The overall goal of the proposed studies is to determine whether chemical carcinogens are directly responsible for the activating mutations in oncogenes detected in the tumors they induce. The in vivo interactions between carcinogens and oncogenes will be examined in animal tumor models in which a given carcinogen is able to reproducibly induce the same type of tumor that frequently contain a specific activated oncogene. Experiments with these tumor models have indicated that the types of mutation leading to oncogene activation often reflect the predicted mutagenic specificity of inducing carcinogen. It has therefore been suggested that the carcinogens are directly responsible for oncogene activation and therefore that the oncogenes are involved in tumor initiation. It must be remembered, however, that a tumor is a clonal population of cells selected for a tumor phenotype. Therefore, by examining the mutations present in the oncogenes from tumors it is not possible to determine whether the oncogenes were activated directly by the carcinogen, or whether biological selection for the tumor phenotype resulted in the preferential expansion of cell populations with given oncogene mutation. In order to distinguish between these two possibilities we will examine the spectrum of mutations present in target and non-target organs prior to carcinogen treatment, and compare these to the spectrum induced mutations. Most importantly, the analysis will be performed prior to any phenotypic selection and will therefore include all possible mutations. The development and use of new techniques that will make it feasible to isolate and sequence mutant cancer gene alleles that are present at a frequency of less than one in 107 cells. These studies will determine the types of mutations induced by carcinogens in cancer genes prior to biological selection and define the role of repair enzymes in the generation of those spectra. The comparison of unselected spontaneous to unselected induced mutational spectra, provides the first opportunity to segregate induction of mutations by the carcinogen from biological selection for mutant oncogenes during tumorigenesis. The studies will also generate in vivo dose response curves based on mutation rather than phenotype, which will be invaluable in risk assessment analyses. This technology will be used to examine mutations in the Ha-ras- 1 and neu oncogenes, as well as the p53 gene, a putative tumor suppressor, before and after mutagenesis.
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