RICERCA ITALIANA     

The biological role of clusterin, a new potential tumor-suppressor gene, in the control of normal and pathological proliferation of prostate epithelium

Università degli Studi di Parma

Abstract

Clusterin (CLU) is the most potently over-expressed gene in regressing rat ventral prostate following several stimuli, including androgen ablation caused either by surgical castration or administration of 5-alfa reductase type II inhibitors (finasteride), and administration of non-hypercalcemic vitamin D analogues. Nearly ubiquitous in the body and involved in many physio-pathological processes among which are membrane remodelling, organ involution and apoptosis, CLU gene is repressed during proliferation and induced in cell atrophy, cell quiescence and tissue regression. CLU is an "enigmatic" gene, whose role in the process of apoptosis has not been defined yet, being still debated in many laboratories. Different functional roles, sometimes antithetic, have been suggested for CLU. This is probably based on the existence of different CLU isoforms possibly exhibiting different functions. One of these isoform could induce cell survival and resistance to apoptosis, while another could be pro-apoptotic. Also CLU involvement in neoplastic transformation is not clear yet. This gene is present as a single copy gene on human chromosome 8 in 8p21, a region frequently subjected to genetic loss occurring as an early event during prostate cancer (CaP) onset. CaP is one of the more frequent tumours in man. Its prevalence increases with age, thus CaP will soon became a major medical, social and economical issue because of constant increase of life expectation in Western countries. Since >>>

Principal Investigator

Saverio BETTUZZI Università degli Studi di PARMA

Research Objectives

The research project here presented is aimed to study the role of CLU in the control of normal and pathological proliferation of the prostate epithelial cells and in the genesis and progression of CaP by pursuing the following objectives:

a) to develop new in vitro experimental models for over-expression of secreted CLU (sCLU) and intracellular CLU (nCLU) through transient trasfection and selection of stable recombinant clones of PNT1A and PC-3 using the multicistronic vector pQUATTRO-tTA or other suitable expression vectors.

b) to identify the molecular partners of CLU. Identification of the genes regulated at transcriptional level by sCLU or nCLU in PNT1A and PC-3 cells through Dna microarray. Validation of the data through real-time PCR analysis.

c) to study the biological modifications induced by over-expression of sCLU or nCLU in PNT1A and PC-3 cells:
- evaluation of proliferation rate, apoptosis induction and changes in cell cycle progression;
- adhesion, migration, invasion, enzymes involved in the degradation of the extra-cellular matrix, MMPs, integrins (alphavbeta3, beta4);
- metabolism of polyamines, genes markers of proliferation and cellular quiescence;
- IGF-1, IGF-II, IGF-R1, IGFBPs systems;
- neuroendocrine differentiation and CrA;
- activation of IRS-1 and Shc;
- responsiveness to androgens and oestrogens;
- changes in the expression of the >>>

First Results

The partial results expected after the first year of research activity will be the following:

1. to generate and characterize sCLU and nCLU over-expressing recombinant clones from parental PNT1A and PC-3 cell lines by taking advantage of the pQUATTRO-tTA multicistronic vector for inducible and auto-regulated expression of CLU gene;

2. to perform transient transfection experiments in the same cell model to analyze the parameters of cell vitality and to compare the data so obtained with those data of point 1.

3. evaluate CLU expression in primary cultures of epithelial and stromal cells from CaP and benign tissue

4. production of adequate amount of transgenic TRAMP mice by inbreeding and characterization of recombinant offspring by RT-PCR for collaborative work;

5. study CLU expression using commercially available antibodies during CaP progression in close comparison to morphological progression (Gleason grade) and with other molecular markers such as MIB1/Ki67, histone H3 e ornithine decarboxyilase (markers of cell proliferation), Gas1 (marker of the quiescent state of the cell), TUNEL, p53 e Bcl-2 (apoptotic markers), integrins, cadherins, metalloproteases (markers of invasion), androgen and estrogen receptors espression (hormone-dependance markers) and growth factors such as IGFs, IGF-R, IGFBPs, trascription factors such as IRS-1, Shc, cell cycle control genes such as RIZ1, RIZ2 and RB. The study >>>

Timescale

24 months

National and international background

CLU is a gene that is transiently over-expressed in regressing rat prostate following castration (1) and administration of anti-androgens (2) or vitamin D analogues (3). Independently identified in many tissues and body fluids by different laboratories, it was named with different synonyms and acronyms (sulphated glycoprotein 2 or SGP-2, TRPM-2, ApoJ, SP-40,40 etc.) before finding that they were all homolog forms of the same gene (4). According to its localization, CLU was found involved in many different physio-pathological processes, among which are lipid transport, complement-cascade regulation, membrane remodelling, tissue degeneration and programmed cell death (4). Generally, CLU gene expression is decreased during cell proliferation, and increased under conditions such as tissue suffering, cell atrophy and organ involution, and also in human normal fibroblasts synchronized in the quiescence state of the cell cycle by serum starvation (5). In rat ventral prostate, that does not develop spontaneously CaP with age, normal circulating androgen levels maintain its expression very low, but the expression of this gene is specifically, markedly and almost linearly induced during ageing (6, 7). In the normal gland, CLU gene is only expressed in cuboidal epithelial cells, a minor cell population subjected physiologically to apoptosis, while is not expressed in the tall, columnar and actively secreting epithelial cells, making up the vast majority of the prostate epithelium (8) >>>