Replicative senescence in human uroepithelial cells.

PURPOSE: Normal human uroepithelial cells (HUCs) proliferate rapidly in culture during early passage and then spontaneously undergo replicative senescence. We previously reported that the cyclin D1-CDK4/6 inhibitor, p16INK4a, is elevated at senescence in HUCs. Hence, we proposed that p16INK4a may play a critical role in mediating senescence in this cell type. In the current study, we further characterized the senescent state in HUCs. We also tested the possible roles of changes in other cell cycle proteins, including p53, p21WAF1, pRb, and cyclin D1 in HUC senescence. METHODS: Normal HUCs cultured from explants of ureteral mucosa were used for these studies. Senescence associated-beta-galactosidase activity (SA-beta-gal) was used to identify cells in senescence. Flow cytometric analysis was used to determine changes in cell cycle distribution at senescence. Response of cells to serum stimulation was determined by Northern analysis of c-fos. Western analysis was used to assess changes in p53, p21WAF, p16INK4a, cyclin D1 and plasminogen activator inhibitor-1 (PAI-1) levels at senescence. RESULTS: beta-gal-positive HUCs were blocked at G1/S in senescence and failed to show c-fos induction in response to serum stimulation. As previously reported, senescent HUCs also showed elevated p16INK4a. However, unlike human fibroblasts, neither p53 nor p21WAF1 elevation accompanied HUCs senescence. PAI-1 levels were also not elevated in HUC senescence. CONCLUSION: These findings support a model in which elevation of p16INK4a, but not p53 or p21WAF1 plays a critical role in HUC replicative senescence. These findings elucidate the tumor suppressor mechanism of p16INK4a and the frequent loss of either p16INK4a or pRb in invasive human bladder tumors.

A molecular genetic model of human bladder cancer pathogenesis.

An understanding of the biological significance of the multiple genetic alterations identified in clinical bladder cancers to the stepwise pathogenesis of the disease is evolving. Alterations in p53 and pRb, products of the chromosomes 17p13 TP53 and 13q14 RB tumor suppressor genes, occur in approximately 50% and approximately 33% of bladder cancers respectively, and are associated with later stage, higher grade disease. p53 and pRb alterations are also known to occur in early stage bladder carcinoma in situ where they are thought to represent a poor prognosis for tumor progression. Allelic loss of genes on 9p21 occurs in approximately 50% of bladder cancers, but whether the only critical gene in this region is the CDKN2/p16 cyclin/CDK inhibitor is at present uncertain. Amplification and/or overexpression of the oncogenes epidermal growth factor receptor and erbB2 are associated with later stage disease. Finally, recent findings generated using in vitro transformation systems with human uroepithelial cells provide strong evidence that loss of genes on 3p, which occurs in approximately 20% of bladder cancers, and/or gain of genes on 20q play an important role in blocking HUC cellular senescence. This latter phenotype should represent a critical step in oncogenesis, as cells that do not senesce can survive to accumulate the multiple genetic alterations associated with invasive and metastatic bladder cancers. Further understanding of the biochemical mechanisms underlying these genetic changes will provide the additional information needed to design better strategies for bladder cancer intervention and treatment.

Apoptosis in human papillomavirus16 E7-, but not E6-immortalized human uroepithelial cells.

We compared the ability of E6-, versus E7-, immortalized human uroepithelial cells (HUC) to undergo apoptosis in response to gamma radiation. Two independent HPV16 E6-immortalized cell lines, alphaE6#1 and alphaE6#2, that showed low or undetectable p53 levels, failed to undergo apoptosis in response to 18 Gray (Gy) gamma radiation as determined by DNA fragmentation. In contrast, two independent HPV16 E7-immortalized cell lines, alphaE7#1 and alphaE7#2, both of which showed stabilized wildtype p53, underwent apoptosis in the same experiment. Interestingly, both alphaE7#1 and alphaE7#2 showed constitutively elevated BAX and lowered BCL-2 levels, compared to either alphaE6#1 or alphaE6#2. However, elevated BAX and reduced BCL-2 per se were insufficient to trigger apoptosis, as apoptosis occurred only after exposure to gamma radiation. These results support a model in which HPV16 E7-immortalized cells are primed to undergo apoptosis, given an appropriate trigger. This apoptotic response was not observed in alphaE6/E7#1 cells which, like alphaE6-HUCs, showed low p53 levels, nor in late passage alphaE7#1 with spontaneously mutated TP53. These results suggest that E7 immortalization primes HUC for apoptosis in response to gamma radiation, and that this enhanced apoptotic response is p53 dependent.

Elevated p16 at senescence and loss of p16 at immortalization in human papillomavirus 16 E6, but not E7, transformed human uroepithelial cells.

CDKN2/p16 inhibits the cyclin D/cyclin-dependent kinase complexes that phosphorylate pRb, thus blocking cell cycle progression. We previously reported that p16 levels are low to undetectable in normal human uroepithelial cells (HUCs) and in immortalized uroepithelial cells with functional pRb, whereas p16 levels are markedly elevated in immortal HUCs with altered pRb (T. Yeager et al., Cancer Res., 55: 493-497, 1995). We now report that elevation of p16 levels occurs at senescence in HUCs, including HUCs transformed by human papillomavirus 16 E7 or E6, whose oncoprotein products lead to functional loss of pRb and p53, respectively. We also report that six of six independently immortalized E7 HUCs show high levels of p16 similar to those observed at HUC senescence, whereas p16 is undetectable in five of five immortal E6 HUCs. Four of the five independent E6 HUCs that lost p16 at immortalization showed hemizygous deletion of the 9p21 region. However, no homozygous CDKN2 deletions were detected, and only one CDKN2 mutation was identified. For the first time, these data associate elevated p16 with senescence in human epithelial cells. These data also suggest that a component of immortalization may be abrogation, either by pRb inactivation (as in the E7-transformed HUCs) or by p16 inactivation (as in the E6-transformed HUCs), of a p16-mediated senescence cell cycle block.