P 01. MULTIPLE LEVELS OF CLUSTERIN REGULATION FOLLOWING PROTEASOME AND LYSOSOME INHIBITION IN NORMAL AND HUMAN CELLS

Eirini Balantinou, Ioannis P. Trougakos, Niki Chondrogianni and Efstathios S. Gonos

Molecular & Cellular Aging Laboratory, Institute of Biological Research & Biotechnology, National Hellenic Research Foundation, Athens, Greece. E-mail: [email protected]

ApoliporoteinJ/Clusterin (CLU) is a secreted with numerous physiological roles. The aim of our work was to examine the effect of proteasome or lysosome inhibition on CLU expression and to determine whether those proteolytic pathways are implicated in CLU degradation. Complete, as well as partial proteasome inhibition increased both mRNA and levels of CLU in U-2 OS and WI38 cells. Since transcriptional elements of HSF-1 and AP-1 are found in CLU promoter, we sought to identify whether CLU mRNA up-regulation after proteasome inhibition is mediated by one of these transcription factors. Pre-treatment of cells with a HSF-1 inhibitor abolished both CLU mRNA and protein induction in U-2 OS cells; whereas, in WI38 cells, although it prevented CLU mRNA up-regulation it further increased CLU protein levels. The AP-1 inhibitor did not abolish proteasome-inhibition- mediated CLU up-regulation in either line. Furthermore, pulse-chase experiments revealed accumulation of the intracellular CLU protein form upon proteasome inhibition, while a CLU-specific antibody immunoprecipitated additional higher molecular weight bands after proteasome inhibition that may correspond to ubiquitinated CLU. Lysosome inhibition led to elevated levels of CLU protein but did not induce any changes in the RNA level. Addition of a CLU antibody to the cell medium resulted in reduced protein levels of the secreted form intracellularly, suggesting that secreted CLU is endocytosed and targeted for degradation at the lysosome. These findings indicate that CLU up-regulation relates to both positive transcriptional regulation by HSF-1, after proteasome inhibition, and post- translational accumulation due to reduced proteasomal and lysosomal degradation.

P 02. CLUSTERIN AS A POSSIBLE BIOMARKER FOR COLORECTAL CARCINOMA. A PROTEOMIC STUDY ON Caco-2 CELL LINE AND HUMAN TISSUE.

Sonia Blanco-Prieto, García-Lorenzo, A., Álvarez-Chaver, P.,Rodríguez-Piñeiro, AM., Rodríguez-Berrocal, FJ., Martínez-Zorzano, VS and Páez de la Cadena, M.

Department of Biochemistry, Genetics and Immunology; University of Vigo, Spain. E-mail: [email protected]

In spite of having been discovered almost three decades ago, no defined biological role has been attributed to CLU, nor a clear understanding of its isoform variability has been achieved. Using proteomic techniques on total serum in order to find that could be used as potential colorectal cancer biomarkers, clusterin (CLU) proved to be differentially expressed and was chosen for a more detailed study. Concanavalin A chromatography of serum samples was performed to separate N- from the remaining, followed by immunodetection, revealing a fraction of CLU with altered associated with the neoplasia. Furthermore, a great variability in clusterin isoforms was found in serum of colorectal patients, in contrast to the classical reported sCLU and nCLU. Looking for a deeper understanding, analysis of CLU expression using an in vitro model was carried out. Caco-2 cells were chosen as they spontaneously differentiate in culture, exhibiting functional and morphological similarities with normal intestine mucosa. Culture media and cell scrapings were investigated by immunodetection for its CLU content, so serum with culture medium and tissue with cell homogenates could be correlated. On cell scrapings much weaker signal than on culture was obtained and mainly two CLU forms, with molecular weights of 38 and 59 kDa, were observed. Denaturing 2- D blots revealed isoforms with more basic pI on undifferentiated cells, which could be due to the altered glycosylation on this particular cell line. On culture media a higher CLU expression was found for undifferentiated cells, with up to fifteen isoforms expanding over a wider range of pI in relation to the reported in serum. These results would confirm its predicted role as an antiapoptotic and proliferative protein. Preliminary results of the isoform pattern of CLU on human tissue showed no uniform behaviour between normal and tumoral mucosa. P 03. THE REGULATION OF THE IGF-1-sCLU PATHWAY AFTER IONIZING RADIATION IN CANCER

Eva M. Goetz, Yonglong Zou, and David A. Boothman

Simmons Comprehensive Cancer Center, Program in Cell Stress and Cancer Nanomedicine, University of Texas Southwestern Medical Center at Dallas, TX. E-mail: [email protected]

