarcinoma due to hypermethylation, and increased significantly with increasing histological grade due to hypomethylation. 14-3-3 sigma expression inversely correlated with estrogen receptor alpha, progesterone receptor and Efp, and positively correlated with myometrial invasion and lymph node metastasis. These results suggest that 14-3-3 sigma was one of the menstrual cycle-related proteins regulated by epigenetic methylation, and its expression was influenced by epigenetic methylation or hormone receptors in progression of endometrial adenocarcinoma, and therefore was more than just a cell-cycle regulator.

    Pathology international 2005;55;11;707-15

  • Towards a proteome-scale map of the human protein-protein interaction network.

    Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, Klitgord N, Simon C, Boxem M, Milstein S, Rosenberg J, Goldberg DS, Zhang LV, Wong SL, Franklin G, Li S, Albala JS, Lim J, Fraughton C, Llamosas E, Cevik S, Bex C, Lamesch P, Sikorski RS, Vandenhaute J, Zoghbi HY, Smolyar A, Bosak S, Sequerra R, Doucette-Stamm L, Cusick ME, Hill DE, Roth FP and Vidal M

    Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA.

    Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

    Funded by: NCI NIH HHS: R33 CA132073; NHGRI NIH HHS: P50 HG004233, R01 HG001715, RC4 HG006066, U01 HG001715; NHLBI NIH HHS: U01 HL098166

    Nature 2005;437;7062;1173-8

  • 14-3-3sigma in endometrial cancer--a possible prognostic marker in early-stage cancer.

    Ito K, Suzuki T, Akahira J, Sakuma M, Saitou S, Okamoto S, Niikura H, Okamura K, Yaegashi N, Sasano H and Inoue S

    Department of Obstetrics and Gynecology, Tohoku University Graduate School of Medicine, Sendai, Japan. kito@mail.tains.tohoku.ac.jp

    Purpose: We examined expression of 14-3-3sigma, a regulator of cell proliferation, and evaluated its clinical significance in endometrioid endometrial carcinoma.

    One hundred three endometrioid endometrial adenocarcinoma cases were examined using immunohistochemistry with archival specimens. We correlated this finding with various clinicopathologic variables, including the status of estrogen receptor, progesterone receptor, and MIB-1 (Ki-57).

    Results: 14-3-3sigma Immunoreactivity was detected in 78 of 103 (75.3%) of carcinoma cases. No statistically significant correlation was detected between status of 14-3-3sigma and any of clinicopathologic variables examined. There was, however, a statistically significant correlation between loss of 14-3-3sigma expression and adverse clinical outcome of the patients (P = 0.0007). In the early stages of cancer (stages I and II), 14-3-3sigma immunoreactivity was absent in 5 of 10 (50.0%) patients who showed recurrence during follow-up, whereas its absence was detected in only 13 of 68 (19.1%) disease-free patients in the same period. In addition, 14-3-3sigma immunoreactivity was absent in 4 of 5 (80.0%) patients who died, whereas its absence was detected in only 14 of 73 (19.2%) patients who had lived during the same period. Patients whose tumors were negative for 14-3-3sigma were at much greater risk to develop recurrent and/or mortal disease (P = 0.0372 and 0.0067). In multivariate analysis using the Cox proportional hazards model, absence of 14-3-3sigma turned out to be statistically independent risk factor in disease-free survival and overall survival even in patients with early-stage disease (P = 0.0321 and 0.0191).

    Conclusions: Results of our study showed that loss or absence of 14-3-3sigma determined by immunohistochemistry may be an important tool to identify endometrial carcinoma cases at high risk of recurrence and/or death, who are otherwise not detected by current clinical and pathologic evaluation, especially in the early stages of the disease. In addition, results of 14-3-3sigma immunohistochemistry in the early stage of endometrial carcinoma could contribute to planning postoperative follow-up and adjuvant therapy.

    Clinical cancer research : an official journal of the American Association for Cancer Research 2005;11;20;7384-91

  • Epigenetic regulation of the cell type-specific gene 14-3-3sigma.

    Oshiro MM, Futscher BW, Lisberg A, Wozniak RJ, Klimecki WT, Domann FE and Cress AE

    Pharmacology and Toxicology, University of Arizona, Arizona Cancer Center, Tucson, AZ 85724, USA.

    Epigenetic control participates in processes crucial in mammalian development, such as X-chromosome inactivation, gene imprinting, and cell type-specific gene expression. We provide evidence that the p53-inducible gene 14-3-3sigma is a new example of a gene important to human cancer, where epigenetic mechanisms participate in the control of normal cell type-specific expression, as well as aberrant gene silencing in cancer cells. Like a previously identified cell type-specific gene maspin, 14-3-3sigma is a p53-inducible gene; however, it participates in G2/M arrest in response to DNA-damaging agents. 14-3-3Sigma expression is restricted to certain epithelial cell types, including breast and prostate, whereas expression is absent in nonepithelial tissues such as fibroblasts and lymphocytes. In this report, we show that in normal cells expressing 14-3-3sigma, the 14-3-3sigma CpG island is unmethylated; associated with acetylated histones, unmethylated histone H3 lysine 9; and an accessible chromatin structure. By contrast, normal cells that do not express 14-3-3sigma have a methylated 14-3-3sigma CpG island with hypoacetylated histones, methylated histone H3 lysine 9, and an inaccessible chromatin structure. These findings extend the spectrum of cell type-specific genes controlled, partly, by normal epigenetic mechanisms, and suggest that this subset of genes may represent important targets of epigenetic dysregulation in human cancer.

    Funded by: NCI NIH HHS: CA56666, CA65662, CA73612, CA75152, P01 CA056666, P01 CA056666-090002, P30 CA023074, P30 CA23074, R01 CA065662, R01 CA073612, R01 CA075152, R01 CA075152-06, R29 CA065662, R29 CA073612, R56 CA073612; NIEHS NIH HHS: ES07091, T32 ES007091

    Neoplasia (New York, N.Y.) 2005;7;9;799-808

  • BCR kinase phosphorylates 14-3-3 Tau on residue 233.

    Clokie SJ, Cheung KY, Mackie S, Marquez R, Peden AH and Aitken A

    School of Biomedical and Clinical Laboratory Sciences, University of Edinburgh, UK.

    The breakpoint cluster region protein, BCR, has protein kinase activity that can auto- and trans-phosphorylate serine, threonine and tyrosine residues. BCR has been implicated in chronic myelogenous leukaemia as well as important signalling pathways, and as such its interaction with 14-3-3 is of major interest. 14-3-3tau and zeta isoforms have been shown previously to be phosphorylated in vitro and in vivo by BCR kinase on serine and threonine residue(s) but site(s) were not determined. Phosphorylation of 14-3-3 isoforms at distinct sites is an important mode of regulation that negatively affects interaction with Raf kinase and Bax, and potentially influences the dimerization of 14-3-3. In this study we have further characterized the BCR-14-3-3 interaction and have identified the site phosphorylated by BCR. We show here that BCR interacts with at least five isoforms of 14-3-3 in vivo and phosphorylates 14-3-3tau on Ser233 and to a lesser extent 14-3-3zeta on Thr233. We have previously shown that these two isoforms are also phosphorylated at this site by casein kinase 1, which, in contrast to BCR, preferentially phosphorylates 14-3-3zeta.

    The FEBS journal 2005;272;15;3767-76

  • Stratifin-induced matrix metalloproteinase-1 in fibroblast is mediated by c-fos and p38 mitogen-activated protein kinase activation.

    Lam E, Kilani RT, Li Y, Tredget EE and Ghahary A

    Wound Healing Research Group, Department of Surgery, University of Alberta, Edmonton, AB, Canada.

    Previously, we have demonstrated that keratinocyte releasable stratifin, also known as 14-3-3 sigma protein, stimulates matrix metalloproteinase (MMP)-1 expression in dermal fibroblasts. In this study, we showed that stratifin induced fibroblast MMP-1 messenger ribonucleic acid (mRNA) and protein levels through p38 mitogen-activated protein kinase (MAPK). Our data indicated that treatment of dermal fibroblasts with stratifin resulted in rapid and transient upregulation of c-jun and c-fos mRNA levels. We also demonstrated that SB203580 (SB), a specific inhibitor of p38 MAPK activity, inhibited the activation of fibroblast MMP-1 mRNA expression by stratifin. Subsequently, western blot analysis revealed phosphorylation of p38 at 90 min after stratifin stimulation and this was decreased to approximately 50% of the maximum value by 120 min. Stratifin was demonstrated to increase MMP-1 protein levels starting at 4 h and reaching its peak at 12-24 h. Furthermore, SB significantly blocked the stratifin induction of MMP-1 protein levels (***p<0.005, n=3). Microarray analysis of stratifin-treated fibroblasts shows an increase in Elk4/Sap1 mRNA expression and this finding was confirmed by northern blot analysis. Our results indicate that stratifin markedly increase Elk4/Sap1 mRNA expression in a time-dependent fashion. In conclusion, stratifin stimulates fibroblast MMP-1 levels through the activation of c-fos and MAPK pathway.

    The Journal of investigative dermatology 2005;125;2;230-8

  • A novel human p53 isoform is an essential element of the ATR-intra-S phase checkpoint.

    Rohaly G, Chemnitz J, Dehde S, Nunez AM, Heukeshoven J, Deppert W and Dornreiter I

    Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie, Universität Hamburg, Martinistrasse 52, D-20251, Hamburg, Germany.

    The archetypal human tumor suppressor p53 is considered to have unique transactivation properties. The assumption is based on the fact that additionally identified human p53 isoforms lack transcriptional activity. However, we provide evidence for the existence of an alternatively spliced p53 isoform (Deltap53) that exerts its transcriptional activity independent from p53. In contrast to p53, Deltap53 transactivates the endogenous p21 and 14-3-3sigma but not the mdm2, bax, and PIG3 promoter. Cell cycle studies showed that Deltap53 displays its differential transcriptional activity only in damaged S phase cells. Upon activation of the ATR-intra-S phase checkpoint, Deltap53, but not p53, transactivates the Cdk inhibitor p21. Induction of p21 results in downregulation of cyclin A-Cdk activity and accordingly attenuation of S phase progression. Data demonstrate that the Deltap53-p21-cyclin A-Cdk pathway is crucial to facilitate uncoupling of repair and replication events, indicating that Deltap53 is an essential element of the ATR-intra-S phase checkpoint.

    Cell 2005;122;1;21-32

  • Targeted proteomic analysis of 14-3-3 sigma, a p53 effector commonly silenced in cancer.

    Benzinger A, Muster N, Koch HB, Yates JR and Hermeking H

    Molecular Oncology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried/Munich, Germany.

