The long term objective of this research is to define molecular genetic changes at different stages of human uroepithelial cell (HUC) carcinogenesis with the hope that this knowledge can be used as a scientifically solid basis for the development of improved strategies to prevent, diagnose and treat human bladder cancers. The immediate goal of the present project is to use uroepithelial cancers generated from an in vitro/in vivo transformation system to analyze the biological significance and mechanisms of genetic losses on chromosome arms 3p, 6q, 13q, 17p and 18q in different steps of HUC tumorigenesis. The rationale for studying 3p, 6q and 18q is that losses of one homologue of these chromosome arms were nonrandom in association with neoplastic transformation in vitro of SV40-immortalized HUC to high grade aggressive cancers. We also propose to study the status of the 13q RB and the 17p p53 genes in this SV40-immortalized transformation system to test the hypothesis that requirements for, or advantages of, losses of one or both of these genes in HUC tumorigenesis are abrogated in the presence of an oncoprotein, such as SV40 T-antigen, that binds their protein products.
The specific aims are: (1) to test the biological significance of losses of 3p and 6q by using chromosome transfer to restore normal genes; (2) to determine if 18q losses represent functional inactivation of the putative DCC cancer suppressor gene by restoration of the DCC gene to high grade cancers using gene transfer, and reciprocally by downregulating DCC gene expression in low grade tumors using DCC antisense constructs; (3) to determine if the formation of T-ag-p53 or T-ag-RB complexes abrogate requirements for, or advantages of, losses of either the p53 or RB gene; (4) to test if gain of function p53 mutants are transforming at any step of tumorigenesis in our system; and (5) to use antisense constructs to the p53 and RB genes to attempt to immortalize HUC without a DNA tumor virus oncoprotein. The significance of these studies is that they will lead to the identification of genetic losses associated with different steps in multistep human uroepithelial tumorigenesis.
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