G2Cdb::Gene report

Gene id
G00001894
Gene symbol
CLU (HGNC)
Species
Homo sapiens
Description
clusterin
Orthologue
G00000645 (Mus musculus)

Databases (8)

Gene
ENSG00000120885 (Ensembl human gene)
1191 (Entrez Gene)
1045 (G2Cdb plasticity & disease)
CLU (GeneCards)
Literature
185430 (OMIM)
Marker Symbol
HGNC:2095 (HGNC)
Protein Expression
476 (human protein atlas)
Protein Sequence
P10909 (UniProt)

Synonyms (4)

  • KUB1
  • SGP-2
  • SP-40
  • TRPM-2

Literature (176)

Pubmed - other

  • Loss of clusterin expression worsens renal ischemia-reperfusion injury.

    Zhou W, Guan Q, Kwan CC, Chen H, Gleave ME, Nguan CY and Du C

    Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia, Canada.

    Prevention of ischemia-reperfusion injury (IRI) is a challenge in clinical care of the patients with kidney transplants or acute kidney injury, and understanding of the intrinsic mechanisms of resistance to injury in the kidney will lead to a novel therapy. Clusterin, a secreted glycoprotein, is an antiapoptotic protein in cancer cells. Our study is to investigate the role of clusterin in renal IRI. Renal IRI in mice was induced by clamping renal vein and artery for 45 or 50 min at 32 degrees C. Apoptosis of renal tubular epithelial cells (TECs) was determined by FACS analysis. Clusterin expression was examined by Western blot or immunohistochemistry. Here, we showed that clusterin protein was induced in TECs following IRI, and more tubules expressed clusterin in the kidneys following ischemia at higher temperatures. In human proximal TEC HKC-8 cultures, clusterin was upregulated by removal of serum and growth factors in medium and was downregulated by TNF-alpha-IFN-gamma mixture. The levels of clusterin were positively correlated with cell survival in these conditions. Knockdown or knockout of clusterin expression enhanced the sensitivity of TECs to apoptosis. In experimental models of renal IRI, deficiency in clusterin expression worsened the injury, as indicated by a significant increase in renal tissue damage with higher levels of serum creatinine and blood urea nitrogen and by a poorer recovery from the injury in clusterin-deficient mice compared with wild-type mice. Our data indicate that the reduction of inducible expression of clusterin results in an increase in TEC apoptosis in the cultures and renders mice susceptibility to IRI, implying a protective role of clusterin in kidney injury.

    Funded by: Canadian Institutes of Health Research

    American journal of physiology. Renal physiology 2010;298;3;F568-78

  • Transcriptome profiling of a TGF-beta-induced epithelial-to-mesenchymal transition reveals extracellular clusterin as a target for therapeutic antibodies.

    Lenferink AE, Cantin C, Nantel A, Wang E, Durocher Y, Banville M, Paul-Roc B, Marcil A, Wilson MR and O'Connor-McCourt MD

    Receptor, Signaling and Proteomics Group, Biotechnology Research Institute, National Research Council of Canada, Montréal, Quebec, Canada. anne.lenferink@cnrc-nrc.gc.ca

    Transforming growth factor (TGF)-beta plays a dual role in tumorigenesis, switching from acting as a growth inhibitory tumor suppressor early in the process, to a tumor promoter in late-stage disease. Since TGF-beta's prometastatic role may be linked to its ability to induce tumor cell epithelial-to-mesenchymal transition (EMT), we explored TGF-beta's EMT-promoting pathways by analysing the transcriptome changes occurring in BRI-JM01 mammary tumor epithelial cells undergoing a TGF-beta-induced EMT. We found the clusterin gene to be the most highly upregulated throughout most of the TGF-beta time course, and showed that this results in an increase of the secreted form of clusterin. By monitoring several hallmark features of EMT, we demonstrated that antibodies targeting secreted clusterin inhibit the TGF-beta-induced EMT of BRI-JM01 cells, as well as the invasive phenotype of several other breast and prostate tumor cell lines (4T1, NMuMG, MDA-MB231LM2 and PC3), without affecting the proliferation of these cells. These results indicate that secreted clusterin is a functionally important EMT mediator that lies downstream within TGF-beta's EMT-promoting transcriptional cascade, but not within its growth-inhibitory pathways. To further investigate the role played by secreted clusterin in tumor metastasis, we assessed the effect of several anti-clusterin monoclonal antibodies in vivo using a 4T1 syngeneic mouse breast cancer model and found that these antibodies significantly reduce lung metastasis. Taken together, our results reveal a role for secreted clusterin as an important extracellular promoter of EMT, and suggest that antibodies targeting clusterin may inhibit tumor metastasis without reducing the beneficial growth inhibitory effects of TGF-beta.

    Oncogene 2010;29;6;831-44

  • Identification of human plasma proteins as major clients for the extracellular chaperone clusterin.

    Wyatt AR and Wilson MR

    School of Biological Sciences, University of Wollongong, Wollongong, New South Wales 2522, Australia.

    Clusterin (CLU) is an extracellular chaperone that is likely to play an important role in protein folding quality control. This study identified three deposition disease-associated proteins as major plasma clients for clusterin by studying CLU-client complexes formed in response to physiologically relevant stress (shear stress, approximately 36 dynes/cm(2) at 37 degrees C). Analysis of plasma samples by size exclusion chromatography indicated that (i) relative to control plasma, stressed plasma contained proportionally more soluble protein species of high molecular weight, and (ii) high molecular weight species were far more abundant when proteins purified by anti-CLU immunoaffinity chromatography from stressed plasma were compared with those purified from control plasma. SDS-PAGE and Western blot analyses indicated that a variety of proteins co-purified with CLU from both stressed and control plasma; however, several proteins were uniquely present or much more abundant when plasma was stressed. These proteins were identified by mass spectrometry as ceruloplasmin, fibrinogen, and albumin. Immunodot blot analysis of size exclusion chromatography fractionated plasma suggested that CLU-client complexes generated in situ are very large and may reach >or=4 x 10(7) Da. Lastly, sandwich enzyme-linked immunosorbent assay detected complexes containing CLU and ceruloplasmin, fibrinogen, or albumin in stressed but not control plasma. We have previously proposed that CLU-client complexes serve as vehicles to dispose of damaged misfolded extracellular proteins in vivo via receptor-mediated endocytosis. A better understanding of these mechanisms is likely to ultimately lead to the identification of new therapies for extracellular protein deposition disorders.

    The Journal of biological chemistry 2010;285;6;3532-9

  • Alzheimer disease: New light on an old CLU.

    Bertram L and Tanzi RE

    Funded by: NIMH NIH HHS: MH060009-10, R37 MH060009, R37 MH060009-10

    Nature reviews. Neurology 2010;6;1;11-3

  • Protective effect of clusterin on oxidative stress-induced cell death of human corneal endothelial cells.

    Shin YJ, Kim JH, Seo JM, Lee SM, Hyon JY, Yu YS and Wee WR

    Myung Dong St. Mary's Eye Center, Seoul National University College of Engineering, Seoul, Korea.

    Purpose: To investigate the protective effect of clusterin on oxidative stress-induced cell death of human corneal endothelial cells.

    Methods: Human corneal endothelial cells (HCECs) were cultured according to previously published methods. With treatment of various concentrations (0-50 mM) of tert-butyl hydroperoxide (tBHP) or clusterin, reactive oxygen species (ROS) producrion was measured using an oxidationsensitive fluorescent probe and 2'7'-dichlorofluorescin diacetate (DCFH-DA). Cell viability was assayed with a Cell Counting Kit-8.

    Results: In HCECs, DCF-DA staining revealed that cells treated with a higher concentration of tBHP had higher fluorescent intensity than cells treated with clusterin, compared to control cells. Clusterin significantly inhibited tBHP-induced ROS production. Cell viability decreased with higher tBHP concentration. Cells treated with clusterin had higher viability than control cells at 5 mM tBHP. Clusterin effectively protected HCECs from ROS-induced cell death.

    Conclusions: Our data suggest that clusterin may protect HCECs from oxidative injury-mediated cell death via inhibition of ROS production.

    Molecular vision 2009;15;2789-95

  • Clusterin as a predictor for chemoradiotherapy sensitivity and patient survival in esophageal squamous cell carcinoma.

    He LR, Liu MZ, Li BK, Rao HL, Liao YJ, Zhang LJ, Guan XY, Zeng YX and Xie D

    State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-Sen University, Guangzhou, China.

    Clusterin (CLU) is frequently overexpressed and correlates closely with chemotherapy and radiotherapy resistance and poor prognosis in many human cancers. However, the significance of CLU expression in chemoradiotherapy (CRT) sensitivity and its effect on the prognosis of esophageal squamous cell carcinoma (ESCC) are still unknown. In the present study, we used the methods of immunohistochemistry and terminal deoxyuridine triphosphate nick-end labeling assay to examine the expression status of CLU and apoptotic index in 110 pretreated biopsy specimens of ESCC patients treated with definitive CRT. High expression of CLU was observed in 42.7% of epithelium and 50.0% of stroma in ESCC. A significant association of high CLU stromal expression with large tumor size (P = 0.012) and locoregional progression (P = 0.001) was observed, and high epithelial expression of CLU showed a significant correlation with the lack of complete response (P = 0.028) and low apoptotic index (P = 0.001). Univariate analysis revealed that high CLU stromal expression was associated with poor locoregional progression-free survival, distant progression-free survival, and overall survival. Furthermore, ESCC patients with high CLU expression in both epithelium and stroma have the shortest survival time among the subgroups of different CLU expression status. In multivariate analysis, CLU stromal expression was evaluated as an independent prognostic factor for locoregional progression-free survival, distant progression-free survival, and overall survival. These findings suggest an important role for CLU, especially in stroma, in ESCC progression, and that high CLU epithelial expression might be a promising predictor of ESCC resistance to CRT.

    Cancer science 2009;100;12;2354-60

  • Gene expression profiling in sinonasal adenocarcinoma.

    Tripodi D, Quéméner S, Renaudin K, Ferron C, Malard O, Guisle-Marsollier I, Sébille-Rivain V, Verger C, Géraut C and Gratas-Rabbia-Ré C

    Inserm, UMR 892, Nantes F-44007, France. dominique.tripodi@chu-nantes.fr

    Background: Sinonasal adenocarcinomas are uncommon tumors which develop in the ethmoid sinus after exposure to wood dust. Although the etiology of these tumors is well defined, very little is known about their molecular basis and no diagnostic tool exists for their early detection in high-risk workers.

    Methods: To identify genes involved in this disease, we performed gene expression profiling using cancer-dedicated microarrays, on nine matched samples of sinonasal adenocarcinomas and non-tumor sinusal tissue. Microarray results were validated by quantitative RT-PCR and immunohistochemistry on two additional sets of tumors.

    Results: Among the genes with significant differential expression we selected LGALS4, ACS5, CLU, SRI and CCT5 for further exploration. The overexpression of LGALS4, ACS5, SRI, CCT5 and the downregulation of CLU were confirmed by quantitative RT-PCR. Immunohistochemistry was performed for LGALS4 (Galectin 4), ACS5 (Acyl-CoA synthetase) and CLU (Clusterin) proteins: LGALS4 was highly up-regulated, particularly in the most differentiated tumors, while CLU was lost in all tumors. The expression of ACS5, was more heterogeneous and no correlation was observed with the tumor type.

    Conclusion: Within our microarray study in sinonasal adenocarcinoma we identified two proteins, LGALS4 and CLU, that were significantly differentially expressed in tumors compared to normal tissue. A further evaluation on a new set of tissues, including precancerous stages and low grade tumors, is necessary to evaluate the possibility of using them as diagnostic markers.

    BMC medical genomics 2009;2;65

  • Overexpression of clusterin in ovarian cancer is correlated with impaired survival.

    Yang GF, Li XM and Xie D

    Department of Gynecology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China. pgf_yang@126.com

    Clusterin has been found to be overexpressed in several human malignancies and also be expressed in ovarian carcinoma tissues. However, to date, no study has investigated the prognostic significance of clusterin expression in ovarian carcinoma. Therefore, we examined the relationship between clusterin overexpression and clinicopathological features to determine its prognostic relevance. Eighty-six patients diagnosed with primary ovarian cancer between 1993 and 2004 were selected and recorded follow-up data and clinicopathological data. The expression of clusterin was detected on the sections of tissue microarray by immunohistochemistry and was evaluated the association with patient's clinical features and prognosis. Overexpression of clusterin protein in ovarian cancer was observed in 46.5% of the patients and was found more often in disease that was in the advanced stage (P = 0.0001). The expression levels of clusterin was associated with International Federation of Gynecology and Obstetrics stage (P = 0.0001) and histologic type (P = 0.002). However, no significant association was observed between clusterin expression and patient age or tumor Silverberg grade (P > 0.05). In addition, the average survival time of the patients with clusterin overexpression was significantly shorter than that with normal expression of clusterin. Clusterin expression was associated with survival of patients with primary ovarian cancer (relative risk for overall survival 1.69; 95% confidence interval, 1.52 to 1.95 (P = 0.033)). Our data show that clusterin is not only a biomarker associated with ovarian cancer, but it also appears to be a prognostic factor associated with adverse outcome.

    International journal of gynecological cancer : official journal of the International Gynecological Cancer Society 2009;19;8;1342-6

  • Candidate genetic analysis of plasma high-density lipoprotein-cholesterol and severity of coronary atherosclerosis.

    Chen SN, Cilingiroglu M, Todd J, Lombardi R, Willerson JT, Gotto AM, Ballantyne CM and Marian AJ

    Center for Cardiovascular Genetics, Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center and Texas Heart Institute, Houston, TX, USA. snchen@bcm.tmc.edu

    Background: Plasma level of high-density lipoprotein-cholesterol (HDL-C), a heritable trait, is an important determinant of susceptibility to atherosclerosis. Non-synonymous and regulatory single nucleotide polymorphisms (SNPs) in genes implicated in HDL-C synthesis and metabolism are likely to influence plasma HDL-C, apolipoprotein A-I (apo A-I) levels and severity of coronary atherosclerosis.

    Methods: We genotyped 784 unrelated Caucasian individuals from two sets of populations (Lipoprotein and Coronary Atherosclerosis Study- LCAS, N = 333 and TexGen, N = 451) for 94 SNPs in 42 candidate genes by 5' nuclease assays. We tested the distribution of the phenotypes by the Shapiro-Wilk normality test. We used Box-Cox regression to analyze associations of the non-normally distributed phenotypes (plasma HDL-C and apo A-I levels) with the genotypes. We included sex, age, body mass index (BMI), diabetes mellitus (DM), and cigarette smoking as covariates. We calculated the q values as indicators of the false positive discovery rate (FDR).

    Results: Plasma HDL-C levels were associated with sex (higher in females), BMI (inversely), smoking (lower in smokers), DM (lower in those with DM) and SNPs in APOA5, APOC2, CETP, LPL and LIPC (each q <or=0.01). Likewise, plasma apo A-I levels, available in the LCAS subset, were associated with SNPs in CETP, APOA5, and APOC2 as well as with BMI, sex and age (all q values <or=0.03). The APOA5 variant S19W was also associated with minimal lumen diameter (MLD) of coronary atherosclerotic lesions, a quantitative index of severity of coronary atherosclerosis (q = 0.018); mean number of coronary artery occlusions (p = 0.034) at the baseline and progression of coronary atherosclerosis, as indicated by the loss of MLD.

    Conclusion: Putatively functional variants of APOA2, APOA5, APOC2, CETP, LPL, LIPC and SOAT2 are independent genetic determinants of plasma HDL-C levels. The non-synonymous S19W SNP in APOA5 is also an independent determinant of plasma apo A-I level, severity of coronary atherosclerosis and its progression.

    BMC medical genetics 2009;10;111

  • Clusterin regulates transthyretin amyloidosis.

    Lee KW, Lee DH, Son H, Kim YS, Park JY, Roh GS, Kim HJ, Kang SS, Cho GJ and Choi WS

    Department of Anatomy and Neurobiology, Institute of Health Sciences, College of Medicine, Gyeongsang National University, Gyeongnam 660-751, South Korea.

    Transthyretin (TTR) is a human disease-associated amyloidogenic protein that has been implicated in senile systemic amyloidosis (SSA) and familial amyloidotic polyneuropathy (FAP). FAP typically results in severe and early-onset disease, and the only therapy established so far is liver transplantation; thus, developing new strategies for treating FAP is of paramount interest. Clusterin has recently been proposed to play a role as an extracellular molecular chaperone, affecting the fibril formation of amyloidogenic proteins. The ability of clusterin to influence amyloid fibril formation prompted us to investigate whether clusterin is capable of inhibiting TTR amyloidosis. Here, we report that clusterin strongly interacts with wild-type TTR and TTR variants V30M and L55P under acidic conditions, and blocks the amyloid fibril formation of TTR variants. In particular, the amyloid fibril formation of V30M TTR in the presence of clusterin is reduced to level similar to wild-type TTR. We also demonstrated that clusterin is an effective inhibitor of L55P TTR amyloidosis, the most aggressive form of TTR diseases. The mechanism by which clusterin inhibits TTR amyloidosis appears to be through stabilization of TTR tetrameric structure. These findings suggest the possibility of using clusterin as a therapeutic agent for TTR amyloidosis.

    Biochemical and biophysical research communications 2009;388;2;256-60

  • Clusterin: a forgotten player in Alzheimer's disease.

    Nuutinen T, Suuronen T, Kauppinen A and Salminen A

    Department of Neuroscience and Neurology, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland.

    Clusterin, also known as apolipoprotein J, is a versatile chaperone molecule which contains several amphipathic and coiled-coil alpha-helices, typical characteristics of small heat shock proteins. In addition, clusterin has three large intrinsic disordered regions, so-called molten globule domains, which can stabilize stressed protein structures. Twenty years ago, it was demonstrated that the expression of clusterin was clearly increased in Alzheimer's disease (AD). Later it was observed that clusterin can bind amyloid-beta peptides and prevent their fibrillization. Clusterin is also involved in the clearance of amyloid-beta peptides and fibrils by binding to megalin receptors and enhancing their endocytosis within glial cells. Clusterin is a complement inhibitor and can suppress complement activation observed in AD. Clusterin is also present in lipoprotein particles and regulates cholesterol and lipid metabolism of brain which is disturbed in AD. Clusterin is a stress-induced chaperone which is normally secreted but in conditions of cellular stress, it can be transported to cytoplasm where it can bind to Bax protein and inhibit neuronal apoptosis. Clusterin can also bind to Smad2/3 proteins and potentiate the neuroprotective TGFbeta signaling. An alternative splicing can produce a variant isoform of clusterin which can be translocated to nuclei where it induces apoptosis. The role of nuclear clusterin in AD needs to be elucidated. We will review here the extensive literature linking clusterin to AD and examine the recent progress in clusterin research with the respect to AD pathology. Though clusterin can be viewed as a multipotent guardian of brain, it is unable to prevent the progressive neuropathology in chronic AD.

    Brain research reviews 2009;61;2;89-104

  • Genome-wide association study identifies variants at CLU and CR1 associated with Alzheimer's disease.

    Lambert JC, Heath S, Even G, Campion D, Sleegers K, Hiltunen M, Combarros O, Zelenika D, Bullido MJ, Tavernier B, Letenneur L, Bettens K, Berr C, Pasquier F, Fiévet N, Barberger-Gateau P, Engelborghs S, De Deyn P, Mateo I, Franck A, Helisalmi S, Porcellini E, Hanon O, European Alzheimer's Disease Initiative Investigators, de Pancorbo MM, Lendon C, Dufouil C, Jaillard C, Leveillard T, Alvarez V, Bosco P, Mancuso M, Panza F, Nacmias B, Bossù P, Piccardi P, Annoni G, Seripa D, Galimberti D, Hannequin D, Licastro F, Soininen H, Ritchie K, Blanché H, Dartigues JF, Tzourio C, Gut I, Van Broeckhoven C, Alpérovitch A, Lathrop M and Amouyel P

    Inserm U744, Lille, France.

    The gene encoding apolipoprotein E (APOE) on chromosome 19 is the only confirmed susceptibility locus for late-onset Alzheimer's disease. To identify other risk loci, we conducted a large genome-wide association study of 2,032 individuals from France with Alzheimer's disease (cases) and 5,328 controls. Markers outside APOE with suggestive evidence of association (P < 10(-5)) were examined in collections from Belgium, Finland, Italy and Spain totaling 3,978 Alzheimer's disease cases and 3,297 controls. Two loci gave replicated evidence of association: one within CLU (also called APOJ), encoding clusterin or apolipoprotein J, on chromosome 8 (rs11136000, OR = 0.86, 95% CI 0.81-0.90, P = 7.5 x 10(-9) for combined data) and the other within CR1, encoding the complement component (3b/4b) receptor 1, on chromosome 1 (rs6656401, OR = 1.21, 95% CI 1.14-1.29, P = 3.7 x 10(-9) for combined data). Previous biological studies support roles of CLU and CR1 in the clearance of beta amyloid (Abeta) peptide, the principal constituent of amyloid plaques, which are one of the major brain lesions of individuals with Alzheimer's disease.

    Nature genetics 2009;41;10;1094-9

  • Genome-wide association study identifies variants at CLU and PICALM associated with Alzheimer's disease.

    Harold D, Abraham R, Hollingworth P, Sims R, Gerrish A, Hamshere ML, Pahwa JS, Moskvina V, Dowzell K, Williams A, Jones N, Thomas C, Stretton A, Morgan AR, Lovestone S, Powell J, Proitsi P, Lupton MK, Brayne C, Rubinsztein DC, Gill M, Lawlor B, Lynch A, Morgan K, Brown KS, Passmore PA, Craig D, McGuinness B, Todd S, Holmes C, Mann D, Smith AD, Love S, Kehoe PG, Hardy J, Mead S, Fox N, Rossor M, Collinge J, Maier W, Jessen F, Schürmann B, Heun R, van den Bussche H, Heuser I, Kornhuber J, Wiltfang J, Dichgans M, Frölich L, Hampel H, Hüll M, Rujescu D, Goate AM, Kauwe JS, Cruchaga C, Nowotny P, Morris JC, Mayo K, Sleegers K, Bettens K, Engelborghs S, De Deyn PP, Van Broeckhoven C, Livingston G, Bass NJ, Gurling H, McQuillin A, Gwilliam R, Deloukas P, Al-Chalabi A, Shaw CE, Tsolaki M, Singleton AB, Guerreiro R, Mühleisen TW, Nöthen MM, Moebus S, Jöckel KH, Klopp N, Wichmann HE, Carrasquillo MM, Pankratz VS, Younkin SG, Holmans PA, O'Donovan M, Owen MJ and Williams J

    Medical Research Council Centre for Neuropsychiatric Genetics and Genomics, Department of Psychological Medicine and Neurology, School of Medicine, Cardiff University, Cardiff, UK.

    We undertook a two-stage genome-wide association study (GWAS) of Alzheimer's disease (AD) involving over 16,000 individuals, the most powerful AD GWAS to date. In stage 1 (3,941 cases and 7,848 controls), we replicated the established association with the apolipoprotein E (APOE) locus (most significant SNP, rs2075650, P = 1.8 x 10(-157)) and observed genome-wide significant association with SNPs at two loci not previously associated with the disease: at the CLU (also known as APOJ) gene (rs11136000, P = 1.4 x 10(-9)) and 5' to the PICALM gene (rs3851179, P = 1.9 x 10(-8)). These associations were replicated in stage 2 (2,023 cases and 2,340 controls), producing compelling evidence for association with Alzheimer's disease in the combined dataset (rs11136000, P = 8.5 x 10(-10), odds ratio = 0.86; rs3851179, P = 1.3 x 10(-9), odds ratio = 0.86).

    Funded by: Intramural NIH HHS; Medical Research Council: G0300429, G0600974, G0601846, G0701075, G0900688, MC_U123160651, MC_U123192748; NCRR NIH HHS: UL1 RR024992; Wellcome Trust: 064354, 082604

    Nature genetics 2009;41;10;1088-93

  • Clusterin facilitates exchange of glycosyl phosphatidylinositol-linked SPAM1 between reproductive luminal fluids and mouse and human sperm membranes.

    Griffiths GS, Galileo DS, Aravindan RG and Martin-DeLeon PA

    Department of Biological Sciences, University of Delaware, Newark, Delaware 19716, USA.

    Glycosyl phosphatidylinositol (GPI)-linked proteins, which are involved in post-testicular maturation of sperm and have a role in fertilization, are acquired on the sperm surface from both vesicular and membrane-free soluble fractions of epididymal luminal fluid (LF) and uterine LF. Herein, we investigate the mechanism of uptake of these proteins from the soluble fraction of LFs using sperm adhesion molecule 1 (SPAM1) as a model. Ultracentrifugation and native Western blot analysis of the soluble fraction revealed that SPAM1 is present in low-molecular-weight (monomeric) and high-molecular-weight (oligomeric) complexes. The latter are incapable of transferring SPAM1 and may serve to produce monomers. Monomers are stabilized by hydrophobic interactions with clusterin (CLU), a lipid carrier that is abundantly expressed in LFs. We show that CLU is involved in the transfer of SPAM1 monomers, whose delivery was decreased by anti-CLU antibody under normal and apolipoprotein-enhanced conditions. Coimmunoprecipitation revealed an intimate association of CLU with SPAM1. Both plasma and recombinant CLU had a dose-related effect on transfer efficiency: high concentrations reduced and low concentrations enhanced delivery of SPAM1 to human and mouse sperm membranes, reflecting physiological states in the epididymal tract. We propose a lipid exchange model (akin to the lipid-poor model for cholesterol efflux) for the delivery of GPI-linked proteins to sperm membranes via CLU. Our investigation defines specific conditions for membrane-free GPI-linked protein transfer in vitro and could lead to technology for improving fertility or treating sperm pathology by the addition of relevant GPI-linked proteins critical for successful fertilization in humans and domestic animals.

    Funded by: NCRR NIH HHS: 5P20RR015588; NICHD NIH HHS: R01 HD38273

    Biology of reproduction 2009;81;3;562-70

  • Roles of clusterin in progression, chemoresistance and metastasis of human ovarian cancer.

    Wei L, Xue T, Wang J, Chen B, Lei Y, Huang Y, Wang H and Xin X

    Department of Obstetrics and Gynecology, Xijing Hospital, the Fourth Military Medical University, People's Republic of China.

    Clusterin (CLU) is a multivalent glycoprotein with ubiquitous tissue distribution. To address the possible differential functional roles assumed by different isoforms of CLU in the progression of human ovarian cancer, we constructed 2 human ovarian cancer cell models that represent examples of contradistinctive CLU expression levels. One is constitutively overexpressing different clusterin isoforms in SKOV3 cells by transfection of the 3 different expression vectors, another is silencing the intrinsically expressing clusterin in cisplatin-resistant human A2780-cis(CP70) tumor cells with the usage of shRNA-mediated CLU gene silencing. Then, the different cellular localization, biological effects, and functional roles played in tumor progression and drug resistances were studied. We found that (i) in the distinct cellular contexts of human ovarian carcinoma SKOV3 and CP70 cells assayed, sCLU is a central molecule in cell homeostasis that functions as a cytoprotective protein, whereas nCLU is proapoptotic; (ii) In SKOV3 cells, nuclear localization of the truncated CLU is NLS dependent, without which the pnCLU protein was sequestrated in cytoplasm to prevent cytotoxicity. (iii) sCLU plays a significant role in the development of the chemoresistance phenotype in ovarian cancer cells. Moreover, with the CLU-specific shRNA oligonucleotides, we successfully sensitized cells for chemotherapy, and inhibited cells' proliferation, migration and invasion. Collectively, our results reveal that, CLU gene expression might play a crucial role in ovarian cancer progression, adaptation and eventual resistance to chemotherapy through differential processing of CLU isoforms. Specifically, sCLU as an antiapoptotic protein, upregulated in an adaptive cell-survival manner by chemotherapy, confers resistance to various cell-death triggers.

    International journal of cancer 2009;125;4;791-806

  • Structural characterization of clusterin-chaperone client protein complexes.

    Wyatt AR, Yerbury JJ and Wilson MR

    School of Biological Sciences, University of Wollongong, Wollongong, New South Wales 2522, Australia.

    Clusterin (CLU) is a potent extracellular chaperone that inhibits protein aggregation and precipitation otherwise caused by physical or chemical stresses (e.g. heat, reduction). This action involves CLU forming soluble high molecular weight (HMW) complexes with the client protein. Other than their unquantified large size, the physical characteristics of these complexes were previously unknown. In this study, HMW CLU-citrate synthase (CS), HMW CLU-fibrinogen (FGN), and HMW CLU-glutathione S-transferase (GST) complexes were generated in vitro, and their structures studied using size exclusion chromatography (SEC), ELISA, SDS-PAGE, dynamic light scattering (DLS), bisANS fluorescence, and circular dichroism spectrophotometry (CD). Densitometry of Coomassie Blue-stained SDS-PAGE gels indicated that all three HMW CLU-client protein complexes had an approximate mass ratio of 1:2 (CLU:client protein). SEC indicated that all three clients formed complexes with CLU>or=4x10(7) Da; however, DLS estimated HMW CLU-FGN to have a diameter of 108.57+/-18.09 nm, while HMW CLU-CS and HMW CLU-GST were smaller with estimated diameters of 51.06+/-6.87 nm and 52.61+/-7.71 nm, respectively. Measurements of bisANS fluorescence suggest that the chaperone action of CLU involves preventing the exposure to aqueous solvent of hydrophobic regions that are normally exposed by the client protein during heat-induced unfolding. CD analysis indicated that, depending on the individual client protein, CLU may interact with a variety of intermediates on protein unfolding pathways with different amounts of native secondary structure. In vivo, soluble complexes like those studied here are likely to serve as vehicles to dispose of otherwise dangerous aggregation-prone misfolded extracellular proteins.

    The Journal of biological chemistry 2009;284;33;21920-7

  • Clusterin is expressed in normal synoviocytes and in tenosynovial giant cell tumors of localized and diffuse types: diagnostic and histogenetic implications.

    Boland JM, Folpe AL, Hornick JL and Grogg KL

    Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55905, USA.

    Tenosynovial giant cell tumors arise from synovium of joints, bursae, or tendon sheaths, and are classified into localized and diffuse types based on the growth pattern and clinical behavior. The mononuclear component of these tumors includes small histiocytoid cells and large mononuclear cells, which are positive for desmin in about 50% of cases. This study seeks to further characterize the immunophenotype of these tumors, and investigates the utility of clusterin as a diagnostic marker. Immunostaining for clusterin was performed on 40 cases of tenosynovial giant cell tumor (11 localized and 29 diffuse). Most cases were also stained for desmin, CD163, CD21, and CD35. Four cases were stained for podoplanin/D2-40 and CXCL13. Clusterin staining was diffuse and strong in the large mononuclear cells in all cases. Desmin positivity in the large cells was identified in 24 out of 34 cases (71%), but was seen in only a subset of cells (<5% to 80%), with 19 out of 24 cases (79%) showing positivity in 10% or less. The large cells were positive for podoplanin in 4 out of 4 cases, but negative for CD163, CD21, CD35, and CXCL13. The smaller histiocytoid cells were positive for CD163 and negative for all other markers. When present, non-neoplastic synoviocytes were positive for clusterin and podoplanin, and focally positive for desmin. Clusterin is a highly sensitive marker for tenosynovial giant cell tumors, which has diagnostic utility in challenging cases. The observed staining patterns provide evidence linking the large mononuclear cells with normal synoviocytes and support that tenosynovial giant cell tumors are neoplasms showing synovial differentiation.

    The American journal of surgical pathology 2009;33;8;1225-9

  • A novel mutation in the complement regulator clusterin in recurrent hemolytic uremic syndrome.

    Ståhl AL, Kristoffersson A, Olin AI, Olsson ML, Roodhooft AM, Proesmans W and Karpman D

    Department of Pediatrics, Clinical Sciences Lund, Lund University, Lund, Sweden.

    A novel heterozygous mutation in the clusterin gene, nucleotide position A1298C (glutamine>proline Q433P), was detected in exon 7 of a child with recurrent hemolytic uremic syndrome (HUS). The same mutation was found in the child's two siblings and mother but not in 120 controls. In addition, a previously described heterozygous mutation was detected in the gene encoding membrane cofactor protein (MCP) causing a 6 base-pair deletion 811-816delGACAGT in exon 6. It was found in the patient, both siblings and the father. One sibling had recovered from post-streptococcal glomerulonephritis. Clusterin levels in the patient, siblings and parents were normal as was the migration pattern in a gel. Patient serum induced C3 and C9 deposition on normal washed platelets, and platelet activation, as detected by flow cytometry. The same phenomenon was found in serum taken from the siblings and the mother but not in the sample from the father and controls. Addition of clusterin to patient serum did not inhibit complement activation on platelets. The Q433P mutant, in isolated form, was further studied by binding to the components of the terminal complement complex. The mutant did not bind to C5b-7 that was immobilized onto a BIAcore chip, whereas wild-type clusterin did, indicating that the mutation could lead to defective inhibition of formation of the membrane attack complex under these conditions. Hemolysis of rabbit erythrocytes was inhibited by wild-type clusterin but not by the mutant. Mutated clusterin could thus not prevent assembly of the membrane attack complex on platelets and erythrocytes.

    Molecular immunology 2009;46;11-12;2236-43

  • Exploring functional candidate genes for genetic association in german patients with pseudoexfoliation syndrome and pseudoexfoliation glaucoma.

    Krumbiegel M, Pasutto F, Mardin CY, Weisschuh N, Paoli D, Gramer E, Zenkel M, Weber BH, Kruse FE, Schlötzer-Schrehardt U and Reis A

    Institute of Human Genetics, University of Erlangen-Nuremberg, Erlangen, Germany.

    Purpose: Pseudoexfoliation (PEX) syndrome is a generalized elastic microfibrillopathy characterized by fibrillar deposits in intra- and extraocular tissues. Genetic and nongenetic factors are known to be involved in its etiopathogenesis. This study was focused on six functional candidate genes involved in PEX material deposition and the analysis of their potential association with PEX syndrome and PEX glaucoma (PEXG).

    Methods: Fifty single-nucleotide polymorphisms (SNPs) capturing >95% of overall genetic variance observed in Europeans at loci for FBN1, LTBP2, MFAP2, TGM2, TGF-b1, and CLU were genotyped in 333 unrelated PEX-affected and 342 healthy individuals of German origin, and a genetic association study was performed. To replicate the findings, two SNPs of the CLU gene were genotyped in a further 328 unrelated German patients with PEX as well as in 209 Italian patients with PEX and 190 Italian control subjects.

    Results: Association with PEX was observed only for the SNP rs2279590 in intron 8 of the CLU gene coding for clusterin (corrected P = 0.0347, OR = 1.34) in our first German cohort. Likewise, a frequent haplotype encompassing the associated risk allele showed nominally significant association. None of remaining SNPs or SNP haplotypes were associated with PEX. The association found was confirmed in a second German cohort (P = 0.0244) but not in the Italian cohort (P = 0.7173). In addition, the association with CLU SNP rs2279590 was more significant in German patients with PEX syndrome than in those with PEXG.

    Conclusions: Genetic variants in the gene encoding clusterin may represent a risk factor for PEX in German patients but not in Italian patients. Variants in FBN1, LTBP2, MFAP2, TGF-b1, and TGM2 do not play a major role in the etiology of PEX syndrome, at least in German patients.

    Investigative ophthalmology & visual science 2009;50;6;2796-801

  • Risk of non-Hodgkin lymphoma in association with germline variation in complement genes.