    To comprehensively identify proteins interacting with 14-3-3 sigma in vivo, tandem affinity purification and the multidimensional protein identification technology were combined to characterize 117 proteins associated with 14-3-3 sigma in human cells. The majority of identified proteins contained one or several phosphorylatable 14-3-3-binding sites indicating a potential direct interaction with 14-3-3 sigma. 25 proteins were not previously assigned to any function and were named SIP2-26 (for 14-3-3 sigma-interacting protein). Among the 92 interactors with known function were a number of proteins previously implicated in oncogenic signaling (APC, A-RAF, B-RAF, and c-RAF) and cell cycle regulation (AJUBA, c-TAK, PTOV-1, and WEE1). The largest functional classes comprised proteins involved in the regulation of cytoskeletal dynamics, polarity, adhesion, mitogenic signaling, and motility. Accordingly ectopic 14-3-3 sigma expression prevented cellular migration in a wounding assay and enhanced mitogen-activated protein kinase signaling. The functional diversity of the identified proteins indicates that induction of 14-3-3 sigma could allow p53 to affect numerous processes in addition to the previously characterized inhibitory effect on G2/M progression. The data suggest that the cancer-specific loss of 14-3-3 sigma expression by epigenetic silencing or p53 mutations contributes to cancer formation by multiple routes.

    Funded by: NCRR NIH HHS: RR11823-08

    Molecular & cellular proteomics : MCP 2005;4;6;785-95

  • A structural basis for 14-3-3sigma functional specificity.

    Wilker EW, Grant RA, Artim SC and Yaffe MB

    Center for Cancer Research, Department of Biology and Division of Biological Engineering, Massachsuetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

    The 14-3-3 family of proteins includes seven isotypes in mammalian cells that play numerous diverse roles in intracellular signaling. Most 14-3-3 proteins form homodimers and mixed heterodimers between different isotypes, with overlapping roles in ligand binding. In contrast, one mammalian isoform, 14-3-3sigma, expressed primarily in epithelial cells, appears to play a unique role in the cellular response to DNA damage and in human oncogenesis. The biological and structural basis for these 14-3-3sigma-specific functions is unknown. We demonstrate that endogenous 14-3-3sigma preferentially forms homodimers in cells. We have solved the x-ray crystal structure of 14-3-3sigma bound to an optimal phosphopeptide ligand at 2.4 angstroms resolution. The structure reveals the presence of stabilizing ring-ring and salt bridge interactions unique to the 14-3-3sigma homodimer structure and potentially destabilizing electrostatic interactions between subunits in 14-3-3sigma-containing heterodimers, rationalizing preferential homodimerization of 14-3-3sigma in vivo. The interaction of the phosphopeptide with 14-3-3 reveals a conserved mechanism for phospho-dependent ligand binding, implying that the phosphopeptide binding cleft is not the critical determinant of the unique biological properties of 14-3-3sigma. Instead, the structure suggests a second ligand binding site involved in 14-3-3sigma-specific ligand discrimination. We have confirmed this by site-directed mutagenesis of three sigma-specific residues that uniquely define this site. Mutation of these residues to the alternative sequence that is absolutely conserved in all other 14-3-3 isotypes confers upon 14-3-3sigma the ability to bind to Cdc25C, a ligand that is known to bind to other 14-3-3 proteins but not to sigma.

    Funded by: NIGMS NIH HHS: GM60594, GM68762

    The Journal of biological chemistry 2005;280;19;18891-8

  • 14-3-3sigma expression is an independent prognostic parameter for poor survival in colorectal carcinoma patients.

    Perathoner A, Pirkebner D, Brandacher G, Spizzo G, Stadlmann S, Obrist P, Margreiter R and Amberger A

    Tyrolean Cancer Research Institute, Innsbruck, Austria.

    Purpose: 14-3-3sigma is an intracellular, dimeric, phosphoserine binding protein that is expressed in epithelial cells and involved in cancer development. In this study, we examined the expression of 14-3-3sigma and evaluated its clinical significance in colorectal carcinoma.

    Expression of 14-3-3sigma was analyzed by Western blot in nine colorectal carcinoma cell lines, eight paired colorectal carcinoma tissues, and normal mucosas. Immunohistochemistry was used to evaluate expression of 14-3-3sigma in tissues of 121 colorectal carcinoma patients and to correlate it with clinical parameters.

    Results: Western blot analysis of colorectal carcinoma cell lines and tissues revealed strong 14-3-3sigma expression in four of eight cell lines and 14-3-3sigma overexpression in carcinomas compared with normal mucosa in six of eight colorectal carcinoma tissue pairs. Immunohistochemical analysis revealed 14-3-3sigma overexpression in 38.8% of colorectal carcinoma samples. Furthermore, highly positive immunoreactivity was significantly correlated with tumor differentiation (P < 0.001) and pT stage (P < 0.003). In Kaplan-Meier analysis, 14-3-3sigma overexpression was associated with a significantly decreased survival time compared with negatively stained or low stained cases (P < 0.0096). In multivariate regression analysis, 14-3-3sigma expression emerged as a significant independent parameter (P < 0.037).

    Conclusions: These results provide evidence that 14-3-3sigma expression increases during carcinoma progression in a subset of colorectal carcinoma. The overexpression of this antigen identifies patients at high risk. It is tempting to suggest that 14-3-3sigma overexpression either promotes tumor proliferation and/or prevents apoptotic signal transduction in colorectal carcinoma. Thus, targeting 14-3-3sigma might be a new therapeutic strategy in colorectal carcinoma.

    Clinical cancer research : an official journal of the American Association for Cancer Research 2005;11;9;3274-9

  • The role of epigenetic inactivation of 14-3-3sigma in human cancer.

    Lodygin D and Hermeking H

    Molecular Oncology, Max-Planck Institute of Biochemistry, Martinsried/Munich, Germany.

    Cancer cells show characteristic alterations in DNA methylation patterns. Aberrant CpG methylation of specific promoters results in inactivation of tumor suppressor genes and therefore plays an important role in carcinogenesis. The p53-regulated gene 14-3-3sigma undergoes frequent epigenetic silencing in several types of cancer, including carcinoma of the breast, prostate, and skin, suggesting that the loss of 14-3-3sigma expression may be causally involved in tumor progression. Functional studies demonstrated that 14-3-3sigma is involved in cell-cycle control and prevents the accumulation of chromosomal damage. The recent identification of novel 14-3-3sigma-associated proteins by a targeted proteomics approach implies that 14-3-3sigma regulates diverse cellular processes, which may become deregulated after silencing of 14-3-3sigma expression in cancer cells.

    Cell research 2005;15;4;237-46

  • 14-3-3 proteins--an update.

    Mhawech P

    Department of Pathology and Laboratory Medicine at Roswell Park Cancer Institute, Buffalo, New York 14263, USA. pmhawech1@yahoo.com

    14-3-3 is a highly conserved acidic protein family, composed of seven isoforms in mammals. 14-3-3 protein can interact with over 200 target proteins by phosphoserine-dependent and phosphoserine-independent manners. Little is known about the consequences of these interactions, and thus are the subjects of ongoing studies. 14-3-3 controls cell cycle, cell growth, differentiation, survival, apoptosis, migration and spreading. Recent studies have revealed new mechanisms and new functions of 14-3-3, giving us more insights on this fascinating and complex family of proteins. Of all the seven isoforms, 14-3-3sigma seems to be directly involved in human cancer. 14-3-3sigma itself is subject to regulation by p53 upon DNA damage and by epigenetic deregulation. Gene silencing of 14-3-3sigma by CpG methylation has been found in many human cancer types. This suggests that therapy-targeting 14-3-3sigma may be beneficial for future cancer treatment.

    Cell research 2005;15;4;228-36

  • JNK phosphorylation of 14-3-3 proteins regulates nuclear targeting of c-Abl in the apoptotic response to DNA damage.

    Yoshida K, Yamaguchi T, Natsume T, Kufe D and Miki Y

    Department of Molecular Genetics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510, Japan. yos.mgen@mri.tmd.ac.jp

    The ubiquitously expressed c-Abl tyrosine kinase localizes to the cytoplasm and nucleus. Nuclear c-Abl is activated by diverse genotoxic agents and induces apoptosis; however, the mechanisms that are responsible for nuclear targeting of c-Abl remain unclear. Here, we show that cytoplasmic c-Abl is targeted to the nucleus in the DNA damage response. The results show that c-Abl is sequestered into the cytoplasm by binding to 14-3-3 proteins. Phosphorylation of c-Abl on Thr 735 functions as a site for direct binding to 14-3-3 proteins. We also show that, in response to DNA damage, activation of the c-Jun N-terminal kinase (Jnk) induces phosphorylation of 14-3-3 proteins and their release from c-Abl. Together with these results, expression of an unphosphorylated 14-3-3 mutant attenuates DNA-damage-induced nuclear import of c-Abl and apoptosis. These findings indicate that 14-3-3 proteins are pivotal regulators of intracellular c-Abl localization and of the apoptotic response to genotoxic stress.

    Funded by: NCI NIH HHS: CA29431, CA98628

    Nature cell biology 2005;7;3;278-85

  • Differentiated keratinocyte-releasable stratifin (14-3-3 sigma) stimulates MMP-1 expression in dermal fibroblasts.

    Ghahary A, Marcoux Y, Karimi-Busheri F, Li Y, Tredget EE, Kilani RT, Lam E and Weinfeld M

    Department of Surgery, Wound Healing Research Group, University of Alberta, Alberta, Canada.

    Through the use of a keratinocyte/fibroblast co-culture system, we have recently identified a potent keratinocyte-derived anti-fibrogenic factor (KDAF) for dermal fibroblasts. A sequential chromatography of the active fractions of keratinocyte-conditioned medium (KCM) and peptide mapping of the candidate proteins identified KDAF as being the keratinocyte-releasable 14-3-3 sigma (14-3-3sigma) protein, which is also known as stratifin. In this study, we hypothesize that differentiated, but not proliferating, keratinocytes are the primary source of releasable 14-3-3sigma in conditioned medium. To address this hypothesis, in a longitudinal study, keratinocyte differentiation was induced by growing these cells in a medium consisting of 50% keratinocyte serum-free medium (KSFM) and 50% Dulbecco's modified eagle's medium without any additives for up to 20 d. When KCM was collected every other day and added to fibroblasts, the level of matrix metalloproteinase (MMP)-1 mRNA expression was markedly increased in fibroblasts receiving KCM and this increase was even greater in cells receiving conditioned media collected at later time points relative to that of controls. The results of a western blot analysis further showed a marked increase in the expression of 14-3-3sigma protein in keratinocytes grown in test medium from day 4 to day 10. This finding was consistent with the levels of 14-3-3sigma mRNA expression in differentiated keratinocytes. In contrast to a very high level of 14-3-3sigma mRNA expression seen in keratinocytes, fibroblasts that are highly responsive to14-3-3sigma were unable to express this factor. Interestingly, the level of 14-3-3sigma mRNA expression was markedly higher in keratinocytes co-cultured with fibroblasts relative to that of mono-cultured keratinocytes. In conclusion, this study provides evidence that keratinocytes express a high level of 14-3-3sigma at the levels of mRNA and protein. But the releasable form of 14-3-3sigma protein was only found in conditioned medium derived from differentiated keratinocytes. Further, our recently purified recombinant 14-3-3sigma protein mimics the collagenase stimulatory effect of KCM in dermal fibroblasts.