Pro-survival proteins induced by ionizing radiation (IR) can promote resistance of tumor cells. One such protein, secretory clusterin (sCLU), protects cells against death induced by IR and chemotherapeutic agents. Due to the importance of sCLU in resistance to therapy, we characterized its induction after IR in cancer cells. We determined that both basal and IR-induced expression of sCLU is dependent on insulin like growth factor 1 (IGF-1) signaling in MCF-7 and other cell types. We have also observed that sCLU expression is generally lower, and IR induction is reduced, in wild-type expressing cells. This is consistent with reports of upregulated sCLU in tumors, since over 50% of have mutations in p53, and those cells that have wild-type p53 commonly have other alterations in the p53 regulatory pathway (, PTEN, Akt). Our recent data suggest that sCLU expression is regulated by the stress-induced synthesis of the IGF-1 ligand. We hypothesize that activation of sCLU after IR and repression by p53 is, in fact, directly due to the regulation of IGF-1 in cancer cells in the following manner: (a) IGF-1 is up-regulated after IR; and (b) p53 suppresses IGF- 1 transcription causing a downstream change in sCLU levels. We show IR-induced expression of IGF-1 ligand and IGF-1 promoter activity, and that the extent of the IR induction is dependent on wild-type p53 expression. Cells that are positive for wild- type p53 have minimal induction of the IGF-1 promoter compared to cells that are knocked-down for p53. We have also observed that Nutlin-3 is a potent inhibitor of sCLU expression in cells with wild-type p53, and we can partially abrogate this by administration of IGF-1. We anticipate this work will uncover new targets that can be altered to decrease sCLU in cells, and lead to radio- and chemo-sensitization.

P 04. CLUSTERIN AS A REGULATOR OF PANCREAS REGENERATION

Song Lee, Seok-Woo Hong, Ranjan K.C, Woo-Chul Lim, Jun-Seok Kim, Young-Jun Shim, Joo-Yea Hwang, Bon-Hong Min and In-Sun Park

Department of Anatomy and Center for Advanced Medical Education by BK 21 project, College of Medicine, Inha University, Shinheung-Dong, Jung-Gu, Incheon, 400-103, Korea. Corresponding author. Tel.: +82 32 890 0911; fax: +82 32 884 2105. E-mail address: [email protected]

Development of pancreas during embryonic stage can be regulated by various transcription factors that are inactivated gradually after postnatal stage, and pancreatic tissue becomes quiescent and is maintained by simple proliferation of the preexisting cells. However, in adult pancreas, new pancreatic tissue can be developed after mechanical or chemical injuries by neogenic regeneration. We hypothesized that some morphogenic factors can be involved in the neogenic formation of the adult pancreatic tissue. We previously reported that over expression of the clusterin strongly induces beta cell transformation from neogenic duct cells and suggested that clusterin plays a pivotal role in pancreas neogenesis. In this study, in order to verify the clusterin action on the neogenic regeneration of the pancreatic tissue, we investigated the morphogenic features both in endocrine and exocrine pancreas during pancreas regeneration using clusterin deficiency mice (CLU-/-) after sub-total pancreatectomy which leads to neogenic regeneration of pancreas in murine species. We confirmed that clusterin expression was absent in CLU-/-, while being over- expressed in developing acini cells and neogenic ductules in wild type mice (CLU+/+) after Px. Regenerating lobule was significantly reduced in CLU-/- whereas dynamic tissue regeneration occurred in CLU+/+ following Px. Neogenic ductules and developing acini, the indicators of exocrine regeneration, were poorly developed in the neogenic lobules of the CLU-/- compared to those of the CLU+/+. The CLU-/- demonstrated, moreover, a considerable reduction of neogenesis of the insulin secreting beta cells in regenerating lobules and lower level of plasma insulin. In addition, we observed an uncontrolled metabolism in the CLU-/- mice showing a hyperglycemia and impaired glucose tolerance after Px. These data lead us to conclude that absence of clusterin during pancreatic regeneration results in insufficient recovery of the tissue after pancreatic injuries, and causes the abnormal glucose metabolism and diabetes.

P 05. CLUSTERIN AND FATTY ACID SYNTHASE: MOLECULAR FOCUS ON A COMPLEX CROSS-TALK

Paola Mazzarelli, Sabina Pucci, Fabiola Sesti, Luigi G. Spagnoli.