    Cerhan JR, Novak AJ, Fredericksen ZS, Wang AH, Liebow M, Call TG, Dogan A, Witzig TE, Ansell SM, Habermann TM, Kay NE and Slager SL

    Division of Epidemiology, Department of Health Sciences Research, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, USA. cerhan.james@mayo.edu

    Germline mutations in complement genes have been associated with susceptibility to infections and autoimmune diseases, conditions that are associated with non-Hodgkin lymphoma (NHL) risk. To test the hypothesis that common genetic variation in complement genes affect risk of NHL, we genotyped 167 single nucleotide polymorphisms (SNPs) from 31 genes in 441 NHL cases and 475 controls. Principal components (PC) and haplotype analyses were used for gene-level tests of NHL risk, while individual SNPs were modelled as having a log-additive effect. In gene level PC analyses, C2 (P = 0.023), C5 (P = 0.0032) and C9 (P = 0.020) were associated with NHL risk; haplotype analyses showed similar results, as well as a haplotype association for C7 (P = 0.046). When all four genes were considered simultaneously, only C5 and C9 remained significant (P < 0.05). In SNP level results from these genes, 10 SNPs had a P < 0.05. However, after correcting for multiple testing, only the C5 SNPs rs7026551 (q = 0.015; OR = 1.54, 95% CI 1.21-1.95) and rs2416810 (q = 0.015; OR = 1.57; 95% CI 1.22-2.01), and the C9 SNP rs187875 (q = 0.015; OR = 0.68; 95% 0.56-0.84) remained noteworthy. Associations were similar for the common NHL subtypes. In summary, we provide evidence for a role of genetic variation in complement genes, particularly C5 and C9, and NHL risk.

    Funded by: NCI NIH HHS: CA092153-06, CA097274-070003, P50 CA097274, P50 CA097274-070003, R01 CA092153, R01 CA092153-06

    British journal of haematology 2009;145;5;614-23

  • Transcriptional and posttranslational regulation of clusterin by the two main cellular proteolytic pathways.

    Balantinou E, Trougakos IP, Chondrogianni N, Margaritis LH and Gonos ES

    National Hellenic Research Foundation, Institute of Biological Research and Biotechnology, 48 Vas. Constantinou Avenue, Athens, Greece.

    Clusterin/apolipoprotein J (CLU) is a secreted glycoprotein associated with many severe physiological disturbances that represent states of increased oxidative stress, such as aging, cancer, atherosclerosis, diabetes, and renal and neurodegenerative diseases. The aim of our work was to examine the effect of proteasome and lysosome inhibition on CLU expression and to determine whether those proteolytic pathways are implicated in CLU gene regulation and protein degradation. To this end we used two different model systems, namely the U-2 OS osteosarcoma cell line and the WI38 primary human embryonic lung fibroblasts. We report that proteasome inhibition promotes both heat-shock factor 1 (HSF-1)-dependent CLU gene expression induction and protein accumulation due to reduced degradation. In contrast, lysosome inhibition results in elevated levels of CLU protein but does not affect the CLU mRNA levels. We also provide direct evidence that both the intracellular precursor, psCLU, and the mature secreted, sCLU, isoforms constitute proteolytic substrates of the proteasome and the lysosome. Overall our findings indicate that CLU overexpression after proteasome inhibition relates to both positive gene transcriptional regulation by HSF-1 and posttranslational protein accumulation due to reduced proteasomal and lysosomal degradation.

    Free radical biology & medicine 2009;46;9;1267-74

  • Clusterin is a short half-life, poly-ubiquitinated protein, which controls the fate of prostate cancer cells.

    Rizzi F, Caccamo AE, Belloni L and Bettuzzi S

    Dipartimento di Medicina Sperimentale, Sezione di Biochimica, Biochimica Clinica e Biochimica dell'Esercizio Fisico, Parma, Italy.

    The Clusterin (CLU) gene produces different forms of protein products, which vary in their biological properties and distribution within the cell. Both the extra- and intracellular CLU forms regulate cell proliferation and apoptosis. Dis-regulation of CLU expression occurs in many cancer types, including prostate cancer. The role that CLU plays in tumorigenesis is still unclear. We found that CLU over-expression inhibited cell proliferation and induced apoptosis in prostate cancer cells. Here we show that depletion of CLU affects the growth of PC-3 prostate cancer cells. Following siRNA targeting all CLU mRNA variants, all protein products quickly disappeared, inducing cell cycle progression and higher expression of specific proliferation markers (i.e., H3 mRNA, PCNA, and cyclins A, B1, and D) as detected by RT-qPCR and Western blot. Quite surprisingly, we also found that the turnover of CLU protein is very rapid and tightly regulated by ubiquitin-proteasome mediated degradation. Inhibition of protein synthesis by cycloheximide showed that CLU half-life is less than 2 h. CLU protein products were found poly-ubiquitinated by co-immuniprecipitation. Proteasome inhibition by MG132 caused stabilization and accumulation of all CLU protein products, including the nuclear form of CLU (nCLU), and committing cells to caspase-dependent death. In conclusion, proteasome inhibition may induce prostate cancer cell death through accumulation of nCLU, a potential tumor suppressor factor.

    Funded by: Worldwide Cancer Research: 06-0711

    Journal of cellular physiology 2009;219;2;314-23

  • Clusterin silencing in human lung adenocarcinoma cells induces a mesenchymal-to-epithelial transition through modulating the ERK/Slug pathway.

    Chou TY, Chen WC, Lee AC, Hung SM, Shih NY and Chen MY

    Institute of Clinical Medicine, National Yang-Ming University, Taiwan. tychou@vghtpe.gov.tw

    The ubiquitously expressed glycoprotein Clusterin (CLU) is implicated in diverse cellular processes, yet its genuine molecular function remains undefined. CLU expression has been associated with various human malignancies, yet the mechanisms by which CLU promotes cancer progression and metastasis are not elucidated. In this study, using human lung adenocarcinoma cell lines as a model, we explored the involvement of CLU in modulating invasiveness of cancer cells. We discovered that CLU levels positively correlated with the degree of invasiveness in human lung adenocarcinoma cell lines. The observation that CLU-rich cells displayed a spindle-shape morphology while those with low CLU levels were cuboidal in shape prompted us to investigate if CLU modulates epithelial-to-mesenchymal transitions (EMT). CLU silencing by siRNA in a highly invasive, CLU-rich lung adenocarcinoma cell line induced a mesenchymal-to-epithelial transition (MET) evidenced by the spindle-to-cuboidal morphological change, increased E-cadherin expression, and decreased fibronectin expression. Compared with the vector-transfected cells, CLU-knocked-down (CLUi) cells showed reduced migration and invasion in vitro, as well as decreased metastatic potential in experimental metastasis. Re-expression of CLU in CLUi cells reversed the MET and restored the mesenchymal and invasive phenotypes. We found that Slug, a zinc-finger-containing transcriptional repressor of E-cadherin, was downregulated in CLUi cells. We also discovered that levels of activated ERK correlated with those of CLU and Slug. Taken together, our data suggest that CLU may regulate EMT and aggressive behaviour of human lung adenocarcinoma cells through modulating ERK signalling and Slug expression.

    Cellular signalling 2009;21;5;704-11

  • Hepatitis delta virus epigenetically enhances clusterin expression via histone acetylation in human hepatocellular carcinoma cells.

    Liao FT, Lee YJ, Ko JL, Tsai CC, Tseng CJ and Sheu GT

    Institute of Medical and Molecular Toxicology, Chung Shan Medical University, Taichung, Taiwan, Republic of China.

    Both isoforms of the hepatitis delta antigen (HDAg) of hepatitis delta virus (HDV) are highly associated with virus proliferation and may act as co-activators of cellular gene expression. Human hepatocellular carcinoma (HCC) cell line Huh7, which stably expresses HDAgs, was differentially screened and the results showed that clusterin gene expression was enhanced. The mechanisms for HDAg-mediated clusterin gene upregulation were investigated. Expression of HDAgs was associated with enhanced histone H3 acetylation within the clusterin promoter in a chromatin immunoprecipitation assay. Transient transfection of HDAg-expressing plasmids into Huh7 cells also enhanced clusterin expression and histone acetylation. Furthermore, HDV replication was associated with histone hyperacetylation and clusterin induction. The effect of increased clusterin expression was determined by a chemosensitivity assay with adriamycin treatment. These data indicated that HDV-induced clusterin protein increases cell survival potential. Thus, it is possible that epigenetic regulation by HDV contributes to a pathological outcome of hepatitis D/hepatitis B viral hepatitis and HCC.

    The Journal of general virology 2009;90;Pt 5;1124-34

  • Clusterin expression correlates with stage and presence of large cells in mycosis fungoides.

    Chandra P, Plaza JA, Zuo Z, Diwan AH, Koeppen H, Duvic M, Medeiros LJ and Prieto VG

    Department of Hematopathology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Box 85, Houston, TX 77030, USA.

    Clusterin expression is common in systemic and cutaneous anaplastic large cell lymphoma (ALCL). Mycosis fungoides (MF) in large cell transformation can resemble ALCL. In this study, we immunohistochemically assessed for clusterin in 97 skin biopsy specimens, including 70 MF cases and 27 other cutaneous neoplasms including ALCL, peripheral T-cell lymphoma unspecified (PTCL), and lymphomatoid papulosis (LyP). Clusterin was positive in 36 (51%) of 70 cases of MF and correlated with clinical stage in 68 cases: 3 of 21 stage I, 11 of 20 stage II, and 23 of 27 stage III/IV. Clusterin expression also correlated with type of skin lesion (3/19 patch, 13/28 plaque, and 20/23 tumor/erythroderma) and number of large cells (6/30 small cell, 12/18 with increased large cells, and 18/22 with large cell transformation). Clusterin expression was not specific for MF as it also was positive in 3 of 3 cases of LyP, 2 of 2 systemic ALCL cases involving skin, 7 of 16 cutaneous ALCLs, and 1 of 6 PTCLs.

    American journal of clinical pathology 2009;131;4;511-5

  • Clusterin expression in cholestasis, hepatocellular carcinoma and liver fibrosis.

    Aigelsreiter A, Janig E, Sostaric J, Pichler M, Unterthor D, Halasz J, Lackner C, Zatloukal K and Denk H

    Institute of Pathology, Medical University of Graz, Graz, Austria.

    Aims: Clusterin shares functional properties with small heat shock proteins. In contrast to other heat shock proteins, it is present in the extracellular space. Its expression is altered in various diseases. The aim was to evaluate the presence and distribution of clusterin in liver diseases associated with cholestasis, in fibrosis and in hepatocellular carcinoma.

    Tissue microarrays and biopsy materials were used to evaluate immunohistochemically the expression of clusterin in hepatocellular carcinoma, primary sclerosing cholangitis, primary biliary cirrhosis, mechanical cholestasis, drug-induced cholestasis, liver fibrosis and cirrhosis. The presence of clusterin in human bile was assessed by Western blotting. Furthermore, real-time reverse transcriptase-polymerase chain reaction was performed on liver tissue with mechanical cholestasis. Clusterin colocalized with elastic fibres, but not with collagen, hepatocytes or bile duct epithelia. It was detected in bile plugs in cholestasis and hepatocellular carcinomas with pseudoglandular features within the lumina. Clusterin was demonstrated in bile by Western blotting and its mRNA was expressed in normal and cholestatic livers.

    Conclusions: Clusterin may protect bile duct epithelium against offensive biliary components or inhibit precipitation of biliary proteins. The association of clusterin with elastic fibres could reflect an extracellular chaperone function by either protecting elastic fibres or shielding abnormal elastic material.

    Histopathology 2009;54;5;561-70

  • Clusterin expression in cutaneous CD30-positive lymphoproliferative disorders and their histologic simulants.

    Olsen SH, Ma L, Schnitzer B and Fullen DR

    Department of Pathology, University of Michigan Medical Center, Ann Arbor, MI 48109-0602, USA.

    Background: Clusterin is a ubiquitous 80 kDa heterodimeric glycoprotein previously shown to be expressed on tumor cells of systemic and, to a lesser extent, primary cutaneous anaplastic large cell lymphoma (PC-ALCL). Lymphomatoid papulosis (LyP), an important differential diagnosis of ALCL, has been studied for clusterin expression in only a small number of cases. The aim of this study was to compare clusterin immunostaining patterns in LyP and other cutaneous histologic simulants with those of PC-ALCL.

    Methods: Formalin-fixed, paraffin-embedded sections of PC-ALCL (6), LyP (20), mycosis fungoides with large cell transformation (MF-LCT, 12), pityriasis lichenoides et varioliformis acuta (PLEVA, 12), arthropod bite reaction (ABR, 12) and lymphomatoid reactions (LR, 9) were immunostained for clusterin and evaluated for staining pattern and distribution. All diagnoses were made with clinicopathologic correlation.

    Results: Characteristic dot-like Golgi staining was identified in 10/20 LyP (50%), 4/6 PC-ALCL (67%) and 9/12 MF-LCT (75%). Two of 12 PLEVA (17%), 1 of 12 ABR (8%) and 2 of 8 LR (25%) had lymphocytes (< 25%) with diffuse cytoplasmic staining. Dermal dendritic cells stained strongly for clusterin. High background staining occurred in some cases.

    Conclusion: Clusterin immunostaining does not reliably distinguish between LyP, PC-ALCL or MF-LCT, but could distinguish LyP from its reactive histologic simulants.

    Journal of cutaneous pathology 2009;36;3;302-7

  • Interleukin-6 affects cell death escaping mechanisms acting on Bax-Ku70-Clusterin interactions in human colon cancer progression.

    Pucci S, Mazzarelli P, Paola M, Sesti F, Fabiola S, Boothman DA, David BA, Spagnoli LG and Luigi SG

    Department of Biopathology, University of Rome Tor Vergata, Rome, Italy. sabinapuc@yahoo.it

    Activation of pro-survival pathways and apoptotic cell death escape are considered hallmarks of oncogenic cell transformation. Tissue microenvironment strongly influences tumorigenesis, redirecting some pathways versus a persisting pro-survival state. Here, we report evidence on the role of interleukin 6 (IL-6) in affecting pro-survival pathways in colon cancer progression, modulating the expression and the molecular interactions among the pro-apoptotic factor Bax, the DNA repair proteins Ku70/86 and Clusterin isoforms. In human colorectal carcinomas (n = 50) at different stages of disease, we found an increased IL-6 production, the loss of Ku86 and Clusterin 50-55 kDa pro-apoptotic isoform. Conversely, we observed the overexpression of Bax and the 40 kDa prosurvival sClusterin (sCLU) isoform. Bax co-localized with Ku70 that was found atypically expressed in the cytoplasm of advanced stage colon cancers (Dukes'C-D; n = 22). IL-6 treatment of a colon cancer cell line, Caco-2, modulated the expression of genes involved in tumor invasion and apoptosis, as observed by microarrays. In particular, IL-6 downmodulated Bax expression at mRNA level. Concomitantly, IL-6 exposure influenced Bax also at protein level acting on the Bax-Ku70-sCLU physical interactions in the cytoplasm, by affecting the Ku70 acetylation and phosphorylation state, thus leading to the inhibition of Bax pro-apoptotic activity. In addition, we found that IL-6 treatment induced a significant downregulation of Ku86 and a strong increase of sCLU, confirming tumor biopsies data. In contrast Somatostatin treatment of Caco-2 cells was able to restore apoptosis, demonstrating that Ku70-Bax-CLU interactions could be dynamically modulated. Hence, IL-6 could favor tumor expansion, promoting cell survival and apoptosis escape throughout the different stages of tumor evolution. Uncovering the molecular mechanisms of action of these factors may offer strategies for selectively manipulate the cancer cells sensitivity to therapy.

    Funded by: NCI NIH HHS: P30 CA013148, P30 CA142543, R01 CA078530, R01 CA102792; NIAID NIH HHS: U19 AI067773, U19 AI067773-05

    Cell cycle (Georgetown, Tex.) 2009;8;3;473-81

  • Genome-wide association study of panic disorder in the Japanese population.

    Otowa T, Yoshida E, Sugaya N, Yasuda S, Nishimura Y, Inoue K, Tochigi M, Umekage T, Miyagawa T, Nishida N, Tokunaga K, Tanii H, Sasaki T, Kaiya H and Okazaki Y

    Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

    Panic disorder (PD) is an anxiety disorder characterized by panic attacks and anticipatory anxiety. Although a number of association studies have been conducted, no gene has been identified as a susceptibility locus. In this study, we conducted a genome-wide association study of PD in 200 Japanese patients and the same number of controls, using the GeneChip Human Mapping 500 K Array Set. Genotypes were determined using the Bayesian Robust Linear Model with Mahalanobis (BRLMM) genotype calling algorithm. The genotype data were data-cleaned using criteria for SNP call rate (>or=95%), Hardy-Weinberg equilibrium (P>or=0.1%) and minor allele frequency (>or=5%). The significance level of the allele P-value was set at 1.0 x 10(-6), to make false discovery rate (FDR) <0.05. As a result, seven SNPs were significantly associated with PD, which were located in or adjacent to genes including PKP1, PLEKHG1, TMEM16B, CALCOCO1, SDK2 and CLU (or APO-J). Studies with other samples are required to confirm the results.

    Journal of human genetics 2009;54;2;122-6

  • Intracellular clusterin inhibits mitochondrial apoptosis by suppressing p53-activating stress signals and stabilizing the cytosolic Ku70-Bax protein complex.

    Trougakos IP, Lourda M, Antonelou MH, Kletsas D, Gorgoulis VG, Papassideri IS, Zou Y, Margaritis LH, Boothman DA and Gonos ES

    Laboratory of Molecular and Cellular Aging, Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece. itrougakos@eie.gr

    Purpose: Secretory clusterin (sCLU)/apolipoprotein J is an extracellular chaperone that has been functionally implicated in DNA repair, cell cycle regulation, apoptotic cell death, and tumorigenesis. It exerts a prosurvival function against most therapeutic treatments for cancer and is currently an antisense target in clinical trials for tumor therapy. However, the molecular mechanisms underlying its function remained largely unknown.

    The molecular effects of small interfering RNA-mediated sCLU depletion in nonstressed human cancer cells were examined by focusing entirely on the endogenously expressed sCLU protein molecules and combining molecular, biochemical, and microscopic approaches.

    Results: We report here that sCLU depletion in nonstressed human cancer cells signals stress that induces p53-dependent growth retardation and high rates of endogenous apoptosis. We discovered that increased apoptosis in sCLU-depleted cells correlates to altered ratios of proapoptotic to antiapoptotic Bcl-2 protein family members, is amplified by p53, and is executed by mitochondrial dysfunction. sCLU depletion-related stress signals originate from several sites, because sCLU is an integral component of not only the secretory pathway but also the nucleocytosolic continuum and mitochondria. In the cytoplasm, sCLU depletion disrupts the Ku70-Bax complex and triggers Bax activation and relocation to mitochondria. We show that sCLU binds and thereby stabilizes the Ku70-Bax protein complex serving as a cytosol retention factor for Bax.

    Conclusions: We suggest that elevated sCLU levels may enhance tumorigenesis by interfering with Bax proapoptotic activities and contribute to one of the major characteristics of cancer cells, that is, resistance to apoptosis.

    Funded by: NCI NIH HHS: P50 CA070907, R01 CA078530

    Clinical cancer research : an official journal of the American Association for Cancer Research 2009;15;1;48-59

  • The role of clusterin (CLU) in malignant transformation and drug resistance in breast carcinomas.

    Redondo M, Tellez T and Roldan MJ

    Department of Biochemistry, Hospital Costa del Sol, CIBER Epidemiología y Salud Pública, Universidad de Málaga, Marbella, Spain.

    Breast cancer is the main cause of cancer-related death among women in Western countries. Current research is focused on identifying antiapoptotic proteins which could be a possible target for novel chemotherapeutic drugs. Secretory clusterin (sCLU) is an extracellular chaperone that has been functionally implicated in DNA repair, cell-cycle regulation, apoptotic cell death and tumorigenesis. The implication of sCLU in carcinogenesis and the progression of breast carcinomas make it an interesting gene, worthy of investigation. It has been reported to present powerful antiapoptotic activity and to perform a prosurvival function with most therapeutic treatments for breast cancer. This review summarizes our current understanding of the role of CLU in tumorigenesis, progression, and response to treatment in breast carcinomas.

    Advances in cancer research 2009;105;21-43

  • Chapter 2: Clusterin (CLU): From one gene and two transcripts to many proteins.

    Rizzi F, Coletta M and Bettuzzi S

    Dipartimento di Medicina Sperimentale, Sezione di Biochimica, Biochimica Clinica e Biochimica dell'Esercizio Fisico, Parma, Italy.

    Clusterin (CLU) has kept many researchers engaged for a long time since its first discovery and characterization in the attempt to unravel its biological role in mammals. Although there is a general consensus on the fact that CLU is supposed to play important roles in nearly all fundamental biological phenomena and in many human diseases including cancer, after about 10 years of work CLU has been defined as an "enigmatic" protein. This sense of frustration among the researchers is originated by the fact that, despite considerable scientific production concerning CLU, there is still a lack of basic information about the complex regulation of its expression. The CLU gene is a single 9-exon gene expressed at very different levels in almost all major tissues in mammals. The gene produces at least three protein forms with different subcellular localization and diverse biological functions. The molecular mechanism of production of these protein forms remains unclear. The best known is the glycosylated mature form of CLU (sCLU), secreted with very big quantitative differences at different body sites. Hormones and growth factors are the most important regulators of CLU gene expression. Before 2006, it was believed that a unique transcript of about 1.9 kb was originated by transcription of the CLU gene. Now we know that alternative transcriptional initiation, possibly driven by two distinct promoters, may produce at least two distinct CLU mRNA isoforms differing in their unique first exon, named Isoform 1 and Isoform 2. A third transcript, named Isoform 11036, has been recently found as one of the most probable mRNA variants. Approaches like cloning, expression, and functional characterization of the different CLU protein products have generated a critical mass of information teaching us an important lesson about CLU gene expression regulation. Nevertheless, further studies are necessary to better understand the tissue-specific regulation of CLU expression and to identify the specific signals triggering the expression of different/alternative transcript isoforms and protein forms in different cell types at appropriate time.

    Funded by: Worldwide Cancer Research: 06-0711

    Advances in cancer research 2009;104;9-23

  • CLU and colon cancer. The dual face of CLU: from normal to malignant phenotype.

    Mazzarelli P, Pucci S and Spagnoli LG

    Department of Biopathology, Institute of Anatomic Pathology, University of Rome Tor Vergata, 00133 Rome, Italy.

    The transition from normal to malignant phenotype implies the activation of some pathways that underlie the aberrant clone expansion. In some way, the conventional function of proteins involved in DNA repair, cell death/growth induction, vascularization, and metabolism is inhibited or shifted toward other pathways by soluble mediators that orchestrate such change depending on the microenvironment conditions. The adenoma-carcinoma sequence of the colon represents one of the most well studied and characterized models of human tumor progression. In this section, we focus our attention on defined pathways that underlie the initiation, promotion, and progression of colon cancer, conferring aggressiveness to the neoplastic cells. Clusterin (CLU) is a pleiotropic protein with a broad range of functions. It has recently drawn much attention because of its association with cancer promotion and metastasis. It is involved in prosurvival and apoptosis processes that are carried out by two different forms. sCLU is cytoprotective and its prosurvival function is the basis of the current Phase I/II clinical trials. In colorectal cancer an increase of sCLU expression occurs, whereas the nuclear proapoptotic form is downregulated. Several controversial data have been published on colon cancer discussing its role as tumor suppressor or prosurvival factor in colon cancer. Here, we report the dynamic interaction of the different forms of CLU with their partners DNA-repair protein Ku70 and proapoptotic factor Bax during colon cancer progression, which seems to be a crucial point for the neoplastic cell fate. We also highlight that the appearance and the progressive increase of the sCLU in colorectal tumors correlate to a significant increase of CLU in serum and stool of patients. On the basis of results obtained by CLU immuno-dosage in blood and stool of colon cancer patients, we report that sCLU could represent a diagnostic molecular marker for colon cancer screening.

    Advances in cancer research 2009;105;45-61

  • Clusterin (CLU) and lung cancer.

    Panico F, Rizzi F, Fabbri LM, Bettuzzi S and Luppi F

    Department of Oncology, Hematology and Respiratory Diseases, Section of Respiratory Diseases, University of Modena and Reggio Emilia, 41100 Modena, Italy.

    Lung cancer is the leading cause of cancer-related mortality. It is categorized into two histological groups that have distinct clinical behaviors, the nonsmall cell lung cancers (NSCLC) and the small cell lung cancer (SCLC). When identified at an early stage, NSCLC is treated by surgical resection. However, patients who undergo surgical resection still have a relative low survival rate, primarily for tumor recurrence. Unfortunately, advances in cytotoxic therapy have reached a plateau and new approaches to treatment are needed together with new and better parameters for more accurate prediction of the outcome and more precise indication of the efficacy of the treatment. Several in vitro studies have examined the role of Clusterin (CLU) in carcinogenesis, lung cancer progression, and response to chemo- and radiotherapy. Studies performed in lung cancer cell lines and animal models showed that CLU is upregulated after exposure to chemo- and radiotherapy. A potential role proposed for the protein is cytoprotective. In vitro, CLU silencing by antisense oligonucleotides (ASO) and small-interfering RNAs (siRNA) directed against CLU mRNA in CLU-rich lung cancer cell lines sensitized cells to chemotherapy and radiotherapy and decreased their metastatic potential. In vivo, a recent work analyzed the prognostic role of CLU in NSCLC, showing that CLU-positive patients with lung cancer had a better overall survival and disease-free survival than those with CLU-negative tumors. These data are contradictory to the promising in vitro results. From the results of these studies we may hypothesize that in early-stage lung cancers CLU represents a positive biomarker correlating with better overall survival. In advanced patients, already treated with chemo- and radiotherapy, the induction of CLU may confer resistance to the treatments. However, many studies are needed to better understand the role of CLU in early-stage and advanced lung cancers with the aim to discriminate patients and specific local conditions that could benefit for a CLU knocking down treatment.

    Funded by: Worldwide Cancer Research: 06-0711

    Advances in cancer research 2009;105;63-76

  • Clusterin (CLU) and prostate cancer.

    Rizzi F and Bettuzzi S

    Dipartimento di Medicina Sperimentale, Sezione di Biochimica, Biochimica Clinica e Biochimica dell'Esercizio Fisico, Università di Parma, Via Volturno 39-43100 Parma, Italy.

    The role of clusterin (CLU) in prostate tumorigenesis is probably the most highly controversial, with evidence that CLU expression is increased or decreased in different cancer models. For example, some studies showed that CLU expression is increased in advanced stages of prostate cancer and that suppression of CLU expression sensitizes prostate cancer cells to chemotherapeutic drugs killing. In contrast with the hypothesis that CLU is a positive modulator of prostate cancer, we and others have observed that CLU is downregulated during human prostate cancer progression. Accordingly, a meta-analysis of available microarray data shows that CLU mRNA is significantly downregulated in prostate cancer tissue compared to normal prostate in 14 out of 15 independent studies. Recently, it was shown that CLU is silenced by promoter methylation in the murine TRAMP-C2 cell line, as well as in the human prostate cancer cell line LNCaP. Consistently, CLU expression was found to be significantly reduced in untreated and hormone-refractory human prostate carcinomas. This data suggest the importance of epigenetic events in the regulation of CLU in prostate cancer, supporting the idea that prostate cell transformation at early stages requires CLU silencing through chromatin remodeling.

    Funded by: Worldwide Cancer Research: 06-0711

    Advances in cancer research 2009;105;1-19

  • Regulation of clusterin expression in human cancer via DNA methylation.

    Serrano A, Redondo M, Tellez T, Castro-Vega I, Roldan MJ, Mendez R, Rueda A and Jimenez E

    Department of Immunology, Hospital Clinico Universitario, Campus Universitario Teatinos, Málaga, Spain.

    Clusterin has attracted much recent attention because of its association with tumorigenesis and the progression of human carcinomas. The present study was designed to examine the role of clusterin methylation as an indicator of clusterin expression in tumor cell lines and breast tissue samples.

    Methods: For this purpose, we used methylation-sensitive restriction analysis followed by PCR.

    Results: None of the non-tumoral breast samples showed expression of clusterin by immunohistochemistry, and a methylated state was found in the promoter region of the gene. However, a demethylated state was found in 5 of 6 analyzed carcinoma cell lines. Four of 5 demethylated cell lines presented moderate to strong expression of clusterin, while no expression was detected in the unmethylated cell line. The inverse correlation found in most cell lines between clusterin expression and promoter methylation was also found in most human tumors analyzed (p < 0.001). Thus, a methylated state was present in 14 carcinomas, 12 of them with a null expression of clusterin, while a demethylated state was detected in 7 breast tumor samples, with 5 of them presenting strong expression.

    Conclusions: We conclude that clusterin expression is under epigenetic control via methylation of its promoter.

    Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 2009;30;5-6;286-91

  • Association between the SERPING1 gene and age-related macular degeneration: a two-stage case-control study.

    Ennis S, Jomary C, Mullins R, Cree A, Chen X, Macleod A, Jones S, Collins A, Stone E and Lotery A

    Genetic Epidemiology and Bioinformatics Group, University of Southampton, Human Genetics Division (Mp 808), Southampton General Hospital, Southampton, UK.

    Background: Age-related macular degeneration is the most prevalent form of visual impairment and blindness in developed countries. Genetic studies have made advancements in establishing the molecular cause of this disease, identifying mutations in the complement factor H (CFH) gene and a locus on chromosome 10 encompassing the HTRA1/LOC387715/ARMS2 genes. Variants in complement 3 (C3) and an HLA locus containing both factor B and C2 genes have also been implicated. We aimed to identify further genetic risk factors for this disease.

    Methods: We used a case-control study design in a UK sample of patients with age-related macular degeneration (n=479) and controls (n=479) and undertook a low-density screen of 32 genes using 93 single nucleotide polymorphisms (SNPs). Genes were selected as candidates on the basis of potential functional relevance to age-related macular degeneration. Significant initial findings were confirmed by replication in an independent US cohort of 248 unrelated patients with disease and 252 controls, and by high-density genotyping around association signals.

    Findings: The SNP variant rs2511989, located within intron six of the SERPING1 gene, showed highly significant genotypic association with age-related macular degeneration (uncorrected p=4.0x10(-5), corrected p=0.00372). We detected no evidence for association between disease and the other 31 candidate genes. The odds ratio for age-related macular degeneration in rs2511989 G/A heterozygotes compared with wild type G/G homozygotes was 0.63 (95% CI 0.47-0.84). A similar comparison of the A/A homozygotes with the wild type yielded an odds ratio of 0.44 (0.31-0.64). We replicated the observed genotypic association in a US cohort (p=0.008). Furthermore, a secondary high-density genotyping study across the SERPING1 gene region identified five additional SNP variants similarly associated with age-related macular degeneration (rs2244169, rs2511990, rs2509897, rs1005510, and rs2511988).

    Interpretation: Genetic variation in SERPING1 significantly alters susceptibility to age-related macular degeneration. SERPING1 encodes the C1 inhibitor, which has a crucial role in inhibition of complement component 1 (C1) and might implicate the classic pathway of complement activation in this disease.

    Funded by: Howard Hughes Medical Institute; NEI NIH HHS: EY-016822, EY-017451; Wellcome Trust: 076169

    Lancet (London, England) 2008;372;9652;1828-34

  • Four potential biomarkers as prognostic factors in stage III serous ovarian adenocarcinomas.

    Partheen K, Levan K, Osterberg L, Claesson I, Fallenius G, Sundfeldt K and Horvath G

    Department of Oncology, University of Gothenburg, Gothenburg, Sweden. karolina.partheen@gu.se

    The mortality rate for patients with ovarian carcinomas is high and the available prognostic factors are insufficient. The use of biomarkers may contribute to better prediction and survival for these patients. We aimed to study the gene and protein expressions for 7 potential biomarkers, to determine if it is possible to use them as prognostic factors. Genes selected from our previous microarray analysis (2006), CLU, ITGB3, TACC1, MUC5B, CAPG, PRAME and TROAP, were analyzed in 19 of the tumors with quantitative real-time polymerase chain reaction (QPCR). We found that CLU and ITGB3 were more expressed in tumors from survivors and PRAME and CAPG were more expressed in tumors from deceased patients. None of the other 3 genes were significantly differently expressed. The protein expressions of CLU, ITGB3, PRAME and CAPG were analyzed in 43 of the tumors with western blot for semiquantitative analysis. We established that the mRNA and protein expressions correlated and that all 4 proteins were significantly differently expressed. Further, immunohistochemistry (IHC) was used to localize the expression of the proteins in the tumor samples. According to our results, the 4 biomarkers CLU, ITGB3, PRAME and CAPG may be used as prognostic factors for patients with stage III serous ovarian adenocarcinomas.

    International journal of cancer 2008;123;9;2130-7

  • Human cellular prion protein interacts directly with clusterin protein.

    Xu F, Karnaukhova E and Vostal JG

    Division of Hematology, Office of Blood Research and Review, Center for Biologics Evaluation and Research, United States Food and Drug Administration, Bethesda, MD, USA.

    Prion protein is a glycosyl-phosphatidyl-inositol anchored glycoprotein localized on the surface and within a variety of cells. Its conformation change is thought to be essential for the proliferation of prion neurodegenerative diseases. Using the yeast two-hybrid assay we identified an interaction between prion protein and clusterin, a chaperone glycoprotein. This interaction was confirmed in a mammalian system by in vivo co-immunoprecipitation and in vitro by circular dichroism analysis. Through deletion mapping analysis we demonstrated that the alpha subunit, but not the beta subunit, of clusterin binds to prion and that the C-terminal 62 amino acid segment of the putative alpha helix region of clusterin is essential for the binding interaction. The full prion protein as well as the N-terminal section (aa 23-95) and C-terminal (aa 96-231) were shown to interact with clusterin. These findings provide new insights into the molecular mechanisms of interaction between prion and clusterin protein and contribute to the understanding of prion protein's physiological function.

    Biochimica et biophysica acta 2008;1782;11;615-20

  • Clusterin is epigenetically regulated in prostate cancer.

    Rauhala HE, Porkka KP, Saramäki OR, Tammela TL and Visakorpi T

    Institute of Medical Technology, University of Tampere, and Tampere University Hospital, Tampere, Finland.

    Lack of good models has complicated investigations on the mechanisms of prostate cancer. By far, the most commonly used transgenic mouse model of prostate cancer is TRAMP, which, however, has not been fully characterized for genetic and epigenetic aberrations. Here, we screened TRAMP-derived C2 cell line for the alterations using different microarray approaches, and compared it to human prostate cancer. TRAMP-C2 had relatively few genomic copy number alterations according to array comparative genomic hybridization (aCGH). However, the gene copy number and expression were significantly correlated (p < 0.001). Screening genes for promoter hypermethylation using demethylation treatment with 5-aza-2'-deoxycytidine and subsequent expression profiling indicated 43 putatively epigenetically silenced genes. Further studies revealed that clusterin is methylated in the TRAMP-C2 cell line, as well as in the human prostate cancer cell line LNCaP. Its expression was found to be significantly reduced (p < 0.01) in untreated and hormone-refractory human prostate carcinomas. Together with known function of clusterin, the data suggest an epigenetic component in the regulation of clusterin in prostate cancer.

    International journal of cancer 2008;123;7;1601-9

  • Clusterin: a potential target for improving response to antiestrogens.

    Toffanin S, Daidone MG, Miodini P, De Cecco L, Gandellini P and Cappelletti V

    Department of Experimental Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, I-20133 Milano, Italy.

    Antiestrogens represent the first line of therapy in the treatment of estrogen receptor-positive (ER+) breast cancer patients. Unfortunately, up to 40% of patients develop resistance associated with progression and frequently die for metastatic breast cancer. The molecular events leading to pharmacological resistance are not completely understood. We attempted to verify in an experimental model the role of cytoplasmic clusterin (CLU), a cytoprotective protein found to be up-regulated in antiestrogen-resistant patients, following neoadjuvant treatment with toremifene. The role of cytoplasmic clusterin in modulating response to two antiestrogens (toremifene and tamoxifen) was studied in two ER+ anti-estrogen-sensitive cell lines (MCF-7, 734B) and one ER+ antiestrogen-resistant cell line (T47D) using siRNA strategy. Resistant cells were characterised by higher levels of cytoplasmic clusterin than sensitive cells, and antiestrogen treatments up-regulated clusterin levels in both sensitive and resistant cell lines. Treatment with siRNA completely abolished cytoplasmic clusterin expression in all cell lines, but its down-regulation resulted in a significant decrease of cell growth only in the resistant line. We therefore concluded that: i) basal clusterin levels are higher in antiestrogen resistant cells, ii) clusterin is up-regulated following antiestrogen treatment independently of the sensitivity of the cell line, iii) down-regulation of cytoplasmic clusterin restores sensitivity to toremifene in the antiestrogen-resistant cell line. Such results support the concept that targeting CLU could represent a promising therapeutic strategy in association with antiestrogen treatment in breast cancer patients.