    The Journal of investigative dermatology 2005;124;1;170-7

  • Extracellular 14-3-3sigma protein: a potential mediator of epithelial-mesenchymal interactions.

    Hermeking H

    Molecular Oncology, Max-Planck-Institute of Biochemistry, Martinsried/Munich, Germany.

    The Journal of investigative dermatology 2005;124;1;ix-x

  • DNA damage-induced downregulation of Cdc25C is mediated by p53 via two independent mechanisms: one involves direct binding to the cdc25C promoter.

    St Clair S, Giono L, Varmeh-Ziaie S, Resnick-Silverman L, Liu WJ, Padi A, Dastidar J, DaCosta A, Mattia M and Manfredi JJ

    Department of Oncological Sciences, Mount Sinai School of Medicine, New York, New York 10029, USA.

    The Cdc25C phosphatase mediates cellular entry into mitosis. The cdc25C gene is a target for transcriptional downregulation by the tumor suppressor protein p53, and this repression can be shown to contribute to p53-dependent cell cycle arrest. Two independent mechanisms have been identified. One involves the direct binding of p53 to a site in the cdc25C promoter, and the second involves a CDE/CHR element. Both of these mediate p53-dependent repression at levels of p53 comparable to those produced by DNA damage. Three CCAAT elements in the cdc25C promoter that were previously implicated in p53-dependent repression fail to do so at physiologically relevant levels of p53. Repression of Cdc25C by p53 represents an additional mechanism for p53-dependent cell cycle arrest in response to DNA damage. Importantly, this is a clear demonstration of p53-mediated transcriptional downregulation that is dependent on sequence-specific DNA binding by p53.

    Funded by: NCI NIH HHS: CA80058, CA86001

    Molecular cell 2004;16;5;725-36

  • Prostate cancer is characterized by epigenetic silencing of 14-3-3sigma expression.

    Lodygin D, Diebold J and Hermeking H

    Molecular Oncology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried/Munich, Germany.

    In order to identify tumor suppressive genes silenced by CpG methylation in prostate carcinoma (PCa), we determined genome-wide expression changes after pharmacological reversal of CpG methylation-mediated transcriptional repression in three PCa cell lines using microarray analysis. Thereby, epigenetic silencing of the 14-3-3sigma gene was detected in the cell line LNCaP. 14-3-3sigma encodes a p53-regulated inhibitor of cell cycle progression. Laser microdissection was used to isolate different cell types present in diseased prostatic tissue. Subsequent methylation-specific PCR analysis showed CpG methylation of 14-3-3sigma in all 41 primary PCa samples analysed, which was accompanied by a decrease or loss of 14-3-3sigma protein expression. In contrast, normal prostate epithelial and benign prostate hyperplasia cells showed high levels of 14-3-3sigma expression. PCa-precursor lesions (prostatic intraepithelial neoplasia) also displayed decreased levels of 14-3-3sigma expression in luminal cells, which are known to contain shortened telomeres. RNA interference-mediated inactivation of 14-3-3sigma compromised a DNA damage-induced G(2)/M arrest in the PCa cell line PC3. The generality of CpG methylation and downregulation of 14-3-3sigma expression in PCa suggests that it significantly contributes to the formation of PCa, potentially by allowing the escape from a DNA damage-induced arrest elicited by telomere shortening.

    Oncogene 2004;23;56;9034-41

  • 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

  • Immunohistochemical expression of 14-3-3 sigma protein in various histological subtypes of uterine cervical cancers.

    Sano T, Shimooka H, Weixa P, Segawa A, Jian Z, Motegi A, Nakayama H, Oyama T and Nakajima T

    Department of Tumor Pathology, Gunma University, Graduate School of Medicine, Faculty of Medicine, Gunma, Japan. sanot@med.gunma-u.ac.jp

    14-3-3 sigma (sigma) has been a major G2/M checkpoint control gene and has demonstrated that its inactivation in various cancers occurs mostly by epigenetic hypermethylation, not by genetic change. In order to confirm 14-3-3sigma protein expression together with p16 and p53 in cervical cancers, immunohistochemistry was performed using various histological subtypes of cervical cancers and dysplasia. Strong and diffuse immunoreactivity for 14-3-3sigma was uniformly observed in all the cervical dysplasia (17/17) and squamous cell carcinomas (29/29) including human papillomavirus (HPV)-negative cases. Even in adenosquamous carcinomas and adenocarcinomas of the cervix, immunohistochemical expression of 14-3-3sigma was shown with relatively high frequency (13/15, 87% and 22/27, 81%). In the in situ hybridization study, mRNA of 14-3-3sigma was expressed in six of eight immunohistochemical-negative cases. Therefore, the undetectable expression of 14-3-3sigma protein in cervical cancers might, at least in part, be due to a proteolysis not epigenetic hypermethylation. It is of interest that cancers without 14-3-3sigma expression were predominantly those lacking HPV DNA, and that there were no cases with concomitant inactivation of 14-3-3sigma and p16 in the present study. These observations are consistent with the hypothesis that inactivation of either 14-3-3sigma or p16 has an effect equivalent to the expression of E6 and E7 oncoproteins of HPV.

    Pathology international 2004;54;10;743-50

  • Protein kinases C and D mediate agonist-dependent cardiac hypertrophy through nuclear export of histone deacetylase 5.

    Vega RB, Harrison BC, Meadows E, Roberts CR, Papst PJ, Olson EN and McKinsey TA

    Department of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9148, USA.

    A variety of stress signals stimulate cardiac myocytes to undergo hypertrophy. Persistent cardiac hypertrophy is associated with elevated risk for the development of heart failure. Recently, we showed that class II histone deacetylases (HDACs) suppress cardiac hypertrophy and that stress signals neutralize this repressive function by triggering phosphorylation- and CRM1-dependent nuclear export of these chromatin-modifying enzymes. However, the identities of cardiac HDAC kinases have remained unclear. Here, we demonstrate that signaling by protein kinase C (PKC) is sufficient and, in some cases, necessary to drive nuclear export of class II HDAC5 in cardiomyocytes. Inhibition of PKC prevents nucleocytoplasmic shuttling of HDAC5 in response to a subset of hypertrophic agonists. Moreover, a nonphosphorylatable HDAC5 mutant is refractory to PKC signaling and blocks cardiomyocyte hypertrophy mediated by pharmacological activators of PKC. We also demonstrate that protein kinase D (PKD), a downstream effector of PKC, directly phosphorylates HDAC5 and stimulates its nuclear export. These findings reveal a novel function for the PKC/PKD axis in coupling extracellular cues to chromatin modifications that control cellular growth, and they suggest potential utility for small-molecule inhibitors of this pathway in the treatment of pathological cardiac gene expression.

    Molecular and cellular biology 2004;24;19;8374-85

  • RACK1 and stratifin target DeltaNp63alpha for a proteasome degradation in head and neck squamous cell carcinoma cells upon DNA damage.

    Fomenkov A, Zangen R, Huang YP, Osada M, Guo Z, Fomenkov T, Trink B, Sidransky D and Ratovitski EA

    Department of Dermatology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

    p53 family members with a transactivation (TA) domain induce cell cycle arrest and promote apoptosis. However, DeltaNp63 isotypes lacking the TA-domain promote cell proliferation and tumorigenesis in vitro and in vgammavo. Although p53, TAp63 or TAp73 are stabilized upon DNA damage, we found that the genotoxic stress agents induced a dramatic decrease and phosphorylation of DeltaNp63alpha in squamous cell carcinoma cells. Further work revealed that RACK1 physically associated with the p63alpha C-terminal domain through its WD40 domain. However, stratifin binds with phosphorylated DeltaNp63alpha in response to cisplatin. Upon DNA damage induced by cisplatin, stratifin mediated a nuclear export of DeltaNp63alpha into cytoplasm and then RACK1 targeted latter into a proteasome degradation pathway possibly serving as an E3 ubiquitin ligase. Moreover, siRNA knockdown of both stratifin and RACK1 inhibited a nuclear export and protein degradation of DeltaNp63alpha, respectively. Our data suggest that modification and down regulation of DeltaNp63alpha is one of the major determinants of the cellular response to DNA damage in human head and neck cancers.

    Funded by: NIAID NIH HHS: R01-AI47224; NIDCR NIH HHS: DE-015834, R01-DE13561

    Cell cycle (Georgetown, Tex.) 2004;3;10;1285-95

  • Frequent downregulation of 14-3-3 sigma protein and hypermethylation of 14-3-3 sigma gene in salivary gland adenoid cystic carcinoma.

    Uchida D, Begum NM, Almofti A, Kawamata H, Yoshida H and Sato M

    Second Department of Oral and Maxillofacial Surgery, Tokushima University School of Dentistry, 3-18-15 Kuramoto, Tokushima 770-8504, Japan. daisuke@dent.tokushima-u.ac.jp

    14-3-3 sigma:, a target gene of the p53 tumour suppressor protein, has been shown to regulate the cell cycle at the G2/M checkpoint. Recent studies have demonstrated that 14-3-3 sigma is downregulated by hypermethylation of the CpG island in several types of cancer. In this study, we investigated the expression and methylation status of 14-3-3 sigma in human salivary gland adenoid cystic carcinoma (ACC) and mucoepidermoid carcinoma (MEC). Immunohistochemical analysis revealed that the positive expression rate of 14-3-3 sigma in ACC (one out of 14) was markedly lower than that in MEC (ten out of 10). Since most of the ACCs carried the wild-type p53 protein, downregulation of 14-3-3 sigma in ACC may not be due to the dysfunction of p53 pathway. Microdissection-methylation-specific PCR revealed that frequent hypermethylation of the 14-3-3 sigma gene was observed in ACC when compared to that in MEC. In cultured-ACC cells, we confirmed the downregulation of 14-3-3 sigma via hemimethylation of the gene by sequencing analysis after sodium bisulphite treatment. Furthermore, re-expression of 14-3-3 sigma in the ACC cells was induced by the treatment with DNA demethylating agent, 5-aza-2'-deoxycytidine. Irradiation apparently induced the enhanced expression of 14-3-3 sigma and G2/M arrest in normal salivary gland cells; however, in the ACC cells, neither induction of 14-3-3 sigma nor G2/M arrest was induced by irradiation. These results suggest that downregulation of 14-3-3 sigma might play critical roles in the neoplastic development and radiosensitivity of ACC.

