SITE-SPECIFIC GENE DAMAGE, MUTATION, AND CANCER

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Evidence suggests that mutations are causally involved in carcinogenesis and recent evidence suggests that UV induced mutations in the p53 gene are involved in squamous cell carcinomas of the skin, but the relative contribution of the major UV photoproducts, cyclobutane pyrimidine dimers (CPD) and 6-4 pyrimidine-pyrimidones (6,4's), to mutagenesis is controversial. To gain insight into the mechanisms by which UV causes mutations, the role DNA repair plays, and the relationship between specific damage and UV induction of cancer, we will determine the relative contribution of these two kinds of photoproducts to mutagenesis in human cells and whether these lesions preferentially induce certain kinds of mutations. We will use synchronized cells that are deficient in repair of CPD but not 6-4's and cells that can remove CPD, but not 6- 4's, irradiating them in early S or G1 to given them little or no time to repair or many hours for repair before DNA replication and compare the frequency, kinds, and location of mutations in the coding and splice site regions of the HPRT gene with those from normal human cells and cells totally incapable of excision. We will measure the rate of excision of each kind of lesion from the specific strands of the HPRT gene in these cells by combining use of T4 endo for CPD, and UvrABC excinuclease for 6-4's with Southern blotting and hybridization with strand-specific probes. To see if "hot spots" for mutation induction correlate with "hot spots" for lesion induction, we will apply ligation mediated-polymerase chain reaction (LM-PCR) to strategic regions of the HPRT gene to determine at the sequence level the initial location of each type of photoproducts and that of lesions still remaining at the time the gene is replicated. We will carry out similar studies with XP variant cells to see if their abnormally high frequency of UV-induced mutations results from defective repair and/or from error-prone bypass of specific types of photoproducts. We will determine the frequency of UV-induced malignant transformation of synchronized MSU-1.1 cells irradiated at S and in early G1 and whether mutations have been induced in the p53 gene and/or RAS oncogenes. Using LM-PCR, we will determine the location of initial UV damage in the p53 gene of those cells and of that remaining after repair. Using LM-PCR, we will determine if the damage induced in these cells by UV254nm is the same as that induced in human skin by simulated sunlight (sunlamps). We will then induce skin tumors in athymic mice by exposure to sunlamps and evaluate cells from such tumors for mutations in the p53 and ras genes.