    International journal of oncology 2008;33;4;791-8

  • Genetic analysis of the clusterin gene in pseudoexfoliation syndrome.

    Burdon KP, Sharma S, Hewitt AW, McMellon AE, Wang JJ, Mackey DA, Mitchell P and Craig JE

    Department of Ophthalmology, Flinders University, Adelaide, Australia.

    Purpose: Pseudoexfoliation syndrome is a major risk factor for the development of glaucoma. Following recent reports of a strong association of coding variants in the lysyl oxidase-like 1 (LOXL1) gene with this syndrome but low penetrance and variable disease frequency between different populations, we aimed to identify additional genetic factors contributing to the disease. The clusterin (CLU) gene has been proposed as a candidate because of the presence of clusterin protein in pseudoexfoliation deposits, its varied levels in aqueous humor of cases compared to controls, and the role of the protein as a molecular chaperone. We investigated the association of genetic variants across CLU in pseudoexfoliation syndrome and analyzed molecular characteristics of the encoded protein in ocular tissues.

    Methods: The expression of clusterin in relevant ocular tissues was assessed using western blotting. Nine tag single nucleotide polymorphisms (SNPs) across CLU were genotyped in 86 cases of pseudoexfoliation syndrome and 2422 controls from the Australian Blue Mountains Eye Study cohort. Each SNP and haplotype was assessed for association with the syndrome.

    Results: Clusterin was identified in normal human iris, the ciliary body, lens capsule, optic nerve, and aqueous humor. Post-translational modification gives rise to a 100 kDa precursor protein in ocular tissues, larger than that reported in non-ocular tissues. One CLU SNP (rs3087554) was nominally associated with pseudoexfoliation syndrome at the genotypic level (p=0.044), although not when the age of controls was restricted to those over 73 years. Only age and the LOXL1 diplotype were significant factors in the logistic regression. One haplotype of all nine CLU SNPs was also associated (p=0.005), but the significance decreased slightly with the use of the age-restricted controls (p=0.011).

    Conclusions: Clusterin is present in ocular anterior segment tissues involved in pseudoexfoliation syndrome. Although one haplotype may contribute in a minor way to genetic risk of pseudoexfoliation syndrome, common variation in this gene is not a major contributor to the risk of pseudoexfoliation syndrome.

    Molecular vision 2008;14;1727-36

  • Clusterin interacts with Paclitaxel and confer Paclitaxel resistance in ovarian cancer.

    Park DC, Yeo SG, Wilson MR, Yerbury JJ, Kwong J, Welch WR, Choi YK, Birrer MJ, Mok SC and Wong KK

    Department of Obstetrics and Gynecology, Saint Vincent's Hospital, The Catholic University of Korea, Suwon, Republic of Korea.

    Optimal debulking followed by chemotherapy is the standard treatment of managing late-stage ovarian cancer, but chemoresistance is still a major problem. In this study, we compared expression profiles of primary tumor tissue from five long-term (>8 years) and five short-term (<2 years) ovarian cancer survivors and identified clusterin as one of the genes that were significantly up-regulated in short-term survivors. We then evaluated the prognostic significance of clusterin and its possible correlation with chemoresistance in ovarian cancer by immunohistostaining of clusterin in 62 tumor samples from patients with stage III, high-grade serous ovarian cancer. After adjusting for debulking status and age, Cox regression analyses showed that high levels of clusterin expression correlate with poor survival (hazard ratio, 1.07; 95% confidence interval, 1.002-1.443; P = .04). We also investigated clusterin in paclitaxel resistance by modulating the endogenous clusterin expression in ovarian cancer cells and treating the cells with purified clusterin. Results indicate that high-clusterin-expressing ovarian cancer cells are more resistant to paclitaxel. Moreover, exposing ovarian cancer cells to exogenous clusterin increases cells' resistance to paclitaxel. Finally, using size exclusion chromatography and fluorescently labeled paclitaxel, we demonstrated that clusterin binds to paclitaxel. In summary, our findings suggest that high levels of clusterin expression increase paclitaxel resistance in ovarian cancer cells by physically binding to paclitaxel, which may prevent paclitaxel from interacting with microtubules to induce apoptosis. Thus, clusterin is a potential therapeutic target for enhancing chemoresponsiveness in patients with a high-level clusterin expression.

    Funded by: NCI NIH HHS: P50 CA083639, P50 CA105009, P50CA105009, P50CA83639, R33 CA103595, R33CA103595

    Neoplasia (New York, N.Y.) 2008;10;9;964-72

  • Differential expression of clusterin according to histological type of endometrial carcinoma.

    Ahn HJ, Bae J, Lee S, Ko JE, Yoon S, Kim SJ and Sakuragi N

    Department of Pathology, College of Medicine, Pochon CHA University, Republic of Korea.

    Objective: Clusterin expression has been associated with various malignancies. Endometrial carcinoma is divided into endometrioid and papillary serous type carcinoma according to the histological characteristics and regarding to the unopposed estrogenic stimulation. In this study, we investigated the expression profiles of clusterin according to the histological types and the effect of estrogen stimulation on its expression in endometrial carcinoma.

    Method: Clusterin expression in endometrial carcinoma tissues was examined by RT-PCR, Western blot analysis, and immunohistochemistry. 63 endometrioid and 10 papillary serous types of fresh cases and 81 endometrioid and 7 papillary serous types of paraffin-embedded cases were studied. Regulation of clusterin expression by beta-estradiol in HEC-1B and HEC-1A cells was investigated using RT-PCR and Western blot analysis. Cell proliferative function of clusterin was examined in 293T cells.

    Results: We found higher expression of clusterin in endometrioid compared to papillary serous carcinoma using both immunohistochemistry (p=0.033) and Western blot analysis (p=0.024). The mRNA and protein expressions of clusterin in endometrioid carcinoma were higher than in benign endometrium (p=0.002). Forced expression of clusterin promoted 293T cell survival in a concentration-dependent manner, and estradiol treatment increased clusterin expression in HEC-1B but not in HEC-1A cells.

    Conclusions: These data suggest that clusterin expression is related to endometrioid carcinoma of endometrium, in which estrogen is involved in the regulatory network of clusterin.

    Gynecologic oncology 2008;110;2;222-9

  • Apolipoprotein J and leptin levels in patients with coronary heart disease.

    Poulakou MV, Paraskevas KI, Wilson MR, Iliopoulos DC, Tsigris C, Mikhailidis DP and Perrea D

    Department of Experimental Surgery and Surgical Research N.S. Christeas, Medical School, National and Kapodistrian University of Athens, Athens, Greece.

    Background: Preliminary data suggest that apolipoprotein J (ApoJ) may play a role in the development and progression of atherosclerosis. Leptin, an adipose tissue hormone, exerts important cardiovascular effects. The association between serum ApoJ and leptin concentrations was assessed in patients with established or suspected coronary heart disease (CHD).

    Serum ApoJ and leptin concentrations were evaluated in 67 CHD patients undergoing coronary angiography [54 individuals with significant (> or =50%) coronary artery stenosis and 13 patients without significant coronary artery stenosis on angiography].

    Results: Serum ApoJ concentrations in patients with significant coronary artery stenosis were significantly higher than in those without (303.9+/-118.6 vs. 121.2+/-37.5 microg/mL, respectively; p<0.001). The reverse pattern was observed for serum leptin levels (8.6+/-5.5 vs. 20.6+/-17.1 ng/ml, respectively; p=0.016). There was a significant negative correlation between ApoJ and leptin levels (r=-0.353; p=0.003).

    Conclusion: ApoJ and leptin may be markers for CHD.

    In vivo (Athens, Greece) 2008;22;4;537-42

  • Cancer cell-derived clusterin modulates the phosphatidylinositol 3'-kinase-Akt pathway through attenuation of insulin-like growth factor 1 during serum deprivation.

    Jo H, Jia Y, Subramanian KK, Hattori H and Luo HR

    Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA.

    Cancer cells in their respective microenvironments must endure various growth-constraining stresses. Under these conditions, the cancer cell-derived factors are thought to modulate the signaling pathways between cell growth and dormancy. Here, we describe a cancer cell-derived regulatory system that modulates the phosphatidylinositol 3'-kinase (PI3K)-Akt pathway under serum deprivation stress. Through the use of biochemical purification, we reveal that cancer cell-secreted insulin-like growth factor 1 (IGF-1) and clusterin, an extracellular stress protein, constitute this regulatory system. We show that secreted clusterin associates with IGF-1 and inhibits its binding to the IGF-1 receptor and hence negatively regulates the PI3K-Akt pathway during serum deprivation. This inhibitory function of clusterin appears to prefer IGF-1, as it fails to exert any effects on epidermal growth factor signaling. We demonstrate furthermore that the constitutive activation of oncogenic signaling downstream of IGF-1 confers insensitivity to the inhibitory effects of clusterin. Thus, the interplay between cancer cell-derived clusterin and IGF-1 may dictate the outcome of cell growth and dormancy during tumorigenic progression.

    Funded by: NIGMS NIH HHS: GM076084, R01 GM076084; NINDS NIH HHS: NS052200, R21 NS052200

    Molecular and cellular biology 2008;28;13;4285-99

  • Increased clusterin expression in Fuchs' endothelial dystrophy.

    Jurkunas UV, Bitar MS, Rawe I, Harris DL, Colby K and Joyce NC

    Schepens Eye Research Institute, Boston, Massachusetts, USA. ula_jurkunas@meei.harvard.edu

    Purpose: To investigate the differential expression of the glycoprotein clusterin/apoJ (CLU) in normal and Fuchs' endothelial dystrophy (FED) corneal endothelium and to compare the expression of various forms of CLU in normal and FED tissue.

    Methods: FED and pseudophakic bullous keratopathy (PBK) corneal buttons were removed during transplantation, and normal corneas were obtained from tissue banks. Human corneal endothelial cells and Descemet's membrane (HCEC-DM) complex was dissected from the stroma. Proteins were separated on 2-D gels and subjected to comparative proteomic analysis. Relative expression of presecretory CLU (pre-sCLU), secretory (s)CLU, and nuclear (n)CLU were compared between normal and FED HCEC-DM by Western blot analysis. Expression of CLU mRNA was compared by using RT-PCR. Subcellular localization of CLU was compared in corneal wholemounts from normal eyes and eyes with FED by immunocytochemistry followed by confocal microscopy.

    Results: Proteomic analysis revealed an apparent increase in CLU expression in FED HCEC-DM compared with the normal control. Western blot analysis demonstrated that pre-sCLU protein expression was 5.2 times higher in FED than in normal samples (P = 3.52E-05), whereas the mature form modified for secretion (sCLU) was not significantly elevated (P = 0.092). Expression of nCLU protein was significantly elevated in FED (P = 0.013). RT-PCR analysis revealed that CLU mRNA was significantly increased (P = 0.002) in FED samples, but not in PBK samples. CLU also had a distinctive localization in FED samples with enhanced intracellular staining around the guttae and in the nuclei of endothelial cells.

    Conclusions: CLU expression is markedly elevated in FED-affected tissue, pointing to a yet undiscovered form of dysregulation of endothelial cell function involved in FED pathogenesis.

    Funded by: NEI NIH HHS: K12 EY016335, K12 EY016335-02

    Investigative ophthalmology & visual science 2008;49;7;2946-55

  • Potential protective role of apoprotein J (clusterin) in atherogenesis: binding to enzymatically modified low-density lipoprotein reduces fatty acid-mediated cytotoxicity.

    Schwarz M, Spath L, Lux CA, Paprotka K, Torzewski M, Dersch K, Koch-Brandt C, Husmann M and Bhakdi S

    Institute of Medical Microbiology and Hygiene, Hochhaus am Augustusplatz, Obere Zahlbacher Strasse 67, 55101 Mainz, Germany.

    Following entrapment in the arterial intima, low-density lipoprotein (LDL) can be modified by hydrolytic enzymes to yield a lipoprotein derivative that binds C-reactive protein, activates complement, and is rapidly taken up by monocytes/macrophages. Free fatty acids contained in enzymatically modified LDL (E-LDL) render the lipoprotein cytotoxic due to their capacity to trigger programmed cell death. Apoprotein J (ApoJ) alias clusterin is a multifunctional glycoprotein with cytoprotective and anti-inflammatory properties. It interacts with diverse substrates, is present in the intima and the media of arteries with atherosclerotic lesions and is also synthesized by smooth muscle cells during development of atherosclerosis. We report that ApoJ binds to E-LDL but not to native LDL. Binding resulted in marked reduction of cytotoxicity of E-LDL on smooth muscle cells, as revealed by determination of caspase activity, annexin binding, and cellular ATP. ApoJ was detected immunohistochemically in early atherosclerotic lesions, where it was found to co-localize with E-LDL. In atherosclerotic lesions, ApoJ may thus subserve protective functions through its capacity to inactivate C5b-9 complement complexes and by reducing the cytotoxic effects of modified LDL on cells that gain contact with the lipoprotein.

    Thrombosis and haemostasis 2008;100;1;110-8

  • TRPM2-mediated Ca2+influx induces chemokine production in monocytes that aggravates inflammatory neutrophil infiltration.

    Yamamoto S, Shimizu S, Kiyonaka S, Takahashi N, Wajima T, Hara Y, Negoro T, Hiroi T, Kiuchi Y, Okada T, Kaneko S, Lange I, Fleig A, Penner R, Nishi M, Takeshima H and Mori Y

    Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura Campus, Nishikyo-ku, Kyoto 615-8510, Japan.

    Reactive oxygen species (ROS) induce chemokines responsible for the recruitment of inflammatory cells to sites of injury or infection. Here we show that the plasma membrane Ca(2+)-permeable channel TRPM2 controls ROS-induced chemokine production in monocytes. In human U937 monocytes, hydrogen peroxide (H(2)O(2)) evokes Ca(2+) influx through TRPM2 to activate Ca(2+)-dependent tyrosine kinase Pyk2 and amplify Erk signaling via Ras GTPase. This elicits nuclear translocation of nuclear factor-kappaB essential for the production of the chemokine interleukin-8 (CXCL8). In monocytes from Trpm2-deficient mice, H(2)O(2)-induced Ca(2+) influx and production of the macrophage inflammatory protein-2 (CXCL2), the mouse CXCL8 functional homolog, were impaired. In the dextran sulfate sodium-induced colitis inflammation model, CXCL2 expression, neutrophil infiltration and ulceration were attenuated by Trpm2 disruption. Thus, TRPM2 Ca(2+) influx controls the ROS-induced signaling cascade responsible for chemokine production, which aggravates inflammation. We propose functional inhibition of TRPM2 channels as a new therapeutic strategy for treating inflammatory diseases.

    Funded by: NIGMS NIH HHS: R01 GM063954, R01 GM063954-06

    Nature medicine 2008;14;7;738-47

  • Clusterin in human gut-associated lymphoid tissue, tonsils, and adenoids: localization to M cells and follicular dendritic cells.

    Verbrugghe P, Kujala P, Waelput W, Peters PJ and Cuvelier CA

    Department of Pathology, University Hospital Ghent, De Pintelaan 185, Ghent, Belgium.

    The follicle-associated epithelium (FAE) overlying the follicles of mucosa-associated lymphoid tissue is a key player in the initiation of mucosal immune responses. We recently reported strong clusterin expression in the FAE of murine Peyer's patches. In this study, we examined the expression of clusterin in the human gut-associated lymphoid tissue (GALT) and Waldeyer's ring. Immunohistochemistry for clusterin in human Peyer's patches, appendix and colon lymphoid follicles revealed expression in M cells and in follicular dendritic cells (FDCs). Using cryo-immunogold electron microscopy in Peyer's patches, we observed cytosolic immunoreactivity in M cells and labeling in the ER/Golgi biosynthetic pathway in FDCs. In palatine tonsils and adenoids, we demonstrated clusterin expression in germinal centers and in the lymphoepithelium in the crypts where M cells are localized. In conclusion, clusterin is expressed in M cells and follicular dendritic cells at inductive sites of human mucosa-associated lymphoid tissue suggesting a role for this protein in innate immune responses. Moreover, the use of clusterin as a human M cell marker could prove to be a valuable tool in future M cell research.

    Histochemistry and cell biology 2008;129;3;311-20

  • Clusterin, a novel modulator of TGF-beta signaling, is involved in Smad2/3 stability.

    Lee KB, Jeon JH, Choi I, Kwon OY, Yu K and You KH

    School of Bioscience and Biotechnology, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejeon 305-764, Republic of Korea.

    Clusterin (CLU) is known as a multifunctional protein involved in a variety of physiological processes including lipid transport, epithelial cell differentiation, tumorigenesis, and apoptosis. It is known that CLU interacts with TGF-beta type ll receptor (TbetaRll). However, the relationship of CLU and TGF-beta signaling is unclear. Here we present that CLU is a novel modulator of TGF-beta signaling by regulating Smad2/3 proteins. Overexpression of CLU enhanced TGF-beta-induced transcriptional activity and increased the amount of Smad2/3 proteins, while CLU siRNA repressed TGF-beta-induced transcriptional activity and decreased the amount of Smad2/3 proteins in Hep3B cells. We also found that CLU was involved in Smad2/3 stability at the protein level. These findings suggest that CLU regulates TGF-beta signaling pathway by modulating the stability of Smad2/3 proteins.

    Biochemical and biophysical research communications 2008;366;4;905-9

  • Clusterin expression in non-neoplastic adenohypophyses and pituitary adenomas: cytoplasmic clusterin localization in adenohypophysis is related to aging.

    Ekici AI, Eren B, Türkmen N, Comunoğlu N and Fedakar R

    Department of Pathology, Yeditepe University School of Medicine, Istanbul, Turkey. isindogan@yahoo.com

    Clusterin is a circulating multifunctional glycoprotein produced in several kinds of epithelial and neuronal cells. Clusterin is upregulated during different physiological and pathological states, such as senescence, type-2 diabetes mellitus, Alzheimer disease, and in various neoplasms. Herein, we investigated the immunohistochemical expression of clusterin in non-neoplastic adenohypophysis of human autopsy subjects and pituitary adenomas. We also investigated the association of clusterin increase with age in adenohypophysis of autopsy subjects. Immunohistochemically, clusterin was found positive in the cytoplasm of all adenoma cases, and in the cytoplasm of parenchymal cells, stellate cells, mixed cell follicles and in colloidal material inside of the follicles of non-neoplastic adenohypophysis as well. Clusterin expression in pituitary adenomas was found significantly higher than in non-neoplastic adenohypophyses. In addition, in non-neoplastic adenohypophysis, a significant increase in clusterin expression levels between young (<or=30 years), middle aged (31 to 60 years), and older (>or=61 years) subjects (p < 0.00001, analysis of variance [ANOVA]) was found. In addition to clusterin accumulation, presence of calcification (p < 0.045, ANOVA) and presence of large follicles with colloid accumulation (p < 0.004, ANOVA) were also statistically significant factors related to aging in non-neoplastic adenohypophysis. In conclusion, the present study demonstrated that clusterin expression was found in non-neoplastic adenohypophysis and in upregulated amounts in pituitary adenomas. This study also demonstrated that in non-neoplastic adenohypophyses, increase of clusterin positive cells; histopathological findings of calcification or presence colloidal material accumulation in large follicles were associated with age. To our knowledge, immunohistochemical localization of clusterin in pituitary adenomas was not reported previously.

    Endocrine pathology 2008;19;1;47-53

  • Ocular clusterin expression in von Hippel-Lindau disease.

    Zhou M, Shen D, Head JE, Chew EY, Chévez-Barrios P, Green WR and Chan CC

    National Eye Institute, National Institutes of Health, Bethesda, MD, USA.

    Purpose: Clusterin is a multifunctional glycoprotein. Its mRNA is ubiquitously expressed, with high levels in von Hippel-Lindau (VHL) target organs such as the brain, liver, kidney, and adrenal medulla. Decreased clusterin secretion has been reported in renal cell carcinoma associated with VHL disease. The purpose of this study was to investigate ocular clusterin expression in VHL disease.

    Methods: This retrospective case series included nine eyes with retinal hemangioblastoma/hemangioma associated with VHL disease, one eye from a patient with a history of VHL disease and central nervous system hemangioblastomas but without ocular lesions, one surgically-excised optic nerve with optic nerve hemangioblastoma/hemangioma, and three normal control eyes. Ocular specimens were evaluated by routine histology, immunohistochemistry for clusterin expression, and molecular detection of clusterin transcripts within ocular VHL hemangioblastomas compared with normal tissue from the same eye using microdissection and quantitative real-time PCR.

    Results: All retinal hemangioblastoma were composed of typical VHL tumor cells admixed with small vascular channels as well as glial cells. Marked decrease of clusterin immunoreactivity was detected in all retinal hemangioblastoma and the optic nerve hemangioblastoma, whereas positive clusterin reactivity of the vascular and glial components was similar to that of normal retina. Quantitative real-time PCR analysis confirmed the decrease of clusterin mRNA in the VHL associated retinal hemangioblastoma and optic nerve hemangioblastoma in five cases.

    Conclusions: Clusterin shows possible important functions in tumor suppression by the VHL gene product (pVHL) and the potential to be a novel biomarker in retinal hemangioblastoma associated VHL disease. Further investigation of clusterin may provide better understanding of retinal hemangioblastoma associated with VHL disease.

    Funded by: Intramural NIH HHS: Z01 EY000222-22, Z99 EY999999

    Molecular vision 2007;13;2129-36

  • Clusterin isoforms differentially affect growth and motility of prostate cells: possible implications in prostate tumorigenesis.

    Moretti RM, Montagnani Marelli M, Mai S, Cariboni A, Scaltriti M, Bettuzzi S and Limonta P

    Institute of Endocrinology, University of Milan, Milan, Italy.

    Besides a fully processed, secreted form of clusterin (sCLU), an alternative proapoptotic form of the protein targeting the nucleus (nCLU) was recently described. The possible differential roles played by the two clusterin forms in growth and motility of nonmalignant and malignant prostate cells are investigated here. sCLU or nCLU was transiently transfected in both androgen-independent prostate cancer cells (PC3 and DU 145) and immortalized prostate epithelial cells (PNT1A, a nontumoral control). Then, cell growth, motility, and cytoskeleton organization were studied. We found that (a) in PNT1A cells, both sCLU and nCLU significantly decreased cell proliferation and motility; (b) in PC3 and DU 145 cancer cells, only nCLU inhibited cell growth and migration, with sCLU being ineffective; and (c) the antimotility effect of nCLU was accompanied by a dramatic dismantling of the actin cytoskeleton. Moreover, transfection with "full-length" CLU cDNA produced both sCLU and nCLU in nonmalignant PNT1A cells, whereas only sCLU was found in cancer cells. Thus, CLU gene expression might play a crucial role in prostate tumorigenesis by exerting differential biological effects on normal versus tumor cells through differential processing of CLU isoforms in the two cell systems. We also found that nCLU binds to alpha-actinin, a key protein for the regulation of actin cytoskeleton, and that nCLU and alpha-actinin colocalize in the cytoplasm. Thus, the antimotility activity of nCLU and its ability to cause dismantling of the actin cytoskeleton seem to be mediated by its binding to alpha-actinin.

    Cancer research 2007;67;21;10325-33

  • Clusterin associates with altered elastic fibers in human photoaged skin and prevents elastin from ultraviolet-induced aggregation in vitro.

    Janig E, Haslbeck M, Aigelsreiter A, Braun N, Unterthor D, Wolf P, Khaskhely NM, Buchner J, Denk H and Zatloukal K

    Institute of Pathology, Medical University of Graz, Graz, Austria.

    Clusterin is a secreted glycoprotein with stress-induced expression in various diseased and aged tissues. It shares basic features with small heat shock proteins because it may stabilize proteins in a folding-competent state. Besides its presence in all human body fluids, clusterin associates with altered extracellular matrix proteins, such as beta-amyloid in Alzheimer senile plaques in the brain. Because dermal connective tissue alterations occur because of aging and UV radiation, we explored the occurrence of clusterin in young, aged, and sun-exposed human skin. Immunohistochemical analysis showed that clusterin is constantly associated with altered elastic fibers in aged human skin. Elastotic material of sun-damaged skin (solar elastosis), in particular, revealed a strong staining for clusterin. Because of the striking co-localization of clusterin with abnormal elastic material, we investigated the interaction of clusterin with elastin in vitro. A chaperone assay was established in which elastin was denatured by UV irradiation in the absence or presence of clusterin. This assay demonstrated that clusterin exerted a chaperone-like activity and effectively inhibited UV-induced aggregation of elastin. The interaction of both proteins was further analyzed by electron microscopy, size exclusion chromatography, and mass spectrometry, in which clusterin was found in a stable complex with elastin after UV exposure.

    The American journal of pathology 2007;171;5;1474-82

  • Cytoplasmic clusterin expression is associated with longer survival in patients with resected non small cell lung cancer.

    Albert JM, Gonzalez A, Massion PP, Chen H, Olson SJ, Shyr Y, Diaz R, Lambright ES, Sandler A, Carbone DP, Putnam JB, Johnson DH and Lu B

    Department of Radiation Oncology, Vanderbilt University, 1301 Medical Center Drive, B-902 The Vanderbilt Clinic, Nashville, TN 37232-5671, USA.

    Background: Clusterin is a glycoprotein that has been implicated in many processes, including apoptosis, cell cycle regulation, and DNA repair. Previous studies have examined the prognostic value of clusterin expression in various malignancies. In the present study, we examined clusterin staining in tumors resected from patients with non-small cell lung cancer (NSCLC).

    Tumor specimens were obtained for 113 patients with completely resected NSCLC from paraffin-embedded tissue microarrays and stained with an antibody specific for clusterin. Staining patterns were observed and graded based on intensity and then correlated with clinical data.

    Results: Positive cytoplasmic clusterin staining was observed in 44 patients, and weak/negative staining was observed in 62 patients. Patients who had tumors that stained positive for cytoplasmic clusterin had significantly longer survival in multivariate analysis (hazard ratio 0.487, 95% confidence interval 0.27-0.89). A correlation was also observed for recurrence-free survival, which approached statistical significance (hazard ratio 0.345, 95% confidence interval 0.12-1.02). In univariate analysis, patients with clusterin-positive tumors had a 63% 3-year survival, whereas patients with clusterin-negative tumors had a 42% 3-year survival (P = 0.0108); clusterin-positive tumors also had significantly less recurrence (P = 0.0231).

    Conclusions: Cytoplasmic clusterin staining is present in a substantial number of NSCLC tumors and may be a biomarker for longer survival in patients with surgically resected NSCLC.

    Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 2007;16;9;1845-51

  • Functional association of the morphogenic factors with the clusterin for the pancreatic beta-cell differentiation.

    Kim SY, Lee S, Min BH and Park IS

    Department of Anatomy and Center for Advanced Medical Education by BK21 project, College of Medicine, Inha University, Choong-Gu, Shinheung-Dong, Incheon 400-103, Korea.

    Several differentiation or morphogenic factors have known to be involved in the developmental process of endocrine pancreas. However, mechanism of action and functional relation of these molecules are not well elucidated particularly in beta-cell formation from adult pancreatic stem cells. We hypothesized that adult pancreatic stem cells could be activated by the functional resumption of the morphogenic factors that were involved in embryonic development of pancreas in the duct system under the specific conditions such as tissue injuries. Besides the well-established genes including Pdx-1 and Ngn-3, we propose the nestin and clusterin as the new morphogenic factors for beta-cell neogenesis and their functional associations. We found extensive in vivo formation of ductules showing a higher replicating ability following the experimental tissue injury. These neogenic ductules were lined with low epithelial cells positive for the nestin, which has been known as neuronal stem cell marker. In in vitro culture, the nestin-rich epithelial cells of the neogenic ductules also displayed extensive self-replication leading to monolayer of epithelial cell explants and transformed into the insulin secreting beta cells as well as duct cells. Thus, we depicted them as nestin-positive duct stem (NPDS) cells. We found a neogenesis specific protein 'clusterin' in the regenerating pancreatic tissues with concomitant increase of Pdx-1 and Ngn-3 expression. The protein is expressed predominantly in the neogenic pancreas undergoing differentiation. In vitro over-expression of the clusterin gene strongly induces beta-cell transformation from neogenic ductal cells. Insulin expression, both insulin mRNA and peptide levels, was increased and showed glucose dependent manner by ectopic expression of clusterin upon the culture of neogenic ductules when compared to the mock-transfected control, implying that the duct cells transformed functional beta cells. We observed that clusterin over-expression led to up-regulation of Pdx-1 and Ngn-3, and clusterin levels were increased upon the transfection of cDNAs of Pdx-1 or Ngn-3, suggesting a close functional association of these morphogenic factors. In conclusion, we suggest that adult pancreatic stem cells can be recapitulated for neogenesis of insulin secreting beta cells not only by reactivation Pdx-1 and Ngn-3, the classical differentiation factors for pancreas development, but also by the intervention of new morphogenic factors including nestin and clusterin. In particular, by modulation of Pdx-1 and Ngn-3, clusterin induces remarkable differentiation of the functional beta cells secreting insulin in response to glucose stimulation.

    Diabetes research and clinical practice 2007;77 Suppl 1;S122-6

  • Multiple pathways regulating the anti-apoptotic protein clusterin in breast cancer.

    Ranney MK, Ahmed IS, Potts KR and Craven RJ

    Department of Molecular and Biomedical Pharmacology, Markey Cancer Center, University of Kentucky, MS-305 UKMC, Lexington, KY 40536, USA.

    Cancer chemotherapy inhibits tumor growth, in part, by triggering apoptosis, and anti-apoptotic proteins reduce the effectiveness of chemotherapy. Clusterin, a chaperone-like protein that binds to apoptotic and DNA repair proteins, is induced by chemotherapy and promotes tumor cell survival. Histone deacetylase inhibitors (HDIs) such as sodium butyrate and suberoylanilide hydroxamic acid (SAHA) are pharmacological agents that induce differentiation and apoptosis in cancer cells by altering chromatin structure, and we have found that combinations of chemotherapeutic drugs such as doxorubicin and HDIs efficiently induce apoptosis, even though they paradoxically induce high levels of clusterin. The hyper-expressed form of clusterin localizes to mitochondria, inhibits cytochrome c release, and is inhibited by the proteasome. When HDIs are used as single agents, clusterin suppresses cytochrome c release and apoptosis. However, doxorubicin/HDI-induced apoptosis is not inhibited by clusterin, and clusterin-resistant apoptosis corresponds with markers of the extrinsic/receptor-mediated apoptotic pathway. Thus, chemotherapy-HDI combinations are capable of overcoming an innate anti-apoptotic pathway of tumor cells, suggesting that chemotherapy-HDI combinations have potential for treating advanced stage breast cancer.

    Funded by: NCRR NIH HHS: P20 RR015592, P20 RR015592-050006, P20 RR15592, RR015592-050006

    Biochimica et biophysica acta 2007;1772;9;1103-11

  • The extracellular chaperone clusterin influences amyloid formation and toxicity by interacting with prefibrillar structures.

    Yerbury JJ, Poon S, Meehan S, Thompson B, Kumita JR, Dobson CM and Wilson MR

    School of Biological Sciences, University of Wollongong, Wollongong, Australia.

    Clusterin is an extracellular chaperone present in all disease-associated extracellular amyloid deposits, but its roles in amyloid formation and protein deposition in vivo are poorly understood. The current study initially aimed to characterize the effects of clusterin on amyloid formation in vitro by a panel of eight protein substrates. Two of the substrates (Alzheimer's beta peptide and a PI3-SH3 domain) were then used in further experiments to examine the effects of clusterin on amyloid cytotoxicity and to probe the mechanism of clusterin action. We show that clusterin exerts potent effects on amyloid formation, the nature and extent of which vary greatly with the clusterin:substrate ratio, and provide evidence that these effects are exerted via interactions with prefibrillar species that share common structural features. Proamyloidogenic effects of clusterin appear to be restricted to conditions in which the substrate protein is present at a very large molar excess; under these same conditions, clusterin coincorporates with substrate protein into insoluble aggregates. However, when clusterin is present at much higher but still substoichiometric levels (e.g., a molar ratio of clusterin:substrate=1:10), it potently inhibits amyloid formation and provides substantial cytoprotection. These findings suggest that clusterin is an important element in the control of extracellular protein misfolding.

    Funded by: Biotechnology and Biological Sciences Research Council: BB/E019927/1; Wellcome Trust

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2007;21;10;2312-22

  • Epigenetic regulation of clusterin/apolipoprotein J expression in retinal pigment epithelial cells.

    Suuronen T, Nuutinen T, Ryhänen T, Kaarniranta K and Salminen A

    Department of Neuroscience and Neurology, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland.

    Age-related macular degeneration (AMD) is the leading cause of blindness worldwide. AMD is characterized by the deposition of drusen aggregates under the retinal pigment epithelium (RPE). Clusterin/apo J, a multifunctional secreted chaperone, is one of the major proteins accumulating in drusen deposits. The regulation of clusterin expression is not well characterized but the promoter of clusterin contains a CpG-rich methylation domain. Since aging affects both DNA methylation and histone acetylation status, the epigenetic regulation might have an important role in clusterin/apo J expression. Our purpose was to elucidate whether the induction of DNA hypomethylation with 5-aza-2'-deoxycytidine (AZA) and histone hyperacetylation with trichostatin A (TSA) could affect the clusterin transcription, protein levels, and secretion in retinal pigment epithelial cells. We observed that both TSA and AZA treatments induced a prominent increase in the expression levels of clusterin mRNA and protein in ARPE-19 cells, as well as in the secretion of clusterin protein. Furthermore, valproic acid, an antiepileptic drug and a recently identified inhibitor of histone deacetylases (HDAC), induced a significant increase in clusterin protein expression and secretion in retinal pigment epithelial cells. HDAC inhibitors are characterized as inhibitors of angiogenesis, and clusterin as a complement inhibitor. Our results indicate that epigenetic factors regulate the clusterin expression of RPE cells and thus might affect the pathogenesis of AMD via the inhibition of angiogenesis and inflammation.

    Biochemical and biophysical research communications 2007;357;2;397-401

  • Clusterin expression in normal mucosa and colorectal cancer.

    Andersen CL, Schepeler T, Thorsen K, Birkenkamp-Demtröder K, Mansilla F, Aaltonen LA, Laurberg S and Ørntoft TF

    Molecular Diagnostic Laboratory, Department of Clinical Biochemistry, Aarhus University Hospital, DK8200 Aarhus N, Denmark. cla@ki.au.dk

    The gene Clusterin is a target for cancer therapy in clinical trials. The indication for intervention is up-regulated Clusterin expression. Clusterin has been reported to be deregulated in multiple cancer types, including colorectal cancer (CRC). However, for CRC the studies have disagreed on whether Clusterin is up- or down-regulated by neoplastic cells. In the present study we sought to clarify the expression and distribution of Clusterin mRNAs and proteins in normal and neoplastic colorectal tissue through laser microdissection, variant-specific real time RT-PCR, immunohistochemistry, immunofluorescence, Western blotting, and array-based transcriptional profiling. At the transcript level we demonstrated the expression of two novel Clusterin transcripts in addition to the known transcript, and at the protein level we demonstrated two Clusterin isoforms. Our analysis of normal epithelial cells revealed that among these, Clusterin was only expressed by rare neuroendocrine subtype. Furthermore our analysis showed that in the normal mucosa the majority of the observed Clusterin protein originated from the stromal compartment. In tumors we found that Clusterin was de novo synthesized by non-neuroendocrine cancer cells in approximately 25% of cases. Moreover we found that the overall Clusterin level in tumors often appeared to be lower than in normal mucosa due to the stromal compartment often being suppressed in tumors. Although Clusterin in normal neuroendocrine cells showed a basal localization, the localization in cancer cells was often apical and in some cases associated with apical secretion. Collectively our results indicate that Clusterin expression is very complex. We conclude that Clusterin expression is associated with neuroendocrine differentiation in normal epithelia and that the Clusterin observed in neoplastic cells is de novo synthesized. The cases with de novo synthesized Clusterin define a distinct subgroup of CRC that may be of clinical importance as anti-Clusterin therapeutics are now in clinical trials.