    British journal of cancer 2004;91;6;1131-8

  • Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization.

    Jin J, Smith FD, Stark C, Wells CD, Fawcett JP, Kulkarni S, Metalnikov P, O'Donnell P, Taylor P, Taylor L, Zougman A, Woodgett JR, Langeberg LK, Scott JD and Pawson T

    Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada.

    Background: 14-3-3 proteins are abundant and conserved polypeptides that mediate the cellular effects of basophilic protein kinases through their ability to bind specific peptide motifs phosphorylated on serine or threonine.

    Results: We have used mass spectrometry to analyze proteins that associate with 14-3-3 isoforms in HEK293 cells. This identified 170 unique 14-3-3-associated proteins, which show only modest overlap with previous 14-3-3 binding partners isolated by affinity chromatography. To explore this large set of proteins, we developed a domain-based hierarchical clustering technique that distinguishes structurally and functionally related subsets of 14-3-3 target proteins. This analysis revealed a large group of 14-3-3 binding partners that regulate cytoskeletal architecture. Inhibition of 14-3-3 phosphoprotein recognition in vivo indicates the general importance of such interactions in cellular morphology and membrane dynamics. Using tandem proteomic and biochemical approaches, we identify a phospho-dependent 14-3-3 binding site on the A kinase anchoring protein (AKAP)-Lbc, a guanine nucleotide exchange factor (GEF) for the Rho GTPase. 14-3-3 binding to AKAP-Lbc, induced by PKA, suppresses Rho activation in vivo.

    Conclusion: 14-3-3 proteins can potentially engage around 0.6% of the human proteome. Domain-based clustering has identified specific subsets of 14-3-3 targets, including numerous proteins involved in the dynamic control of cell architecture. This notion has been validated by the broad inhibition of 14-3-3 phosphorylation-dependent binding in vivo and by the specific analysis of AKAP-Lbc, a RhoGEF that is controlled by its interaction with 14-3-3.

    Funded by: NIDDK NIH HHS: DK44239

    Current biology : CB 2004;14;16;1436-50

  • Exportin 7 defines a novel general nuclear export pathway.

    Mingot JM, Bohnsack MT, Jäkle U and Görlich D

    ZMBH, INF 282, Heidelberg, Germany.

    Most transport pathways between cell nucleus and cytoplasm are mediated by nuclear transport receptors of the importin beta family. These receptors are in continuous circulation between the two compartments and transfer cargo molecules from one side of the nuclear envelope to the other. RanBP16 is a family member from higher eukaryotes of so far unknown function. We now show that it exports p50RhoGAP from the nucleus and thereby confines this activity to the cytoplasm. It also accounts for nuclear exclusion of 14-3-3sigma, which in turn is known to anchor, for example, cyclin-dependent kinases in the cytoplasm. Our data further suggest that RanBP16 exports several additional cargoes. It thus appears to be a nuclear export mediator with broad substrate specificity and we will therefore refer to it as exportin 7 (Exp7). Finally, we demonstrate that Exp7-dependent nuclear export signals differ fundamentally from the leucine-rich, CRM1-dependent ones: First, they are not just short linear sequences, but instead include folded motifs. Second, basic residues are critical for Exp7 recruitment.

    The EMBO journal 2004;23;16;3227-36

  • 14-3-3sigma mediation of cell cycle progression is p53-independent in response to insulin-like growth factor-I receptor activation.

    Zhang Y, Karas M, Zhao H, Yakar S and LeRoith D

    Section on Molecular and Cellular Physiology, Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases/NIH, Bethesda, MD 20892-1758, USA.

    We investigated the role of 14-3-3sigma protein in insulin-like growth factor-I (IGF-I) receptor signaling. It has been previously shown that 14-3-3sigma negatively regulates cell cycle especially in response to p53-sensitive DNA damage. In this study we demonstrated that 14-3-3sigma is a positive mediator of IGF-I receptor-induced cell proliferation. Treatment with IGF-I increased 14-3-3sigma mRNA and protein levels about 4-fold, in a time-dependent manner in MCF-7 breast cancer cells. Preincubation with the phosphoinositide 3'-kinase inhibitor LY294002 significantly reduced the effects of IGF-I on 14-3-3sigma gene expression in these cells, suggesting that this effect of IGF-I occurs via the phosphoinositide 3'-kinase pathway. 14-3-3sigma is induced by IGF-I in MCF-7 cells, which express wild-type p53, as well as in MCF-7 cells transfected with a small interference RNA targeting duplex that reduced p53 expression levels. These results suggest that IGF-I induces 14-3-3sigma expression in a manner that is independent of p53. Using the small interference RNA strategy, we demonstrated that a 70-75% reduction of 14-3-3sigma mRNA levels resulted in a similar decrease in the effects of IGF-I on cell cycle progression and proliferation in MCF-7 cells. This effect was also associated with a reduction in IGF-I-induced cyclin D1 expression. Taken together, these results suggest that 14-3-3sigma positively mediates IGF-I-induced cell cycle progression.

    The Journal of biological chemistry 2004;279;33;34353-60

  • Immunohistochemical analysis of 14-3-3 sigma and related proteins in hyperplastic and neoplastic breast lesions, with particular reference to early carcinogenesis.

    Simooka H, Oyama T, Sano T, Horiguchi J and Nakajima T

    Department of Tumor Pathology, Gunma University, Graduate School of Medicine, Gunma, Japan.

    In order to confirm the role of 14-3-3 sigma (sigma) as a tumor suppressor in breast carcinogenesis, we have studied the expression of 14-3-3sigma immunohistochemically in usual ductal hyperplasia (UDH), ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) breast lesions. Immunostaining for estrogen receptor alpha (ERalpha), p53 and estrogen-responsive RING finger protein (Efp) was also carried out. Immunohistochemically, expression of 14-3-3sigma was seen in 92% UDH lesions and gradually decreased from 65% in DCIS to 23% in IDC. The expression of ERalpha decreased gradually from UDH to DCIS to IDC, while p53 showed an inverse staining pattern to that of ERalpha. The expression of Efp showed no significant difference among the three breast lesions. Hence, the present immunohistochemical study confirmed 14-3-3sigma as a tumor suppressor in breast carcinogenesis. A similar immunohistochemical analysis was then carried out on columnar cell hyperplasia with atypia (CCHA), in which the expression pattern of tumor suppressor 14-3-3sigma, ERalpha and p53 suggested that it might be possible that CCHA is a precancerous lesion.

    Pathology international 2004;54;8;595-602

  • 14-3-3sigma is down-regulated in human prostate cancer.

    Urano T, Takahashi S, Suzuki T, Fujimura T, Fujita M, Kumagai J, Horie-Inoue K, Sasano H, Kitamura T, Ouchi Y and Inoue S

    Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.

    The 14-3-3sigma is a negative regulator of the cell cycle, which is induced by p53 in response to DNA damage. It has been characterized as an epithelium-specific marker and down-regulation of the protein has been shown in breast cancers, suggesting its tumor-suppressive activity in epithelial cells. Here we demonstrate that 14-3-3sigma protein is down-regulated in human prostate cancer cell lines, LNCaP, PC3, and DU145 compared with normal prostate epithelial cells. Immunohistochemical analysis of primary prostate cells shows that the expression of 14-3-3sigma protein is epithelial cell-specific. Among prostate pathological specimens, > 95% of benign hyperplasia samples show significant and diffuse immunostaining of 14-3-3sigma in the cytoplasm whereas < 20% of carcinoma samples show positive staining. In terms of mechanisms for the down-regulation of 14-3-3sigma in prostate cancer cells, hypermethylation of the gene promoter plays a causal role in LNCaP cells as 14-3-3sigma mRNA level was elevated by 5-aza-2'-deoxycytidine demethylating treatment. Intriguingly, the proteasome-mediated proteolysis is responsible for 14-3-3sigma reduction in DU145 and PC3 cells, as 14-3-3sigma protein expression was increased by treatment with a proteasome inhibitor MG132. Furthermore, tumor necrosis factor-related apoptosis-inducing ligand enhances 14-3-3sigma gene and protein expression in DU145 and PC3 cells. These data suggest that 14-3-3sigma expression is down-regulated during the neoplastic transition of prostate epithelial cells.

    Biochemical and biophysical research communications 2004;319;3;795-800

  • Binding of 14-3-3beta but not 14-3-3sigma controls the cytoplasmic localization of CDC25B: binding site preferences of 14-3-3 subtypes and the subcellular localization of CDC25B.

    Uchida S, Kuma A, Ohtsubo M, Shimura M, Hirata M, Nakagama H, Matsunaga T, Ishizaka Y and Yamashita K

    Division of Life Science, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192, Japan.

    The dual specificity phosphatase CDC25B positively controls the G2-M transition by activating CDK1/cyclin B. The binding of 14-3-3 to CDC25B has been shown to regulate the subcellular redistribution of CDC25B from the nucleus to the cytoplasm and may be correlated with the G2 checkpoint. We used a FLAG-tagged version of CDC25B to study the differences among the binding sites for the 14-3-3 subtypes, 14-3-3beta, 14-3-3epsilon and 14-3-3sigma, and the relationship between subtype binding and the subcellular localization of CDC25B. All three subtypes were found to bind to CDC25B. Site-directed mutagenesis studies revealed that 14-3-3beta bound exclusively near serine-309 of CDC25B1, which is within a potential consensus motif for 14-3-3 binding. By contrast, 14-3-3sigma bound preferentially to a site around serine-216, and the presence of serine-137 and -309 enhanced the binding. In addition to these binding-site differences, we found that the binding of 14-3-3beta drove CDC25B to the cytoplasm and that mutation of serine-309 to alanine completely abolished the cytoplasmic localization of CDC25B. However, co-expression of 14-3-3sigma and CDC25B did not affect the subcellular localization of CDC25B. Furthermore, serine-309 of CDC25B was sufficient to produce its cytoplasmic distribution with co-expression of 14-3-3beta, even when other putative 14-3-3 binding sites were mutated. 14-3-3epsilon resembled 14-3-3beta with regard to its binding to CDC25B and the control of CDC25B subcellular localization. The results of the present study indicate that two 14-3-3 subtypes can control the subcellular localization of CDC25B by binding to a specific site and that 14-3-3sigma has effects on CDC25B other than the control of its subcellular localization.