Lab. of Molecular Pathology, Dept. of Biopathology, University of Rome Tor Vergata, 00133, Rome, Italy. E-mail: [email protected]

Clusterin is a nearly ubiquitous glycoprotein and upregulation of clusterin mRNA and protein levels are detected in response to injury and in diverse states. It functions in membrane recycling, in apoptotic cell death and as a stress-induced secreted protein, amongst others. In tumours, the secreted isoform (s-CLU) acts as cytoprotective protein promoting the survival of malignant cells. Lipid metabolism is markedly altered in the tumoral context. Neoplastic cells use endogenously synthetic fatty acids to support membrane synthesis. The long-chain fatty acids accumulated can be toxic and also induce cell death. Clusterin, which binds hydrophobic macromolecules, would act to "clear" potentially harmful cellular components, enhancing survival of cancerous cell. The major enzyme required for the synthesis of fatty acids is the fatty acid synthase (FASN). FASN is up-regulated in a wide array of solid tumours. Further, FASN inhibition triggers in human cancer cells and experiments of RNA interference-knockdown confirmed its role as a novel metabolic oncogene. Gene promoter of FASN is induced by ErbB2 receptor oncogene (amplified in 25% of breast cancers) and its effector PI3K. We hypothesized that FASN and Clusterin protein levels could be correlated, thus promoting cancer cell survival. We studied human breast carcinomas and in vitro cells (MCF-7, BT474). We found a significant increase of FASN protein in breast human carcinomas. The breast cancer tissues analyzed, which had ErbB2 amplification (characterized by IHC and FISH) displayed FASN over-expressed in the 64% only of the cases. The expression levels of FASN would be an indicator of Her2 transduction activity. In our breast cancer model, increasing endogenous synthesized fatty acids together with increasing levels of IL-6 (also induced by Her2/Neu signalling and sustained in breast cancer by autocrine loop), cooperate to induce pro- survival s-Clusterin. In breast cancer, lipid as steroid hormone dexamethasone, used in anti-cancer therapy, induces s-CLU, contrasting the beneficial effects of the classical antitumoral drugs. Our observation makes attractive the hypothesis for new combined experimental therapies in breast cancers, which target as the metabolic oncogene FASN, as the pro-survival s-CLU.

P 06. POSSIBLE CLUSTERIN EFFECTS IN THE MODULATION OF NF-kB SIGNAL PATHWAY IN THE PROSTATE GLAND

S. Astancolle, G. Fregni, M. Marchetti, Davide Pellacani, P. Davalli, S. Bettuzzi and A. Corti

Dipartimento di Scienze Biomediche, Universita' di Modena e Reggio Emilia, Via G. Campi 287-41100 Modena, Italy. E-mail: [email protected]

The aim of this project is to understand the possible influence exerted by clusterin (Clu) isoforms on the principal components of the NF-kB signal pathway in the prostate gland. Growing data show that activation of NF-kB signalling may play a critical role in cancer development and progression. This role resides in its ability to modulate the expression of involved in apoptosis inhibition, cell proliferation, cell migration and adhesion. NF-kB is activated in human and the expression of its target genes, involved in tumorigenesis (Bcl-2, cyclin-D1, MMP-9, VEGF), is higher in cancer specimens than benign tissues. Human prostate epithelial cells, immortalised with SV40 (PNT1A cells), that were transfected either with empty vector (controls) or with the vector containing the cDNA coding for full length Clu (Clu clones) indicate that: a) Clu clones show a diminished level of phosphorylation of IkB alpha and thence higher abundance of IkB alpha (an inhibitor of NF-kB) in the . This permits a greater availability for the formation of the inactive complex with NF-kB. b) As expected, the immunocytochemistry analysis indicated in Clu clones a prevalent localization of NF-kB (inactive form) to the cytoplasm, while with controls the nuclear form (active) prevails. This was confirmed by Western analysis showing higher levels of pNF-kB in controls as compared to Clu clones. c) To confirm the minor activation of NF-kB factor in Clu clones in comparison to mock ones, the expression level of two NF-kB target genes, Bcl-2 and cyclin-D1, was analyzed. Western analysis indicated a higher level of these two genes in controls than in Clu ones. These data strongly suggest an involvement of clu isoforms in the modulation of the principal components of NF-kB pathway and corroborate our previous observations indicating an early role in the onset and progression of prostatic neoplasia.