    Molecular & cellular proteomics : MCP 2007;6;6;1039-48

  • Anaplastic large cell lymphomas: a study of 75 pediatric patients.

    d'Amore ES, Menin A, Bonoldi E, Bevilacqua P, Cazzavillan S, Donofrio V, Gambini C, Forni M, Gentile A, Magro G, Boldrini R, Pillon M, Rosolen A and Alaggio R

    Department of Pathology, Ospedale San Bortolo, Vicenza, Italy. emanuele.damore@unipd.it

    In this article, we describe the morphologic and immunophenotypic features of 75 cases of pediatric anaplastic large cell lymphoma (ALCL). According to the World Health Organization classification, 49 cases were common subtype ALCL, and respectively, 3, 6, and 17 cases were small cell, lymphohistiocytic, or mixed histologic variants. Anaplastic lymphoma kinase positivity was detected in 90.7% of the tumors and, using a panel of 9 T-cell surface markers, 88% could be assigned to the T-cell lineage. A molecular analysis for the T-cell receptor gamma (TCR- gamma) and the heavy chain of the immunoglobulin H rearrangements was performed on 6/9 ALCLs with a null immunophenotype, and a TCR clonal pattern was detected in 5/6 cases. In addition, 94.1% were immunoreactive for 1 or more cytotoxic proteins (Tia1, granzyme B, or perforin), and 15% expressed CD56. Clusterin, CD83, and Pax5, respectively, expressed in 91.3%, 1.7%, and 0% of the ALCLs, were useful biomarkers for the differential diagnosis with Hodgkin's lymphomas.

    Pediatric and developmental pathology : the official journal of the Society for Pediatric Pathology and the Paediatric Pathology Society 2007;10;3;181-91

  • Stress-induced retrotranslocation of clusterin/ApoJ into the cytosol.

    Nizard P, Tetley S, Le Dréan Y, Watrin T, Le Goff P, Wilson MR and Michel D

    UMR6026 CNRS Université de Rennes1, Intracellular Protein Homeostasis, IFR 140, Campus de Beaulieu, Bat. 13, 35042 Rennes cedex, France.

    Clusterin is a usually secreted glycoprotein with chaperone properties. Recently, it has been suggested that clusterin isoforms reside in the nuclear and cytosolic compartments of human cell types, where they can influence various cellular programs including DNA repair, transcription and apoptosis. Several mechanisms have been proposed to explain this atypical location, including alternative transcription initiation and alternative splicing. However, none of these have been unequivocally established as occurring in live cells. Here we provide direct experimental evidence that in live intact cells, under certain stress conditions, clusterin can evade the secretion pathway and reach the cytosol. This was demonstrated using several complementary approaches. Flow cytometry and selective permeabilization of U251 cell membranes with digitonin allowed detection of cytosolic clusterin in stressed U251 cells. In addition, a stringent enzymatic assay reliant upon the exclusively cytosolic deubiquitinase enzymes confirmed that clusterin synthesized with its hydrophobic secretion signal sequence can reach the cytosol of U251 cells. The retrotranslocation of clusterin is likely to occur through a mechanism similar to the endoplasmic reticulum (ER)-associated protein degradation pathway and involves passage through the Golgi apparatus. We also report that the ER-associated ubiquitin ligase Hrd1/synoviolin can interact with, and ubiquitinate clusterin. The possible biological functions of these novel behaviours of clusterin are discussed.

    Traffic (Copenhagen, Denmark) 2007;8;5;554-65

  • Low-power millimeter wave radiations do not alter stress-sensitive gene expression of chaperone proteins.

    Zhadobov M, Sauleau R, Le Coq L, Debure L, Thouroude D, Michel D and Le Dréan Y

    Institute of Electronics and Telecommunications of Rennes (IETR), University of Rennes I, Campus de Beaulieu, bâtiment 11D, 263 avenue du Général Leclerc, 35042 Rennes Cedex, France. maxim.zhadobov@ieee.org

    This article reports experimental results on the influence of low-power millimeter wave (MMW) radiation at 60 GHz on a set of stress-sensitive gene expression of molecular chaperones, namely clusterin (CLU) and HSP70, in a human brain cell line. Selection of the exposure frequency is determined by its near-future applications for the new broadband civil wireless communication systems including wireless local area networks (WLAN) for domestic and professional uses. Frequencies around 60 GHz are strongly attenuated in the earth's atmosphere and such radiations represent a new environmental factor. An exposure system operating in V-band (50-75 GHz) was developed for cell exposure. U-251 MG glial cell line was sham-exposed or exposed to MMW radiation for different durations (1-33 h) and two different power densities (5.4 microW/cm(2) or 0.54 mW/cm(2)). As gene expression is a multiple-step process, we analyzed chaperone proteins induction at different levels. First, using luciferase reporter gene, we investigated potential effect of MMWs on the activation of transcription factors (TFs) and gene promoter activity. Next, using RT-PCR and Western blot assays, we verified whether MMW exposure could alter RNA accumulation, translation, or protein stability. Experimental data demonstrated the absence of significant modifications in gene transcription, mRNA, and protein amount for the considered stress-sensitive genes for the exposure durations and power densities investigated. The main results of this study suggest that low-power 60 GHz radiation does not modify stress-sensitive gene expression of chaperone proteins.

    Bioelectromagnetics 2007;28;3;188-96

  • Clusterin over-expression modulates proapoptotic and antiproliferative effects of 1,25(OH)2D3 in prostate cancer cells in vitro.

    Shannan B, Seifert M, Boothman DA, Tilgen W and Reichrath J

    Department of Dermatology, The Saarland University Hospital, Building 18, D-66421 Homburg, Germany. bshannan@ureach.com

    Prostate cancer is the most commonly diagnosed cancer in the majority of western countries. Due to their antiproliferative and proapoptotic activity, vitamin D analogues have been introduced recently as an experimental therapy for prostate cancer. Clusterin (CLU) is a glycoprotein that has two known isoforms generated in human cells. A nuclear form of CLU protein (nCLU) is pro-apoptotic, and a secretory form (sCLU) is pro-survival. In this study, we analyzed whether proapoptotic and antiproliferative effects of 1,25(OH)(2)D(3) on LNCaP prostate cancer cells are modulated by expression of sCLU. Using colony forming assay, we studied the effect of treatment with different doses of 1,25(OH)(2)D(3) (10(-6), 10(-7), 10(-10)M) on proliferation of LNCaP cells that were stable transfected and over-express sCLU (LNT-1) as compared to empty vector-transfected cells (LN/C). We also measured apoptosis using TUNEL assay. sCLU over-expression protected against both antiproliferative (30%) and proapoptotic (15%) effects of 1,25(OH)(2)D(3), although this effect was statistically not significant. In conclusion, our findings demonstrate that expression of sCLU modulates growth regulatory effects of 1,25(OH)(2)D(3) in prostate cancer indicating that CLU interferes with vitamin D signalling pathways.

    Funded by: NCI NIH HHS: P30 CA142543, R01 CA078530

    The Journal of steroid biochemistry and molecular biology 2007;103;3-5;721-5

  • Identifying leukocyte gene expression patterns associated with plasma lipid levels in human subjects.

    Ma J, Dempsey AA, Stamatiou D, Marshall KW and Liew CC

    ChondroGene, Inc., 800 Petrolia Road, Unit 15, Toronto, Ont., Canada M3J 3K4.

    Plasma lipid levels have been known to be risk factors for atherosclerosis for decades, and in recent years it has become accepted that inflammation is a crucial event in the pathogenesis of atherosclerosis. In this study, we investigated the relationship between plasma lipids and leukocytes by profiling and analyzing leukocyte gene expression in response to plasma lipid levels. We discovered several interesting patterns of leukocyte gene expression: (1) the expression of a number of immune response- and inflammation-related genes are correlated with plasma lipid levels; (2) genes involved in lipid metabolism and in the electron transport chain were positively correlated with triglycerides and low-density lipoprotein cholesterol (LDL) levels, and negatively correlated with high-density lipoprotein cholesterol (HDL) levels; (3) genes involved in platelet activation were negatively correlated with HDL levels; (4) transcription factors regulating lipogenesis-related genes were correlated with plasma lipid levels; (5) a number of genes correlated with plasma lipid levels were found to be located in the regions of known quantitative trait loci (QTLs) associated with hyperlipemia. Our findings suggest that leukocytes respond to changing plasma lipid levels by regulating a network of genes, including genes involved in immune response, and lipid and fatty acid metabolism.

    Atherosclerosis 2007;191;1;63-72

  • Effects of glycosylation on the structure and function of the extracellular chaperone clusterin.

    Stewart EM, Aquilina JA, Easterbrook-Smith SB, Murphy-Durland D, Jacobsen C, Moestrup S and Wilson MR

    School of Biological Sciences and the Institute for Biomolecular Sciences, University of Wollongong, Northfields Avenue, Wollongong 2522, NSW, Australia.

    Clusterin is the first well characterized, constitutively secreted extracellular chaperone that binds to exposed regions of hydrophobicity on non-native proteins. It may help control the folding state of extracellular proteins by targeting them for receptor-mediated endocytosis and intracellular lysosomal degradation. A notable feature of secreted clusterin is its heavy glycosylation. Although carbohydrate comprises approximately 20-25% of the total mass of the mature molecule, its function is unknown. Results from the current study demonstrate that deglycosylation of human serum clusterin had little effect on its overall secondary structure content but produced a small increase in solvent-exposed hydrophobicity and enhanced the propensity of the molecule to aggregate in solution. These changes were associated with increased binding to a variety of ligands but did not substantially impact the ability of clusterin to inhibit heat-induced precipitation of citrate synthase. Evidence suggesting that the normally conjugated sugars are important in the interaction of secreted clusterin with a lectin-type receptor on liver cells is also presented. Bulk expression of fully processed, glycosylated clusterin in mammalian cells is difficult, often producing inappropriately disulfide-bonded high molecular weight aggregates; this has hampered previous studies aimed at identifying those regions of the molecule important in its chaperone action. The current results suggest that it may be possible in the future to study the structure and chaperone function of clusterin using recombinant protein (lacking sugars) conveniently bulk-expressed in bacteria.

    Biochemistry 2007;46;5;1412-22

  • Differential regulation of clusterin and its isoforms by androgens in prostate cells.

    Cochrane DR, Wang Z, Muramaki M, Gleave ME and Nelson CC

    Department of Genetics, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.

    Clusterin mRNA levels were shown to increase dramatically in rat ventral prostate following castration, and clusterin was therefore originally thought to be repressed by androgens. It was later discovered that the increased clusterin levels are most likely due to castration-induced apoptosis of the prostatic epithelium rather than direct action of the androgen receptor (AR). In the studies presented here, LNCaP cells in culture and rat prostate organ culture were treated with androgens. Clusterin mRNA and protein are shown to increase with androgen treatment in a time- and dose-dependent manner. This induction of clusterin requires AR and can be inhibited by casodex, an AR antagonist. We have found that the first intron of the clusterin gene contains putative androgen response elements. The intronic region is shown to be bound by AR in chromatin immunoprecipitation assays and is transactivated by AR in reporter assays. Two isoforms of clusterin result from alternate transcriptional start sites. Both isoforms are cytoprotective; however, Isoform 1 has the capacity to produce a splice variant that is apoptotic. Real time PCR was used to determine the response of the two isoforms to androgens. Intriguingly, these results illustrated that Isoform 2 was up-regulated, whereas Isoform 1 was down-regulated by androgens. Isoform 2 was also increased as the LNCaP xenograft tumor progressed to androgen-independence, whereas Isoform 1 was unaltered. This androgen regulation of clusterin may underline the cytoprotective role of androgens in normal prostate physiology as well as play an antiapoptotic role in prostate cancer progression.

    The Journal of biological chemistry 2007;282;4;2278-87

  • Disrupted in Schizophrenia 1 Interactome: evidence for the close connectivity of risk genes and a potential synaptic basis for schizophrenia.

    Camargo LM, Collura V, Rain JC, Mizuguchi K, Hermjakob H, Kerrien S, Bonnert TP, Whiting PJ and Brandon NJ

    Merck Research Labs, Merck & Co., Boston, MA 02115, USA. miguel_camargo@merck.com

    Disrupted in Schizophrenia 1 (DISC1) is a schizophrenia risk gene associated with cognitive deficits in both schizophrenics and the normal ageing population. In this study, we have generated a network of protein-protein interactions (PPIs) around DISC1. This has been achieved by utilising iterative yeast-two hybrid (Y2H) screens, combined with detailed pathway and functional analysis. This so-called 'DISC1 interactome' contains many novel PPIs and provides a molecular framework to explore the function of DISC1. The network implicates DISC1 in processes of cytoskeletal stability and organisation, intracellular transport and cell-cycle/division. In particular, DISC1 looks to have a PPI profile consistent with that of an essential synaptic protein, which fits well with the underlying molecular pathology observed at the synaptic level and the cognitive deficits seen behaviourally in schizophrenics. Utilising a similar approach with dysbindin (DTNBP1), a second schizophrenia risk gene, we show that dysbindin and DISC1 share common PPIs suggesting they may affect common biological processes and that the function of schizophrenia risk genes may converge.

    Molecular psychiatry 2007;12;1;74-86

  • Modulation of Ku70/80, clusterin/ApoJ isoforms and Bax expression in indocyanine-green-mediated photo-oxidative cell damage.

    Ricci F, Pucci S, Sesti F, Missiroli F, Cerulli L and Spagnoli LG

    Section of Ophthalmology, University of Rome Tor Vergata, Rome, Italy.

    Purpose: In order to characterize the biological effects and molecular mechanism underlying indocyanine-green (ICG)-mediated photo-oxidative cell damage, human cultured retinal pigmented epithelium (RPE) cells preloaded with ICG were exposed to 810-nm laser irradiation. Cell viability and death induction were examined, as well as the modulation of proteins involved in cell death and DNA repair.

    Methods: ARPE-19 cells preloaded with 100 microM ICG were irradiated using continuous and micropulsed 810-nm laser for the dye photoactivation, and cell viability and apoptosis were evaluated. The expression and subcellular localization of Bax, Ku70, Ku80 and clusterin/ApoJ were analyzed by immunocytochemistry and Western blot.

    Results: ICG photoactivation induced apoptosis in RPE cells. The micropulsed laser irradiation induced a higher percentage of cell killing as compared to continuous wave. Cell killing was inhibited by sodium azide, suggesting the involvement of reactive oxygen species in the laser-induced cell damage. Bax was strongly induced after 4 and up to 24 h of treatment. The nuclear proapoptotic isoform of clusterin/ApoJ was selectively upregulated after 24 h of treatment. The DNA repair machinery was upregulated after 4 and up to 24 h.

    Conclusion: These data elucidate some molecular mechanisms involved in cell death induced by ICG photosensitization. The increase and relocalization of Bax into the mitochondria and the upregulation and translocation of the proapoptotic isoform of clusterin/ApoJ in the nucleus demonstrated the involvement of these proteins in the photo-oxidative cell death pathway. These data point out new molecular targets and suggest potential applications in the therapy of the retinal diseases that could benefit by selective RPE treatment.

    Ophthalmic research 2007;39;3;164-73

  • Prognostic significance of clusterin immunoreactivity in breast cancer.

    Krüger S, Ola V, Fischer D, Feller AC and Friedrich M

    Institute of Pathology and Department of Gynecology and Obstetrics, Lübeck, Germany. krueger@patho.uni-luebeck.de

    Clusterin (CLU) is involved in a variety of biological processes and has been found to be expressed even in many human malignancies, including breast cancer. Currently, there are only few data on the prognostic value of CLU in breast cancer. We therefore evaluated the relationship between CLU expression and clinicopathological parameters as well as relapse-free survival (RFS) and metastasis-free survival (MFS) of 141 breast cancer patients using the monoclonal antibody 7D1. CLU expression was found in 26% of cases and correlated significantly with high histological tumor grade and high Ki-67 labeling index (p=0.026 and p=0.010, respectively). Univariate Cox regression analysis revealed that CLU expression was tendentiously associated with RFS (p=0.068; relative risk [RR]: 1.77) and MFS (p=0.122; RR: 1.57). In a multivariate analysis, tumor grade, stage, estrogen receptor status and patients age (concerning RFS) as well as grade and lymph node status (concerning MFS) were identified as significant independent prognosticators. CLU expression showed an independent prognostic relevance concerning prediction of RFS by trend (p=0.110; RR: 1.81). We conclude from our data that estimation of CLU immunoreactivity may be helpful as a supplementary criterion to better assess the tumors propensity to relapse in selected cases of breast carcinoma.

    Neoplasma 2007;54;1;46-50

  • Characterization and functional consequences of underexpression of clusterin in rheumatoid arthritis.

    Devauchelle V, Essabbani A, De Pinieux G, Germain S, Tourneur L, Mistou S, Margottin-Goguet F, Anract P, Migaud H, Le Nen D, Lequerré T, Saraux A, Dougados M, Breban M, Fournier C and Chiocchia G

    Institut Cochin, Département d'Immunologie, 27 rue du Faubourg Saint-Jacques, 75674 Paris Cedex 14, France.

    We previously compared by microarray analysis gene expression in rheumatoid arthritis (RA) and osteoarthritis (OA) tissues. Among the set of genes identified as a molecular signature of RA, clusterin (clu) was one of the most differentially expressed. In the present study we sought to assess the expression and the role of CLU (mRNA and protein) in the affected joints and in cultured fibroblast-like synoviocytes (FLS) and to determine its functional role. Quantitative RT-PCR, Northern blot, in situ hybridization, immunohistochemistry, and Western blot were used to specify and quantify the expression of CLU in ex vivo synovial tissue. In synovial tissue, the protein was predominantly expressed by synoviocytes and it was detected in synovial fluids. Both full-length and spliced isoform CLU mRNA levels of expression were lower in RA tissues compared with OA and healthy synovium. In synovium and in cultured FLS, the overexpression of CLU concerned all protein isoforms in OA whereas in RA, the intracellular forms of the protein were barely detectable. Transgenic overexpression of CLU in RA FLS promoted apoptosis within 24 h. We observed that CLU knockdown with small interfering RNA promoted IL-6 and IL-8 production. CLU interacted with phosphorylated IkappaBalpha. Differential expression of CLU by OA and RA FLS appeared to be an intrinsic property of the cells. Expression of intracellular isoforms of CLU is differentially regulated between OA and RA. We propose that in RA joints, high levels of extracellular CLU and low expression of intracellular CLU may enhance NF-kappaB activation and survival of the synoviocytes.

    Journal of immunology (Baltimore, Md. : 1950) 2006;177;9;6471-9

  • Clusterin up-regulation following sub-lethal oxidative stress and lipid peroxidation in human neuroblastoma cells.

    Strocchi P, Smith MA, Perry G, Tamagno E, Danni O, Pession A, Gaiba A and Dozza B

    Department of Pharmacology, University of Bologna, Bologna 40126, Italy. strocchi@biocfarm.unibo.it

    Clusterin/apolipoprotein J is a multifunctional protein up-regulated during various pathophysiological states. Since oxidative stress plays an important role in brain aging, and in many neurodegenerative disorders, to further understand the mechanistic underpinnings of clusterin expression, in this study, we examined clusterin expression in human neuroblastoma cells under conditions of increased production of reactive oxygen species and lipid peroxidation. Specifically, we analyzed clusterin mRNA and protein levels in human neuroblastoma IMR-32 and SH-SY5Y cells following exposure to sub-lethal amounts of iron-ascorbate to induce an increase in reactive oxygen species generation and lipid peroxidation. Under such conditions, we observed a time-dependent up-regulation of clusterin protein and mRNA levels, detected by immunoblot analysis and RT-PCR, respectively. Given the known roles of clusterin, the results of the present study support the notion that an increase in clusterin expression may be a physiological defence mounted to reduce cell damage and maintain cell viability during periods of increased oxidative stress.

    Neurobiology of aging 2006;27;11;1588-94

  • Clusterin/Apolipoprotein J up-regulation after zinc exposure, replicative senescence or differentiation of human haematopoietic cells.

    Trougakos IP, Pawelec G, Tzavelas C, Ntouroupi T and Gonos ES

    Laboratory of Molecular & Cellular Ageing, Institute of Biological Research & Biotechnology, National Hellenic Research Foundation, 48 Vas. Constantinou Ave., Athens, 11635, Greece.

    Clusterin/Apolipoprotein J (CLU) is a cellular senescence biomarker implicated in several physiological processes. In this work we have investigated CLU expression and function in human haematopoietic cells. We found that early passage human T cell clones (TCC) express minimal endogenous amounts of CLU, which are significantly elevated in late passage cells. Moreover, exposure of TCC to increased levels of the essential micronutrient zinc in culture resulted in intense induction of CLU. Because haematopoietic cells cease proliferation following induction of terminal differentiation, we also studied the expression profile of CLU in the leukemic progenitor cell lines K562 and HL-60. We found that, like TCC, both cell lines express minimal endogenous levels of CLU in their actively proliferating state. However, when induced to differentiate into their distinct cell types, CLU was found to be up-regulated specifically in those cells expressing the main differentiation markers. Enforced stable over-expression of CLU in K562 cells inhibited the expression of the CD14 differentiation marker and blocked differentiation to either monocytes/megacaryoblasts or to erythrocytes. Overall, our results suggest that CLU is actively involved in both replicative senescence and terminal differentiation in different types of human haematopoietic cells.

    Biogerontology 2006;7;5-6;375-82

  • Clusterin and DNA repair: a new function in cancer for a key player in apoptosis and cell cycle control.

    Shannan B, Seifert M, Boothman DA, Tilgen W and Reichrath J

    Department of Dermatology, The Saarland University Hospital, Building 18, Homburg/Saar 66421, Germany.

    The glycoprotein clusterin (CLU), has two known isoforms generated in human cells. A nuclear form of CLU protein (nCLU) is pro-apoptotic, while a secretory form (sCLU) is pro-survival. Both forms are implicated in various cell functions, including DNA repair, cell cycle regulation, and apoptotic cell death. CLU expression has been associated with tumorigenesis and the progression of various malignancies. In response to DNA damage, cell survival can be enhanced by activation of DNA repair mechanisms, while simultaneously stimulating energy-expensive cell cycle checkpoints that delay the cell cycle progression to allow more time for DNA repair. This review summarizes our current understanding of the role of clusterin in DNA repair, apoptosis, and cell cycle control and the relevance.

    Funded by: NCI NIH HHS: P30 CA013148, P30 CA142543, R01 CA078530

    Journal of molecular histology 2006;37;5-7;183-8

  • Decrease of both stem cell factor and clusterin mRNA levels in testicular biopsies of azoospermic patients with constitutive or idiopathic but not acquired spermatogenic failure.

    Plotton I, Sanchez P, Durand P and Lejeune H

    INSERM, U 418, Hôpital Debrousse, INRA, Université Claude Bernard Lyon 1, Lyon, France.

    Background: Sertoli cells nurse germ cells during spermatogenesis, and alterations of Sertoli cell functions have been suggested in cases of spermatogenic failures.

    Methods: In this work, we measured stem cell factor (SCF) and clusterin mRNA levels, by quantitative RT-PCR, in RNA extracted from testicular biopsies of 49 azoospermic patients classified according to testicular histology as having normal spermatogenesis or spermatogenic failure.

    Results: When related to the percentage of Sertoli cells counted on a histological section of a neighbouring tissue sample, SCF and clusterin mRNA levels were significantly lower in the 'spermatogenic failure' group compared with the control group (P = 0.0297 and P = 0.0043, respectively). These levels were also significantly lower in the cases of 'constitutive' (cryptorchidism and Yq microdeletion) and 'idiopathic' spermatogenic failures when compared with the control group; conversely, they were not significantly decreased in the group with 'acquired spermatogenic failure' (orchitis, testicular traumatism, chemoradiotherapy and varicocele).

    Conclusions: These data further demonstrate an alteration of Sertoli cell functions in some human spermatogenic failures and suggest that a lack of Sertoli cell maturation may be involved in cases of constitutive or idiopathic spermatogenic failures.

    Human reproduction (Oxford, England) 2006;21;9;2340-5

  • Clusterin decreases oxidative stress in lung fibroblasts exposed to cigarette smoke.

    Carnevali S, Luppi F, D'Arca D, Caporali A, Ruggieri MP, Vettori MV, Caglieri A, Astancolle S, Panico F, Davalli P, Mutti A, Fabbri LM and Corti A

    Department of Oncology, Haematology, and Pulmonology, University of Modena and Reggio Emilia, Via del Pozzo 71, 41100 Modena, Italy.

    Rationale: Cigarette smoke causes injury to lung fibroblasts, partly by means of oxidative stress, and oxidative stress can lead to various lung diseases, such as chronic obstructive pulmonary disease. Clusterin is a widely distributed protein with many functions, including cellular protection in response to oxidative stress.

    Objectives: To determine whether clusterin is involved in the defense of the lung against cigarette smoke, we investigated the effects of cigarette smoke extract on clusterin expression and its protective effect, if any, against oxidative stress.

    Methods: Fibroblasts were coincubated with conditioned medium and cigarette smoke extract, and bronchial biopsy specimens obtained from nonsmokers, smokers, and ex-smokers were analyzed by immunohistochemistry.

    At concentrations of 2.5 and 5.0%, cigarette smoke extract induced oxidative stress. It also markedly increased the expression of two clusterin isoforms (60 and 76-80 kD) and the 76-80-kD isoform was secreted in the incubation medium. Coincubation of fibroblasts with conditioned medium significantly decreased the cellular oxidation caused by the cigarette smoke extract. Immunohistochemical analysis of clusterin on bronchial biopsy specimens obtained from smokers and ex-smokers showed localization of clusterin mainly in the submucosa.

    Conclusions: We conclude that clusterin may have a protective effect against cigarette smoke-induced oxidative stress in lung fibroblasts.

    American journal of respiratory and critical care medicine 2006;174;4;393-9

  • Glomerular clusterin is associated with PKC-alpha/beta regulation and good outcome of membranous glomerulonephritis in humans.

    Rastaldi MP, Candiano G, Musante L, Bruschi M, Armelloni S, Rimoldi L, Tardanico R, Sanna-Cherchi S, Cherchi SS, Ferrario F, Montinaro V, Haupt R, Parodi S, Carnevali ML, Allegri L, Camussi G, Gesualdo L, Scolari F and Ghiggeri GM

    Renal Immunopathology Laboratory, Fondazione D'Amico per la Ricerca sulle Malattie Renali, Nuova Nefrologia Research Association, c/o San Carlo Borromeo Hospital, Milan, Italy.

    Mechanisms for human membranous glomerulonephritis (MGN) remain elusive. Most up-to-date concepts still rely on the rat model of Passive Heymann Nephritis that derives from an autoimmune response to glomerular megalin, with complement activation and membrane attack complex assembly. Clusterin has been reported as a megalin ligand in immunodeposits, although its role has not been clarified. We studied renal biopsies of 60 MGN patients by immunohistochemistry utilizing antibodies against clusterin, C5b-9, and phosphorylated-protien kinase C (PKC) isoforms (pPKC). In vitro experiments were performed to investigate the role of clusterin during podocyte damage by MGN serum and define clusterin binding to human podocytes, where megalin is known to be absent. Clusterin, C5b-9, and pPKC-alpha/beta showed highly variable glomerular staining, where high clusterin profiles were inversely correlated to C5b-9 and PKC-alpha/beta expression (P=0.029), and co-localized with the low-density lipoprotein receptor (LDL-R). Glomerular clusterin emerged as the single factor influencing proteinuria at multivariate analysis and was associated with a reduction of proteinuria after a follow-up of 1.5 years (-88.1%, P=0.027). Incubation of podocytes with MGN sera determined strong upregulation of pPKC-alpha/beta that was reverted by pre-incubation with clusterin, serum de-complementation, or protein-A treatment. Preliminary in vitro experiments showed podocyte binding of biotinilated clusterin, co-localization with LDL-R and specific binding inhibition with anti-LDL-R antibodies and with specific ligands. These data suggest a central role for glomerular clusterin in MGN as a modulator of inflammation that potentially influences the clinical outcome. Binding of clusterin to the LDL-R might offer an interpretative key for the pathogenesis of MGN in humans.

    Kidney international 2006;70;3;477-85

  • Isolation and characterization of human apolipoprotein M-containing lipoproteins.

    Christoffersen C, Nielsen LB, Axler O, Andersson A, Johnsen AH and Dahlbäck B

    Department of Clinical Biochemistry, Rigshospital, University of Copenhagen, Copenhagen DK-2100, Denmark.

    Apolipoprotein M (apoM) is a novel apolipoprotein with unknown function. In this study, we established a method for isolating apoM-containing lipoproteins and studied their composition and the effect of apoM on HDL function. ApoM-containing lipoproteins were isolated from human plasma with immunoaffinity chromatography and compared with lipoproteins lacking apoM. The apoM-containing lipoproteins were predominantly of HDL size; approximately 5% of the total HDL population contained apoM. Mass spectrometry showed that the apoM-containing lipoproteins also contained apoJ, apoA-I, apoA-II, apoC-I, apoC-II, apoC-III, paraoxonase 1, and apoB. ApoM-containing HDL (HDL(apoM+)) contained significantly more free cholesterol than HDL lacking apoM (HDL(apoM-)) (5.9 +/- 0.7% vs. 3.2 +/- 0.5%; P < 0.005) and was heterogeneous in size with both small and large particles. HDL(apoM+) inhibited Cu(2+)-induced oxidation of LDL and stimulated cholesterol efflux from THP-1 foam cells more efficiently than HDL(apoM-). In conclusion, our results suggest that apoM is associated with a small heterogeneous subpopulation of HDL particles. Nevertheless, apoM designates a subpopulation of HDL that protects LDL against oxidation and stimulates cholesterol efflux more efficiently than HDL lacking apoM.

    Journal of lipid research 2006;47;8;1833-43

  • Clusterin in cerebrospinal fluid: analysis of carbohydrates and quantification of native and glycosylated forms.

    Nilselid AM, Davidsson P, Nägga K, Andreasen N, Fredman P and Blennow K

    Institute of Clinical Neuroscience, The Sahlgrenska Academy at Göteborg University, Sahlgrenska University Hospital, SE 431 80 Mölndal, Sweden. aml_mag@hotmail.com

    Clusterin is suggested to be involved in the pathogenesis of Alzheimer's disease. Clusterin expression is increased in brain tissue in affected regions of Alzheimer patients, and intense clusterin staining is found in both senile plaques and in neuronal and glia cells. In contrast, the cerebrospinal fluid level of clusterin in Alzheimer patients has, thus far, been found unchanged. Clusterin is a glycosylated protein, and an alteration of its glycosylation in Alzheimer's disease might influence accurate quantification in cerebrospinal fluid through interference of antibody binding to the protein. Using enzymatic deglycosylation of clusterin isolated from cerebrospinal fluid, we found that the carbohydrates attached to clusterin were of the N-linked type and sialic acids. Based on this finding, cerebrospinal fluid samples from Alzheimer patients (n=99) and controls (n=39) were analysed. The samples were treated with peptide: N-glycanase F, cleaving off N-linked carbohydrates, and clusterin was quantified before and after deglycosylation using a new sandwich enzyme-linked immunosorbent assay. Clusterin was significantly increased in Alzheimer patients, in both native (7.17+/-2.43 AU versus 5.73+/-2.09 AU; p=0.002), and deglycosylated samples (12.19+/-5.00 AU versus 9.68+/-4.38 AU; p=0.004). Deglycosylation led to increased measured levels of clusterin by 70% (p<0.001) in Alzheimer patients and 67% (p<0.001) in controls. These findings indicate that glycosylation of proteins may interfere with their quantification. The results show that clusterin is significantly increased in cerebrospinal fluid from Alzheimer patients as a group, supporting that clusterin might be involved in the pathogenesis of Alzheimer's disease. However, the individual clusterin levels overlap between the two groups, and thus cerebrospinal fluid clusterin measurement is not suitable as a biochemical marker in the diagnosis of Alzheimer's disease.

    Neurochemistry international 2006;48;8;718-28

  • Identification of N-linked glycoproteins in human saliva by glycoprotein capture and mass spectrometry.

    Ramachandran P, Boontheung P, Xie Y, Sondej M, Wong DT and Loo JA

    Department of Chemistry and Biochemistry, University of California-Los Angeles, Los Angeles, California, USA.

    Glycoproteins make up a major and important part of the salivary proteome and play a vital role in maintaining the health of the oral cavity. Because changes in the physiological state of a person are reflected as changes in the glycoproteome composition, mapping the salivary glycoproteome will provide insights into various processes in the body. Salivary glycoproteins were identified by the hydrazide coupling and release method. In this approach, glycoproteins were coupled onto a hydrazide resin, the proteins were then digested and formerly N-glycosylated peptides were selectively released with the enzyme PNGase F and analyzed by LC-MS/MS. Employing this method, coupled with in-solution isoelectric focusing separation as an additional means for pre-fractionation, we identified 84 formerly N-glycosylated peptides from 45 unique N-glycoproteins. Of these, 16 glycoproteins have not been reported previously in saliva. In addition, we identified 44 new sites of N-linked glycosylation on the proteins.

    Funded by: NIDCR NIH HHS: U01 DE016275; NIGMS NIH HHS: GM07185

    Journal of proteome research 2006;5;6;1493-503

  • Nuclear clusterin accumulation during heat shock response: implications for cell survival and thermo-tolerance induction in immortalized and prostate cancer cells.

    Caccamo AE, Desenzani S, Belloni L, Borghetti AF and Bettuzzi S

    Dipartimento di Medicina Sperimentale, Sezione di Biochimica, Biochimica Clinica e Biochimica dell'Esercizio Fisico, Università di Parma, Via Volturno Parma, Italy.

    Clusterin (CLU), whose role is still debated, is differentially regulated in several patho-physiological processes and invariably induced during apoptosis. In heat shock response, CLU is considered a stress-inducible, pro-survival/cyto-protective factor via an HSE element present in his promoter. In both human prostate PNT1A and PC-3 epithelial cells we found that apoptotic stimuli induced nuclear localization of CLU (nCLU), and that overexpression of nCLU is pro-apoptotic. We show here that CLU time-course accumulation kinetic is different from that of HSP70 in these cells, thus other factor(s) might mediate HSF-1 activation and CLU expression. Sub-lethal heat shock inhibited the secretion of CLU (sCLU), leading to increased cytoplasm accumulation of CLU (cCLU) in association to cell survival. At difference, lethal heat stress caused massive accumulation of pro-apoptotic nCLU in cells dying by caspase-3-dependent apoptosis. Double heat stress (sub-lethal heat shock followed by recovery and lethal stress) induced HSP70 and thermo-tolerance in PNT1A cells, but not in PC-3 cells. In PNT1A cells, CLU secretion was inhibited and cCLU was accumulated, suggesting that cCLU might be pro-survival, while in PC-3 cells accumulation of nCLU was concomitant to caspase-3 induction and PARP activation instead. Thus, CLU expression/sub-cellular localization is strictly related to cell fate. In particular, nCLU and physiological levels of HSP70 affected cell survival in an antagonistic fashion. Prevalence of heat-induced nCLU, not allowing PC-3 cells to cope with heat shock, could be the rational explaining why malignant cells are more sensitive to heat when delivered by minimally invasive procedures for ablation of localized prostate cancer.

    Journal of cellular physiology 2006;207;1;208-19

  • Transcriptomic and proteomic analyses of rhabdomyosarcoma cells reveal differential cellular gene expression in response to enterovirus 71 infection.

    Leong WF and Chow VT

    Human Genome Laboratory, Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Kent Ridge, Singapore 117597.