    Journal of cell science 2004;117;Pt 14;3011-20

  • Loss of 14-3-3sigma in prostate cancer and its precursors.

    Cheng L, Pan CX, Zhang JT, Zhang S, Kinch MS, Li L, Baldridge LA, Wade C, Hu Z, Koch MO, Ulbright TM and Eble JN

    Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA. lcheng@iupui.edu

    Purpose: The 14-3-3 family proteins are highly conserved over many mammalian species. The sigma isoform (also called HME-1 or stratifin) is expressed in epithelial cells. Loss of 14-3-3sigma is associated with failure to arrest the cell cycle at the G(2)-M phase checkpoint after DNA damage that leads to increased G(2)-type chromosomal aberrations. The role of 14-3-3sigma in prostatic carcinogenesis is uncertain.

    We studied one hundred and eleven specimens of invasive prostate adenocarcinoma with paired, adjacent high-grade prostatic intraepithelial neoplasia and normal prostate epithelium. Immunohistochemistry was used to detect the expression of 14-3-3sigma. The findings were correlated with various clinical pathological parameters.

    Results: 14-3-3sigma is ubiquitously expressed at high levels in normal prostate epithelium. Its expression is significantly decreased in prostatic intraepithelial neoplasia and prostatic adenocarcinoma. Ninety percent of samples of prostatic intraepithelial neoplasia had no or low 14-3-3sigma expression. Ninety-seven percent of invasive adenocarcinomas had no or low 14-3-3sigma expression. In most specimens (90%), suppression of 14-3-3sigma expression occurred during the development of prostatic intraepithelial neoplasia from normal epithelium.

    Conclusions: Our data suggest that loss of 14-3-3sigma contributes to the development of prostate adenocarcinoma. 14-3-3sigma expression is significantly decreased during the progression of normal prostatic epithelium to prostatic intraepithelial neoplasia and invasive cancer.

    Clinical cancer research : an official journal of the American Association for Cancer Research 2004;10;9;3064-8

  • Keratinocyte-releasable stratifin functions as a potent collagenase-stimulating factor in fibroblasts.

    Ghahary A, Karimi-Busheri F, Marcoux Y, Li Y, Tredget EE, Taghi Kilani R, Li L, Zheng J, Karami A, Keller BO and Weinfeld M

    Department of Surgery, Wound Healing Research Group, University of Alberta, Edmonton, Alberta, Canada. aghahary@ualberta.ca

    Termination of wound healing requires a fine balance between collagen deposition and its hydrolysis. To dissect the underlying control mechanisms for this process, we established a keratinocyte/fibroblast co-culture system and subsequently demonstrated more than a 10-fold increase in collagenase expression in fibroblasts co-cultured with keratinocytes relative to that of control cells. This finding was further confirmed in fibroblasts grown in a keratinocyte/fibroblast collagen-GAG gel. The efficacy of keratinocyte-derived collagenase stimulatory factors on collagenase activity was evaluated, and the results showed that only conditioned medium derived from fibroblasts co-cultured with keratinocytes was able to break down markedly type I collagen to its one-quarter and three-quarter fragments of both alpha (alpha1 and alpha2) and beta (beta1.1 and beta1.2) chains. The results of a dose-response experiment showed that keratinocyte-conditioned medium (KCM) stimulates the expression of collagenase mRNA by dermal fibroblasts in a concentration-dependent fashion. In a similar experiment, the results of a time-response experiment revealed that KCM treatment increases the expression of collagenase mRNA in dermal fibroblasts as early as 6 h and reaches its maximum level within 24-48 h. Considering that this keratinocyte-releasable factor has a potent collagenase stimulatory effect on fibroblasts, which favors the resolution of accumulated type I and type III collagen found in fibrotic tissue, we referred to this protein as a keratinocyte-derived anti-fibrogenic factor (KDAF). In a series of chromatography experiments and a direct trypsin digestion of the proteins and subsequent peptide mapping, a keratinocyte-derived collagenase-stimulating factor turned out to be a releasable form of stratifin, also known as 14-3-3 sigma protein. To validate this finding, stratifin cDNA was cloned into a pGEX-6P-1 expressing vector and more than 50 mg of recombinant stratifin was generated and used to treat fibroblasts with various concentrations for 24 h. The results of northern analysis showed a remarkable dose-response increase in the expression of collagenase mRNA in stratifin-treated fibroblasts relative to that of the control. This finding was consistent with that obtained from collagenase activity assay. In conclusion, we identified a keratinocyte-releasable form of stratifin in KCM that mimics the collagenase stimulatory effect of KCM for dermal fibroblasts. This finding suggests that stratifin is likely to be, at least, one of the KDAFs found in KCM.

    The Journal of investigative dermatology 2004;122;5;1188-97

  • Decreased expression of 14-3-3 sigma is associated with advanced disease in human epithelial ovarian cancer: its correlation with aberrant DNA methylation.

    Akahira J, Sugihashi Y, Suzuki T, Ito K, Niikura H, Moriya T, Nitta M, Okamura H, Inoue S, Sasano H, Okamura K and Yaegashi N

    Department of Obstetrics and Gynecology, Tohoku University Graduate School of Medicine, Sendai, Japan. jakahiro-tohoku@umin.ac.jp

    Purpose: In this study, we examined the promoter methylation status and expression of 14-3-3 sigma and evaluated its clinical significance in epithelial ovarian cancer.

    Twelve ovarian cancer cell lines; 2 ovarian surface epithelial cell lines; and 8 normal, 8 benign, 12 borderline, and 102 ovarian cancer tissues were examined. Methylation-specific PCR, quantitative reverse transcription-PCR, and immunohistochemistry were used to evaluate methylation status and expression of 14-3-3 sigma gene and protein.

    Results: Among the 12 ovarian cancer cell lines, the presence of a methylated band was detected in seven cell lines. Median values of relative 14-3-3 sigma gene expression in cancers with methylation (3.27) were significantly lower than those without methylation (16.4; P < 0.001). Treatment of 5-aza-2'-deoxycitidine resulted in the demethylation of the promoter CpG islands and reexpression. All of the normal, benign, and borderline tissues were positive for 14-3-3 sigma protein, and in ovarian cancer tissues, 73.5% (75 of 102) were positive for 14-3-3 sigma protein and was almost consistent with methylation status. Negative immunoreactivity of 14-3-3 sigma was significantly correlated with high age and serous histology, high-grade, advanced-stage residual tumor of >2 cm, high serum CA125, high Ki-67 labeling index, and positive p53 immunoreactivity. 14-3-3 sigma immunoreactivity was significantly associated with overall survival (P = 0.0058).

    Conclusions: Our findings suggest that 14-3-3 sigma is inactivated mainly by aberrant DNA methylation and that it may play an important role in the pathogenesis of epithelial ovarian cancer.

    Clinical cancer research : an official journal of the American Association for Cancer Research 2004;10;8;2687-93

  • Interaction of apoptosis signal-regulating kinase 1 with isoforms of 14-3-3 proteins.

    Subramanian RR, Zhang H, Wang H, Ichijo H, Miyashita T and Fu H

    Department of Pharmacology, Emory University School of Medicine, Atlanta, GA 30322, USA.

    Apoptosis signal-regulating kinase 1 (ASK1) is a critical mediator of apoptotic signaling pathways initiated by a variety of death stimuli. Its activity is tightly controlled by various mechanisms such as covalent modification and protein-protein interaction. One of the proteins that control ASK1 function is 14-3-3zeta, a member of the 14-3-3 protein family. Here, we report that ASK1 is capable of binding to other isoforms of 14-3-3, suggesting that binding ASK1 is a general property of the 14-3-3 family. In support of this notion, mutational analysis revealed that the ASK1/14-3-3 interaction was mediated by the conserved amphipathic groove of 14-3-3 with some residue selectivity. Functionally, expression of various isoforms of 14-3-3 suppressed ASK1-induced apoptosis. To understand how 14-3-3 controls the ASK1 activity, we examined intracellular localization of ASK1 upon 14-3-3 co-expression. We found that 14-3-3 co-expression is correlated with the translocation of ASK1 from the cytoplasm to a perinuclear localization, likely the ER compartment. Consistent with this notion, ASK1(S967A), a 14-3-3 binding defective mutant of ASK, showed no change in intracellular distribution upon 14-3-3 co-expression. These data support a model that 14-3-3 proteins regulate the proapoptotic function of ASK1 in part by controlling its subcellular distribution.

    Funded by: NIGMS NIH HHS: GM53165, GM60033

    Experimental cell research 2004;294;2;581-91

  • 14-3-3 sigma possibly plays a constitutive role in papillary carcinoma, but not in follicular tumor of the thyroid.

    Ito Y, Miyoshi E, Uda E, Yoshida H, Uruno T, Takamura Y, Miya A, Kobayashi K, Matsuzuka F, Matsuura N, Kakudo K, Kuma K and Miyauchi A

    Department of Surgery, Kuma Hospital, 8-2-35, Shimoyamate-dori, Chuo-ku, Kobe 650-0011, Japan. ito01@kuma-h.or.jp

    14-3-3 sigma is a negative regulator of the cell cycle and contributes to G2 arrest. Thus far, the lack of its expression due to hypermethylation of the CpG islands has been reported in some carcinomas. In this study, we investigated the expression of 14-3-3 sigma in thyroid neoplasms by means of immunohistochemistry as well as Western blot analysis. Normal follicules did not express 14-3-3 sigma. In 82 papillary carcinomas, all the cases expressed 14-3-3 sigma and its expression was not reduced but even enhanced in the advanced stage and in poorly differentiated types. Furthermore, 21 of the 23 anaplastic carcinomas expressed 14-3-3 sigma and its expression level tended to be higher than in papillary carcinoma. On the other hand, none of the 34 follicular carcinomas or 29 follicular adenomas expressed 14-3-3 sigma. These results suggest that 14-3-3 sigma plays a constitutive role in papillary carcinoma rather than acting as a cell cycle regulator, whereas it is not required for the occurrence and development of follicular tumor.

    Cancer letters 2003;200;2;161-6

  • 14-3-3 sigma positively regulates p53 and suppresses tumor growth.

    Yang HY, Wen YY, Chen CH, Lozano G and Lee MH

    Department of Molecular and Cellular Oncology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.

