Alterations inp53 andp16/pRb pathways in human uroepithelial cell immortalization

This thesis investigated the relevance of p53 and p16-Cyclin D1-Cdk4/6-pRb alterations in immortalization of human uroepithelial cells (HUC). Bypassing senescence (or immortalization) is a putative early step in transformation, when cells overcome the checkpoints created by limited life span of somatic cells. To understand the mechanism of senescence in human uroepithelial cells [HUCs] further, a detailed characterization of human uroepithelial cell senescence was undertaken first. We found that some biomarkers of senescence in fibroblasts such as plasminogen activator inhibitor-1 and p21Cip1 were uninformative in senescent HUC, while others such as elevation of p16 at senescence and absence of c-fos induction following serum stimulation were also seen in HUCs. Using Human Papillomavirus (HPV) E6- and E7-immortalized HUCs, we investigated abrogation of the p53 and p16/pRb pathways during immortalization. E6- and E7-HUCs did not exhibit elevation or transcriptional activation of p53 following gamma irradiation, nor did they arrest in G1. E7-HUCs, however did retain the capacity to undergo p53-dependent apoptosis, which was absent in E6-HUCs. Both E6-HUCs and E7-HUCs showed a p16/pRb pathway alteration. p16 was lost in all E6-HUCs, while pRb was compromised in E7-HUC by the inactivation of pRb by E7. It has been suggested that p16-Cyclin D1-Cdk4/6-pRb pathway functions as a single unit in mediating cell cycle arrest at senescence. To test this, we next examined the hypothesis that a mutant Cdk4 (R24C) that is resistant to inhibition by p16 is capable of overcoming p16-mediated inhibition of pRb-phosphorylation in the presence of non-limiting amounts of Cyclin D1. HUCs were transformed with HPV16 E6, Cdk4-R24C and Cyclin D1. Such transformants immortalized, yet retained high senescence-associated levels of p16. This is the first report showing that mutant Cdk4-R24C suffices as a p16/pRb pathway alteration in relieving p16-mediated inhibition of pRb-phosphorylation. These studies add to our understanding of the biological significance of genetic alterations in bladder cancer pathogenesis.