    Insights into the host antiviral strategies as well as viral disease manifestations can be achieved through the elucidation of host- and virus-mediated transcriptional responses. An oligo-based microarray was employed to analyse mRNAs from rhabdomyosarcoma cells infected with the MS/7423/87 strain of enterovirus 71 (EV71) at 20 h post infection. Using Acuity software and LOWESS normalization, 152 genes were found to be downregulated while 39 were upregulated by greater than twofold. Altered transcripts include those encoding components of cytoskeleton, protein translation and modification; cellular transport proteins; protein degradation mediators; cell death mediators; mitochondrial-related and metabolism proteins; cellular receptors and signal transducers. Changes in expression profiles of 15 representative genes were authenticated by real-time reverse transcription polymerase chain reaction (RT-PCR), which also compared the transcriptional responses of cells infected with EV71 strain 5865/Sin/000009 isolated from a fatal case during the Singapore outbreak in 2000. Western blot analyses of APOB, CLU, DCAMKL1 and ODC1 proteins correlated protein and transcript levels. Two-dimensional proteomic maps highlighted differences in expression of cellular proteins (CCT5, CFL1, ENO1, HSPB1, PSMA2 and STMN1) following EV71 infection. Expression of several apoptosis-associated genes was modified, coinciding with apoptosis attenuation observed in poliovirus infection. Interestingly, doublecortin and CaM kinase-like 1 (DCAMKL1) involved in brain development, was highly expressed during infection. Thus, microarray, real-time RT-PCR and proteomic analyses can elucidate the global view of the numerous and complex cellular responses that contribute towards EV71 pathogenesis.

    Cellular microbiology 2006;8;4;565-80

  • Elucidation of N-glycosylation sites on human platelet proteins: a glycoproteomic approach.

    Lewandrowski U, Moebius J, Walter U and Sickmann A

    Protein Mass Spectrometry and Functional Proteomics Group, Rudolf Virchow Center for Experimental Biomedicine, Versbacher Strasse 9, 97078 Wuerzburg, Germany.

    Among known platelet proteins, a prominent and functionally important group is represented by glycoprotein isoforms. They account e.g. for secretory proteins and plasma membrane receptors including integrins and glycoprotein VI as well as intracellular components of cytosol and organelles including storage proteins (multimerin 1 etc.). Although many of those proteins have been studied for some time with regard to their function, little attention has been paid with respect to their glycosylation sites. Here we report the analysis of N-glycosylation sites of human platelet proteins. For the enrichment of glycopeptides, lectin affinity chromatography as well as chemical trapping of protein bound oligosaccharides was used. Therefore, concanavalin A was used for specific interaction with carbohydrate species along with periodic acid oxidation and hydrazide bead trapping of glycosylated proteins. Derivatization by peptide:N-glycosidase F yielded deglycosylated peptides, which provided the basis for the elucidation of proteins and their sites of modification. Using both methods in combination with nano-LC-ESI-MS/MS analysis 70 different glycosylation sites within 41 different proteins were identified. Comparison with the Swiss-Prot database established that the majority of these 70 sites have not been specifically determined by previous research projects. With this approach including hydrazide bead affinity trapping, the immunoglobulin receptor G6f, which is known to couple to the Ras-mitogen-activated protein kinase pathway in the immune system, was shown here for the first time to be present in human platelets.

    Molecular & cellular proteomics : MCP 2006;5;2;226-33

  • DNA sequence and analysis of human chromosome 8.

    Nusbaum C, Mikkelsen TS, Zody MC, Asakawa S, Taudien S, Garber M, Kodira CD, Schueler MG, Shimizu A, Whittaker CA, Chang JL, Cuomo CA, Dewar K, FitzGerald MG, Yang X, Allen NR, Anderson S, Asakawa T, Blechschmidt K, Bloom T, Borowsky ML, Butler J, Cook A, Corum B, DeArellano K, DeCaprio D, Dooley KT, Dorris L, Engels R, Glöckner G, Hafez N, Hagopian DS, Hall JL, Ishikawa SK, Jaffe DB, Kamat A, Kudoh J, Lehmann R, Lokitsang T, Macdonald P, Major JE, Matthews CD, Mauceli E, Menzel U, Mihalev AH, Minoshima S, Murayama Y, Naylor JW, Nicol R, Nguyen C, O'Leary SB, O'Neill K, Parker SC, Polley A, Raymond CK, Reichwald K, Rodriguez J, Sasaki T, Schilhabel M, Siddiqui R, Smith CL, Sneddon TP, Talamas JA, Tenzin P, Topham K, Venkataraman V, Wen G, Yamazaki S, Young SK, Zeng Q, Zimmer AR, Rosenthal A, Birren BW, Platzer M, Shimizu N and Lander ES

    Broad Institute of MIT and Harvard, 320 Charles St, Cambridge, Massachusetts 02141, USA. chad@broad.mit.edu

    The International Human Genome Sequencing Consortium (IHGSC) recently completed a sequence of the human genome. As part of this project, we have focused on chromosome 8. Although some chromosomes exhibit extreme characteristics in terms of length, gene content, repeat content and fraction segmentally duplicated, chromosome 8 is distinctly typical in character, being very close to the genome median in each of these aspects. This work describes a finished sequence and gene catalogue for the chromosome, which represents just over 5% of the euchromatic human genome. A unique feature of the chromosome is a vast region of approximately 15 megabases on distal 8p that appears to have a strikingly high mutation rate, which has accelerated in the hominids relative to other sequenced mammals. This fast-evolving region contains a number of genes related to innate immunity and the nervous system, including loci that appear to be under positive selection--these include the major defensin (DEF) gene cluster and MCPH1, a gene that may have contributed to the evolution of expanded brain size in the great apes. The data from chromosome 8 should allow a better understanding of both normal and disease biology and genome evolution.

    Funded by: Medical Research Council: G0000107

    Nature 2006;439;7074;331-5

  • Challenge and promise: roles for clusterin in pathogenesis, progression and therapy of cancer.

    Shannan B, Seifert M, Leskov K, Willis J, Boothman D, Tilgen W and Reichrath J

    Department of Dermatology, The Saarland University Hospital, Homburg 66421, Germany.

    Clusterin (CLU) has been implicated in various cell functions involved in carcinogenesis and tumour progression. There are two known CLU protein isoforms generated in human cells. A nuclear form of CLU protein (nCLU) is proapoptotic, and a secretory form (sCLU) is prosurvival. CLU expression has been associated with tumorigenesis of various malignancies, including tumours of prostate, colon, and breast. Furthermore, CLU expression is modulated by many factors that are believed to regulate tumour growth and/or apoptosis, including 1,25-dihydroxyvitamin D3, transforming growth factor beta-1, ultraviolet radiation, and IR. sCLU upregulation appears to be a general molecular stress response. Presently, preliminary results indicate that therapeutic modalities targeting CLU may be effective in cancer treatment. However, such strategies should make sure that nCLU is not eliminated or reduced. This review summarizes our present understanding of the importance of CLU in various physiological functions including tumour growth, and discusses its relevance to future cancer therapy.

    Funded by: NCI NIH HHS: R01 CA078530, R01 CA102792

    Cell death and differentiation 2006;13;1;12-9

  • Expression of the antiapoptotic proteins clusterin and bcl-2 in laryngeal squamous cell carcinomas.

    Redondo M, Esteban F, González-Moles MA, Delgado-Rodríguez M, Nevado M, Torres-Muñoz JE, Tellez T, Villar E, Morell M and Petito CK

    Departamento de Bioquímica, Hospital Costa del Sol, Universidad de Málaga, Marbella, Spain. mredondo@hcs.es

    Bcl-2 and clusterin genes have been related to the inhibition of apoptosis, an event that plays a key role in malignant transformation and in invasive disease. In this work, we determine the significance of clusterin and bcl-2 expression in a large series of laryngeal carcinomas. We used immunohistochemical methods and in situ hybridization to examine the expression of these proteins. Nontumoral epithelial laryngeal tissues did not express clusterin and bcl-2 proteins. However, 9% (14 out of 154) and 25% of these tumors (39 of 154) had positive clusterin and bcl-2 staining, respectively. Clusterin expression was significantly related to the degree of local invasion and higher bcl-2 expression was found in these clusterin-positive tumors (p < 0.05). Bcl-2 expression was significantly correlated with supraglottic localization, nodal metastases, invasion in depth, and poorly differentiated tumors. However, by multivariate analysis, bcl-2 was shown to be an independent predictor of good prognosis in these tumors (OR = 0.12, 95% CI = 0.02-0.91). These findings indicate that clusterin and bcl-2 are upregulated in laryngeal carcinomas and their expression is related to the invasiveness of these tumors.

    Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 2006;27;4;195-200

  • Human plasma N-glycoproteome analysis by immunoaffinity subtraction, hydrazide chemistry, and mass spectrometry.

    Liu T, Qian WJ, Gritsenko MA, Camp DG, Monroe ME, Moore RJ and Smith RD

    Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99354, USA.

    The enormous complexity, wide dynamic range of relative protein abundances of interest (over 10 orders of magnitude), and tremendous heterogeneity (due to post-translational modifications, such as glycosylation) of the human blood plasma proteome severely challenge the capabilities of existing analytical methodologies. Here, we describe an approach for broad analysis of human plasma N-glycoproteins using a combination of immunoaffinity subtraction and glycoprotein capture to reduce both the protein concentration range and the overall sample complexity. Six high-abundance plasma proteins were simultaneously removed using a pre-packed, immobilized antibody column. N-linked glycoproteins were then captured from the depleted plasma using hydrazide resin and enzymatically digested, and the bound N-linked glycopeptides were released using peptide-N-glycosidase F (PNGase F). Following strong cation exchange (SCX) fractionation, the deglycosylated peptides were analyzed by reversed-phase capillary liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Using stringent criteria, a total of 2053 different N-glycopeptides were confidently identified, covering 303 nonredundant N-glycoproteins. This enrichment strategy significantly improved detection and enabled identification of a number of low-abundance proteins, exemplified by interleukin-1 receptor antagonist protein (approximately 200 pg/mL), cathepsin L (approximately 1 ng/mL), and transforming growth factor beta 1 (approximately 2 ng/mL). A total of 639 N-glycosylation sites were identified, and the overall high accuracy of these glycosylation site assignments as assessed by accurate mass measurement using high-resolution liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR) is initially demonstrated.

    Funded by: NCRR NIH HHS: P41 RR018522, RR18522; NIGMS NIH HHS: U54 GM-62119-02, U54 GM062119

    Journal of proteome research 2005;4;6;2070-80

  • 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

  • Clusterin inhibits apoptosis by interacting with activated Bax.

    Zhang H, Kim JK, Edwards CA, Xu Z, Taichman R and Wang CY

    Laboratory of Molecular Signaling and Apoptosis, Department of Biological and Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor, MI 48109-1078, USA.

    Clusterin is an enigmatic glycoprotein that is overexpressed in several human cancers such as prostate and breast cancers, and squamous cell carcinoma. Because the suppression of clusterin expression renders human cancer cells sensitive to chemotherapeutic drug-mediated apoptosis, it is currently an antisense target in clinical trials for prostate cancer. However, the molecular mechanisms by which clusterin inhibits apoptosis in human cancer cells are unknown. Here we report that intracellular clusterin inhibits apoptosis by interfering with Bax activation in mitochondria. Intriguingly, in contrast to other inhibitors of Bax, clusterin specifically interacts with conformation-altered Bax in response to chemotherapeutic drugs. This interaction impedes Bax oligomerization, which leads to the release of cytochrome c from mitochondria and caspase activation. Moreover, we also find that clusterin inhibits oncogenic c-Myc-mediated apoptosis by interacting with conformation-altered Bax. Clusterin promotes c-Myc-mediated transformation in vitro and tumour progression in vivo. Taken together, our results suggest that the elevated level of clusterin in human cancers may promote oncogenic transformation and tumour progression by interfering with Bax pro-apoptotic activities.

    Nature cell biology 2005;7;9;909-15

  • Insertion/deletion polymorphism in clusterin gene influences serum lipid levels and carotid intima-media thickness in hypertensive Japanese females.

    Miwa Y, Takiuchi S, Kamide K, Yoshii M, Horio T, Tanaka C, Banno M, Miyata T, Sasaguri T and Kawano Y

    Division of Hypertension and Nephrology, National Cardiovascular Center, Suita, Japan. ymiwa@clipharm.med.kyushu-u.ac.jp

    Clusterin has been implicated in lipid metabolism and atherogenesis, however, the influence of genetic variation has not been examined in Japanese. In this study, we identified 11 single nucleotide polymorphisms (SNPs) of clusterin gene by direct sequencing. Among them, one promoter SNP (-4453T>G), one missense SNP (4183G>A), and 2 common SNPs (5608T>C and 6316delT) were genotyped in 525 asymptomatic hypertensives not treated with lipid lowering agents. -4453T>G, 4183G>A, and 5608T>C showed no correlation with the clinical characteristics, however, in the 6316delT, an insertion (I)/deletion (D) polymorphism, D/D subjects had significantly higher levels of total cholesterol and low-density lipoprotein (LDL)-cholesterol than I/I subjects in females but not in males. Female subjects with the D allele (D/D+I/D) had greater intima-media thickness of the carotid artery than I/I subjects. In a multiple logistic regression analysis, the D allele of 6316delT was detected as an independent predictor for the plaque prevalence. In conclusion, the clusterin gene polymorphism may contribute to the serum lipid levels and the progression of carotid atherosclerosis in hypertensive Japanese females.

    Biochemical and biophysical research communications 2005;331;4;1587-93

  • Oncogenic role of clusterin overexpression in multistage colorectal tumorigenesis and progression.

    Xie D, Sham JS, Zeng WF, Che LH, Zhang M, Wu HX, Lin HL, Wen JM, Lau SH, Hu L and Guan XY

    Department of Clinical Oncology, The University of Hong Kong, Room 109, School of Chinese Medicine Building, 10 Sassoon Road, Hong Kong, China.

    Aim: To investigate the expression pattern of clusterin in colorectal adenoma-carcinoma-metastasis series, and to explore the potential role of clusterin in multistage colorectal tumorigenesis and progression.

    Methods: A colorectal carcinoma (CRC)-tissue microarray (TMA), which contained 85 advanced CRCs including 43 cases of Dukes B, 21 of Dukes C and 21 of Dukes D tumors, were used for assessing the expression of clusterin (clone 41D) and tumor cell apoptotic index (AI) by immunohistochemistry and TUNEL assay, respectively. Moreover the potential correlation of clusterin expression with the patient's clinical-pathological features were also examined.

    Results: The positive staining of clusterin in different colorectal tissues was primarily a cytoplasmic pattern. Cytoplasmic overexpression of clusterin was detected in none of the normal colorectal mucosa, 17% of the adenomas, 46% of the primary CRCs, and 57% of the CRC metastatic lesions. In addition, a significant positive correlation between overexpression of clusterin and advanced clinical (Dukes) stage was observed (P<0.01). Overexpression of cytoplasmic clusterin in CRCs was inversely correlated with tumor apoptotic index (P<0.01), indicating the anti-apoptotic function of cytoplasmic clusterin in CRCs.

    Conclusion: These data suggests that overexpression of cytoplasmic clusterin might be involved in the tumorigenesis and/or progression of CRCs. The anti-apoptotic function of cytoplasmic clusterin may be responsible, at least in part, for the development and biologically aggressive behavior of CRC.

    World journal of gastroenterology 2005;11;21;3285-9

  • Clusterin regulates drug-resistance in melanoma cells.

    Hoeller C, Pratscher B, Thallinger C, Winter D, Fink D, Kovacic B, Sexl V, Wacheck V, Gleave ME, Pehamberger H and Jansen B

    Department of Dermatology, Division of General Dermatology and Medical University Vienna, Vienna, Austria. christoph.hoeller@univie.ac.at

    Clusterin has recently been shown to act as an antiapoptotic protein that confers drug-resistance in models of epithelial tumors. The aim of our work was to provide an insight into a possible role of clusterin in the regulation of drug-resistance in melanoma. In tissue samples, clusterin expression was low in nevi, but high in primary melanoma and melanoma metastases. Clusterin was also strongly expressed in melanoma cell lines, but was barely detectable in cultured melanocytes. To elucidate a possible role of clusterin in drug-resistance of melanoma, clusterin expression was regulated by either plasmid-driven overexpression or by antisense-mediated downregulation. Clusterin overexpression was associated with an increase in drug-resistance, i.e., with an increased survival of melanoma cells in the presence of cytotoxic drugs. In contrast, downregulation of clusterin by 2'-O-(2-methoxy)ethyl (2'MOE)-modified antisense oligonucleotides (AS-ODN) directed against clusterin mRNA significantly reduced drug-resistance, i.e., decreased survival of melanoma cells in the presence of cytotoxic drugs. To evaluate the effects of clusterin-antisense treatment in vivo, we applied an SCID-mouse/human-melanoma xenotransplantation model. Pre-treatment of mice with the 2'MOE-modified clusterin AS-ODN was associated with a significantly improved tumor response to dacarbazine as compared with animals pretreated with a scrambled control oligonucleotide. Taken together, we show that clusterin is strongly expressed in melanoma. Downregulation of clusterin reduces drug-resistance, i.e., reduces melanoma cell survival in response to cytotoxic drugs in vitro and in vivo. Thus, reducing clusterin expression may provide a novel tool to overcome drug-resistance in melanoma.

    The Journal of investigative dermatology 2005;124;6;1300-7

  • Dys-regulation of clusterin in human hepatoma is not associated with tumorigenesis but is secondary to cell response to external tresses.

    Hsieh SY, Chen WY, Shih TC, Yeh JY and Jeng JT

    Liver Research Unit, Chang Gung Memorial Hospital, Tao-Yuan 333, Taiwan.

    It becomes feasible to perform genome-wide differential gene or protein expression in the post genome era. However, little has been addressed on the effects of external stresses and microenvironment alterations on the outcomes of gene and protein expression. To identify downregulated genes during hepatoma development, we combined the cDNA representational difference analysis (RDA) and reverse Northern blot analysis identifying eight genes. Of interest, the expression of the clusterin gene was either down or upregulated in 8 and 7 out of the 20 hepatoma tissues, respectively. Further analysis revealed that its expression was independent of patients' age, gender, causes of liver disease, tumor size, tumor histological stage, or clinical outcome, but was strongly associated with the methods of hepatectomy procedures. In vitro studies disclosed that the clusterin mRNA was increased twofold in early exponential phase of cell proliferation followed by downregulation in the subsequent quiescence phase, whereas it was rapidly increased up to twelvefold upon UV-induced apoptosis. These results suggest that dys-regulation of the clusterin gene in human hepatoma was most likely due to cellular responses to external stresses especially during the procedures for sample collection rather than any correlation to hepatoma development or progression. Our findings that external stresses or microenvironmental changes could greatly affect gene or protein expression offer a general caution to all the studies conducted via genomic and proteomic approaches.

    Molecular carcinogenesis 2005;43;2;100-7

  • Enhanced radiosensitivity by inhibition of the anti-apoptotic gene clusterin using antisense oligodeoxynucleotide in a human bladder cancer model.

    Yamanaka K, Gleave M, Muramaki M, Hara I and Miyake H

    Department of Urology, Kobe University School of Medicine, Kobe 650-0017, Japan.

    Clusterin has been shown to be implicated in the acquisition of resistant phenotype to various kinds of apoptotic stimuli, including radiation. In bladder cancer, our previous study demonstrated that overexpression of clusterin is closely associated with disease progression and recurrence. The objective of this study was to investigate whether radiation sensitivity was enhanced by suppressing clusterin gene expression with antisense (AS) oligodeoxynucleotide (ODN) in the human bladder cancer KoTCC-1 model. Clusterin mRNA in KoTCC-1 cells after radiation was up-regulated in a dose-dependent manner; however, AS clusterin ODN treatment resulted in a marked inhibition of clusterin mRNA even after irradiation. Combined treatment of KoTCC-1 cells with radiation and AS clusterin ODN synergistically decreased plating efficacy and induced apoptotic cell death compared with either radiation or AS clusterin ODN treatment alone. In vivo systemic administration of AS clusterin ODN enhanced radiation sensitivity, significantly reducing subcutaneous KoTCC-1 tumor volume in nude mice, compared with that of mismatch control ODN. Moreover, additional administration of cisplatin to this combined regimen further achieved potential antitumor effects on subcutaneous KoTCC-1 tumor growth in nude mice. Collectively, these findings suggest that clusterin acts as a cell survival protein mediating radioresistance through the inhibition of apoptosis, and that inactivation of clusterin using AS technology might offer a novel strategy to improve the outcome of radiation therapy for patients with bladder cancer.

    Oncology reports 2005;13;5;885-90

  • Delayed activation of insulin-like growth factor-1 receptor/Src/MAPK/Egr-1 signaling regulates clusterin expression, a pro-survival factor.

    Criswell T, Beman M, Araki S, Leskov K, Cataldo E, Mayo LD and Boothman DA

    Department of Radiation Oncology and Program in Molecular and Cellular Basis of Disease, Laboratory of Molecular Stress Responses, Case Comprehensive Cancer Center, Cleveland, Ohio 44106-7285, USA.

    Secretory clusterin protein (sCLU) is a general genotoxic stress-induced, pro-survival gene product implicated in aging, obesity, heart disease, and cancer. However, the regulatory signal transduction processes that control sCLU expression remain undefined. Here, we report that induction of sCLU is delayed, peaking 72 h after low doses of ionizing radiation, and is dependent on the up-regulation of insulin-like growth factor-1 as well as phosphorylation-dependent activation of its receptor (IGF-1 and IGF-1R, respectively). Activated IGF-1R then stimulates the downstream Src-Mek-Erk signal transduction cascade to ultimately transactivate the early growth response-1 (Egr-1) transcription factor, required for sCLU expression. Thus, ionizing radiation exposure causes stress-induced activation of IGF-1R-Src-Mek-Erk-Egr-1 signaling that regulates the sCLU pro-survival cascade pathway, important for radiation resistance in cancer therapy.

    Funded by: NCI NIH HHS: P30 CA142543, P30 CA43703, R01 CA078530

    The Journal of biological chemistry 2005;280;14;14212-21

  • Differential effects of clusterin/apolipoprotein J on cellular growth and survival.

    Trougakos IP, Lourda M, Agiostratidou G, Kletsas D and Gonos ES

    Laboratory of Molecular & Cellular Aging, Institute of Biological Research & Biotechnology, National Hellenic Research Foundation, 48 Vas. Constantinou Ave., Athens 11635, Greece.

    The secreted clusterin/apolipoprotein J (CLU) protein form is a ubiquitously expressed heterodimeric glycoprotein which is differentially regulated in many severe physiological disturbance states including cell death, ageing, cancer progression, and various neurological diseases. Despite extensive efforts CLU function remains an enigma, the main cause being the intriguingly distinct and usually opposed functions in various cell types and tissues. In the current report we investigated the effects of CLU on cellular growth and survival in three human osteosarcoma (OS) cell lines, namely KH OS, Sa OS, and U-2 OS that express very low, moderate, and high endogenous steady-state CLU amounts, respectively. We found that exposure of these established OS cell lines or primary OS cells to genotoxic stress results in CLU gene induction at distinct levels that correlate negatively to CLU endogenous amounts. Following CLU-forced overexpression by means of an artificial transgene, we found that although extracellular CLU inhibits cell death in all three OS cell lines, intracellular CLU has different effects on cellular proliferation and survival in these cell lines. Transgenic KH OS cell lines adapted to moderate intracellular CLU levels were growth-retarded and became resistant to genotoxic and oxidative stress. In contrast, transgenic Sa OS and U2 OS cell lines adapted to high intracellular CLU amounts were sensitive to genotoxic and oxidative stress. In these two cell lines, the proapoptotic CLU function could be rescued by caspase inhibition. To monitor the immediate effects of heterologous CLU overexpression prior to cell adaptation, we performed transient transfections in all three OS cell lines. We found that induction of high intracellular CLU amounts increases spontaneous apoptosis in KH OS cells and reduces DNA synthesis in all three cell lines assayed. On the basis of these novel findings we propose that although extracellular CLU as well as intracellular CLU at low/moderate levels is cytoprotective, CLU may become highly cytostatic and/or cytotoxic if it accumulates intracellularly in high amounts either by direct synthesis or by uptake from the extracellular milieu.

    Free radical biology & medicine 2005;38;4;436-49

  • Modulation of clusterin isoforms is associated with all-trans retinoic acid-induced proliferative arrest and apoptosis of intimal smooth muscle cells.

    Orlandi A, Pucci S, Ciucci A, Pichiorri F, Ferlosio A and Spagnoli LG

    Anatomic Pathology Institute, Department of Biopathology and Image Diagnostics, Tor Vergata University of Rome, Via Montpellier 1, 00133 Rome, Italy. orlandi@uniroma2.it

    Objective: Clusterin is a heterodimeric glycoprotein which is implicated in several biological processes. The nuclear (n-CLU) and cytoplasmic secreted (s-CLU) isoforms have recently been described, but their role is still unclear. The aim of this study is to investigate the expression of clusterin and its isoforms during proliferative arrest and apoptosis of vascular smooth muscle cells (SMCs).

    Clusterin expression was evaluated by immunohistochemistry and Western blotting in human arteries and rat aortas. In human diffuse myointimal thickening, clusterin was detected in cell cytoplasm and extracellular space, whereas it was practically absent in the media. In rat aortas 15 days after ballooning, intimal cells (IT cells) overexpressed s-CLU and n-CLU, the latter mainly in the inner neointima; clusterin expression decreased at 60 days. In vitro, IT cells maintained high clusterin expression and its antisense markedly reduced proliferation and increased apoptosis. Western blotting showed that all-trans retinoic acid-induced proliferative arrest and increased alpha-smooth muscle actin expression did associate to s-CLU and B-myb reduction, whereas bax-related apoptosis was associated to a shift from the s-CLU to n-CLU isoform.

    Conclusions: Clusterin overexpression characterized neointimal SMCs; s-CLU expression decreased in IT cells during all-trans retinoic acid-induced proliferative arrest and redifferentiation, whereas n-CLU overexpression was characteristic of apoptosis. Clusterin was detected in human arterial myointimal thickening and absent in the underlying media. Rat neointimal cells overexpressed clusterin and clusterin antisense oligonucleotide reduced proliferation and increased apoptosis. All-trans retinoic acid-induced proliferative arrest showed association with s-CLU reduction and n-CLU overexpression with apoptosis, supporting a different biological role of these isoforms.

    Arteriosclerosis, thrombosis, and vascular biology 2005;25;2;348-53

  • Ca2+ depletion induces nuclear clusterin, a novel effector of apoptosis in immortalized human prostate cells.

    Caccamo AE, Scaltriti M, Caporali A, D'Arca D, Corti A, Corvetta D, Sala A and Bettuzzi S

    Cell death and differentiation 2005;12;1;101-4

  • Association of apolipoprotein J gene 866C-->T polymorphism with preeclampsia and essential hypertension.

    Chen M, Yuan Z and Shan K

    Department of Obstetrics and Gynecology, Affiliated Hospital of Guiyang Medical College, Guiyang, China.

    The etiology of preeclampsia (PE) is unknown, but endothelial cell injury plays a pivotal role. Moreover, there is a significantly positive association between PE and later hypertension. Apolipoprotein J (apo J) plays an important role in stabilizing cell membranes at diverse fluid-tissue interfaces and might protect vascular endothelium against an attack by some factors in plasma such as active complement complexes. This study investigated the prevalence and possible association of apo J gene in PE and essential hypertension (EH). To screen for polymorphisms of apo J gene in a population in Guizhou, China, by employing polymerase chain reaction combined with denaturing gradient gel electrophoresis, we identified 866C-->T polymorphism in exon 5 and a rare variant 1061C-->T in exon 7. The CT genotype and T-allele frequencies in PE (CT genotype 26%; T allele 13%) and EH (CT genotype 28.57%; T allele 17.14%) were significantly lower than that in the control group (CT geno type 50%; T allele 27%). The results suggested that the 866C-->T polymorphism might be associated with PE and EH. It is plausible that apo J may play a certain role in the predisposition to PE and EH.

    Gynecologic and obstetric investigation 2005;60;3;133-8

  • Isolation and characterization of a novel gene CLUAP1 whose expression is frequently upregulated in colon cancer.

    Takahashi M, Lin YM, Nakamura Y and Furukawa Y

    1Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai Minato-ku, Tokyo 108-8639, Japan.

    To disclose mechanisms of colorectal carcinogenesis and identify novel diagnostic markers and drug targets for treatment of these tumors, we previously analysed the expression profiles of 11 colorectal cancers using a genome-wide cDNA microarray containing 23,040 genes. Among the genes commonly transactivated in the cancers, we identified a novel human gene, which we termed CLUAP1 (clusterin-associated protein 1). It encodes a nuclear protein of 413 amino acids containing a coiled-coil domain. To investigate its function, we searched for CLUAP1-interacting proteins using yeast two-hybrid system and identified nuclear Clusterin. Expression of CLUAP1 was gradually increased in the late S to G2/M phases of cell cycle and it returned to the basal level in the G0/G1 phases. Suppression of this gene by short interfering RNAs resulted in growth retardation in the transfected cells. These data provide better understanding of colorectal carcinogenesis, and inactivation of CLUAP1 may conceivably serve in the future as a novel therapeutic intervention for treatment of colon cancer.

    Oncogene 2004;23;57;9289-94

  • Protection of androgen-dependent human prostate cancer cells from oxidative stress-induced DNA damage by overexpression of clusterin and its modulation by androgen.

    Miyake H, Hara I, Gleave ME and Eto H

    Department of Urology, Hyogo Medical Center for Adults, Akashi, Japan. hideakimiyake

    Background: Recent studies reported that oxidative stress is one of the major factors associated with the progression of prostate cancer through the accumulation of DNA damage. In the present study, we investigated the effect of oxidative stress on cell injury using androgen-dependent human prostate cancer LNCaP cells overexpressing clusterin, which has been shown to play crucial roles in the acquisition of resistance to several apoptotic stimuli.

    Methods: We introduced clusterin cDNA into LNCaP cells which do not express a detectable level of clusterin expression, and generated a clusterin-overexpressing cell line (LNCaP/Cl) and a control vector only-transfected cell line (LNCaP/Co). The effects of hydrogen peroxide (H2O2) treatment on the LNCaP sublines with and without the addition of dihydrotestosterone (DHT) were analyzed using the in vitro mitogenic assay and lipid peroxidation assay, and morphological changes in the LNCaP sublines after H2O2 treatment were examined by staining with Hoechst 33258. The degrees of DNA damage induced by H2O2 into the LNCaP sublines were evaluated by the measurement of 8-hydroxy-2'-deoxyguanosine (8-OHdG) level.

    Results: H2O2-induced apoptosis in LNCaP/Cl was significantly suppressed compared with that in LNCaP/Co through the inhibition of membrane damage; however, the measurement of 8-OHdG level demonstrated that DNA damage was more intensively accumulated in LNCaP/Cl cells than LNCaP/Co cells. Furthermore, DHT suppressed the incidence of apoptotic cell death and enhanced the formation of 8-OHdG in both LNCaP/Cl and LNCaP/Co cells after H2O2 treatment in a dose-dependent manner.

    Conclusions: These findings suggest that clusterin may contribute to conferring resistance to oxidative stress-mediated cellular injury on prostate cancer cells, especially in the presence of androgen.

    The Prostate 2004;61;4;318-23

  • Clusterin-mediated apoptosis is regulated by adenomatous polyposis coli and is p21 dependent but p53 independent.

    Chen T, Turner J, McCarthy S, Scaltriti M, Bettuzzi S and Yeatman TJ

    Department of Interdisciplinary Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA.

    Clusterin is a widely expressed glycoprotein that has been paradoxically observed to have both pro- and antiapoptotic functions. Recent reports suggest this apparent dichotomy of function may be related to two different isoforms, one secreted and cytoplasmic, the other nuclear. To clarify the functional role of clusterin in regulating apoptosis, we examined its expression in human colon cancer tissues and in human colon cancer cell lines. We additionally explored its expression and activity using models of adenomatous polyposis coli (APC)- and chemotherapy-induced apoptosis. Clusterin RNA and protein levels were decreased in colon cancer tissues largely devoid of wild-type APC when compared with matched normal tissue controls, suggesting a means for invasive cancers to avoid apoptosis. Conversely, induction of apoptosis by expression of wild-type APC or by treatment with chemotherapy led to increased clusterin RNA and protein levels localizing to apoptotic nuclei. We found that transient transfection of clusterin to colon cancer cell lines directly enhanced basal and chemotherapy-induced apoptosis. Clusterin-induced apoptosis was inhibited by antisense clusterin and was found to be highly dependent on p21 but not p53 expression, yet a deficit in p21 can be subverted by clusterin transfection. Collectively, these data support the hypothesis that nuclear clusterin function is proapoptotic when induced by APC or chemotherapy in the context of p21 expression. Absent of p21, clusterin in not induced, and apoptosis is significantly inhibited. These data support a potential therapeutic role for clusterin in enhancing chemotherapy-induced apoptosis and in promoting apoptosis in cells deficient in p21.

    Cancer research 2004;64;20;7412-9

  • 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

  • Ectopic expression of clusterin/apolipoprotein J or Bcl-2 decreases the sensitivity of HaCaT cells to toxic effects of ropivacaine.

    Kontargiris E, Kolettas E, Vadalouca A, Trougakos IP, Gonos ES and Kalfakakou V

    Laboratory of Experimental Physiology, University of Ioannina Medical School, 45110 Ioannina, Greece.

    Local anesthetics inhibit cell proliferation and induce apoptosis in various cell types. Ropivacaine, a unique, novel tertiary amine-type anesthetic, was shown to inhibit the proliferation of several cell types including keratinocytes. We found that Ropivacaine could inhibit the proliferation and induce apoptosis in an immortalized human keratinocyte line, HaCaT, in a dose- and time-dependent manner and with the deprivation of serum. The dose-dependent induction of apoptosis by ropivacaine was demonstrated by DNA fragmentation analysis and the proteolytic cleavage of a caspase-3 substrate-poly (ADP-ribose) polymerase (PARP). In addition, ropivacaine downregulated the expression of clusterin/ apoliporotein J, a protein with anti-apoptotic properties, in a dose-dependent manner, which well correlated with the induction of apoptosis of HaCaT cells. To investigate the role of clusterin/apoliporotein J in ropivacaine-induced apoptosis, HaCaT cells overexpressing clusterin/apoliporotein J were generated and compared to cells expressing the well established anti-apoptotic Bcl-2 protein. Ectopic overexpression of the secreted form of clusterin/apoliporotein J or Bcl-2 decreased the sensitivity of HaCaT cells to toxic effects of ropivacaine as demonstrated by DNA fragmentation, the proteolytic cleavage of PARP and by a reduction in procaspase-3 expression. Furthermore, the downregulation of endogenous clusterin/apolipoprotein J levels by ropivacaine suggested that this might be one mechanism by which ropivacaine induced cell death in HaCaT cells. In conclusion, the ability of ropivacaine to induce antiproliferative responses and to suppress the expression of the anti-apoptotic protein clusterin/apolipoprotein J, combined with previously reported anti-inflammatory activity and analgesic property of the drug, suggests that ropivacaine may have potential utility in the local treatment of tumors.

    Cell research 2004;14;5;415-22

  • Sequence comparison of human and mouse genes reveals a homologous block structure in the promoter regions.

    Suzuki Y, Yamashita R, Shirota M, Sakakibara Y, Chiba J, Mizushima-Sugano J, Nakai K and Sugano S

    Human Genome Center, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, 108-8639, Japan. ysuzuki@ims.u-tokyo.ac.jp

    Comparative sequence analysis was carried out for the regions adjacent to experimentally validated transcriptional start sites (TSSs), using 3324 pairs of human and mouse genes. We aligned the upstream putative promoter sequences over the 1-kb proximal regions and found that the sequence conservation could not be further extended at, on average, 510 bp upstream positions of the TSSs. This discontinuous manner of the sequence conservation revealed a "block" structure in about one-third of the putative promoter regions. Consistently, we also observed that G+C content and CpG frequency were significantly different inside and outside the blocks. Within the blocks, the sequence identity was uniformly 65% regardless of their length. About 90% of the previously characterized transcription factor binding sites were located within those blocks. In 46% of the blocks, the 5' ends were bounded by interspersed repetitive elements, some of which may have nucleated the genomic rearrangements. The length of the blocks was shortest in the promoters of genes encoding transcription factors and of genes whose expression patterns are brain specific, which suggests that the evolutional diversifications in the transcriptional modulations should be the most marked in these populations of genes.