    The 14-3-3 sigma (sigma) protein, a negative regulator of the cell cycle, is a human mammary epithelium-specific marker that is downregulated in transformed mammary carcinoma cells. It has also been identified as a p53-inducible gene product involved in cell cycle checkpoint control after DNA damage. Although 14-3-3 sigma is linked to p53-regulated cell cycle checkpoint control, detailed mechanisms of how cell cycle regulation occurs remain unclear. Decreased expression of 14-3-3 sigma was recently reported in several types of carcinomas, further suggesting that the negative regulatory role of 14-3-3 sigma in the cell cycle is compromised during tumorigenesis. However, this possible tumor-suppressive role of 14-3-3 sigma has not yet been characterized. Here, we studied the link between 14-3-3 sigma activities and p53 regulation. We found that 14-3-3 sigma interacted with p53 in response to the DNA-damaging agent adriamycin. Importantly, 14-3-3 sigma expression led to stabilized expression of p53. In studying the molecular mechanism of this increased stabilization of p53, we found that 14-3-3 sigma antagonized the biological functions of Mdm2 by blocking Mdm2-mediated p53 ubiquitination and nuclear export. In addition, we found that 14-3-3 sigma facilitated the oligomerization of p53 and enhanced p53's transcriptional activity. As a target gene of p53, 14-3-3 sigma appears to have a positive feedback effect on p53 activity. Significantly, we also showed that overexpression of 14-3-3 sigma inhibited oncogene-activated tumorigenicity in a tetracycline-regulated 14-3-3 sigma system. These results defined an important p53 regulatory loop and suggested that 14-3-3 sigma expression can be considered for therapeutic intervention in cancers.

    Molecular and cellular biology 2003;23;20;7096-107

  • Identification and characterization of a cell cycle and apoptosis regulatory protein-1 as a novel mediator of apoptosis signaling by retinoid CD437.

    Rishi AK, Zhang L, Boyanapalli M, Wali A, Mohammad RM, Yu Y, Fontana JA, Hatfield JS, Dawson MI, Majumdar AP and Reichert U

    Veterans Affairs Medical Center, Department of Internal Medicine and Karmanos Cancer Institute, Wayne State University, Detroit, Michigan 48201, USA. Rishia@Karmanos.org

    CD437, a novel retinoid, causes cell cycle arrest and apoptosis in a number of cancer cells including human breast carcinoma (HBC) by utilizing an undefined retinoic acid receptor/retinoid X receptor-independent mechanism. To delineate mediators of CD437 signaling, we utilized a random antisense-dependent functional knockout genetic approach. We identified a cDNA that encodes approximately 130-kDa HBC cell perinuclear protein (termed CARP-1). Treatments with CD437 or chemotherapeutic agent adriamycin, as well as serum deprivation of HBC cells, stimulate CARP-1 expression. Reduced levels of CARP-1 result in inhibition of apoptosis by CD437 or adriamycin, whereas increased expression of CARP-1 causes elevated levels of cyclin-dependent kinase inhibitor p21WAF1/CIP1 and apoptosis. CARP-1 interacts with 14-3-3 protein as well as causes reduced expression of cell cycle regulatory genes including c-Myc and cyclin B1. Loss of c-Myc sensitizes cells to apoptosis by CARP-1, whereas expression of c-Myc or 14-3-3 inhibits CARP-1-dependent apoptosis. Thus, apoptosis induction by CARP-1 involves sequestration of 14-3-3 and CARP-1-mediated altered expression of multiple cell cycle regulatory genes. Identification of CARP-1 as a key mediator of signaling by CD437 or adriamycin allows for delineation of pathways that, in turn, may prove beneficial for design and targeting of novel antitumor agents.

    Funded by: NCI NIH HHS: CA51993

    The Journal of biological chemistry 2003;278;35;33422-35

  • The MSP receptor regulates alpha6beta4 and alpha3beta1 integrins via 14-3-3 proteins in keratinocyte migration.

    Santoro MM, Gaudino G and Marchisio PC

    Department of Medical Sciences, University of Piemonte Orientale "A. Avogadro", 28100, Novara, Italy. msantoro@med.unipmn.it

    Growth factors, integrins, and the extracellular matrix (ECM) are known to play key roles in epidermal wound healing, although the interplay between these proteins is not fully understood. We show that growth factor macrophage stimulating protein (MSP)- and its receptor Ron-mediated PI3K activation in keratinocytes induces phosphorylation of both Ron and alpha6beta4 integrin at specific 14-3-3 binding sites. Consequently, a Ron/alpha6beta4 complex formed via 14-3-3 binding displaces alpha6beta4 from its location at hemidesmosomes (structures supporting cell adhesion) and relocalizes it to lamellipodia. Concomitant activation of alpha3beta1 and keratinocyte spreading/migration on laminin-5 occurs. Further, MSP-dependent beta4 tyrosine phosphorylation evokes p38 and NF-kappaB signaling required for keratinocyte wound closure. Based on these results, we propose a mechanism based on MSP-Ron-dependent phosphorylation and 14-3-3 association, whereby the function of alpha6beta4 switches from a mechanical adhesive device into a signaling component, and might be critically involved in human epidermal wound healing.

    Developmental cell 2003;5;2;257-71

  • Protein 14-3-3sigma interacts with and favors cytoplasmic subcellular localization of the glucocorticoid receptor, acting as a negative regulator of the glucocorticoid signaling pathway.

    Kino T, Souvatzoglou E, De Martino MU, Tsopanomihalu M, Wan Y and Chrousos GP

    Pediatric and Reproductive Endocrinology Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA. kinot@mail.nih.gov

    The glucocorticoid receptor (GR) alpha interacts with the highly conserved 14-3-3 family proteins. The latter bind phosphorylated serine/threonine residues of "partner" molecules and influence many signal transduction events by altering their subcellular localization and/or protecting them from proteolysis. To examine the physiologic role of 14-3-3 on the glucocorticoid-signaling pathway, we studied the nucleocytoplasmic shuttling and transactivation properties of GRalpha in a cell line replete with or devoid of 14-3-3sigma. We found that endogenous 14-3-3sigma helped localize green fluorescent protein-fused GRalpha in the cytoplasm in the absence of ligand and potentiated its nuclear export after ligand withdrawal. 14-3-3sigma also suppressed the transcriptional activity of GRalpha on a glucocorticoid-responsive promoter. Disruption of the classic nuclear export signal of 14-3-3sigma inactivated its ability to influence the nucleocytoplasmic trafficking and transactivation activity of GRalpha, whereas introduction of a mutation inactivating the binding activity of 14-3-3sigma to some of its partner proteins did not. 14-3-3sigma bound the ligand-binding domain of GRalpha through its COOH-terminal portion, in a partially ligand-dependent fashion, while it did not interact with "ligand-binding domain" of GRbeta at all. These results suggest that 14-3-3sigma functions as a negative regulator in the glucocorticoid signaling pathway, possibly by shifting the subcellular localization/circulation of this receptor toward the cytoplasm through its nuclear export signal. Since 14-3-3 proteins play significant roles in numerous cellular activities, such as cell cycle progression, growth, differentiation, and apoptosis, these actions might indirectly influence the transcriptional activity of GRalpha. Conversely, through its 14-3-3 protein interactions, GRalpha may influence these processes.

    The Journal of biological chemistry 2003;278;28;25651-6

  • Immunohistochemical demonstration of 14-3-3 sigma protein in normal human tissues and lung cancers, and the preponderance of its strong expression in epithelial cells of squamous cell lineage.

    Nakajima T, Shimooka H, Weixa P, Segawa A, Motegi A, Jian Z, Masuda N, Ide M, Sano T, Oyama T, Tsukagoshi H, Hamanaka K and Maeda M

    Second Department of Pathology and The First Department of Surgery, Gunma University School of Medicine, and Immuno-Biological Laboratories Co Ltd, Gunma, Japan. nakajima@med.gunma-u.ac.jp

    In order to confirm 14-3-3 sigma (sigma) protein distribution in human tissues, immunohistochemistry was performed using various paraffin-embedded human tissues. In normal human tissues, the strongest immunoreactivity for 14-3-3sigma protein was observed in squamous epithelia at various sites, followed by basal cells of the trachea, bronchus and basal or myoepithelial cells of various glands. Moderate to weak 14-3-3sigma immunoreactivity was seen in the epithelial cells of the alimentary tract, gall bladder, urinary tract and endometrium. In the lung, 14-3-3sigma immunoreactivity was also observed in hyperplastic type II alveolar cells and metaplastic squamous cells. Immunohistochemical study using non-small-cell lung cancers revealed that 14-3-3sigma immunoreactivity was stronger in squamous cell carcinomas than in adenocarcinomas. The present study revealed that 14-3-3sigma expression was exclusively present in various epithelial cells and had a tendency to be stronger in cells destined for squamous epithelium or differentiating toward squamous cells in human normal and neoplastic cells.

    Pathology international 2003;53;6;353-60

  • The tumor suppressor gene 14-3-3 sigma is commonly methylated in normal and malignant lymphoid cells.

    Bhatia K, Siraj AK, Hussain A, Bu R and Gutiérrez MI

    KFNCCC&R, Research Centre, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia, 11211. Kishor_Bhatia@kfshrc.edu.sa

    14-3-3 sigma/Stratifin was first identified as an epithelial cell antigen (HME-1) exclusively expressed in epithelia. However, the functional role of sigma in cell proliferation and apoptosis would suggest that this protein could be relevant to the regulation of growth and differentiation of multiple cell types. Recent evidence demonstrates that sigma acts as a tumor suppressor gene that is inactivated by methylation of its 5' CpG islands in epithelial tumor cells. In normal epithelia, sigma is commonly unmethylated. The objective of this study was to determine the methylation status of sigma in lymphoid cells. We now demonstrate by methylation-specific PCR analysis that sigma is also methylated in normal and malignant lymphocytes. Such methylation, however, fails to completely silence its expression. Compared with the robust expression in epithelial cells, lymphocytes showed basal, but clearly evident, levels of sigma as determined by reverse transcription-PCR and Western blot. The finding of sigma 5' region methylation in lymphocytes has direct implications in the use of body fluids on methylation tests for noninvasive monitoring of occult epithelial tumor cells and suggests that sigma may not be an adequate biomarker for methylation-specific PCR analysis.

    Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 2003;12;2;165-9

  • Regulation of TSC2 by 14-3-3 binding.

    Li Y, Inoki K, Yeung R and Guan KL

    Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.

