The hypothesis that wild-type p53 (wt-p53) modulates repair of DNA damage caused by the human bladder carcinogen, 4-aminobiphenyl (ABP), will be tested in its actual in vivo target cell types, namely the human uroepithelial cells (HUC). Using a unique set of isogeneic cell lines, differing in wt-p53 functional status (due to the expression of HPV-E6 oncoprotein or a transdominant mutant of p53), the following questions will be addressed. What types of DNA damage (covalent adducts and strand breaks) are caused by the N-hydroxy metabolites of ABP in HUC? Does the loss of function of wt-p53 cause a reduction in the rate of DNA repair either at the level of overall genomic DNA or at the level of an individual gene, such as hypoxanthine guanine phosphoribosyl transferase (HGPRT) gene?; and if so, does the effect of wt-p53 dependent upon the transcription status? To answer the above, DNA strand breaks and covalent adduct formation will be analyzed by alkaline elution, sucrose gradient sedimentation and by 32P-postlabeling methods. The effect of loss of function of wt-p53 on DNA lesion will be determined by comparison of the kinetics of DNA repair between the set of isogeneic cell lines, differing in wt-p53 activity. The kinetics of DNA repair will be measured by: 1) monitoring the disappearance of ABP-DNA adducts by 32P-postlabeling; 2) by quantifying the unrepaired DNA adducts by digestion with E. Coli UvrABC nuclease; and 3) by estimating the DNA repair patch synthesis. The effect of wt-p53 on the transcription dependent versus -independent repair will be determined by measuring the kinetics of repair synthesis using riboprobes selective for the transcribed versus nontranscribed strand of HGPRT. A rare feature of our HUC system is that it permits us to test the effect of the environmental carcinogen ABP, directly on its in vivo target cells, and thus surmount the serious limitations of the fibroblast or rodent culture system. The results of these studies have important etiologic, mechanistic and therapeutic implications in human cancers.
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