    Genome research 2004;14;9;1711-8

  • Clusterin expression distinguishes follicular dendritic cell tumors from other dendritic cell neoplasms: report of a novel follicular dendritic cell marker and clinicopathologic data on 12 additional follicular dendritic cell tumors and 6 additional interdigitating dendritic cell tumors.

    Grogg KL, Lae ME, Kurtin PJ and Macon WR

    Division of Anatomic Pathology, Department of Laboratory Medicine and Pathology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA.

    While tumors of dendritic cell lineage may have overlapping histomorphologic features, most but not all cases can be classified using an immunohistochemical panel, including CD21, CD23, CD35, CD1a, and S-100. Based on observations that clusterin is expressed in benign follicular dendritic cells, clusterin expression in 32 dendritic cell tumors was evaluated. Diffuse strong staining for clusterin was seen in 12 of 12 follicular dendritic cell tumors. Two of these cases were negative for traditional markers (CD21, CD23, CD35); they were classified based on characteristic ultrastructural features. Three of 6 interdigitating dendritic cell tumors were negative for clusterin and 3 showed focal weak positivity. Clusterin staining in Langerhans cell histiocytosis ranged from negative (6 of 14) to weak/moderate (8 of 14). Follicular dendritic cell tumors behaved as benign tumors or low-grade sarcomas. Interdigitating dendritic cell tumors demonstrated a widely variable behavior, ranging from benign to rapidly fatal disease. Based on this initial study, strong clusterin staining supports a diagnosis of follicular dendritic cell tumor. Thus, staining for clusterin is useful in classification of dendritic cell tumors, particularly when the more common markers of follicular dendritic cells are not expressed.

    The American journal of surgical pathology 2004;28;8;988-98

  • Transcriptional down-regulation of the platelet ADP receptor P2Y(12) and clusterin in patients with systemic lupus erythematosus.

    Wang L, Erling P, Bengtsson AA, Truedsson L, Sturfelt G and Erlinge D

    Department of Cardiology, Lund University Hospital, S-221 85 Lund, Sweden.

    Cardiovascular complications are common in systemic lupus erythematosus (SLE) and myocardial infarctions are the leading cause of increased mortality. The ADP receptor P2Y(12) plays a central role in platelet activation and the P2Y(12) blocker clopidogrel reduces the incidence of cardiovascular events. Clusterin, a complement inhibitory protein suggested to be involved in the pathogenesis of SLE, has been found recently in a microarray study to be expressed at very high levels in platelets. Using a new protocol for mRNA quantification in platelets we set out to study if gene expression is altered in SLE patients compared with a healthy control group. Quantitative assay based on real-time PCR was used to measure mRNA expression, Western blot for P2 receptor protein expression and PFA-100 for platelet aggregation. The P2Y(12) receptor expression was decreased in SLE compared to the controls (P < 0.05), while expression of P2Y(1) and P2X(1) were unaltered. These findings were consistent at the protein level. The clusterin mRNA expression was very high. However, SLE patients had significantly lower levels than controls (P < 0.05). Platelet aggregation was similar in both groups. It may be suggested that a decreased level of P2Y(12) receptors could represent a protective response in SLE against thrombotic complications. Lowered clusterin levels could be involved in the pathogenesis of SLE due to decreased protective effects. These findings could help to achieve a better understanding of the platelet function in SLE and serve as a guide for further research and drug use.

    Journal of thrombosis and haemostasis : JTH 2004;2;8;1436-42

  • Involvement of clusterin in 15-deoxy-delta12,14-prostaglandin J2-induced vascular smooth muscle cell differentiation.

    Miwa Y, Takahashi-Yanaga F, Morimoto S and Sasaguri T

    Department of Clinical Pharmacology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan. ymiwa@clipharm.med.kyushu-u.ac.jp

    To establish an in vitro model of vascular smooth muscle cell (VSMC) differentiation, we examined the effect of 15-deoxy-delta12,14-prostaglandin J(2) (15d-PGJ(2)) on the expression of VSMC differentiation markers. After the addition of 15d-PGJ(2) to confluent human umbilical artery smooth muscle cells synchronized in the G(0) phase, cells showed a "hill and valley" appearance and thereafter aggregated and formed macroscopic nodules. Cells forming nodules expressed high levels of SM2, the most specific VSMC differentiation marker, comparable to medial VSMCs in vivo. 15d-PGJ(2) significantly increased the mRNA and protein expression levels of clusterin, a secreted glycoprotein reported to induce nodule formation and differentiation of VSMCs. Moreover, addition of an anti-clusterin antibody completely inhibited the nodule formation induced by 15d-PGJ(2) and induced apoptosis. Our results suggested that clusterin is involved in 15d-PGJ(2)-induced nodule formation and cell differentiation in VSMCs.

    Biochemical and biophysical research communications 2004;319;1;163-8

  • Functional analysis of clusterin/apolipoprotein J in cellular death induced by severe genotoxic stress.

    Trougakos IP and Gonos ES

    Laboratory of Molecular and Cellular Aging, Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens 11635, Greece.

    Clusterin/apolipoprotein J (CLU) is a secreted heterodimeric glycoprotein that is reportedly upregulated during tumorigenesis, as well as during cell injury or death. Despite extensive efforts, CLU function during cellular death remains largely elusive. We are using as a model system to study CLU function three human osteosarcoma (OS) cell lines, namely, Sa OS, KH OS, and U-2 OS cells, induced to die after exposure to severe genotoxic stress mediated by the chemotherapeutic drug doxorubicin (DXR). We initially applied small interfering RNA (siRNA)-mediated specific knockdown of the CLU protein in OS cells. In all three cell lines, CLU knockdown resulted in increased sensitization to DXR-induced apoptosis. Supportively, moderate levels of forced transgene-mediated CLU stable overexpression in KH OS cells could rescue them from DXR-mediated apoptosis. In contrast, stable overexpression of high CLU levels in Sa OS and U-2 OS cells augmented apoptosis induced by cell exposure to severe DXR-mediated genotoxic stress. In summary, our data provide evidence that, although CLU is essential for cellular homeostasis, it may become highly cytotoxic in certain cellular contexts when it accumulates in high amounts intracellularly either by direct synthesis or by uptake from the extracellular milieu.

    Annals of the New York Academy of Sciences 2004;1019;206-10

  • Time-controlled transcardiac perfusion cross-linking for the study of protein interactions in complex tissues.

    Schmitt-Ulms G, Hansen K, Liu J, Cowdrey C, Yang J, DeArmond SJ, Cohen FE, Prusiner SB and Baldwin MA

    Institute for Neurodegenerative Disease, San Francisco, California 94143, USA. g.schmittulms@utoronto.ca

    Because of their sensitivity to solubilizing detergents, membrane protein assemblies are difficult to study. We describe a protocol that covalently conserves protein interactions through time-controlled transcardiac perfusion cross-linking (tcTPC) before disruption of tissue integrity. To validate tcTPC for identifying protein-protein interactions, we established that tcTPC allowed stringent immunoaffinity purification of the gamma-secretase complex in high salt concentrations and detergents and was compatible with mass spectrometric identification of cross-linked aph-1, presenilin-1 and nicastrin. We then applied tcTPC to identify more than 20 proteins residing in the vicinity of the cellular prion protein (PrPC), suggesting that PrP is embedded in specialized membrane regions with a subset of molecules that, like PrP, use a glycosylphosphatidylinositol anchor for membrane attachment. Many of these proteins have been implicated in cell adhesion/neuritic outgrowth, and harbor immunoglobulin C2 and fibronectin type III-like motifs.

    Funded by: NCRR NIH HHS: NCRR RR01614; NIA NIH HHS: AG010770, AG02132

    Nature biotechnology 2004;22;6;724-31

  • Alterations in expression, proteolysis and intracellular localizations of clusterin in esophageal squamous cell carcinoma.

    He HZ, Song ZM, Wang K, Teng LH, Liu F, Mao YS, Lu N, Zhang SZ, Wu M and Zhao XH

    National Laboratory of Molecular Oncology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China.

    Aim: To investigate biogenesis and intracellular localizations of clusterin to elucidate the potential molecular mechanisms implicated in tumorigenesis of esophageal mucosa.

    Methods: Semi-quantitative RT-PCR for multi-region alteration analysis, Western blot for different transcriptional forms and immunohistochemical staining for intracellular localizations of clusterin were carried out in both tissues and cell lines of ESCC.

    Results: The N-terminal deletions of the clusterin gene and the appearance of a 50-53 ku nuclear clusterin, an uncleaved, nonglycosylated, and disulfide-linked isoform, were the major alterations in cancer cells of esophagus. Naturally the 40 ku clusterin was located in the connective tissue of the lamina propria of epithelial mucosa and right under the basal membrane of epithelia, but it was disappeared in stromal mucosa of esophagus and the pre-matured clusterin was found positive in cancerous epithelia.

    Conclusion: The N-terminal deletion of clusterin may be essential for its alterations of biogenesis in ESCC.

    World journal of gastroenterology 2004;10;10;1387-91

  • The human plasma proteome: a nonredundant list developed by combination of four separate sources.

    Anderson NL, Polanski M, Pieper R, Gatlin T, Tirumalai RS, Conrads TP, Veenstra TD, Adkins JN, Pounds JG, Fagan R and Lobley A

    The Plasma Proteome Institute, Washington DC 20009-3450, USA. leighanderson@plasmaproteome.org

    We have merged four different views of the human plasma proteome, based on different methodologies, into a single nonredundant list of 1175 distinct gene products. The methodologies used were 1) literature search for proteins reported to occur in plasma or serum; 2) multidimensional chromatography of proteins followed by two-dimensional electrophoresis and mass spectroscopy (MS) identification of resolved proteins; 3) tryptic digestion and multidimensional chromatography of peptides followed by MS identification; and 4) tryptic digestion and multidimensional chromatography of peptides from low-molecular-mass plasma components followed by MS identification. Of 1,175 nonredundant gene products, 195 were included in more than one of the four input datasets. Only 46 appeared in all four. Predictions of signal sequence and transmembrane domain occurrence, as well as Genome Ontology annotation assignments, allowed characterization of the nonredundant list and comparison of the data sources. The "nonproteomic" literature (468 input proteins) is strongly biased toward signal sequence-containing extracellular proteins, while the three proteomics methods showed a much higher representation of cellular proteins, including nuclear, cytoplasmic, and kinesin complex proteins. Cytokines and protein hormones were almost completely absent from the proteomics data (presumably due to low abundance), while categories like DNA-binding proteins were almost entirely absent from the literature data (perhaps unexpected and therefore not sought). Most major categories of proteins in the human proteome are represented in plasma, with the distribution at successively deeper layers shifting from mostly extracellular to a distribution more like the whole (primarily cellular) proteome. The resulting nonredundant list confirms the presence of a number of interesting candidate marker proteins in plasma and serum.

    Molecular & cellular proteomics : MCP 2004;3;4;311-26

  • Characterization of the proteins released from activated platelets leads to localization of novel platelet proteins in human atherosclerotic lesions.

    Coppinger JA, Cagney G, Toomey S, Kislinger T, Belton O, McRedmond JP, Cahill DJ, Emili A, Fitzgerald DJ and Maguire PB

    Department of Clinical Pharmacology, Royal College of Surgeons in Ireland, 123 St Stephen's Green, Dublin 2, Ireland.

    Proteins secreted by activated platelets can adhere to the vessel wall and promote the development of atherosclerosis and thrombosis. Despite this biologic significance, however, the complement of proteins comprising the platelet releasate is largely unknown. Using a proteomics approach, we have identified more than 300 proteins released by human platelets following thrombin activation. Many of the proteins identified were not previously attributed to platelets, including secretogranin III, a potential monocyte chemoattractant precursor; cyclophilin A, a vascular smooth muscle cell growth factor; calumenin, an inhibitor of the vitamin K epoxide reductase-warfarin interaction, as well as proteins of unknown function that map to expressed sequence tags. Secretogranin III, cyclophilin A, and calumenin were confirmed to localize in platelets and to be released upon activation. Furthermore, while absent in normal vasculature, they were identified in human atherosclerotic lesions. Therefore, these and other proteins released from platelets may contribute to atherosclerosis and to the thrombosis that complicates the disease. Moreover, as soluble extracellular proteins, they may prove suitable as novel therapeutic targets.

    Blood 2004;103;6;2096-104

  • Screening for N-glycosylated proteins by liquid chromatography mass spectrometry.

    Bunkenborg J, Pilch BJ, Podtelejnikov AV and Wiśniewski JR

    MDS Proteomics Odense, Denmark.

    In the last few years mass spectrometry has become the method of choice for characterization of post-translationally modified proteins. Whereas most protein chemical modifications are binary in the sense that only one change can be associated with a given residue, many different oligosaccharides can be attached to a glycosylation site residue. The detailed characterization of glycoproteins in complex biological samples is extremely challenging. However, information on N-glycosylation can be gained at an intermediary level. Here we demonstrate a procedure for mapping N-glycosylation sites in complex mixtures by reducing sample complexity and enriching glycoprotein content. Glycosylated proteins are selected by an initial lectin chromatography step and digested with endoproteinase Lys-C. Glycosylated peptides are then selected from the digest mixture by a second lectin chromatography step. The glycan components are removed with N-glycosidase F and the peptides digested with trypsin before analysis by on-line reversed-phase liquid chromatography mass spectrometry. Using two different lectins, concanavalin A and wheat germ agglutinin, this procedure was applied to human serum and a total of 86 N-glycosylation sites in 77 proteins were identified.

    Proteomics 2004;4;2;454-65

  • Clusterin (SGP-2, ApoJ) expression is downregulated in low- and high-grade human prostate cancer.

    Scaltriti M, Brausi M, Amorosi A, Caporali A, D'Arca D, Astancolle S, Corti A and Bettuzzi S

    Dipartimento di Scienze Biomediche, Università di Modena e Reggio Emilia, Modena, Italy.

    Clusterin is overexpressed during tissue and cell involution and downregulated in proliferating cells. Its role in cell survival, cell death and neoplastic transformation remains debated. We studied the expression and distribution of clusterin mRNA and protein in human prostate carcinoma (CaP) specimens of different degrees of malignancy. Fresh CaP specimens were obtained from 25 patients subjected to long-term androgen ablation before surgery. Clusterin expression was studied by Northern and Western analysis, in situ hybridization and immunohistochemistry, in comparison with Gas1 and histone H3 mRNA (markers of cell quiescence and S phase of the cell cycle, respectively). Clusterin is downregulated in CaP in comparison with matched benign controls. In low-grade CaP, clusterin colocalized with Gas1 to the stromal compartment, and in some glands, the basal lamina was heavily stained. In high-grade CaP clusterin stained the remnants of stromal matrix while histone H3 localized to cancer cells, which were very rarely clusterin positive. High clusterin expression was found in the branches of a nerve infiltrated by tumor. The periglandular clusterin expression found in low-grade CaP could result from induction of quiescence and/or apoptosis of prostatic fibroblasts lining those glands in which tumor invasion is at an initial stage, involving basal lamina. In advanced CaP, the staining of the remnants of the extracellular matrix suggests a role for clusterin in the process of dismantling the stromal organization caused by cancer progression.

    International journal of cancer 2004;108;1;23-30

  • Complete sequencing and characterization of 21,243 full-length human cDNAs.

    Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, Wakamatsu A, Hayashi K, Sato H, Nagai K, Kimura K, Makita H, Sekine M, Obayashi M, Nishi T, Shibahara T, Tanaka T, Ishii S, Yamamoto J, Saito K, Kawai Y, Isono Y, Nakamura Y, Nagahari K, Murakami K, Yasuda T, Iwayanagi T, Wagatsuma M, Shiratori A, Sudo H, Hosoiri T, Kaku Y, Kodaira H, Kondo H, Sugawara M, Takahashi M, Kanda K, Yokoi T, Furuya T, Kikkawa E, Omura Y, Abe K, Kamihara K, Katsuta N, Sato K, Tanikawa M, Yamazaki M, Ninomiya K, Ishibashi T, Yamashita H, Murakawa K, Fujimori K, Tanai H, Kimata M, Watanabe M, Hiraoka S, Chiba Y, Ishida S, Ono Y, Takiguchi S, Watanabe S, Yosida M, Hotuta T, Kusano J, Kanehori K, Takahashi-Fujii A, Hara H, Tanase TO, Nomura Y, Togiya S, Komai F, Hara R, Takeuchi K, Arita M, Imose N, Musashino K, Yuuki H, Oshima A, Sasaki N, Aotsuka S, Yoshikawa Y, Matsunawa H, Ichihara T, Shiohata N, Sano S, Moriya S, Momiyama H, Satoh N, Takami S, Terashima Y, Suzuki O, Nakagawa S, Senoh A, Mizoguchi H, Goto Y, Shimizu F, Wakebe H, Hishigaki H, Watanabe T, Sugiyama A, Takemoto M, Kawakami B, Yamazaki M, Watanabe K, Kumagai A, Itakura S, Fukuzumi Y, Fujimori Y, Komiyama M, Tashiro H, Tanigami A, Fujiwara T, Ono T, Yamada K, Fujii Y, Ozaki K, Hirao M, Ohmori Y, Kawabata A, Hikiji T, Kobatake N, Inagaki H, Ikema Y, Okamoto S, Okitani R, Kawakami T, Noguchi S, Itoh T, Shigeta K, Senba T, Matsumura K, Nakajima Y, Mizuno T, Morinaga M, Sasaki M, Togashi T, Oyama M, Hata H, Watanabe M, Komatsu T, Mizushima-Sugano J, Satoh T, Shirai Y, Takahashi Y, Nakagawa K, Okumura K, Nagase T, Nomura N, Kikuchi H, Masuho Y, Yamashita R, Nakai K, Yada T, Nakamura Y, Ohara O, Isogai T and Sugano S

    Helix Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan.

    As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

    Nature genetics 2004;36;1;40-5

  • Nuclear translocation of a clusterin isoform is associated with induction of anoikis in SV40-immortalized human prostate epithelial cells.

    Caccamo AE, Scaltriti M, Caporali A, D'Arca D, Scorcioni F, Candiano G, Mangiola M and Bettuzzi S

    Department of Medicina Sperimentale, University of Parma, 43100 Parma, Italy.

    Clusterin gene expression is potently induced in experimental models in which apoptosis is activated, such as rat prostate involution following castration. Nevertheless, its precise physiological role has not yet been established, and both anti-apoptotic and pro-apoptotic functions have been suggested for this gene. Clusterin expression level depends on cell proliferation state, and we recently showed that its over-expression inhibited cell cycle progression of SV40-immortalized human prostate epithelial cells PNT2 and PNT1a. Here we studied clusterin expression in PNT1a cells subjected to serum-starvation with the aim of defining clusterin early molecular changes following apoptosis induction. Under serum-starvation conditions, decreased growth rate, slow rounding-up of cells, cell detachment, and formation of apoptotic bodies indicative of anoikis (detachment-induced apoptosis) were preceded by significant downregulation of 70 kDa clusterin precursor and upregulation of 45-40 kDa isoforms. On the 8th day of serum-free culturing, only the higher molecular weight protein-band of about 45 kDa was clearly induced and accumulated in detached cells and apoptotic bodies in which PARP was activated. Anoikis was preceded by induction and transloction of a 45-kDa clusterin isoform to the nucleus. Thus, nuclear targeting of a specific 45-kDa isoform of clusterin appeared to be an early and specific molecular signal triggering anoikis-death. Considering also that clusterin is downregulated during prostate cancer onset and progression, and that its upregulation has inhibited DNA synthesis and cell cycle progression of immortalized human prostate epithelial cells, we suggest that clusterin might be a new anti-oncogene in the prostate.

    Annals of the New York Academy of Sciences 2003;1010;514-9

  • Essential requirement of apolipoprotein J (clusterin) signaling for IkappaB expression and regulation of NF-kappaB activity.

    Santilli G, Aronow BJ and Sala A

    Molecular Haematology and Cancer Biology Unit, Institute of Child Health, University College London, London WC1N, United Kingdom.

    Apolipoprotein J/clusterin is an enigmatic protein highly regulated in inflammation, apoptosis, and cancer. Despite extensive studies, its biological function has remained obscure. Here we show that apolipoprotein J inhibits neuroblastoma cell invasion. Since this function can be regulated by NF-kappaB, we explored the possibility that apolipoprotein J might interfere with NF-kappaB signaling. Ectopic apolipoprotein J expression strongly inhibited NF-kappaB activity in human neuroblastoma cells and murine embryonic fibroblasts by stabilizing inhibitors of NF-kappaB (IkappaBs). Steady state levels of IkappaB proteins are drastically reduced in mouse embryo fibroblasts after disruption of the apolipoprotein J gene. Absence of apolipoprotein J causes reduction of IkappaB stability, a tumor necrosis factor-dependent increase in NF-kappaB activity, increased transcription of the NF-kappaB target gene c-IAP and down-modulation of p53 protein. These results suggest that an unexpected physiological role of apolipoprotein J is to inhibit NF-kappaB signaling through stabilization of IkappaBs and that this activity may result in suppression of tumor cell motility.

    The Journal of biological chemistry 2003;278;40;38214-9

  • Clusterin, an abundant serum factor, is a possible negative regulator of MT6-MMP/MMP-25 produced by neutrophils.

    Matsuda A, Itoh Y, Koshikawa N, Akizawa T, Yana I and Seiki M

    Division of Cancer Cell Research, Institute of Medical Science, the University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.

    MT6-MMP/MMP-25 is the latest member of the membrane-type matrix metalloproteinase (MT-MMP) subgroup in the MMP family and is expressed in neutrophils and some brain tumors. The proteolytic activity of MT6-MMP has been studied using recombinant catalytic fragments and shown to degrade several components of the extracellular matrix. However, the activity is possibly modulated further by the C-terminal hemopexin-like domain, because some MMPs are known to interact with other proteins through this domain. To explore the possible function of this domain, we purified a recombinant MT6-MMP with the hemopexin-like domain as a soluble form using a Madin-Darby canine kidney cell line as a producer. Mature and soluble MT6-MMP processed at the furin motif was purified as a 45-kDa protein together with a 46-kDa protein having a single cleavage in the hemopexin-like domain. Interestingly, 73- and 70-kDa proteins were co-purified with the soluble MT6-MMP by forming stable complexes. They were identified as clusterin, a major component of serum, by N-terminal amino acid sequencing. MT1-MMP that also has a hemopexin-like domain did not form a complex with clusterin. MT6-MMP forming a complex with clusterin was detected in human neutrophils as well. The enzyme activity of the soluble MT6-MMP was inactive in the clusterin complex. Purified clusterin was inhibitory against the activity of soluble MT6-MMP. On the other hand, it had no effect on the activities of MMP-2 and soluble MT1-MMP. Because clusterin is an abundant protein in the body fluid in tissues, it may act as a negative regulator of MT6-MMP in vivo.

    The Journal of biological chemistry 2003;278;38;36350-7

  • A model for modulation of leptin activity by association with clusterin.

    Bajari TM, Strasser V, Nimpf J and Schneider WJ

    Institute of Medical Biochemistry, Department of Molecular Genetics, Dr. Bohr Gasse 9/2, A-1030 Vienna, Austria. wjs@mol.univie.ac.at

    Transport, biological action, and clearance of leptin are subject to modulation by plasma components responsible for the formation of the so-called "bound" fraction of serum leptin. Candidates for modulators have been identified previously, but mechanisms for their action, and thus their physiological roles, have remained unclear. Here we have obtained evidence for a role of serum-borne clusterin in leptin biology and have delineated a possible mechanism for its action. We demonstrate complex formation between clusterin and leptin by several approaches and show that the binary complex retains the ability to transduce the leptin signal via binding to the leptin receptor and activation of the Janus kinase/signal transducer and activator of transcription pathway. The interaction of leptin with clusterin does not require additional serum components. Furthermore, and importantly for modulation of the bioactivity of leptin, uptake of leptin present in the complex can be mediated by members of the low density lipoprotein (LDL) receptor family, i.e., apolipoprotein receptor type-2 and the very LDL receptor, which here are shown to efficiently endocytose both free and leptin-associated clusterin. Thus, bioavailability of leptin at a given tissue site may be determined by the levels of clusterin and/or by the relative distribution of certain relatives of the LDL receptor vis-à-vis active leptin receptors.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2003;17;11;1505-7

  • Alterations in the post-translational modification and intracellular trafficking of clusterin in MCF-7 cells during apoptosis.

    O'Sullivan J, Whyte L, Drake J and Tenniswood M

    Department of Biological Sciences, University of Notre Dame, Notre Dame IN 46556, USA.

    Clusterin is a heterodimeric, disulfide-linked 70-80 kDa glycoprotein that is induced during regression of most, if not all, hormone-dependent epithelial tissues. These studies describe the biogenesis and intracellular trafficking of clusterin in MCF-7 cells before and after the initiation of apoptosis with antiestrogens and TNF alpha. Under physiological conditions, clusterin is modified in the endoplasmic reticulum (ER), and proteolytically cleaved in the Golgi to generate discrete alpha and beta chains prior to secretion. Treatment with TNFalpha or the antiestrogen, ICI 182,780, induces apoptosis in MCF-7 cells and leads to substantial changes in the activity of Golgi-resident enzymes, significantly altering the biogenesis of clusterin. This leads to the appearance of a 50-53 kDa uncleaved, nonglycosylated, disulfide-linked isoform of clusterin that accumulates in the nucleus. While clusterin contains a cryptic SV-40-like nuclear localization signal, mutation of this sequence does not affect the nuclear accumulation of the disulfide-linked nuclear isoform. Confocal microscopy demonstrates that the nuclear accumulation of clusterin is coincident with DNA fragmentation. These data suggest that, at least in secretory epithelial cells, retrograde transport from the Golgi to the ER of a nonglycosylated, uncleaved isoform and the subsequent translocation of clusterin to the nucleus occur in dying cells.

    Cell death and differentiation 2003;10;8;914-27

  • Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry.

    Zhang H, Li XJ, Martin DB and Aebersold R

    Institute for Systems Biology, 1441 N 34th Street, Seattle, Washington 98103-8904, USA.

    Quantitative proteome profiling using stable isotope protein tagging and automated tandem mass spectrometry (MS/MS) is an emerging technology with great potential for the functional analysis of biological systems and for the detection of clinical diagnostic or prognostic marker proteins. Owing to the enormous complexity of proteomes, their comprehensive analysis is an as-yet-unresolved technical challenge. However, biologically or clinically important information can be obtained if specific, information-rich protein classes, or sub-proteomes, are isolated and analyzed. Glycosylation is the most common post-translational modification. Here we describe a method for the selective isolation, identification and quantification of peptides that contain N-linked carbohydrates. It is based on the conjugation of glycoproteins to a solid support using hydrazide chemistry, stable isotope labeling of glycopeptides and the specific release of formerly N-linked glycosylated peptides via peptide- N-glycosidase F (PNGase F). The recovered peptides are then identified and quantified by MS/MS. We applied the approach to the analysis of plasma membrane proteins and proteins contained in human blood serum.

    Funded by: NCI NIH HHS: K08CA97282-01, R33 CA93302

    Nature biotechnology 2003;21;6;660-6

  • Loss of clusterin both in serum and tissue correlates with the tumorigenesis of esophageal squamous cell carcinoma via proteomics approaches.

    Zhang LY, Ying WT, Mao YS, He HZ, Liu Y, Wang HX, Liu F, Wang K, Zhang DC, Wang Y, Wu M, Qian XH and Zhao XH

    National Laboratory of Molecular Oncology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, Beijing, China.

    Aim: To identify the differentially secreted proteins or polypeptides associated with tumorigenesis of esophageal squamous cell carcinoma (ESCC) from serum and to find potential tumor secreted biomarkers.

    Methods: Proteins from human ESCC tissue and its matched adjacent normal tissue; pre-surgery and post-surgery serum; and pre-surgery and normal control serum were separated by two-dimensional electrophoresis (2-DE) to identify differentially expressed proteins. The silver-stained 2-DE were scanned with digital ImageScanner and analyzed with ImageMaster 2D Elite 3.10 software. A cluster of protein spots differentially expressed were selected and identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). One of the differentially expressed proteins, clusterin, was down-regulated in cancer tissue and pre-surgery serum, but it was reversed in post-surgery serum. The results were confirmed by semi-quantitative reverse-transcription (RT)-PCR and western blot.

    Results: Comparisons of the protein spots identified on the 2-DE maps from human matched sera showed that some proteins were differentially expressed, with most of them showing no differences in composition, shape or density. Being analyzed by MALDI-TOF-MS and database searching, clusterin was differentially expressed and down-regulated in both cancer tissue and pre-surgery serum compared with their counterparts. The results were also validated by RT-PCR and western blot.

    Conclusion: The differentially expressed clusterin may play a key role during tumorigenesis of ESCC. The 2DE-MS based proteomic approach is one of the powerful tools for discovery of secreted markers from peripheral.

    World journal of gastroenterology 2003;9;4;650-4

  • Synthesis and functional analyses of nuclear clusterin, a cell death protein.

    Leskov KS, Klokov DY, Li J, Kinsella TJ and Boothman DA

    Department of Radiation Oncology, Laboratory of Molecular Stress Responses, Ireland Comprehensive Cancer Center and Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4942, USA.

    Nuclear clusterin (nCLU) is an ionizing radiation (IR)-inducible protein that binds Ku70, and triggers apoptosis when overexpressed in MCF-7 cells. We demonstrate that endogenous nCLU synthesis is a product of alternative splicing. Reverse transcriptase-PCR analyses revealed that exon II, containing the first AUG and encoding the endoplasmic reticulum-targeting peptide, was omitted. Exons I and III are spliced together placing a downstream AUG in exon III as the first available translation start site. This shorter mRNA produces the 49-kDa precursor nCLU protein. Ku70 binding activity was localized to the C-terminal coiled-coil domain of nCLU. Leucine residues 357, 358, and 361 of nCLU were necessary for Ku70-nCLU interaction. The N- and C-terminal coiled-coil domains of nCLU interacted with each other, suggesting that the protein could dimerize or fold. Mutation analyses indicate that the C-terminal NLS was functional in nCLU with the same contribution from N-terminal NLS. The C-terminal coiled-coil domain of nCLU was the minimal region required for Ku binding and apoptosis. MCF-7 cells show nuclear as well as cytoplasmic expression of GFP-nCLU in apoptotic cells. Cytosolic aggregation of GFP-nCLU was found in viable cells. These results indicate that an inactive precursor of nCLU exists in the cytoplasm of non-irradiated MCF-7 cells, translocates into the nucleus following IR, and induces apoptosis.

    Funded by: NCI NIH HHS: CA78530, CA84578, P30 CA43703, R01 CA078530

    The Journal of biological chemistry 2003;278;13;11590-600

  • Isolation of differentially expressed genes in human heart tissues.

    Sun G, Yuen Chan S, Yuan Y, Wang Chan K, Qiu G, Sun K and Ping Leung M

    Department of Medical Genetics, Institute of Basic Medicine, China Medical University, Shenyang, PR China. guifengs@uci.edu

    We applied RNA arbitrarily primed-PCR (RAP-PCR) to screen the genes differentially expressed between common congenital heart defects (CHD) [atrial septal defect, ventricular septal defect, Tetrology of Fallot (TOF)] and normal human heart samples. Three of these differentially amplified fragments matched cDNA sequences coding for proteins of unknown function in humans: hCALO (human homologue of calossin), NP79 (coding for a nuclear protein of 79KD) and SUN2 (Sad-1 unc-84 domain protein 2). The other four fragments were from known human genes: apolipoprotein J, titin, dystrophin and protein kinase C-delta. Northern blot analysis confirmed that all of these genes are expressed in the human heart. The results of RAP-PCR were reconfirmed by quantitative RT-PCR in TOF and control heart samples. Both techniques showed the levels of expression of hCALO, NP79 and SUN2 to be comparable in TOF and control samples and the level of expression of dystrophin and titin, both coding for cytoskeletal proteins, to be significantly upregulated in TOF samples. In summary, we have shown that the RAP-PCR technique is useful in the identification of differentially expressed gene from biopsy samples of human CHD tissues. In this manner, we have identified three novel genes implicated in the normal function of the human heart and two known genes upregulated in TOF samples.

    Biochimica et biophysica acta 2002;1588;3;241-6

  • Depletion of clusterin in renal diseases causing nephrotic syndrome.

    Ghiggeri GM, Bruschi M, Candiano G, Rastaldi MP, Scolari F, Passerini P, Musante L, Pertica N, Caridi G, Ferrario F, Perfumo F and Ponticelli C

    Laboratory on Pathophysiology of Uremia and Unit of Nephrology, Istituto Giannina Gaslini, Genova, Italy. labnefro@ospedale-gaslini.ge.it

    Background: Clusterin is a lipoprotein that has anti-complement effects in membranous nephropathy (MN). In focal segmental glomerulosclerosis (FSGS), it inhibits permeability plasma factor activity and could influence proteinuria. Moreover, with aging, knockout mice for clusterin develop a progressive glomerulopathy with sclerosis.

    Methods: Since little is known about clusterin metabolism in humans, we determined clusterin levels and composition in the sera and urine of 23 patients with MN, 25 with FSGS and 23 with steroid-responsive nephrotic syndrome (NS). Renal localization was evaluated by immunofluorescence and morphometry.

    Results: Serum clusterin was markedly reduced in active MN, in FSGS and in children with NS compared to controls; after stable remission of proteinuria, nearly normal levels were restored. Among various biochemical variables, serum clusterin was inversely correlated with hypercholesterolemia. Urinary clusterin, representing a 0.01 fraction of serum, was higher in the urine from normal subjects and FSGS patients in remission with proteinuric MN, FSGS and idiopathic NS; clusterin was inversely correlated with proteinuria. In all cases, urinary and serum clusterin was composed of the same 80 kD isoforms. Finally, a decrease in focal segmental or global clusterin staining was found in FSGS glomeruli, especially in areas of sclerosis. Instead, in MN an overall increment of staining was observed that ranged from mild/focal to very intense/diffuse.

    Conclusions: The overall pool of clusterin is reduced in glomerular diseases causing nephrotic syndrome, with hypercholesterolemia appearing as the unifying feature. Depletion of clusterin should negatively affect the clinical outcome in nephrotic patients and efforts should be aimed at normalizing clusterin overall pool.

    Kidney international 2002;62;6;2184-94

  • Clusterin/apolipoprotein J in human aging and cancer.

    Trougakos IP and Gonos ES

    Laboratory of Molecular & Cellular Aging, Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, 48 Vas Constantinou Avenue, Athens 11635, Greece.

    Clusterin/Apolipoprotein J (ApoJ) is a heterodimeric highly conserved secreted glycoprotein being expressed in a wide variety of tissues and found in all human fluids. Despite being cloned since 1989, no genuine function has been attributed to ApoJ so far. The protein has been reportedly implicated in several diverse physiological processes such as sperm maturation, lipid transportation, complement inhibition, tissue remodeling, membrane recycling, cell-cell and cell-substratum interactions, stabilization of stressed proteins in a folding-competent state and promotion or inhibition of apoptosis. ApoJ gene is differentially regulated by cytokines, growth factors and stress-inducing agents, while another defining prominent and intriguing ApoJ feature is its upregulation in many severe physiological disturbances states and in several neurodegenerative conditions mostly related to advanced aging. Moreover, ApoJ accumulates during the viable growth arrested cellular state of senescence, that is thought to contribute to aging and to tumorigenesis suppression; paradoxically ApoJ is also upregulated in several cases of in vivo cancer progression and tumor formation. This review focuses on the reported data related to ApoJ cell-type and signal specific regulation, function and site of action in normal and cancer cells. We discuss the role of ApoJ during cellular senescence and tumorigenesis, especially under the light of the recently demonstrated various ApoJ intracellular protein forms and their interaction with molecules involved in signal transduction and DNA repair, raising the possibility that its overexpression during cellular senescence might cause a predisposition to cancer.

    The international journal of biochemistry & cell biology 2002;34;11;1430-48

  • Clusterin expression in malignant lymphomas: a survey of 266 cases.

    Saffer H, Wahed A, Rassidakis GZ and Medeiros LJ

    Department of Pathology, The University of Texas-Houston Health Sciences Center, The University of Texas-M.D. Anderson Cancer Center, Houston, Texas 77030, USA.