    Mutation in either the TSC1 or TSC2 tumor suppressor gene is responsible for the inherited genetic disease of tuberous sclerosis complex. TSC1 and TSC2 form a physical and functional complex to regulate cell growth. Recently, it has been demonstrated that TSC1.TSC2 functions to inhibit ribosomal S6 kinase and negatively regulate cell size. TSC2 is negatively regulated by Akt phosphorylation. Here, we report that TSC2, but not TSC1, associates with 14-3-3 in vivo. Phosphorylation of Ser(1210) in TSC2 is required for its association with 14-3-3. Our data indicate that 14-3-3 association may inhibit the function of TSC2 and represents a possible mechanism of TSC2 regulation.

    The Journal of biological chemistry 2002;277;47;44593-6

  • 14-3-3 interacts with the tumor suppressor tuberin at Akt phosphorylation site(s).

    Liu MY, Cai S, Espejo A, Bedford MT and Walker CL

    Department of Carcinogenesis, Science Park-Research Division, The University of Texas M. D. Anderson Cancer Center, Smithville, Texas 78957, USA.

    Tuberin, the product of the tuberous sclerosis complex 2 tumor suppressor gene, is a phosphoprotein that negatively regulates phosphatidylinositol 3'-kinase signaling downstream of Akt. Several high stringency 14-3-3 binding sites that overlapped with Akt phosphorylation sites were identified in tuberin in silico. Recognition of tuberin by an alpha-14-3-3 binding site-specific antibody correlated with mitogen-induced phosphorylation of tuberin and recognition of tuberin by an alpha-Akt phosphorylation substrate antibody. Recognition of tuberin by both antibodies was blocked by inhibiting phosphatidylinositol 3'-kinase activity. Using a protein domain microarray, a tuberin peptide containing Ser(939) demonstrated phospho-specific binding to 14-3-3. Glutathione S-transferase pull-down assays with 14-3-3 fusion proteins revealed that all seven 14-3-3 isoforms (beta, gamma, zeta, epsilon, tau, eta, and sigma) could bind tuberin, and this interaction was abrogated by competition with phosphorylated but not unphosphorylated Ser(939) tuberin peptide. Tuberin also coimmunoprecipitated with 14-3-3, confirming the interaction between endogenous 14-3-3 and tuberin. These data establish the presence of functional and overlapping 14-3-3 and Akt recognition site(s) in tuberin.

    Funded by: NCI NIH HHS: CA63613; NIDDK NIH HHS: DK62268; NIEHS NIH HHS: ES07784, ES08263

    Cancer research 2002;62;22;6475-80

  • Interaction with 14-3-3 proteins promotes functional expression of the potassium channels TASK-1 and TASK-3.

    Rajan S, Preisig-Müller R, Wischmeyer E, Nehring R, Hanley PJ, Renigunta V, Musset B, Schlichthörl G, Derst C, Karschin A and Daut J

    Institute of Physiology, Marburg University, Deutschhausstrasse 2, 35037 Marburg, Germany.

    The two-pore-domain potassium channels TASK-1, TASK-3 and TASK-5 possess a conserved C-terminal motif of five amino acids. Truncation of the C-terminus of TASK-1 strongly reduced the currents measured after heterologous expression in Xenopus oocytes or HEK293 cells and decreased surface membrane expression of GFP-tagged channel proteins. Two-hybrid analysis showed that the C-terminal domain of TASK-1, TASK-3 and TASK-5, but not TASK-4, interacts with isoforms of the adapter protein 14-3-3. A pentapeptide motif at the extreme C-terminus of TASK-1, RRx(S/T)x, was found to be sufficient for weak but significant interaction with 14-3-3, whereas the last 40 amino acids of TASK-1 were required for strong binding. Deletion of a single amino acid at the C-terminal end of TASK-1 or TASK-3 abolished binding of 14-3-3 and strongly reduced the macroscopic currents observed in Xenopus oocytes. TASK-1 mutants that failed to interact with 14-3-3 isoforms (V411*, S410A, S410D) also produced only very weak macroscopic currents. In contrast, the mutant TASK-1 S409A, which interacts with 14-3-3-like wild-type channels, displayed normal macroscopic currents. Co-injection of 14-3-3zeta cRNA increased TASK-1 current in Xenopus oocytes by about 70 %. After co-transfection in HEK293 cells, TASK-1 and 14-3-3zeta (but not TASK-1DeltaC5 and 14-3-3zeta) could be co-immunoprecipitated. Furthermore, TASK-1 and 14-3-3 could be co-immunoprecipitated in synaptic membrane extracts and postsynaptic density membranes. Our findings suggest that interaction of 14-3-3 with TASK-1 or TASK-3 may promote the trafficking of the channels to the surface membrane.

    The Journal of physiology 2002;545;1;13-26

  • Efp targets 14-3-3 sigma for proteolysis and promotes breast tumour growth.

    Urano T, Saito T, Tsukui T, Fujita M, Hosoi T, Muramatsu M, Ouchi Y and Inoue S

    Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.

    Oestrogen exerts its influence on target organs through activating oestrogen receptors (ERs) and regulating downstream genes by means of their oestrogen-responsive elements. Efp, a target gene product of ER alpha, is a member of the RING-finger B-box coiled-coil (RBCC) motif family. Efp is predominantly expressed in various female organs as well as in breast cancers, and is thought to be essential for oestrogen-dependent cell proliferation and organ development Efp-disrupted mice display underdeveloped uteri and reduced oestrogen responsiveness. Here we show that Efp is a RING-finger-dependent ubiquitin ligase (E3) that targets proteolysis of 14-3-3 sigma, a negative cell cycle regulator that causes G2 arrest. We demonstrate that tumour growth of breast cancer MCF7 cells implanted in female athymic mice is reduced by treatment with antisense Efp oligonucleotide. Efp-overexpressing MCF7 cells in ovariectomized athymic mice generate tumours in the absence of oestrogen. Loss of Efp function in mouse embryonic fibroblasts results in an accumulation of 14-3-3 sigma, which is responsible for reduced cell growth. These data provide an insight into the cell-cycle machinery and tumorigenesis of breast cancer by identifying 14-3-3 sigma as a target for proteolysis by Efp, leading to cell proliferation.

    Nature 2002;417;6891;871-5

  • Cytoplasmic localization of tristetraprolin involves 14-3-3-dependent and -independent mechanisms.

    Johnson BA, Stehn JR, Yaffe MB and Blackwell TK

    Center for Blood Research and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.

    The immediate early gene tristetraprolin (TTP) is induced transiently in many cell types by numerous extracellular stimuli. TTP encodes a zinc finger protein that can bind and destabilize mRNAs that encode tumor necrosis factor-alpha (TNFalpha) and other cytokines. We hypothesize that TTP also has a broader role in growth factor-responsive pathways. In support of this model, we have previously determined that TTP induces apoptosis through the mitochondrial pathway, analogously to certain oncogenes and other immediate-early genes, and that TTP sensitizes cells to the pro-apoptotic signals of TNFalpha. In this study, we show that TTP and the related proteins TIS11b and TIS11d bind specifically to 14-3-3 proteins and that individual 14-3-3 isoforms preferentially bind to different phosphorylated TTP species. 14-3-3 binding does not appear to inhibit or promote induction of apoptosis by TTP but is one of multiple mechanisms that localize TTP to the cytoplasm. Our results provide the first example of 14-3-3 interacting functionally with an RNA binding protein and binding in vivo to a Type II 14-3-3 binding site. They also suggest that 14-3-3 binding is part of a complex network of stimuli and interactions that regulate TTP function.

    Funded by: NCI NIH HHS: CA84418; NIGMS NIH HHS: GM60594

    The Journal of biological chemistry 2002;277;20;18029-36

  • Keratin binding to 14-3-3 proteins modulates keratin filaments and hepatocyte mitotic progression.

    Ku NO, Michie S, Resurreccion EZ, Broome RL and Omary MB

    Department of Medicine, Veterans Affairs Palo Alto Health Care System, 3801 Miranda Avenue, 154J, Palo Alto, CA 94304, USA.

    Keratin polypeptides 8 and 18 (K8/18) are the major intermediate filament proteins of simple-type epithelia. K18 Ser-33 phosphorylation regulates its binding to 14-3-3 proteins during mitosis. We studied the significance of keratin binding to 14-3-3 in transgenic mice that overexpress wild-type or Ser-33-->Ala (S33A) K18. In S33A but not wild-type K18-overexpressing mice, pancreatic acinar cell keratin filaments retracted from the basal nuclear region and became apically concentrated. In contrast, K18 S33A had a minimal effect on hepatocyte keratin filament organization. Partial hepatectomy of K18-S33A-overexpressing mice did not affect liver regeneration but caused limited mitotic arrest, accumulation of abnormal mitotic figures, dramatic fragmentation of hepatocyte keratin filaments, with retention of a speckled 14-3-3zeta mitotic cell nuclear-staining pattern that usually becomes diffuse during mitosis. Hence, K18 Ser-33 phosphorylation regulates keratin filament organization in simple-type epithelia in vivo. Keratin binding to 14-3-3 may partially modulate hepatocyte mitotic progression, in association with nuclear redistribution of 14-3-3 proteins during mitosis.

    Funded by: NIDDK NIH HHS: DK52951, DK56339

    Proceedings of the National Academy of Sciences of the United States of America 2002;99;7;4373-8

  • Radiation-induced phosphorylation of Chk1 at S345 is associated with p53-dependent cell cycle arrest pathways.

    Tian H, Faje AT, Lee SL and Jorgensen TJ

    Department of Radiation Medicine, Lombardi Cancer Center, Georgetown University Medical Center, 3970 Reservoir Road, N.W., Washington, DC 20007-2197, USA.

    Because DNA damage-inducible cell cycle checkpoints are thought to protect cells from the lethal effects of ionizing radiation, a better understanding of the mechanistic functions of cell cycle regulatory proteins may reveal new molecular targets for cancer therapy. The two major regulatory proteins of G2 arrest are Chk1 and p53. Yet, it is unclear how these two proteins interact and coordinate their functional roles during radiation-induced G2 arrest. To determine Chk1's role in p53-dependent G2 arrest, we used p53 proficient cells and examined expression of G2 arrest proteins under conditions in which G2 arrest was inhibited by the staurosporine analog, UCN-01. We found that UCN-01 inhibited both G1 and G2 arrest in irradiated p53 proficient cells. The arrest inhibition was associated with suppression of radiation-induced expression of both p21 and 14-3-3 sigma -- two known p53-dependent G2 arrest proteins. The suppression occurred despite normal induction of p53 and normal phosphorylation of p53 at S20 and Cdc25C at S216 -- the two known substrates of Chk1 kinase activity. In contrast, we showed that radiation-induced phosphorylation of Chk1 at S345 was associated with binding of Chk1 to p53, p21, and 14-3-3 sigma, and that UCN-01 inhibited S345 phosphorylation. We suggest that DNA damage-induced phosphorylation of Chk1 at S345, and subsequent p53 binding, links Chk1 with p53 downstream responses and may provide a coordinated interaction between DNA damage responses and cell cycle arrest functions.