P 07. ANTICLUSTERIN TREATMENT OF BREAST CANCER CELLS IN CREASES THE SENSITIVITIES OF AND TAMOXIFEN AND COUNTERACTS THE INHIBITORY ACTION OF DEXAMETHASONE ON CHEMOTHERAPY-INDUCED CYTOTOXICITY

Roldán MJ, Téllez T, Serrano A, García-Aranda M, Gleave ME, Hortas M, Jiménez E, Maximino Redondo

Department of Biochemistry, Hospital Costa del Sol, Carretera de Cádiz Km 187, 29600 Marbella, Málaga, Spain. E-mail: [email protected]

Overexpression of the apoptosis-related protein clusterin is associated with breast cancer development and tumor progression. We describe the use of clusterin-specific antisense oligonucleotides and antibodies to sensitize breast carcinoma cells to anticancer drugs routinely used in breast cancer therapy. MCF-7 cells and MDA-MB 231 were treated with the oligonucleotide or antibody, chemotherapeutic agents (doxorubicin or paclitaxel), tamoxifen, or with combinations of these. Treatments that include antisense clusterin oligonucleotide or antibody to clusterin have been shown to reduce the number of viable cells more effectively than treatment with the drugs alone. We also demonstrate that dexamethasone pretreatment of breast cancer cell lines inhibits chemotherapy-induced cytotoxicity and is associated with the transcriptional induction of clusterin. However, anticlusterin treatment increases chemotherapy-induced cytotoxicity, even in the presence of glucocorticoids, suggesting a possible role for these proteins in glucocorticoid-mediated survival. These data suggest that combined treatment with antibodies to clusterin or antisense clusterin oligodeoxynucleotides and paclitaxel, doxorubicin or tamoxifen could be a novel and attractive strategy to inhibit the progression of breast carcinoma by regulation of the clusterin function. Moreover, glucocorticoid activation in breast cancer cells regulates survival signaling by the direct transactivation of genes like clusterin which encode proteins that decrease susceptibility to apoptosis. Given the widespread clinical administration of dexamethasone before chemotherapy, understanding glucocorticoid-induced survival mechanisms is essential for achieving optimal therapeutic responses.

P 08. CLUSTERIN EXPRESSION IS MODULATED BY PROMOTOR METHYLATION IN HUMAN CARCINOMAS

Maria J. Roldán, Redondo M, Martín E, Serrano A, Téllez T, Méndez R, Hortas ML, Jiménez E.

Department of Biochemistry, Hospital Costa del Sol, Carretera de Cádiz Km 187, 29600 Marbella, Málaga, Spain. E-mail: [email protected]

Because production of clusterin is associated with cancer progression, a better understanding of how clusterin expression is regulated may yield new strategies for diagnosis and treatment. The present study was designated to examine the role of clusterin methylation as an indicator of clusterin production in tumor cell lines and breast tissue samples. For this purpose, we used methylation-sensitive restriction analysis followed by polymerase chain reaction (PCR). Non tumoral breast samples showed no expression for clusterin by immunohistochemistry and RT-PCR, respectively, and a methylated state was found in the promoter region of the gene. However, a demethylated state was found in four of six cell lines analyzed. The mammary tumor cell line MDA and PC-3 (prostate carcinoma), presented a demethylated state with a strong constitutive expression of clusterin. MCF-7 (-dependent mammary carcinoma) also showed no methylation in the promoter regions but presented a slight expression of clusterin. However this expression can be stimulated. Finally, MSR3 () presented a null expression of clusterin and the promoter region was methylated. This inverse correlation 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 and two presenting focal expression of clusterin. A demethylated state was detected in 7 breast tumor samples with five of them presenting a strong expression with more than 50% of the cells stained by immunohistochemistry. The importance of DNA methylation in the control of clusterin expression in cancer cells is further strengthened by demonstration of re- expression of clusterin mRNA in clusterin-negative melanoma cell line MSR3 with a demethylating agent 5-aza-2'-deoxycytidine. We conclude clusterin expression is under epigenetic control via methylation of its promoter.

P 09. AUTOANTIBODIES AGAINST CLUSTERIN IN PROSTATE CANCER PATIENTS

Göran Ronquist, L Carlsson, G Ronquist, A Semjonow, C Wülfing, A Larsson

Göran Ronquist, Department of Medical Science, University Hospital, Uppsala, Sweden. E-mail: [email protected]

Prostasomes are small membrane-surrounded extracellular vesicles secreted by the epithelial cells of the prostate contributing to the composition of prostatic fluid as part of semen. The average diameter is 150 nm and the bilayered membrane has a highly ordered structure, resembling lipid rafts. The highly ordered prostasome membrane constitutes a good environment for signalling proteins and GPI-anchored proteins. Moreover, the ability to synthesize and release prostasomes is preserved by prostate cancer cells. It has previously been reported that prostasomes derived from malignant cells raises autoantibodies and an exploration of the immunogenic proteins of these prostasomes revealed clusterin as a major autoantigen. Here we examined the Spearman rank-correlation between serum autoantibody levels against clusterin and prostate cancer related variables in 391 consecutive patients with suspected prostate cancer. A significant “inverse” correlation existed between anti-clusterin ELISA titres and lymph node metastases (p=0.047), but only 11 out of 161 patients had metastases. Also, these titres correlated significantly to total prostate (p=0.021) and transitional zone (p=0.015) volumes of the patients.

