Role of cyclin-dependent kinase inhibitors in the growth arrest at senescence in human prostate epithelial and uroepithelial cells

Cellular senescence has been proposed to be an in vitro and in vivo block that cells must overcome in order to immortalize and become tumorigenic. To characterize these pathways, we focused on changes in the cyclin-dependent kinase inhibitors and their binding partners that underlie the cell cycle arrest at senescence. As a model, we utilized normal human prostate epithelial cell (HPEC) and human uroepithelial cell (HUC) cultures. After 30–40 population doublings cells became growth-arrested in G0/1 with a threefold decrease in Cdk2-associated activity, a point defined as pre-senescence. Temporally following this growth arrest, the cells develop a senescence morphology and express senescence-associated β-galactosidase (SA-β-gal). Levels of p16INK4a and p57KIP2 rise in HUCs during progressive passages, whereas only p16 increases in HPEC cultures. The induced expression of p57, similar to p16, produces a senescent-like phenotype. pRB, cyclin D, p19INK4d and p27KIP1 decrease in both cell types. We find that p53, p21CIP1 and p15INK4b are transiently elevated in HPECs and HUCs at the pre-senescent growth arrest, then return to low proliferating levels at terminal senescence. Analysis of p53, p21CIP1, p15INK4b, p16INK4a, and p57KIP2 reveals altered expression in immortalized, non-tumorigenic HPV16 E6 and E7 prostate lines and in tumorigenic prostate cancer cells. These results indicate: (i) the existence of a subset of growth inhibiting genes elevated at the onset of the senescence, (ii) a distinct class of genes involved in the maintenance of senescence, and (iii) the frequent inactivation of these pathways during immortalization.