    Funded by: NCI NIH HHS: 2P30-CA51008, P01-CA74175

    Neoplasia (New York, N.Y.) 2002;4;2;171-80

  • The G2/M regulator 14-3-3sigma prevents apoptosis through sequestration of Bax.

    Samuel T, Weber HO, Rauch P, Verdoodt B, Eppel JT, McShea A, Hermeking H and Funk JO

    Laboratory of Molecular Tumor Biology, Department of Dermatology, University of Erlangen-Nuremberg, 91052 Erlangen, Germany.

    In response to DNA damage and genotoxic stress, the p53 tumor suppressor triggers either cell cycle arrest or apoptosis. The G(2) arrest after damage is, in part, mediated by the p53 target, 14-3-3final sigma (final sigma). Colorectal tumor cells lacking final sigma are exquisitely sensitive to DNA damage. Here we analyzed the mechanism of this sensitivity in final sigma(-/-) as compared with final sigma(+/+) human colorectal tumor cells. Exposure to adriamycin resulted in rapid apoptosis only in final sigma(-/-) cells. This was further characterized by caspase-3 activation, p21(CIP1) cleavage, and CDK2 activation. Moreover, Bax was rapidly translocated out of the cytoplasm, and cytochrome c was released in final sigma(-/-) cells. Transient adenovirus-mediated reconstitution of final sigma in the final sigma(-/-) cells led to effective rescue of this phenotype and protected cells against apoptosis. The association of final sigma, Bax, and CDK1 in protein complexes may be the basis for this antiapoptotic mechanism. In conclusion, final sigma not only enforces the p53-dependent G(2) arrest but also delays the apoptotic signal transduction.

    The Journal of biological chemistry 2001;276;48;45201-6

  • Association of the cyclin-dependent kinases and 14-3-3 sigma negatively regulates cell cycle progression.

    Laronga C, Yang HY, Neal C and Lee MH

    Departments of Surgical Oncology/Molecular and Cellular Oncology and the Breast Cancer Research Program, the University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.

    14-3-3 sigma, implicated in cell cycle arrest by p53, was cloned by expression cloning through cyclin-dependent kinase 2 (CDK2) association. 14-3-3 sigma shares cyclin-CDK2 binding motifs with different cell cycle regulators, including p107, p130, p21(CIP1), p27(KIP1), and p57(KIP2), and is associated with cyclin.CDK complexes in vitro and in vivo. Overexpression of 14-3-3 sigma obstructs cell cycle entry by inhibiting cyclin-CDK activity in many breast cancer cell lines. Overexpression of 14-3-3 sigma can also inhibit cell proliferation and prevent anchorage-independent growth of these cell lines. These findings define 14-3-3 sigma as a negative regulator of the cell cycle progression and suggest that it has an important function in preventing breast tumor cell growth.

    The Journal of biological chemistry 2000;275;30;23106-12

  • 14-3-3Sigma is required to prevent mitotic catastrophe after DNA damage.

    Chan TA, Hermeking H, Lengauer C, Kinzler KW and Vogelstein B

    The Johns Hopkins Oncology Center, Program in Human Genetics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, USA.

    14-3-3Sigma is a member of a family of proteins that regulate cellular activity by binding and sequestering phosphorylated proteins. It has been suggested that 14-3-3sigma promotes pre-mitotic cell-cycle arrest following DNA damage, and that its expression can be controlled by the p53 tumour suppressor gene. Here we describe an improved approach to the generation of human somatic-cell knockouts, which we have used to generate human colorectal cancer cells in which both 14-3-3sigma alleles are inactivated. After DNA damage, these cells initially arrested in the G2 phase of the cell cycle, but, unlike cells containing 14-3-3sigma, the 14-3-3sigma-/- cells were unable to maintain cell-cycle arrest. The 14-3-3sigma-/- cells died ('mitotic catastrophe') as they entered mitosis. This process was associated with a failure of the 14-3-3sigma-deficient cells to sequester the proteins (cyclin B1 and cdc2) that initiate mitosis and prevent them from entering the nucleus. These results may indicate a mechanism for maintaining the G2 checkpoint and preventing mitotic death.

    Nature 1999;401;6753;616-20

  • 14-3-3 sigma is a p53-regulated inhibitor of G2/M progression.

    Hermeking H, Lengauer C, Polyak K, He TC, Zhang L, Thiagalingam S, Kinzler KW and Vogelstein B

    Johns Hopkins Oncology Center, Baltimore, Maryland, USA.

    Exposure of colorectal cancer (CRC) cells to ionizing radiation results in a cell-cycle arrest in G1 and G2. The G1 arrest is due to p53-mediated induction of the cyclin-dependent kinase inhibitor p21WAF1/CIP1/SDI1, but the basis for the G2 arrest is unknown. Through a quantitative analysis of gene expression patterns in CRC cell lines, we have discovered that 14-3-3 sigma is strongly induced by gamma irradiation and other DNA-damaging agents. The induction of 14-3-3 sigma is mediated by a p53-responsive element located 1.8 kb upstream of its transcription start site. Exogenous introduction of 14-3-3 sigma into cycling cells results in a G2 arrest. As the fission yeast 14-3-3 homologs rad24 and rad25 mediate similar checkpoint effects, these results document a molecular mechanism for G2/M control that is conserved throughout eukaryotic evolution and regulated in human cells by p53.

    Funded by: NCI NIH HHS: CA 43460, CA 57345

    Molecular cell 1997;1;1;3-11

  • Molecular cloning and expression of the transformation sensitive epithelial marker stratifin. A member of a protein family that has been involved in the protein kinase C signalling pathway.

    Leffers H, Madsen P, Rasmussen HH, Honoré B, Andersen AH, Walbum E, Vandekerckhove J and Celis JE

    Institute of Medical Biochemistry, Danish Centre for Human Genome Research, Aarhus University.

    We have identified a family of abundant acidic human keratinocyte proteins with apparent molecular masses ranging between 30,000 and 31,100 (isoelectric focussing sample spot proteins 9109 (epithelial marker stratifin), 9124, 9125, 9126 and 9231 in the master two-dimensional gel database of human keratinocyte proteins) that share peptide sequences with each other, with protein 14-3-3 and with the kinase C inhibitory protein. Immunofluorescence staining of keratinocytes showed that two of these proteins (IEF SSPs 9124 and 9126) localize to the Golgi apparatus, while stratifin is distributed diffusely in the cytoplasm. Significant levels of stratifin, and in smaller amount the sample spot proteins 9124, 9125 and 9126, were detected in the medium of cultured human keratinocytes suggesting that they are partially secreted by these cells. Two-dimensional gel analysis of proteins from cultured human cells and fetal tissues showed that polypeptides comigrating with proteins 9124, 9125 and 9126 are ubiquitous and highly expressed in the brain. Stratifin, however, was present only in cultured epithelial cells and was most abundant in fetal and adult human tissues enriched in stratified squamous keratinising epithelium. We have cloned and sequenced cDNAs coding for members of this family. The complete identity of the sequenced peptides from stratifin with the amino acid sequence translated from the stratifin cDNA clone indicated that this cDNA codes for stratifin. The identity of clones 1054, HS1 and AS1 is less clear as, with few exceptions, none of the individual peptide sequences fits the predicted protein sequences. The polypeptides synthesized by clones 1054 and HS1 in the vaccinia expression system, on the other hand, comigrate with proteins 9126 and 9124, suggesting cell-type-specific expression of members of the protein family. Database searches indicated that clone HS1 corresponds to a human T-cell cDNA 14-3-3 clone, while the high level of similarity of clones 1054 and AS1 with the 14-3-3 beta and eta sequences respectively, suggested that they code for the human equivalent of the two bovine proteins. Microsequence data indicated that IEF SSP 9124 corresponds to the human homolog of bovine 14-3-3 gamma.

    Journal of molecular biology 1993;231;4;982-98

  • Microsequences of 145 proteins recorded in the two-dimensional gel protein database of normal human epidermal keratinocytes.

    Rasmussen HH, van Damme J, Puype M, Gesser B, Celis JE and Vandekerckhove J

    Institute of Medical Biochemistry, Aarhus University, Denmark.

    Microsequencing of proteins recovered from two-dimensional (2-D) gels is being used systematically to identify proteins in the master human keratinocyte 2-D gel database. To date, about 250 protein spots recorded in human 2-D gel databases have been microsequenced and, of these, 145 are recorded in the keratinocyte database under the entry partial amino acid sequence. Coomassie Brilliant Blue-stained protein spots cut from several (up to 40) dry gels were concentrated by elution-concentration gel electrophoresis, electroblotted onto PVDF membranes and digested in situ with trypsin. Eluting peptides were separated by reversed-phase HPLC, collected individually and sequenced. Computer search using the FASTA and TFASTA programs from Genetics Computer Group indicated that 110 of the microsequenced polypeptides shared significant similarity with proteins contained in the PIR, Mipsx or GenEMBL databases. Only 35 polypeptides corresponded to hitherto unknown proteins. Peptide sequences of all 145 proteins are listed together with their coordinates (apparent molecular weight and pI) in the keratinocyte database.

    Electrophoresis 1992;13;12;960-9

  • Complementary DNA cloning of a novel epithelial cell marker protein, HME1, that may be down-regulated in neoplastic mammary cells.

    Prasad GL, Valverius EM, McDuffie E and Cooper HL

    Cell and Molecular Physiology Section, National Cancer Institute, Bethesda, Maryland 20892.

    A full-length complementary DNA clone from a normal human mammary epithelial cell (strain 184) encoding a 25-kilodalton protein, HME1, was isolated. Expression of HME1 RNA appears to be limited to epithelial cells. The HME1 sequence has extensive sequence homology with bovine 14-3-3 protein, which is an activator of tyrosine and tryptophan hydroxylase. However, the tissue distribution, arrangement of charged amino acids, and location of potential phosphorylation sites of HME1 differ from those of 14-3-3. Compared with normal mammary epithelial cells, expression of HME1 RNA was dramatically low in two cell lines derived from human mammary carcinoma that were examined, and in a line of normal mammary epithelial cells transformed by oncogenes. HME1 therefore appears to be a cellular differentiation marker that may be down-regulated during neoplastic transformation.

    Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research 1992;3;8;507-13

Gene lists (6)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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