    Clusterin expression has been reported to be characteristic of systemic anaplastic large cell lymphoma and usually negative in cutaneous anaplastic large cell lymphoma as well as other lymphoma types. We surveyed clusterin expression using immunohistochemical methods in 266 cases of non-Hodgkin's lymphoma and Hodgkin's disease to further assess the diagnostic utility of this marker. Clusterin immunostaining was observed in 40 of 49 (82%) systemic anaplastic large cell lymphomas and 12 of 29 (41%) cutaneous anaplastic large cell lymphomas. Clusterin also was expressed in 5 of 43 (12%) diffuse large B-cell lymphomas (4 of 5 CD30+), 1 of 14 (7%) peripheral T-cell lymphomas, 1 of 32 (3%) cases of nodular sclerosis Hodgkin's disease, and 1 case of mycosis fungoides in large cell transformation. Clusterin was negative in all other neoplasms assessed including follicular lymphoma of all grades (n = 24), mantle cell lymphoma (n = 13), marginal zone B-cell lymphoma (n = 12), precursor T-cell or B-cell lymphoblastic leukemia/lymphoma (n = 10), mixed cellularity Hodgkin's disease (n = 8), chronic lymphocytic leukemia/small lymphocytic lymphoma (n = 7), Burkitt lymphoma (n = 7), mycosis fungoides (n = 4), nodular lymphocyte predominant Hodgkin's disease (n = 3), lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia (n = 2), and plasmacytoma (n = 2). We conclude that clusterin is a marker of anaplastic large cell lymphoma and that addition of clusterin to antibody panels designed to distinguish systemic anaplastic large cell lymphoma from classical Hodgkin's disease is useful. However, clusterin is also positive in a substantial subset of cutaneous anaplastic large cell lymphomas, a smaller subset of diffuse large B-cell lymphomas, and rarely in cases of peripheral T-cell lymphoma and nodular sclerosis Hodgkin's disease.

    Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 2002;15;11;1221-6

  • Mildly acidic pH activates the extracellular molecular chaperone clusterin.

    Poon S, Rybchyn MS, Easterbrook-Smith SB, Carver JA, Pankhurst GJ and Wilson MR

    Department of Biological Sciences, University of Wollongong, Northfields Ave., Wollongong, New South Wales 2522, Australia.

    Many features of the chaperone action of clusterin are similar to those of the intracellular small heat shock proteins (sHSPs) that, like clusterin, exist in solution as heterogeneous aggregates. Increased temperature induces dissociation of some sHSP aggregates and an enhanced chaperone action, suggesting that a dissociated form is the active chaperone species. We recently reported that clusterin aggregates dissociate at mildly acidic pH. To further explore the similarities between clusterin and the sHSPs, we tested the effects of temperature and pH on the structure of clusterin and its chaperone action. Our results demonstrate that increased temperature does not induce dissociation of clusterin aggregates, or other major structural changes, and has little effect on its chaperone action. However, we show that the chaperone action of clusterin is enhanced at mildly acidic pH. Clusterin is the first chaperone shown to be activated by reduced pH. This unique mode of activation appears to result from an increase in regions of solvent-exposed hydrophobicity, which is independent of any major changes in secondary or tertiary structure. We propose a model in which low pH-induced dissociation of clusterin aggregates increases the abundance of the heterodimeric chaperone-active species, which has greater hydrophobicity exposed to solution.

    The Journal of biological chemistry 2002;277;42;39532-40

  • Serum levels of the senescence biomarker clusterin/apolipoprotein J increase significantly in diabetes type II and during development of coronary heart disease or at myocardial infarction.

    Trougakos IP, Poulakou M, Stathatos M, Chalikia A, Melidonis A and Gonos ES

    Laboratory of Molecular and Cellular Ageing, Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, 48 Vas. Constantinou Avenue, Athens 11635, Greece.

    Clusterin/apolipoprotein J (hereafter ApoJ) is a conserved secreted glycoprotein expressed by a wide array of tissues and being implicated in several physiological processes. ApoJ has been shown to associate with both normal in vitro aging, namely replicative senescence, as well as with stress induced premature senescence. In vivo, the protein is up-regulated in many severe physiological disturbances that relate to advanced aging, including accumulation in the artery wall during the development of atherosclerosis. In the current report we have expanded our previous studies that focus in the biological role of ApoJ during aging by addressing two interrelated issues: (a) we have examined the potential ApoJ association with in vivo aging and (b) we have studied whether its accumulation in the artery wall during the development of atherosclerosis is combined with a measurable increase of its serum levels, as well as, whether a similar effect occurs in diseases, such as diabetes type II, known to represent major risk factors of atherosclerosis. By combining a sandwich ELISA assay and immunoblotting analysis we demonstrate a measurable increase of ApoJ serum levels with age in males and provide evidence that, as compared to healthy donors, the serum ApoJ amount increases significantly in diabetic type II patients and in patients suffering from either a developing coronary heart disease, or myocardial infarction. The highest serum ApoJ levels were found during myocardial infarction but no correlation was observed with the number of vessels with documented atherosclerotic damage. In conclusion, this report illustrates that ApoJ accumulation in serum is probably coupled to a generalized stress mediated induction mechanism that is specifically related to certain diseases; moreover these data raise the possibility that elevated ApoJ levels in serum may represent a strong indication of vascular damage.

    Experimental gerontology 2002;37;10-11;1175-87

  • Clusterin/apolipoprotein J is associated with cortical Lewy bodies: immunohistochemical study in cases with alpha-synucleinopathies.

    Sasaki K, Doh-ura K, Wakisaka Y and Iwaki T

    Department of Neuropathology, Neurological Institute, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan. ksasaki@np.med.kyushu-u.ac.jp

    Clusterin/apolipoprotein J protein expression in cases with "alpha-synucleinopathies", such as Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), was investigated using an immunohistochemical method for the labeling of multiple antigens. About 50% of the cortical Lewy bodies in the cases with DLB were immunoreactive for clusterin, whereas brain-stem Lewy bodies in PD and DLB were rarely associated with clusterin. Clusterin was also immunopositive in around 10% of the glial cytoplasmic inclusions (GCIs) in the cases with MSA. Colocalization of clusterin with alpha-synuclein in such bodies or inclusions was clearly correlated with the immunostaining pattern of alpha-synuclein. Subcellular localization of clusterin was almost completely overlapped with the homogeneous immunoreaction of alpha-synuclein in the cortical Lewy bodies; however, clusterin immunoreactivity was not detected in the halo or ring-like structures of the brain-stem Lewy bodies. Furthermore, some Lewy bodies with intense immunoreactivity for clusterin showed only a weak signal for alpha-synuclein. These results suggest that clusterin may modify the formation of alpha-synuclein-positive inclusion bodies such as Lewy bodies and GCIs, through a previously proposed chaperone property of clusterin.

    Acta neuropathologica 2002;104;3;225-30

  • Clusterin (SGP-2) transient overexpression decreases proliferation rate of SV40-immortalized human prostate epithelial cells by slowing down cell cycle progression.

    Bettuzzi S, Scorcioni F, Astancolle S, Davalli P, Scaltriti M and Corti A

    Dipartimento di Medicina Sperimentale, Università di Parma, Via Volturno, 39-43100 Parma, Italy. saverio.bettuzzi@unipr.it

    Clusterin is a highly conserved, widely distributed glycoprotein whose biological significance is still debated. Involved in many biological processes and disease states, clusterin is induced by cell injury and tissue regression, but is repressed during cell proliferation. We have previously reported that clusterin mRNA induction is associated with epithelial cell atrophy in the rat prostate and both clusterin transcript and protein accumulated in quiescent normal human skin fibroblasts. Here we show that transient clusterin overexpression, in SV40-immortalized human prostate epithelial cells (PNT2), resulted in increased accumulation of cells in the G(0)/G(1) phases of the cell cycle, accompanied by slowdown of cell cycle progression and decrease of DNA synthesis. The activities of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), and the level of histone H3 mRNA (markers of cell proliferation) concomitantly decreased, while Gas1 mRNA (a marker of cell quiescence) accumulated. Thus it appears that clusterin, by opposing the effect of SV40 on the proliferation rate of PNT2 cells, acts as an anti-oncogene in the prostate, suggesting a role for this gene in controlling proliferation of normal and transformed prostate epithelial cells.

    Oncogene 2002;21;27;4328-34

  • Clusterin.

    Jones SE and Jomary C

    Retinitis Pigmentosa Research Unit, Division of Pharmacology & Therapeutics, GKT School of Biomedical Sciences, The Rayne Institute, St Thomas' Hospital, Londoon SE1 7EH, UK. sejones@hgmp.mrc.ac.uk

    Clusterin is an enigmatic glycoprotein with a nearly ubiquitous tissue distribution and an apparent involvement in biological processes ranging from mammary gland involution to neurodegeneration in Alzheimer's disease. Its major form, a 75-80 kDa heterodimer, is secreted and present in physiological fluids, but truncated forms targeted to the nucleus have also been identified. Upregulation of clusterin mRNA and protein levels detected in diverse disease states and in in vitro systems have led to suggestions that it functions in membrane lipid recycling, in apoptotic cell death, and as a stress-induced secreted chaperone protein, amongst others. Recent studies of knockout mice have further complicated the picture by implicating clusterin in exacerbating neuronal death in hypoxia-ischemia. The question of whether clusterin is a multifunctional protein, or deploys a single primary function influenced by cellular context, remains a central issue continuing to stimulate interest in this unusual molecule.

    The international journal of biochemistry & cell biology 2002;34;5;427-31

  • Clusterin expression is significantly enhanced in prostate cancer cells following androgen withdrawal therapy.

    July LV, Akbari M, Zellweger T, Jones EC, Goldenberg SL and Gleave ME

    The Prostate Centre, Vancouver General Hospital, University of British Columbia, Vancouver, British Columbia, Canada.

    Progression of prostate cancer to androgen independence (AI) results in part from the upregulation of anti-apoptotic genes following androgen withdrawal, and androgen-independent disease remains the primary obstacle to improved survival. Testosterone-repressed prostate message-2 (TRPM-2) encodes the anti-apoptotic protein clusterin, which is upregulated in response to cellular compromise as observed in normal and malignant tissues undergoing apoptosis. Systemic administration of antisense clusterin oligonucleotides in prostate cancer xenograft models delays progression to AI and enhances chemosensitivity. The objective of this study was to define changes in clusterin expression following neoadjuvant hormone therapy (NHT) in prostate cancer patients.

    Archival radical prostatectomy (RP) specimens were obtained for 128 patients who received either no NHT or treatment for 2-8 weeks, 3 months, or 8 months. Paired needle biopsy specimens were acquired for 30 patients and all tissues were subjected to clusterin immunohistochemistry. Western blot analysis was performed on frozen tissue from 5 untreated and 5 treated patients.

    Results: Clusterin expression in malignant prostatic tissue was significantly greater in patients who underwent preoperative NHT (P < 0.001). Needle biopsies obtained prior to NHT consistently demonstrated lower staining intensity than corresponding RP specimens (P < 0.001). Western blot analysis confirmed clusterin levels increased 17-fold beginning within 4 weeks after androgen withdrawal.

    Conclusions: Upregulation of clusterin levels following androgen ablation therapy may represent an adaptive cell survival response following apoptotic signals like androgen withdrawal. These findings support clusterin as a valid therapeutic target in strategies employing novel multimodality therapy for advanced prostate cancer.

    The Prostate 2002;50;3;179-88

  • Overexpression of apolipoprotein J in human fibroblasts protects against cytotoxicity and premature senescence induced by ethanol and tert-butylhydroperoxide.

    Dumont P, Chainiaux F, Eliaers F, Petropoulou C, Remacle J, Koch-Brandt C, Gonos ES and Toussaint O

    Unit of Cellular Biochemistry and Biology, Department of Biology, The University of Namur (FUNDP), Belgium.

    Human diploid fibroblasts (HDFs) exposed to subcytotoxic stresses under H2O2, tert-butylhydroperoxide (t-BHP), and ethanol (EtOH) undergo stress-induced premature senescence (SIPS) characterized by many biomarkers of HDFs replicative senescence. Among these biomarkers are a growth arrest, an increase in the senescence-associated beta-galactosidase activity, a senescent morphology, an overexpression of p21waf-1 and the subsequent inability to phosphorylate pRb, the presence of the common 4977-bp mitochondrial deletion, and an increase in the steady-state level of several senescence-associated genes such as apolipoprotein J (apo J). Apo J has been described as a survival gene against cytotoxic stress. In order to study whether apo J would be protective against cytotoxicity SIPS and replicative senescence in human fibroblasts, a full-length complementary deoxyribonucleic acid of apo J was transfected into WI-38 HDFs and SV40-transformed WI-38 HDFs. The overexpression of apo J resulted in an increased cell survival after t-BHP and EtOH stresses at cytotoxic concentrations. In addition, when WI-38 HDFs were exposed to 5 subcytotoxic stresses with EtOH or t-BHP, in conditions that were previously shown to induce SIPS, a lower induction of 2 biomarkers of SIPS was observed in HDFs overexpressing apo J. No effect of apo J overexpression was observed on the proliferative life span of HDFs, even if apo J overexpression triggered osteonectin (SPARC) overexpression, which was shown to decrease the mitogenic potential of platelet-derived growth factor but not of other common growth-inducing conditions. Apo J senescence-related overexpression is proposed to have antiapoptotic rather than antiproliferative effects.

    Cell stress & chaperones 2002;7;1;23-35

  • Clusterin expression in adult human normal and osteoarthritic articular cartilage.

    Connor JR, Kumar S, Sathe G, Mooney J, O'Brien SP, Mui P, Murdock PR, Gowen M and Lark MW

    Department of Musculoskeletal Diseases, GlaxoSmithKline Pharmaceuticals, King of Prussia, Pennsylvania 19406, USA. Janicr_R_Connor@gsk.com

    Objective: To characterize the expression pattern of clusterin in adult human normal and osteoarthritic cartilage.

    Methods: Clusterin mRNA expression in adult human normal and osteoarthritic cartilage was investigated by analysis of cDNA libraries, TaqMan quantitative RT-PCR, microarray and in situ hybridization.

    Results: Sequence analysis of ESTs from adult human normal and osteoarthritic cartilage cDNA libraries demonstrated that the abundance of clusterin in these libraries was equivalent to genes which have been more commonly associated with cartilage. To examine tissue distribution, TaqMan Quantitative PCR analysis was performed using RNA from a panel of individual normal tissues. Clusterin was expressed at significant levels in cartilage, brain, liver, and pancreas. The expression of clusterin mRNA was up-regulated in early osteoarthritic vs normal cartilage when analysed by microarray analysis. Using in situ hybridization, chondrocytes of normal cartilage expressed moderate levels of clusterin. Upper mid-zone chondrocytes in cartilage with early stages of osteoarthritic disease expressed high levels of clusterin mRNA. In advanced osteoarthritic cartilage, the overall expression of clusterin was reduced.

    Conclusion: The induction of clusterin has been associated with a variety of disease states where it appears to provide a cytoprotective effect. The increased expression of clusterin mRNA in the early stages of osteoarthritis (OA) may reflect an attempt by the chondrocytes to protect and repair the tissue. In contrast, the decrease in clusterin mRNA in the advanced osteoarthritic cartilage accompanies the final degenerative stages of the disease. An understanding of the expression of clusterin in osteoarthritis may allow consideration of this protein as a marker for cartilage changes in this chronic degenerative condition.

    Osteoarthritis and cartilage 2001;9;8;727-37

  • Introduction of clusterin gene into human renal cell carcinoma cells enhances their resistance to cytotoxic chemotherapy through inhibition of apoptosis both in vitro and in vivo.

    Hara I, Miyake H, Gleave ME and Kamidono S

    Department of Urology, Kobe University School of Medicine, Chuo-ku, Kobe 650-0017. hara@med.kobe-u.ac.jp

    Recent studies have revealed the powerful antiapoptotic activity of clusterin in various malignant tumors; however, the significance of clusterin expression in the acquisition of a resistant phenotype against several kinds of treatment in human renal cell carcinoma (RCC) has not been well characterized. We, therefore, transfected the clusterin cDNA into RCC ACHN cells, that scarcely express clusterin protein, to examine whether overexpression of clusterin inhibits chemotherapy-induced apoptosis both in vitro and in vivo. Although no significant differences were observed in the in vitro growth rates between clusterin-transfected ACHN (ACHN/CL) and the vector only-transfected cell line (ACHN/Co), ACHN/CL exhibited high resistance to cisplatin treatment compared with ACHN/Co, with a greater than 5-fold higher IC(50) through the inhibition of apoptotic cell death, which was demonstrated by DNA fragmentation analysis and western blotting of PARP protein. Moreover, intravenous administration of cisplatin into athymic nude mice bearing ACHN/CL tumors resulted in 2- to 3-times faster tumor growth compared with ACHN/Co tumors. These findings suggest that clusterin overexpression helps confer a chemoresistant phenotype through inhibition of apoptosis in human RCC cells.

    Japanese journal of cancer research : Gann 2001;92;11;1220-4

  • Clusterin, a binding protein with a molten globule-like region.

    Bailey RW, Dunker AK, Brown CJ, Garner EC and Griswold MD

    School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4660,USA.

    Clusterin is a heterodimeric glycoprotein found in many tissues of the body and is the most abundant protein secreted by cultured rat Sertoli cells. The function of clusterin is unknown, but it has been associated with cellular injury, lipid transport, apoptosis, and it may be involved in the clearance of cellular debris caused by cell injury or death. Consistent with this last idea, clusterin has been shown to bind to a variety of molecules with high affinity including lipids, peptides, and proteins and the hydrophobic probe 1-anilino-8-naphthalenesulfonate (ANS). Given this variety of ligands, clusterin must have specific structural features that provide the protein with its promiscuous binding activity. Using sequence analyses, we show that clusterin likely contains three long regions of natively disordered or molten globule-like structures containing putative amphipathic alpha-helices. These disordered regions were highly sensitive to trypsin digestion, indicating a flexible nature. The effects of denaturation on the fluorescence of the clusterin-ANS complex were compared between proteins with structured binding pockets and molten globular forms of proteins. Clusterin bound ANS in a manner that was very similar to that of molten globular proteins. Furthermore, we found that, when bound to ANS, at least one cleavage site within the protease-sensitive disordered regions of clusterin was protected from trypsin digestion. In addition, we show that clusterin can function as a biological detergent that can solubilize bacteriorhodopsin. We propose that natively disordered regions with amphipathic helices form a dynamic, molten globule-like binding site and provide clusterin the ability to bind to a variety of molecules.

    Funded by: NICHD NIH HHS: R01 HD 30692

    Biochemistry 2001;40;39;11828-40

  • Localisation of clusterin in normal human sperm by immunogold electron microscopy.

    Atlas-White M, Murphy BF and Baker HW

    University of Melbourne, Department of Obstetrics and Gynaecology, The Royal Women's Hospital, Carlton, Victoria, Australia.

    In the human male reproductive tract, two forms of clusterin have been detected: the conventional heterodimeric form and a novel acrosomal form. On human sperm the novel form of clusterin is present in the acrosomal region of acrosome intact sperm only. The aim of this study was to determine the site of localisation of the acrosomal form of clusterin using immunogold electron microscopy on normal human sperm. Using the E5 anticlusterin mAb and a preembedding technique, acrosomal clusterin was localised in the acrosomal contents. Immunogold particles were detected on ethanol fixed spermatozoa that were subjected to Triton X-100 permeabilisation treatment. These sperm had lost their plasmalemma and outer acrosomal membrane. Specific immunogold labeling was present over the surface mainly of the acrosomal contents exposed by the loss of the plasma-lemma and outer acrosomal membrane. Immunogold particles were also detected in the equatorial segment of the sperm. These data confirm that the acrosomal form of clusterin is associated with the contents of the acrosome.

    Pathology 2000;32;4;258-61

  • Isolation of Ku70-binding proteins (KUBs).

    Yang CR, Yeh S, Leskov K, Odegaard E, Hsu HL, Chang C, Kinsella TJ, Chen DJ and Boothman DA

    Department of Radiation Oncology and Department of Pharmacology and the Ireland Cancer Center,Laboratory of Molecular Stress Responses, Case Western Reserve University, BRB-326 East,10900 Euclid Avenue, Cleveland, OH 44106-4942, USA.

    DNA-dependent protein kinase (DNA-PK) plays a critical role in resealing DNA double-stand breaks by non-homologous end joining. Aside from DNA-PK, XRCC4 and DNA ligase IV, other proteins which play a role(s) in this repair pathway remain unknown; DNA-PK contains a catalytic subunit (DNA-PKcs) and a DNA binding subunit (Ku70 and Ku80). We isolated Ku70-binding proteins (KUB1-KUB4) using yeast two-hybrid analyses. Sequence analyses revealed KUB1 to be apolipoprotein J (apoJ), also known as X-ray-inducible transcript 8 (XIP8), testosterone-repressed prostate message-2 (TRPM-2) and clusterin. KUB2 is Ku80. KUB3 and KUB4 are unknown, >10 kb trans-cripts. Interactions of apoJ/XIP8 or KUB3 with Ku70 were confirmed by co-immunoprecipitation analyses in MCF-7:WS8 breast cancer or IMR-90 normal lung fibroblast cells, respectively. The interaction of apoJ/XIP8 with Ku70 was confirmed by far-western analyses. Stable over-expression of full-length apoJ/XIP8 in MCF-7:WS8 caused decreased Ku70/Ku80 DNA end binding that was restored by apoJ/XIP8 monoclonal antibodies. The role of apoJ/XIP8 in ionizing radiation resistance/sensitivity is under investigation.

    Funded by: NCI NIH HHS: CA-50595, CA-ES78530, CA50519, R01 CA139217

    Nucleic acids research 1999;27;10;2165-74

  • Binding of gelsolin, a secretory protein, to amyloid beta-protein.

    Chauhan VP, Ray I, Chauhan A and Wisniewski HM

    New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, New York 10314-6399, USA. chauhanvps@aol.com

    Soluble amyloid beta-protein (Abeta) is normally present in the cerebrospinal fluid (CSF) and plasma. However, it is fibrillized and deposited as plaques in the brains of patients with Alzheimer's disease. Cerebrospinal fluid (CSF) contains several circulating proteins (apolipoprotein E, apolipoprotein J, and transthyretin) that bind to Abeta. We report here that gelsolin, a secretory protein, also binds to Abeta in a concentration-dependent manner. Under similar conditions, other proteins such as G-actin, protein kinase C, polyglutamic acid, and gelatin did not bind to Abeta. Solid phase binding assays showed two Abeta binding sites on gelsolin that have dissociation constants (Kd) of 1.38 and 2.55 microM. Abeta was found to co-immunoprecipitate along with gelsolin from the plasma, suggesting that gelsolin-Abeta complex exists under physiological conditions. The gelsolin-Abeta complex was sodium dodecyl sulfate (SDS)stable in the absence of reducing agent, but was dissociated when the SDS stop solution contained dithiothreitol (reducing agent). This study suggests that the function of secretory gelsolin in the CSF and plasma is to bind and sequester Abeta.

    Funded by: NIA NIH HHS: AG 14199

    Biochemical and biophysical research communications 1999;258;2;241-6

  • Characterization of the heparin-binding properties of human clusterin.

    Pankhurst GJ, Bennett CA and Easterbrook-Smith SB

    Department of Biochemistry University of Sydney, Sydney NSW 2006, Australia.

    Clusterin is a highly conserved mammalian glycoprotein which has been predicted to contain heparin-binding sites. We tested this prediction by studying the interactions between heparin and clusterin using ELISA and heparin affinity chromatography methodologies. Two forms of biotinylated heparin were used in ELISA: heparin which had been directly biotinylated with a biotin-N-hydroxysuccinimide ester and heparin which had been activated using epichlorohydrin and 1,6-diaminohexane prior to biotinylation. Both gave dose-dependent increases in ELISA signal with increasing concentrations of biotinylated heparin, with the latter giving signals an order of magnitude greater than the former. There was a dose-dependent increase in the ELISA signal from bound biotinylated heparin with increasing concentrations of plate-bound clusterin. The apparent affinity constant for binding of biotinylated heparin to plate-bound clusterin at pH 6.0 was estimated as 0.06 +/- 0.02 microM. Unlabeled heparin blocked the binding of biotinylated heparin to clusterin over a concentration range similar to that of the binding of biotinylated heparin to plate-bound clusterin. The binding of biotinylated heparin to clusterin was independent of the presence or absence of Ca2+. The binding of biotinylated heparin to plate-bound clusterin increased with decreasing pH over the range 5.5-8.0 and was characterized by an apparent pKa of 6.9. Clusterin in human serum bound to heparin-Sepharose at pH 6.0 but not at pH 7.4. Dot-blot experiments showed that one of the polypeptide chains of clusterin which had been reduced and alkylated under denaturing conditions bound to heparin-Sepharose. This chain was identified as the alpha chain from its N-terminal amino acid sequence.

    Biochemistry 1998;37;14;4823-30

  • Identification and characterization of glycosylation sites in human serum clusterin.

    Kapron JT, Hilliard GM, Lakins JN, Tenniswood MP, West KA, Carr SA and Crabb JW

    W. Alton Jones Cell Science Center, Lake Placid, New York 12946, USA.

    Clusterin is a ubiquitous, heterodimeric glycoprotein with multiple possible functions that are likely influenced by glycosylation. Identification of oligosaccharide attachment sites and structural characterization of oligosaccharides in human serum clusterin has been performed by mass spectrometry and Edman degradation. Matrix-assisted laser desorption ionization mass spectrometry revealed two molecular weight species of holoclusterin (58,505 +/- 250 and 63,507 +/- 200). Mass spectrometry also revealed molecular heterogeneity associated with both the alpha and beta subunits of clusterin, consistent with the presence of multiple glycoforms. The data indicate that clusterin contains 17-27% carbohydrate by weight, the alpha subunit contains 0-30% carbohydrate and the beta subunit contains 27-30% carbohydrate. Liquid chromatography electrospray mass spectrometry with stepped collision energy scanning was used to selectively identify and preparatively fractionate tryptic glycopeptides. Edman sequence analysis was then used to confirm the identities of the glycopeptides and to define the attachment sites within each peptide. A total of six N-linked glycosylation sites were identified, three in the alpha subunit (alpha 64N, alpha 81N, alpha 123N) and three in the beta subunit (beta 64N, beta 127N, and beta 147N). Seven different possible types of oligosaccharide structures were identified by mass including: a monosialobiantennary structure, bisialobiantennary structures without or with one fucose, trisialotriantennary structures without or with one fucose, and possibly a trisialotriantennary structure with two fucose and/or a tetrasialotriantennary structure. Site beta 64N exhibited the least glycosylation diversity, with two detected types of oligosaccharides, and site beta 147N exhibited the greatest diversity, with five or six detected types of oligosaccharides. Overall, the most abundant glycoforms detected were bisialobiantennary without fucose and the least abundant were monosialobiantennary, trisialotriantennary with two fucose and/or tetrasialotriantennary. Clusterin peptides accounting for 99% of the primary structure were identified from analysis of the isolated alpha and beta subunits, including all Ser- and Thr-containing peptides. No evidence was found for the presence of O-linked or sulfated oligosaccharides. The results provide a molecular basis for developing a better understanding of clusterin structure-function relationships and the role clusterin glycosylation plays in physiological function.

    Funded by: NEI NIH HHS: EY06603; NIDDK NIH HHS: DK38639

    Protein science : a publication of the Protein Society 1997;6;10;2120-33

  • Interaction of apolipoprotein J-amyloid beta-peptide complex with low density lipoprotein receptor-related protein-2/megalin. A mechanism to prevent pathological accumulation of amyloid beta-peptide.

    Hammad SM, Ranganathan S, Loukinova E, Twal WO and Argraves WS

    Cell Biology and Anatomy Department, Medical University of South Carolina, Charleston, South Carolina 29425-2204, USA.

    Apolipoprotein J (apoJ) has been shown to be the predominant amyloid beta-peptide (Abeta)-binding protein in cerebrospinal fluid. We have previously demonstrated that the endocytic receptor low density lipoprotein receptor-related protein-2/megalin (LRP-2), which is expressed by choroid plexus epithelium and ependymal cells lining the brain ventricles and neural tube, binds and mediates cellular uptake of apoJ (Kounnas, M. Z., Loukinova, E. B., Stefansson, S., Harmony, J. A., Brewer, B., Strickland, D. K., and Argraves, W. S. (1995) J. Biol. Chem. 270, 13070-13075). In the present study, we evaluated the ability of apoJ to mediate binding of Abeta1-40-apoJ complex to LRP-2 in vitro. Immunoblot analysis showed that incubation of apoJ with Abeta1-40 resulted in the formation of Abeta1-40-apoJ complex and the inhibition of the formation of Abeta1-40 aggregates. Using an enzyme-linked immunosorbent assay, an estimated dissociation constant (Kd) of 4.8 nM was derived for the interaction between Abeta1-40 and apoJ. Enzyme-linked immunosorbent assay was also used to study the interaction of the Abeta1-40-apoJ complex with LRP-2. The results showed that Abeta alone did not bind directly to LRP-2; however, when Abeta1-40 was combined with apoJ to form a complex, binding to LRP-2 took place. The binding interaction could be blocked by inclusion of the receptor-associated protein, an antagonist of apoJ binding to LRP-2. When LRP-2-expressing cells were given 125I-Abeta1-40, cellular uptake of the radiolabeled peptide was promoted by co-incubation with apoJ. When the cells were provided purified 125I-Abeta1-40-apoJ complex, the complex was internalized and degraded, and both processes were inhibited with polyclonal LRP-2 antibodies. Furthermore, chloroquine treatment inhibited the cellular degradation of the complex. The data indicate that apoJ facilitates Abeta1-40 binding to LRP-2 and that the receptor mediates cellular clearance of Abeta1-40-apoJ complex leading to lysosomal degradation of Abeta1-40. The findings support the possibility that LRP-2 can act in vivo to mediate clearance of the complex from biological fluids such as cerebrospinal fluid and thereby play a role in the regulation of Abeta accumulation.

    Funded by: NIDDK NIH HHS: DK45598

    The Journal of biological chemistry 1997;272;30;18644-9

  • Apolipoprotein J expression in human ocular surface epithelium.

    Nishida K, Kawasaki S, Adachi W and Kinoshita S

    Department of Ophthalmology, Kyoto Prefectural University of Medicine, Japan.

    Purpose: Apolipoprotein J (apoJ) expression has been detected in various mouse mucosal epithelial cells, as well as in the human ciliary body, retina, vitreous humor, and aqueous humor. The purpose of this study was to determine the expression and localization pattern of apoJ mRNA transcripts and protein in the human ocular surface epithelium.

    Methods: The expression of apoJ mRNA in corneal and conjunctival epithelial cells was investigated by reverse-transcriptase polymerase chain reaction (RT-PCR). mRNA localization in the corneal epithelium and protein localization in corneal and conjunctival epithelia were analyzed by in situ hybridization and immunohistochemistry, respectively.

    Results: The RT-PCR studies demonstrated the expression of apoJ mRNA transcripts in corneal and conjunctival epithelial cells. In situ hybridization analysis revealed that apoJ mRNA signals were detected in all layers of the corneal epithelium, most prominently in the basal cells. Immunohistochemical analysis revealed positive immunostaining for apoJ in the apical cell layers of corneal and conjunctival epithelia.

    Conclusions: ApoJ is synthesized by and localized in the ocular surface epithelium. This suggests a role for this protein at the tear-ocular surface interface.

    Investigative ophthalmology & visual science 1996;37;11;2285-92

  • Clusterin (Apo J) protects against in vitro amyloid-beta (1-40) neurotoxicity.

    Boggs LN, Fuson KS, Baez M, Churgay L, McClure D, Becker G and May PC

    Lilly Research Laboratories, CNS Research Division, Eli Lilly and Company, Indianapolis, Indiana 46285, USA.

    Clusterin is a secreted glycoprotein that is markedly induced in many disease states and after tissue injury. In the CNS, clusterin expression is elevated in neuropathological conditions such as Alzheimer's disease (AD), where it is found associated with amyloid-beta (A beta) plaques. Clusterin also coprecipitates with A beta from CSF, suggesting a physiological interaction with A beta. Given this interaction with A beta, the goal of this study was to determine whether clusterin could modulate A beta neurotoxicity. A mammalian recombinant source of human clusterin was obtained by stable transfection of hamster kidney fibroblasts with pADHC-9, a full-length human cDNA clone for clusterin. Recombinant clusterin obtained from this cell line, as well as a commercial source of native clusterin purified from serum, afforded dose-dependent neuroprotection against A beta (1-40) when tested in primary rat mixed hippocampal cultures. Clusterin afforded substoichiometric neuroprotection against several lots of A beta (1-40) but not against H2O2 or kainic acid excitotoxicity. These results suggest that the elevated expression of clusterin found in AD brain may have effects on subsequent amyloid-beta plaque pathology.

    Journal of neurochemistry 1996;67;3;1324-7

  • A gene expression profile of human corneal epithelium and the isolation of human keratin 12 cDNA.

    Nishida K, Adachi W, Shimizu-Matsumoto A, Kinoshita S, Mizuno K, Matsubara K and Okubo K

    Department of Ophthalmology, Kyoto Prefectural University of Medicine, Japan.

    Purpose: To describe the quantitative and qualitative aspects of gene expression in human corneal epithelium and to discover novel cornea-specific genes.

    Methods: A 3'-directed cDNA library was constructed with messenger RNA prepared from normal human corneal epithelial cells, and inserts in 1069 randomly chosen clones were sequenced. These sequences were compared with each other to determine the frequency of appearance and were searched against GenBank for identification. The resultant expression profile, a list of gene species and their recurrences, reflected the composition of mRNA in the cornea. Recurrently appearing sequences, representing abundant transcripts, were compared with sequences in expression profiles obtained from seven other tissues and from those in dbEST to discover cornea-specific genes.

    Results: The expression profile of human corneal epithelium showed that the most abundant transcript in this tissue was that for apolipoprotein J. Altogether 62 genes were suggested to be very active, including calcyclin, alpha-enolase, keratin 3, connexin 43, and 12 novel genes. The expression of four of these 12 novel genes seemed to be limited to cornea because they were not found in seven other expression profiles nor in dbEST. Full-length cDNA corresponding to one of these (GS8025), isolated from a separately made cDNA library, contained open reading frame highly homologous to mouse keratin 12, which is known to be cornea specific.

    Conclusions: An expression profile of corneal epithelium provides probes to monitor physiological and pathologic conditions of this tissue in terms of gene expression. Furthermore, by comparing this profile with those of other tissues, probes to isolate genes uniquely transcribed in corneal epithelium are determined. These genes are assumed to carry unique functions for this tissue and are candidate genes for inherited diseases that manifest only in cornea. As an example, human cornea-specific keratin was isolated, and partial cDNA sequences for three more cornea-specific genes were presented.

    Investigative ophthalmology & visual science 1996;37;9;1800-9

  • Transforming growth factor beta (TGF beta)-induced nuclear localization of apolipoprotein J/clusterin in epithelial cells.

    Reddy KB, Jin G, Karode MC, Harmony JA and Howe PH

    Department of Cell Biology, Cleveland Clinic Research Institute, Ohio 44195-5245, USA.

    Apolipoprotein J (apoJ)/clusterin was first identified as an 80 kDa secretory glycoprotein present in most body fluids. It has been implicated in a variety of physiological processes including cellular differentiation and apoptosis. We demonstrate here that in addition to the well characterized secreted form of the protein, there exists an intracellular, nuclear form of apoJ. This intracellular form of the protein is induced to accumulate in the nucleus of two epithelial cell lines (HepG2 and CCL64) in response to treatment with transforming growth factor beta (TGF beta). We demonstrate in vitro that apoJ protein can be translated from two in-frame ATG sites. Initiation from the first ATG encodes for the secretory form of apoJ and initiation from the second ATG, located 33 amino acids downstream of the first and lacking the hydrophobic signal sequence, encodes for a truncated apoJ protein. This shorter form of apoJ is not recognized by microsomes and therefore not glycosylated, and we postulate that it is retained intracellularly and targeted to the nucleus due to the presence of an SV40-like nuclear localization sequence (NLS). This mechanism of nuclear targeting of apoJ occurs in cells since the protein isolated from nuclei of TGF beta-treated cells and the in vitro-translated truncated form are identical by V8 protease analysis. These results suggest that the diverse physiological responses attributed to apoJ may be elicited through a common molecular mechanism involving a previously uncharacterized intracellular form of the protein.

