The goals of this proposal are to continue our studies into the mechanisms and molecular biology of metal-salt transformation of C3H/10T1/2 cells and diploid human foreskin fibroblasts. We will finalize inducing nickel subsulfide and hexavalent chromium salt transformed 10T1/2 cell lines and characterizing them for anchorage independence and tumorigenicity. Then, we will study the molecular biology of transformation in arsenic, nickel, and chromium salt transformed 10T1/2 cell lines. Poly A+ mRNA extracted from arsenic, nickel, and chromium transformed 10T1/2 cell lines will be probed with cloned murine and avian RNA tumor virus oncogene probes in RNA dot and Northern blotting analyses to determine whether proto-oncogenes are expressed at higher steady-state levels in metal-transformed 10T1/2 cell lines. DNA from metal salt transformed 10T1/2 cell lines will be probed with cloned RNA tumor virus oncogenes in restriction enzyme-Southern blotting analyses to determine whether proto-oncogenes are amplified, rearranged, or under-methylated in metal-transformed 10T1/2 cells. DNA from metal-transformed 10T1/2 cell lines will be transfected into NIH3T3 cells to determine whether metal-induced morphological transformation is encoded on DNA. Patterns of restriction endonuclease sensitivity of ability of DNA's from metal-transformed cell lines to transfect transformed phenotypes will be studied. Cotransfection of DNA from metal transformed cell lines and PBr322 and construction of total genomic libraries from DNA of secondary transfectants will be used to clone metal-induced transforming genes. These genes will be probed with RNA tumor virus oncogenes in restriction endonuclease-Southern blotting analyses to identify them. These genes will be used to isolate their normal homologs from a total genomic mouse DNA library. We will also determine whether there are perturbations in oncogene expression in arsenic, nickel, and chromium induced anchorage-independent human cell strains we previously derived and whether DNA from these cell strains can transfect anchorage-independence to NIH3T3 cells. We will also attempt to provoke full transformation of these anchorage-independent human cell strains to focus formation, immortality, and tumorigenicity by a) transfecting cloned, mutated, transforming myc genes into these cell strains or by b) treating these cell strains with tumor promoters or with colcemid to induce chromosomal aneuploidy.
Showing the most recent 10 out of 15 publications
