p16/pRb pathway alterations are required for bypassing senescence in humanprostate epithelial cells

The cell cycle regulatory genes p16/CDKN2 RE are frequently deleted in pros tate cancers. In this study, We examined the role of alterations in p16 and pRb during growth, senescence, and immortalization in vitro of human prost ate epithelial cells (HPECs), HPECs are established from normal prostate ti ssues and cultured on collagen-coated dishes, Our results show that p16 is reproducibly elevated at senescence in HPECs, HPECs are immortalized using human papilloma virus 16 E6 and/or E7 as molecular tools to inactivate p53 and/or pRb, respectively. Immortalization occurs infrequently in this syste m and only after a latent period during which additional genetic/epigenetic changes are thought to occur. Notably;, all of the E6-immortalized HPEC li nes but none of the E7 lines show, inactivation oof p16/CDKN2 by deletion, methylation, or mutation in association with immortalization. In contrast, E7 lines, in which pRb function is abrogated bg E7 binding, retain the high levels of p16 observed at senescence. Thus, all lines show either a p16 or pRb inactivation. Analysis of six independent lines from metastatic prosta te cancers reveals a similar loss of either p16 or pRb, Comparative genomic hybridization of HPECs shows that gains of chromosomes 5q, so, and 20 are nonrandomly associated with; bSpassing senescence (probability = 0.95). The se results suggest that high levels of the cyclin-dependent kinase inhibito r p16 mediate senescence G(1), arrest in HPECs and that bypassing this bloc k by a p16/pRb pathway alteration is required for immortalization in vitro and possibly tumorigenesis in vivo. Our results further indicate that inact ivation of the p16/pRb pathway alone is not sufficient to immortalize HPECs and that additional genetic alterations are required for this process.