    Funded by: NCI NIH HHS: CA55536; NHLBI NIH HHS: HL41496

    Biochemistry 1996;35;19;6157-63

  • Interaction of transforming growth factor beta receptors with apolipoprotein J/clusterin.

    Reddy KB, Karode MC, Harmony AK and Howe PH

    Department of Cell Biology, Cleveland Clinic Research Institute, Ohio 44195-5245, USA.

    Proteins mediating the transmission of the signal from an activated transforming growth factor beta (TGF beta) receptor complex have not been identified. Using a yeast interaction screen to search for proteins that associate with the type II TGF beta receptor (RII), we isolated a protein which was identical to apolipoprotein J (apoJ)/clusterin. ApoJ interacts with both the type I (RI) and type II (RII) TGF beta receptors but does not interact with the epidermal growth factor (EGF) receptor. The interaction between RII and apoJ occurs through the C-terminal 127 amino acids of RII. Deletion of this region, which contains the kinase insert 2 domain, abrogates binding to apoJ. The binding of apoJ to either the RI and the RII receptors is direct, not requiring other proteins, and is not specific for the alpha or beta subunit of apoJ since both subunits are effective in competing for binding. RI and RII fusion proteins are capable of precipitating the 60 kDa intracellular form of apoJ from [35S]methionine-labeled cellular lysates, suggesting that this form of the protein may play some role in TGF beta signaling or TGF beta receptor processing.

    Funded by: NCI NIH HHS: CA55536; NHLBI NIH HHS: HL41496

    Biochemistry 1996;35;1;309-14

  • Identification of glycoprotein 330 as an endocytic receptor for apolipoprotein J/clusterin.

    Kounnas MZ, Loukinova EB, Stefansson S, Harmony JA, Brewer BH, Strickland DK and Argraves WS

    J. H. Holland Laboratory, Biochemistry Department American Red Cross, Rockville, Maryland 20855, USA.

    Glycoprotein 330 (gp330) is a member of a family of endocytic receptors related to the low density lipoprotein receptor. gp330 has previously been shown to bind a number of ligands in common with its family member, the low density lipoprotein receptor-related protein (LRP). To identify ligands specific for gp330 and relevant to its localization on epithelia such as in the mammary gland, gp330-Sepharose affinity chromatography was performed. As a result, a 70-kDa protein was selected from human milk and identified by protein sequencing to be apolipoprotein J/clusterin (apoJ). Solid-phase binding assays confirmed that gp330 bound to apoJ with high affinity (Kd = 14.2 nM). Similarly, gp330 bound to apoJ transferred to nitrocellulose after SDS-polyacrylamide gel electrophoresis. LRP, however, showed no binding to apoJ in either type of assay. The binding of gp330 to apoJ could be competitively inhibited with excess apoJ as well as with the gp330 ligands apolipoprotein E, lipoprotein lipase, and the receptor-associated protein, a 39-kDa protein that acts to antagonize binding of all known ligands for gp330 and LRP. Several cultured cell lines that express gp330 and ones that do not express the receptor were examined for their ability to bind and internalize 125I-apoJ. Only cells that expressed gp330 endocytosed and degraded radiolabeled apoJ. Furthermore, F9 cells treated with retinoic acid and dibutyryl cyclic AMP to increase expression levels of gp330 displayed an increased capacity to internalize and degrade apoJ. Cellular internalization and degradation of radiolabeled apoJ could be inhibited with unlabeled apoJ, receptor-associated protein, and gp330 antibodies. The results indicate that gp330 but not LRP can bind to apoJ in vitro and that gp330 expressed by cells can mediate apoJ endocytosis leading to lysosomal degradation.

    Funded by: NHLBI NIH HHS: HL41496; NIDDK NIH HHS: DK45598; NIGMS NIH HHS: GM42581; ...

    The Journal of biological chemistry 1995;270;22;13070-5

  • Expression of apolipoprotein J in the uterus is associated with tissue remodeling.

    Brown TL, Moulton BC, Baker VV, Mira J and Harmony JA

    Department of Obstetrics & Gynecology, University of Cincinnati College of Medicine, Ohio 45267-0575, USA.

    The endometrium is a dynamic tissue that, in response to hormonal cues, undergoes cycles of growth and involution. Extracellular factors required for this remodeling are poorly understood. The potential role in endometrial turnover of apolipoprotein J (apoJ), a secretory glycoprotein that can bind lipids and membrane-active proteins, is proposed on the basis of its spatial and temporal patterns of expression during normal cycling, after ovariectomy, and in response to hormone manipulation. In the mouse, apoJ mRNA was expressed in uterine luminal and glandular epithelial cells coincident with the presence of apoJ protein. The apoJ gene was differentially expressed in the glandular and uterine luminal epithelial cells during the estrous cycle and following hormone depletion. Expression of apoJ was not induced in ovariectomized mice by estrogen, progesterone, or dexamethasone treatment alone. Progesterone administration after an initial estrogen pretreatment, however, resulted in dramatic induction of apoJ as the progesterone level declined. In contrast, apoJ was not induced when a long-lived progesterone analog, medroxyprogesterone, was substituted for progesterone. In the human menstrual cycle, apoJ was present in glandular lumens only during the late secretory phase. Declining progesterone levels, causing substantial tissue reorganization, are characteristic of the times of marked apoJ induction in uterine epithelial cells. These expression patterns are consistent with apoJ functioning as an extracellular cytoprotectant by mediating clearance of and/or neutralizing cytolytic tissue debris.

    Funded by: NHLBI NIH HHS: HL07527, HL41496

    Biology of reproduction 1995;52;5;1038-49

  • Local synthesis of apolipoprotein J in the eye.

    Reeder DJ, Stuart WD, Witte DP, Brown TL and Harmony JA

    Department of Ophthalmology, University of Cincinnati College of Medicine, OH, USA.

    Apolipoprotein J (apoJ), a secretory glycoprotein known to transport lipids and to regulate terminal complement function, is present in the human eye in both aqueous and vitreous, as well as in the retina. Ocular apoJ is the product of local synthesis, rather than plasma contamination, as demonstrated by its distinct structural properties and the presence of abundant apoJ mRNA in retina and retina pigment epithelium. ApoJ mRNA is also present in mouse eye, with a developmentally regulated pattern of expression. In fetal mouse, apoJ mRNA is present in retina, lens and cornea. In contrast, adult eye apoJ mRNA is present in retina and ciliary body. We propose that apoJ is important in tissue remodeling and in stabilizing hydrophobic molecules which are required for vision and/or which would otherwise be deleterious and membrane-active.

    Funded by: NHLBI NIH HHS: HL07527, HL41496

    Experimental eye research 1995;60;5;495-504

  • Molecular characterization of human TRPM-2/clusterin, a gene associated with sperm maturation, apoptosis and neurodegeneration.

    Wong P, Taillefer D, Lakins J, Pineault J, Chader G and Tenniswood M

    Department of Biochemistry, University of Ottawa, Canada.

    The TRPM-2/clusterin gene and its cognate protein has been characterized in a number of species. Although the functional role, or roles, of the TRPM-2/clusterin protein remains to be firmly established, the gene has been implicated in a variety of physiological processes, including sperm maturation, lipid transport, membrane remodelling and inhibition of the complement cascade. TRPM-2/clusterin is induced de novo during the regression of the prostate and other hormone-dependent tissues after hormone ablation, and is over-expressed in several human neurodegenerative diseases including Alzheimer's disease, epilepsy and retinitis pigmentosa. We describe the genomic structure of the human TRPM-2/clusterin gene which is organized into nine exons, ranging in size from 47 bp (exon I) to 412 bp (exon V), spanning a region of 16,580 bp. Comparison with sequences registered in the databases shows that it has extensive similarity to the human protein designated as SP-40,40 or complement-lysis inhibitor (CLI), a protein that appears to block the membrane-attack complex of complement. However, the cDNA sequences reported for SP-40,40 and CLI diverge significantly in the 5' untranslated region of the mRNA (coded for by exon I), raising the possibility that the TRPM-2/clusterin gene is present in the human genome as a small multi-gene family or that there are several alternate exon I sequences in the TRPM-2 gene. Southern analysis and fluorescent in situ hybridization suggest that the clusterin gene is a single-copy gene, and that, if alternative exon I sequences are present in the genome, they lie outside of the lambda clones that have been characterized. Analysis of the promoter region of the human TRPM-2/clusterin gene shows many similarities to the rat TRPM-2/clusterin promoter including a putative control region containing several potential regulatory elements that may regulate the complex tissue-specific control of the gene which must be constitutively expressed in some tissues but is inducible in others.

    European journal of biochemistry 1994;221;3;917-25

  • Apolipoprotein J is associated with paraoxonase in human plasma.

    Kelso GJ, Stuart WD, Richter RJ, Furlong CE, Jordan-Starck TC and Harmony JA

    Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, Ohio 45267.

    Apolipoprotein J (apoJ)-containing high-density lipoproteins (HDL), isolated from human plasma by immunoaffinity chromatography, are associated with apoAI and a protein of approximately 44 kDa. In order to advance our understanding of apoJ's role in the vasculature, a comprehensive investigation was performed to identify and characterize this 44-kDa protein and to study its interaction with apoJ. The 44-kDa protein, a monomeric glycoyslated polypeptide, was identified by N-terminal sequencing as serum paraoxonase. Paraoxonase exists in two oxidation states: one contains all free cysteines while the other has one disulfide bond between Cys42 and Cys284. Northern analysis of eight human tissues shows paraoxonase message present only in the liver. The majority of apoJ/paraoxonase-HDL are 90-140 kDa; however, not all of the plasma paraoxonase is associated with apoJ. The specificity of the apoJ/paraoxonase interaction, inferred by the constant mole ratio of the two proteins in affinity-purified apoJ-HDL, is confirmed in direct binding assays. For purified proteins, there is more than a 5-fold increase in the apparent affinity of apoJ for immobilized paraoxonase as the paraoxonase coating concentration is increased from 0.5 to 2.0 micrograms/mL. Both oxidation states of paraoxonase bind to apoJ with equal affinity. Our data combined with other evidence suggest that the plasma link of apoJ with paraoxonase will be implicated as a predictor of vascular damage.

    Funded by: NHLBI NIH HHS: HL07382, HL41496; NIEHS NIH HHS: ES05194; ...

    Biochemistry 1994;33;3;832-9

  • Clusterin, the human apolipoprotein and complement inhibitor, binds to complement C7, C8 beta, and the b domain of C9.

    Tschopp J, Chonn A, Hertig S and French LE

    Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland.

    Clusterin is a heterodimeric multifunctional protein expressed in a variety of tissues and cells. It forms high density lipid complexes in plasma and participates in the control of the lytic activity of the late complement complex (TCC, C5b-9). Together with vitronectin, clusterin binds to the nascent amphiphilic C5b-9 complex, rendering it water soluble and lytically inactive. To define the interactions that underlie the complement-inhibitory function of clusterin, we have examined the binding interactions between [125I]clusterin and the isolated components of the complex, C5b-6, C7, C8, and C9 and vitronectin. By using ligand blotting in the presence of Tween, specific binding of the labeled clusterin with C7, the beta-subunit of C8 and C9 was detected. Binding to C9 was competed by polymerized C9, but not by C8, C7, C6, and CD59, suggesting that the conformational change occurring during the hydrophilic-amphiphilic transition of C9 exposes the interaction site for clusterin. When thrombin-treated C9 was analyzed, clusterin was found to recognize the C9b fragment containing the hydrophobic membrane interaction segment. Both subunits of clusterin interact with C9 and are similarly potent in inhibiting C5b-9-mediated hemolysis and Zn+(+)-induced C9 polymerization. These results show that clusterin exerts its inhibitory effect by interacting with a structural motif common to C7, C8 alpha, and C9b.

    Journal of immunology (Baltimore, Md. : 1950) 1993;151;4;2159-65

  • The cerebrospinal-fluid soluble form of Alzheimer's amyloid beta is complexed to SP-40,40 (apolipoprotein J), an inhibitor of the complement membrane-attack complex.

    Ghiso J, Matsubara E, Koudinov A, Choi-Miura NH, Tomita M, Wisniewski T and Frangione B

    Department of Pathology, New York University Medical Center, NY 10016.

    The amyloid fibrils deposited in Alzheimer's neuritic plaque cores and cerebral blood vessels are mainly composed of aggregated forms of a unique peptide, 39-42 amino acids long, named amyloid beta (A beta). A similar, although soluble, A beta ('sA beta') has been identified in cerebrospinal fluid, plasma and cell supernatants, indicating that it is normally produced by proteolytic processing of its precursor protein, amyloid precursor protein (APP). Using direct binding experiments we have isolated and characterized an 80 kDa circulating protein that specifically interacts with a synthetic peptide identical with A beta. The protein was unmistakably identified as SP-40,40 or ApoJ, a cytolytic inhibitor and lipid carrier, by means of amino acid sequence and immunoreactivity with specific antibodies. Immunoprecipitation with anti-SP-40,40 retrieved soluble A beta from cerebrospinal fluid, indicating that the interaction occurs in vivo.

    Funded by: NIA NIH HHS: AG05891, AG08721, AG10953

    The Biochemical journal 1993;293 ( Pt 1);27-30

  • Elevation of human cerebrospinal fluid clusterin concentration is associated with acute neuropathology.

    Polihronis M, Paizis K, Carter G, Sedal L and Murphy B

    Department of Nephrology, St. Vincent's Hospital, Melbourne, Australia.

    Clusterin is a serum glycoprotein which is an inhibitor of complement and is expressed in many tissues in cell injury and death. It has been identified normal and pathological brain tissue and is a component of normal human cerebrospinal fluid (CSF). We have measured the clusterin concentration of 115 abnormal and normal human CSF samples and related these data to the patient's clinical diagnoses. CSF clusterin levels in patients with neurodegenerative and meningeal disease were within the normal range. Twelve of 15 patients with demyelination, however, had significant elevation of CSF clusterin concentration. This was not a specific finding for multiple sclerosis as elevated clusterin levels were also seen in patients with other acute neuropathology. Determination of CSF clusterin concentration may be of clinical value in neurological diagnosis.

    Journal of the neurological sciences 1993;115;2;230-3

  • Genomic organization and expression of the rat TRPM-2 (clusterin) gene, a gene implicated in apoptosis.

    Wong P, Pineault J, Lakins J, Taillefer D, Léger J, Wang C and Tenniswood M

    Department of Biochemistry, University of Ottawa, Ontario, Canada.

    We describe the genomic structure of the rat TRPM-2 gene. This gene is induced de novo during the regression of the prostate and other hormone-dependent tissues after hormone ablation and plays an important role in apoptosis, or active cell death. TRPM-2 is a single copy gene, organized into nine exons, ranging in size from 47 base pairs (exon I) to 412 base pairs (exon IV), spanning a region of 13,750 base pairs. Comparison with sequences registered in the data bases shows that it has extensive homology to SGP-2, a gene that is expressed constitutively in the testis, although the sequences diverge dramatically in the 5'-untranslated region of the mRNA (coded for by exon I in TRPM-2), raising the possibility of alternative exon I usage in SGP-2. Primer extension, S1 nuclease protection, and extensive polymerase chain reaction analysis suggest that the TRPM-2 transcript from the prostate and the SGP-2 transcript from the testis are in fact identical and only contain the exon I sequence identified in the TRPM-2 genomic clone. Analysis of the promoter region of the TRPM-2 gene demonstrates that the putative control region contains several potential regulatory elements that may regulate the complex tissue-specific control of a gene which must be constitutively expressed in some tissues but repressed in others until induced during active cell death.

    The Journal of biological chemistry 1993;268;7;5021-31

  • Identification of the disulfide bonds in human plasma protein SP-40,40 (apolipoprotein-J).

    Choi-Miura NH, Takahashi Y, Nakano Y, Tobe T and Tomita M

    Department of Physiological Chemistry, School of Pharmaceutical Sciences, Showa University, Tokyo.

    SP-40,40, a human plasma protein, is a modulator of the membrane attack complex formation of the complement system as well as a subcomponent of high-density lipoproteins. In the present study, the positions of the disulfide bonds in SP-40,40 were determined. SP-40,40 was purified from human seminal plasma by affinity chromatography using an anti-SP-40,40 monoclonal antibody and reversed-phase, high-performance liquid chromatography (HPLC). The protein was digested with trypsin and the fragments were separated by reversed-phase HPLC. The peptides containing disulfide bonds were fluorophotometrically detected with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). The peptides containing more than two disulfide bonds were further digested with Staphylococcus aureus V8 protease and lysylendopeptidase, and the fragments were isolated by HPLC. The amino acid compositions and the amino acid sequences of the peptides containing only a disulfide bond were determined. Disulfide bonds thus determined were between Cys58(alpha)-Cys107(beta), Cys68(alpha)-Cys99(beta), Cys75(alpha)-Cys94(beta), and Cys86(alpha)-Cys80(beta). Since there was no free sulfhydryl groups in the SP-40,40 molecule, Cys78(alpha) and Cys91(beta) should also be linked by a disulfide bond. It is notable that all of the disulfide bonds in SP-40,40 are not only formed by inter-chain pairing, but also appear to form an antiparallel ladder-like structure between the two chains. The unique structure could be related to the functions of SP-40,40.

    Journal of biochemistry 1992;112;4;557-61

  • Clusterin: the intriguing guises of a widely expressed glycoprotein.

    Jenne DE and Tschopp J

    Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland.

    The glycoprotein clusterin has recently entered the scientific arena in diverse guises. It forms high-density lipoprotein complexes with apolipoprotein A-I, participates in the terminal complement reaction and serves as a granule constituent in neuronal and endocrine cells. Apically secreted, it is also found in the male reproductive tract and the tubular lumen of epithelial ducts. Thus, it may serve important functions in tissue remodelling, immune defense and transport of biologically active peptides.

    Trends in biochemical sciences 1992;17;4;154-9

  • SP-40,40, a protein involved in the control of the complement pathway, possesses a unique array of disulphide bridges.

    Kirszbaum L, Bozas SE and Walker ID

    Department of Veterinary Sciences, University of Melbourne, Parkville, Australia.

    SP-40,40 is a two-chain serum protein which acts in vitro as a potent inhibitor of the assembly of the membrane attack complex of human complement. It contains 10 cysteine residues, the numbers and locations of which are conserved in several mammalian species. Evidence is presented that all the cysteine residues are involved in interchain (alpha-beta) disulphide bonds. There are no free cysteine residues. The disulphide bond motif established in this study for SP-40,40 is unique and bears no obvious homology to those complement components whose disulphide bonds have been assigned, nor is there any homology apparent between SP-40,40 and other multi-chain proteins containing disulphide bonds.

    FEBS letters 1992;297;1-2;70-6

  • Regional localization of the gene for clusterin (SP-40,40; gene symbol CLI) to human chromosome 8p12-->p21.

    Dietzsch E, Murphy BF, Kirszbaum L, Walker ID and Garson OM

    University of Melbourne Department of Medicine, St. Vincent's Hospital, Fitzroy, Victoria, Australia.

    The human clusterin (SP-40,40) gene, designated CLI (complement lysis inhibitor) by the Human Gene Nomenclature Committee, has previously been assigned to chromosome 8. In situ hybridization allowed us to map the locus at 8p12-->p21.

    Cytogenetics and cell genetics 1992;61;3;178-9

  • Human gliomas and epileptic foci express high levels of a mRNA related to rat testicular sulfated glycoprotein 2, a purported marker of cell death.

    Danik M, Chabot JG, Mercier C, Benabid AL, Chauvin C, Quirion R and Suh M

    Département de Médecine, Université de Montréal, Qc, Canada.

    Clone pTB16 has been isolated by differential screening of a human glioma cDNA library. Northern blot analysis has shown that pTB16 expression is several times (greater than 11-fold) higher in gliomas than in a primitive neuroectodermal tumor. This observation was supported by in situ hybridization and extended to nine other gliomas. Expression was virtually absent in adenocarcinoma cells metastasized to brain. Malignant gliomas showed stronger hybridization than benign gliomas, while blood capillaries did not show hybridization. pTB16 mRNA was also shown to be expressed in established glioma cell lines and at high levels in epileptic foci, indicating that expression of the gene may be limited to certain cell types and that its upregulation is not merely a consequence of cellular proliferation. Nucleotide sequence analysis identified pTB16 as the human counterpart for rat testicular sulfated glycoprotein 2 (SGP-2), whose function in the reproductive system remains unknown. Although SGP-2 transcripts, and hence pTB16, were recently shown to be increased in neurodegenerative diseases such as scrapie in hamsters and Alzheimer disease in humans, our observations with brain tumors and epilepsy are suggestive of a role for pTB16 in neuropathologies in general and support the hypothesis of its involvement in tissue remodeling and cell death.

    Proceedings of the National Academy of Sciences of the United States of America 1991;88;19;8577-81

  • Characterization of a human high density lipoprotein-associated protein, NA1/NA2. Identity with SP-40,40, an inhibitor of complement-mediated cytolysis.

    James RW, Hochstrasser AC, Borghini I, Martin B, Pometta D and Hochstrasser D

    Department of Medicine, University Hospital, Geneva, Switzerland.

    Two peptides, NA1 and NA2, which we previously suggested to be associated with high density lipoproteins (HDLs), have been purified. Polyclonal antibodies against each peptide and a monoclonal antibody against NA2 have been used to further characterize them and their association with HDL. Immunoblotting studies revealed that the peptides form a complex of molecular mass of approximately 80 kd. Agarose gel filtration showed coelution of NA1/NA2 and apolipoprotein (apo) A-I, the structural protein of HDL. This was confirmed by fast protein liquid chromatography, which further indicated that up to 60% of NA1/NA2 was located within the lower density range of the HDL spectrum. Complementary studies with anti-apo A-I immunoaffinity columns provided evidence that at least 40% of NA1/NA2 was associated with HDL, an association easily disrupted by ultracentrifugal manipulation. Finally, partial amino acid sequences showed virtually complete homology with a recently identified protein, SP-40,40, or cytolysis inhibitor. The protein is suggested to have a powerful inhibitory effect on complement-mediated cell lysis. Our results could thus furnish an explanation for the previously observed modulating influence of HDL on complement activity.

    Arteriosclerosis and thrombosis : a journal of vascular biology 1991;11;3;645-52

  • Purification and characterization of apolipoprotein J.

    de Silva HV, Stuart WD, Park YB, Mao SJ, Gil CM, Wetterau JR, Busch SJ and Harmony JA

    Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, Ohio 45267-0575.

    Apolipoprotein J (apoJ), a unique 70-kDa component of high density lipoproteins in human plasma, consists of two disulfide-linked subunits designated apoJ alpha (34-36 kDa), and apoJ beta (36-39 kDa) which share pI values of 4.9-5.4 and which are recognized by a monoclonal antibody (mAb) 11. ApoJ and its subunits were purified to homogeneity from plasma by a combination of immunoaffinity chromatography, using mAb11 linked to Affi-Gel, and reverse-phase high performance liquid chromatography. ApoJ alpha and apoJ beta are both glycoproteins. When deglycosylated, the molecular mass of apoJ alpha is 24 kDa and that of apoJ beta is 28 kDa, suggesting that approximately 30% of the mass of each subunit is carbohydrate. The amino acid compositions of apoJ alpha and apoJ beta are very similar; however, the sequences of the first 30-amino acid residues are distinct. A comparison of peptide maps suggests that apoJ alpha and apoJ beta are not identical but share limited regions of homology. This possibility is supported by immunochemical data. Five additional mAb specific for apoJ were characterized. One of the mAb, like mAb11, reacts with both apoJ alpha and apoJ beta; the others react with apoJ alpha only. All mAb, including those which recognize both apoJ alpha and apoJ beta and those which recognize apoJ alpha only, immunoprecipitate a approximately 50-kDa protein synthesized from a liver mRNA template translated in a rabbit reticulocyte lysate. We propose that the apoJ alpha and apoJ beta subunits, which have limited homology, are derived by proteolytic cleavage of a common precursor.

    Funded by: NHLBI NIH HHS: HL22619, HL30999, HL41496; ...

    The Journal of biological chemistry 1990;265;24;14292-7

  • Apolipoprotein J: structure and tissue distribution.

    de Silva HV, Harmony JA, Stuart WD, Gil CM and Robbins J

    Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Ohio 45267-0575.

    The primary structure of apolipoprotein J (apoJ) was deduced by the combined strategies of protein sequencing and cDNA cloning and sequencing. ApoJ, an apolipoprotein associated with discrete subclasses of high-density lipoproteins, is encoded by a single gene in both the human and mouse genomes. ApoJ is synthesized as a 427 amino acid polypeptide that is posttranslationally cleaved at an internal bond between Arg-205 and Ser-206. The subunits of apoJ are designated apoJ alpha, corresponding to residues 1-205, and apoJ beta, corresponding to resides 206-427. The subunits are associated through disulfide bonds. Analysis of the primary structure of apoJ predicts the existence of amphiphilic helices, which may account for the association of apoJ with lipoproteins, and heparin-binding motifs in both subunits. ApoJ appears to be the human analogue of a rat protein present in high concentrations in the testis, sulfated glycoprotein 2. ApoJ mRNA (1.9 kb) is expressed in all but one tissue examined. The mRNA is present in relatively high levels in brain, ovary, testis, and liver, is less abundant in heart, spleen, lung, and breast, and is absent in T-lymphocytes. ApoJ is unique among previously characterized human apolipoproteins in its structure and tissue distribution.

    Funded by: NHLBI NIH HHS: HL30999, HL41496; NICHD NIH HHS: HD13225; ...

    Biochemistry 1990;29;22;5380-9

  • Human seminal clusterin (SP-40,40). Isolation and characterization.

    O'Bryan MK, Baker HW, Saunders JR, Kirszbaum L, Walker ID, Hudson P, Liu DY, Glew MD, d'Apice AJ and Murphy BF

    St. Vincent's Hospital, Melbourne, Australia.

    Molecular cloning of the human complement inhibitor SP-40,40, has revealed strong homology to a major rat and ram Sertoli cell product, sulfated glycoprotein-2, known also as clusterin. This study reports the purification and characterization of human seminal clusterin. Two-dimensional gel electrophoresis revealed charge differences between clusterin purified from semen and the serum-derived material. Both preparations demonstrate comparable hemagglutination (clustering) activity and inhibition of C5b-6 initiated hemolysis. The average clusterin concentration in normal seminal plasma is considerably higher than that found in serum. Mean seminal plasma clusterin concentrations were significantly lower in azoospermia caused by obstruction or seminiferous tubule failure than with oligospermia or normospermia. Only men with vasal agenesis had undetectable seminal clusterin, suggesting that some of the seminal clusterin is produced by the seminal vesicles. Immunofluorescence of human spermatozoa revealed that clusterin was detected on 10% of spermatozoa, predominantly those that were immature or had abnormal morphology. A pilot study of 25 patients suggests that seminal clusterin concentration, together with sperm motility and morphology, is correlated with the fertilization rate in vitro. The function of seminal clusterin is unknown. Its extensive distribution in the male genital tract and its high concentration in seminal plasma suggests an important role in male fertility.

    The Journal of clinical investigation 1990;85;5;1477-86

  • A serum protein SP40,40 modulates the formation of membrane attack complex of complement on erythrocytes.

    Choi NH, Mazda T and Tomita M

    Department of Physiological Chemistry, School of Pharmaceutical Sciences, Showa University, Tokyo, Japan.

    SP40,40 was isolated from the soluble membrane attack complex (SC5b-9) by HPLC using a reverse-phase column. Amino acid compositions of its alpha- and beta-subunits were similar to each other with the exception of glycine content. Amino-terminal sequences of its alpha- and beta-subunits were identical to those of the subunits prepared from human serum, respectively, indicating that there was no degradation of SP40,40 during incorporation into SC5b-9. When guinea pig erythrocytes were incubated with C56f, C7, C8 and C9 in the presence of SP40,40, SP40,40 enhanced the hemolysis. The protein, however, inhibited the hemolysis when erythrocytes were pre-incubated with C56f and SP40,40 prior to the addition of C7, C8 and C9. These findings indicate that SP40,40 modulates the formation of membrane attack complex by interacting with C56f at the first step, and that the co-existence of other factors, besides C56f, is required for the enhancing activity of SP40,40.

    Molecular immunology 1989;26;9;835-40

  • Changes in brain gene expression shared by scrapie and Alzheimer disease.

    Duguid JR, Bohmont CW, Liu NG and Tourtellotte WW

    Geriatric Research, Education and Clinical Center, Edith N. Rogers Memorial Veterans Hospital, Bedford, MA 01730.

    We have isolated two recombinant cDNAs whose corresponding RNAs have an increased abundance in scrapie-infected hamster brain. DNA sequence analysis has shown that these two recombinants represent the genes for sulfated glycoprotein 2 and transferrin. The abundance of sulfated glycoprotein 2 RNA is increased in hippocampus from patients with Alzheimer disease and Pick disease, whereas transferrin RNA is not strongly modulated in these conditions. Expression of two previously identified scrapie-modulated genes, encoding glial fibrillary acidic protein and metallothionein, is also increased in both of these neurodegenerative diseases.

    Funded by: NIA NIH HHS: R01 AG05894-16

    Proceedings of the National Academy of Sciences of the United States of America 1989;86;18;7260-4

  • Molecular structure and functional characterization of a human complement cytolysis inhibitor found in blood and seminal plasma: identity to sulfated glycoprotein 2, a constituent of rat testis fluid.

    Jenne DE and Tschopp J

    Institut de Biochimie, Université de Lausanne, Switzerland.

    A component of soluble terminal complement complexes was identified and affinity-purified to homogeneity by using a monoclonal antibody previously developed against the soluble C5b-9 complex. The protein, which we have designated complement cytolysis inhibitor (CLI), has a molecular mass of 70 kDa and consists of two nonidentical, disulfide-linked subunits of 35 kDa. Partial amino acid sequences determined for the amino-termini of the two subunits were identical with those of a recently characterized serum protein called SP-40,40. An almost full-length cDNA clone of 1651 base pairs was isolated from a human liver cDNA library by using long synthetic oligonucleotides as probes. The encoded amino acid sequence of CLI consists of 427 amino acid residues preceded by a 21-residue-long typical signal peptide and shows an overall 75.6% amino acid sequence homology to sulfated glycoprotein 2 (SGP-2), a major Sertoli cell-derived protein of rat testis fluid. As in SGP-2, proteolytic processing between residues 206 and 207 yields the two disulfide-linked subunits of plasma CLI. CLI and SGP-2 were shown to be orthologous single-copy genes in humans and rats by Southern blotting experiments. In addition, CLI was immunologically identified in human seminal plasma. Functional studies with purified terminal complement components showed that CLI suppresses the cytolytic potential of nascent C5b-7 complexes at physiological blood plasma concentrations (approximately 50 micrograms/ml). Its presence on the surface of mature sperm cells and its relative abundance in seminal plasma (approximately 250 micrograms/ml) suggest that CLI protects sperm cells and epithelial tissues against complement attack in the male reproductive tract.

    Proceedings of the National Academy of Sciences of the United States of America 1989;86;18;7123-7

  • Molecular cloning and characterization of the novel, human complement-associated protein, SP-40,40: a link between the complement and reproductive systems.

    Kirszbaum L, Sharpe JA, Murphy B, d'Apice AJ, Classon B, Hudson P and Walker ID

    Department of Veterinary Preclinical Sciences, University of Melbourne, Victoria, Australia.

    The cDNA sequence encoding the human complement-associated protein, SP-40,40, is reported. The two chains of SP-40,40 are coded in a single open reading frame on the same mRNA molecule, indicating the existence of a biosynthetic precursor protein which matures post-synthetically by the proteolysis of at least one peptide bond. The precursor is preceded by a signal sequence for vectorial export and contains six N-linked glycosylation sites distributed equally between the two chains of the structure. The sequence of the SP-40,40 precursor bears a 77% identity to a rat sulphated glycoprotein-2 (SGP-2) which is the major secreted product of Sertoli cells. The presence of SP-40,40 within human seminal plasma at levels comparable to those in serum was demonstrated, indicating that SP-40,40 and SGP-2 are serum and seminal forms of the same protein. A sequence of 23 amino acids within the beta-chain of SP-40,40 exhibited significant homology to corresponding segments located within complement components C7, C8 and C9. The short cysteine-containing motif represented the only evidence of a possible vestigial relationship between SP-40,40 and other complement components. The precise role of SP-40,40 is not known in either blood or semen but the present findings document an intriguing link between the immune and the reproductive systems.

    The EMBO journal 1989;8;3;711-8

  • Isolation and characterization of sulfated glycoprotein from human pancreatic juice.

    Yokoyama M, Matsue H, Muramoto K, Sasaki M, Ono K and Endo M

    Department of Biochemistry, Hirosaki University School of Medicine, Japan.

    Sulfated glycoprotein was isolated by precipitation from dialyzed human pancreatic juice and purified by ion-exchange chromatography followed by repeated gel chromatography. The sulfated glycoprotein was obtained as a sulfated glycoprotein-lipid complex by Sepharose CL-2B chromatography. Lipids aggregating with the sulfated glycoprotein were glycolipids such as ceramide trihexoside, and simple lipids such as cholesterol and cholesterol ester. This glycoprotein was resistant to digestion with mucopolysaccharidases or alpha-amylase, and consisted of 60% (w/w) protein and 40% sugars. The polypeptide core was characterized by a high content of serine, threonine, aspartic acid and glycine, but lacked cysteine. Its sugar components were N-acetylglucosamine, N-acetylgalactosamine, galactose, fucose and sialic acid. Absorption at 1240 cm-1 and 820 cm-1 by infrared spectroscopy indicated the presence of a sulfate ester group. All the carbohydrate chains of this sulfated glycoprotein, which are polydisperse and heterogeneous, were O-glycosidically linked through N-acetylgalactosamine to a protein core.

    Biochimica et biophysica acta 1988;967;1;34-42

  • SP-40,40, a newly identified normal human serum protein found in the SC5b-9 complex of complement and in the immune deposits in glomerulonephritis.

    Murphy BF, Kirszbaum L, Walker ID and d'Apice AJ

    Department of Nephrology, Royal Melbourne Hospital, Australia.

    We report herein the isolation and initial characterization of a novel protein, termed SP-40,40, which is present at moderate levels (35-105 micrograms/ml) in normal human serum. SP-40,40 is deposited in the renal glomeruli of patients with glomerulonephritis but is not found in normal glomeruli. The protein is a heterodimeric structure of relative molecular mass 80 kD, both chains of which are of a similar size (40 kD). The amino-terminal sequences of both chains are unrelated to one another and possess no significant homology to any known protein sequence. The tissue distribution of SP-40,40 closely resembles that of the terminal complement components and its physicochemical properties are similar to, but distinct from, those of the S protein of complement. We have identified SP-40,40 in the SC5b-9 complex of complement and have demonstrated incorporation of labeled SP-40,40 into this complex. These data suggest that SP-40,40 is an additional component of SC5b-9.

    The Journal of clinical investigation 1988;81;6;1858-64

  • HDL particle associated proteins in plasma and cerebrospinal fluid: identification and partial sequencing.

    Hochstrasser AC, James RW, Martin BM, Harrington M, Hochstrasser D, Pometta D and Merril CR

    Biochemical Genetics Section, National Institute of Mental Health, Bethesda, Maryland 20892.

    The proteins from plasma HDL particles isolated by immunoaffinity chromatography on anti-apolipoprotein A-1 affinity columns have been analysed and purified by high resolution two dimensional gel electrophoresis. Two of the lipoprotein-associated proteins found in the HDL plasma fraction, previously referred to as NA1 and NA2, have also been found in cerebrospinal fluid. After separation by 2DGE, these two proteins were transferred to PVDF membranes, stained and cut out for N-terminal sequencing. The partial sequences (11 and 13 amino acids) obtained for the two HDL particle associated proteins do not match any of those included in the December 1987 National Biomedical Research Foundation (NBRF) database, and there are no significant sequence similarities.

    Applied and theoretical electrophoresis : the official journal of the International Electrophoresis Society 1988;1;1;73-6

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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