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 Sadenosylmethionine 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 antioncogene in the prostate, suggesting a role for this gene in controlling proliferation of normal and transformed prostate epithelial cells.

Clusterin, ubiquitously distributed in mammalians, was cloned and identified as the most potently induced gene during rat prostate involution following androgen deprivation. Also found to be involved in many other patho-physiological processes, its biological significance is still controversial, particularly with regard to apoptosis. We previously showed that transient over-expression of clusterin blocked cell cycle progression of simian-virus-40-immortalized human prostate epithelial cell lines PNT1A and PNT2. We show in the present study that the accumulation of an intracellular 45 kDa clusterin isoform was an early event closely associated with death of PNT1A cells caused by cell detachment followed by apoptosis induction (anoikis). Cell morphological changes, decreased proliferation rate and cell cycle arrest at G0/G1-S-phase checkpoint were all strictly associated with the production and early translocation to the nucleus of a 45 kDa clusterin isoform. Later, nuclear clusterin was found accumulated in detached cells and apoptotic bodies. These results suggest that a 45 kDa isoform of clusterin, when targeted to the nucleus, can decrease cell proliferation and promotes cell-detachment-induced apoptosis, suggesting a possible major role for clusterin as an anti-proliferative gene in human prostate epithelial cells.

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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.

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