The long-term objective of our research program is to understand the pathogenesis of human bladder cancer with the hope that this information will be valuable for bladder cancer prevention, diagnosis, prognosis, or treatment. The immediate goal of the present application is to determine the biological, biochemical, and genetic consequences of p53 loss in multistep chemical transformation of human uroepithelial cells (HUC). The rationale for studying the effects of p53 loss early in HUC transformation is based on observations that TP53 is the most commonly mutated tumor suppressor gene in clinical bladder cancers and its loss correlates with invasive cancers and a poor clinical prognosis. It has been hypothesized that p53 loss leads to genome instability at both the DNA and chromosome levels. However, direct experimental evidence in a human epithelial system associating p53 loss of function with chromosome instability, increased DNA mutability, or tumor progression is lacking. In this research, the predicted phenotypes for p53 loss of function will be tested. For these studies, we have established a unique new in vitro transformation system consisting of an isogeneic series of HUC cell lines immortalized after transformation in vitro with Human Papilloma Virus 16 E6 or E7 genes. E6 binds wildtype (wt) p53 leading to rapid p53 degradation, while E7 binds and alters Hb (and other proteins). Thus, the E6-HUC mimic HUC that have sustained a TP53 mutation early in tumorigenesis, while the E7-HUC represent HUC that have not yet acquired such a change. We will answer the following specific questions: (1) Do transdominant p53 mutants (p53TDM) that override (wt)p53 generate chromosome instability? (2) What is the biological significance of the genetic alterations associated with immortalization? (3) Does loss of (wt)p53 function potentiate spontaneous or carcinogen induced mutability? (4) Is TP53 a target gene for arylamine mutagenesis in HUC immortalization or tumorigenic transformation? The significance of these studies is that they directly address fundamental and timely questions concerning the biological and biochemical consequence of an early or late loss of p53 function in the pathogenesis of human bladder cancer. Furthermore, they will elucidate mechanisms by which HPV16 E6 and E7 genes contribute to transformation of urogenital epithelium.