P 10. THE DETERMINATION OF SERUM AND CEREBROSPINAL FLUID CLUSTERINE UTILIZED IN THE DIAGNOSTICS OF THE CNS AFFECTION – A PILOT STUDY

David Stejskal, Karpíšek M, Vavroušková J

Department of Laboratory Medicine, Sternberk Hospital, Jivavská 20, 785 16 Sternberk, Czech Republic. E-mail: [email protected]

Background: Recently, there has appeared information concerning the possibility of determining the presence and extent of the brain disorder by means of examining selected parameters in the cerebrospinal fluid (CSF) or blood. One of these potential indices could be clusterine, an apoptosis inhibitor. Aim: The study was aimed at the determination of the diagnostic effectiveness of examining clusterine in serum and CSF of patients with their CNS involvement. Methods: Thirty-eight probands underwent the examination. The subjects were divided into two groups according to the presence of the CNS affection due to various causes (without/with involvement). Cerebral CT examination, lumbar puncture with morphological and biochemical investigations of CSF and determining the indices of albumins, immunoglobulins, cystatine and clusterine were carried out in all the probands. Results: Subjects with signs of the CNS involvement showed higher concentrations of clusterine in CSF and its index in comparison with probands without the CNS affection (medians of clusterine, or index Csf/S 5760 vs 3435 kU/l, p < 0.01). There were revealed significant correlations between CSF clusterine and indices of albumin (r = 0.58), immunoglobulin G (r = 0.66), immunoglobulin M (r = 0.24), immunoglobulin A (r = 0.66), and cystatine C in CSF ( r = 0.43). After the classification of patients based on the presence of CNS disorder, it was found out that the determination of clusterine in CSF had the sensitivity of 79 % and specificity of 86 % at the value of clusterine > 4230 kU/l, and the sensitivity of clusterine index was 65 % and specificity 93 % at the value > 0.03 kU/l. Conclusion: Clusterine seems to be a promising laboratory index of a damage to the brain cells with satisfactory diagnostic effectiveness.

P 11. STUDY OF THE ROLE OF J IN THE PATHOGENESIS OF ALZHEIMER’S DISEASE IN A TRANSGENIC MOUSE MODEL

Kalliopi Thanopoulou and Spiros Georgopoulos

Laboratory of Cellular Neurobiology, Centre of Basic Research I, Biomedical Research Foundation of the Academy of Athens. E-mail: [email protected]

Alzheimer’s disease (AD) is a progressive neurodegenerative disease that attacks the basic cognitive functions of the brain. Amyloid plaques and neurofibrillary tangles are established as the hallmarks of AD. Important factors in the pathogenesis of AD are the APP (Amyloid Precursor Protein), the presenilins 1 and 2 and the ApoE allele, ε4 [1]. Apolipoprotein J (apoJ) is a multifunctional disulfide linked heterodimeric glycoprotein, encoded by a gene located on 8. It is present in almost all mammalian tissues and physiological fluids, such as plasma, milk, urine, CSF and semen [2] and it is primarily distributed within High Density Lipoproteins. Apolipoprotein J has been shown to be involved in spermatogenesis, development, complement inhibition, lipid transport, maintenance of AD’s Aβ peptides solubility and apoptosis. Recent studies have implicated apoJ in the pathogenesis of Alzheimer’s disease and have further elucidated its role in amyloid plaque formation and its additive effect with apoE in this process [3]. ApoJ has been found in AD cortex in both diffuse and compact plaques [4] and it may act intracellularly as a protective agent against the formation of neurofibrillary tangles [5]. In order to study the role of the Apolipoprotein J in the pathogenesis of Alzheimer’s Disease, we have generated transgenic mouse lines that express apoJ. ‘Conventional’ transgenic mice where the human apoJ has been expressed in the brain under the PDGF promoter have been generated. Also conditional tissue specific transgenic mice, with the Cre-loxP technology, have been generated that express human ApoJ. These mice models are currently under evaluation to explore a more detailed role for apoJ in and Alzheimer’s disease.

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