The long-range goal is to understand the mechanisms by which carcinogens induce mutations in human cells and the role such mutations play in the neoplastic transformation of human cells in culture and in human carcinogenesis. Mutations in the p53 gene are very frequently found in a variety of types of human tumors, including bone and soft tissue sarcomas, and are distributed non-randomly in characteristic """"""""hot spots"""""""" in evolutionarily conserved regions of the gene. Such mutations are known to predispose cells to malignancy. We are interested in factors that determine the kinds and distribution of these mutations (spectrum). we have a quantitative system for carcinogen-induced transformation of a near- diploid, karyotypically stable human fibroblast cell strain that yields malignantly-transformed cells with p53 mutations. We will use it to determine the relationship between the frequency and spectrum of mutations induced in the coding region of the p53 gene of the cell strains and the spectrum of carcinogen adducts initially formed in the gene, or still remaining unrepaired in the gene at the time S-phase replication occurs. LM-PCR will be used to determine the spectrum of carcinogen adducts at the sequence level. We will determine the relationship between these carcinogen-induced mutations in the p53 gene of the cells, loss of p53 transactivating function, and the ability of the focus-derived cell strains to form tumors in athymic mice. The spectrum of p53 mutations induced in the cell strains that are malignant will be compared to those reported for human tumors. We will analyze the ability of selected p53 mutated alleles, generated in this study and assayed for the status of transactivating function of the protein, to exert their transforming effect in the presence or absence of a wild type allele. (Evidence shows that certain p53 mutations allow the mutant p53 protein to sequester the wild type protein, but that others can stabilize the p53 protein only when there is not wild type allele.) Selected cell strains from the proposed study will be transfected with these alleles carried on an expression vector with a modulatable promoter. The ability of a transfected wild type allele to suppress transformation of MSU-1.1 cells to focus formation and tumorigenicity will be similarly tested.
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