METAL INDUCED TRANSFORMATION IN C3H10T1/2 CELLS

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The goal of this proposed research is to use carcinogenic metal salts to induce neoplastic transformation in C3H/10T1/2 CI 8 mouse fibroblasts and to study the molecular biology of neoplastic transformation induced by carcinogenic metal salts. Arsenic, chromium, and nickel salts, metal compounds that are strongly suspected to be human carcinogens, will be used to induce transformation in C3H/10T1/2 cells in this proposal. I will isolate a number of transformed C3H/10T1/2 cell lines derived from induction of Type II and Type III foci by carcinogenic metal salts. These cell lines will be seeded into soft agorose to determine whether they are anchorage independent and injected into nude mice to determine whether they are tumorigenic. Poly A containing mRNA will be extracted from metal-transformed cell lines and a battery of cloned murine and avian RNA tumor virus oncogene probes will be used in dot blotting and Northern blotting analyses to determine whether any particular proto-oncogenes are expressed at higher steady-state levels in metal-transformed C3H/10T1/2 cell lines. Where this is the case, I will use known cloned RNA tumor virus oncogene probes in restriction enzyme-Southern blotting analyses to determine whether any of the analogous proto-oncogenes are transposed in metal-transformed C3H/10T1/2 cells of whether they are differentially under-methylated in metal-transformed compared to in nontransformed C3H/10T1/2 cells. To study genes involved in transformation by carcinogenic metal salts, I will extract DNA from metal-transformed C3H/10T1/2 cell lines and determine whether the metal-transformed phenotypes of morphological transformation and anchorage independence can be transfected on DNA to nontransformed NIH3T3 and C3H/10T1/2 cells. The pattern of restriction endonuclease sensitivity of the ability of DNA's extracted from various metal-transformed cell lines to transfect transformed phenotypes will be studied. Contransfection of DNA from metal transformed cell lines and PBr322 and the construction of total genomic libraries from the DNA of secondary transfectants will be used to clone metal-transformed genes, and these gene probes will be used to isolate their normal homologs from a library of total genomic mouse DNA. Further restriction and Southern blotting analyses utilizing these genes and RNA tumor virus oncogenes will be done to provide information on the identify of these genes.