The two basic objectives of this research are to: 1) biochemically characterize the function of glutathione (GSH) S-transferase in regulating the cytotoxic and transforming events induced by electrophilic carcinogen metabolites (e.g. benzo(a)pyrene anti 7,8-dihydrodiol 9,10-epoxide), and 2) identify a pool of carcinogen-induced genetic events which are permissive for 10T1/2 cell transformation. We will use integrated biochemical techniques (affinity and isoelectric point chromatography) and molecular techniques (plasmid reconstruction, DNA transfection) to characterize GSH-transferase in benzo(a)pyrene-transformable 10T1/2 mouse cells and to augment its detoxifying function by stably integrating additional cDNA copies of specific GSH-transferase isozymes into 10T1/2 cells. In our second objective, we will use cellular techniques (cell transformation) and molecular techniques (oncogene-plasmid transfection, hybridization analysis) to characterize the morphology and tumorigenicity of 10T1/2 cells purposely transformed with known aberrant oncogenes or transformed with carcinogens. We will use DNA transfection, restriction endonuclease analysis and nucleic acid hybridization to define a pool of genetic events which are linked to 10T1/2 cell transformation.
Our aim here is to integrate biochemical observations of carcinogen metabolism and its regulation by GSH-transferase, to molecular and genetic observations of the results of carcinogen-DNA interaction in 10T1/2 cells, in order to develop an understanding of neoplastic transformation.
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| Manoharan, T H; Puchalski, R B; Burgess, J A et al. (1987) Promoter-glutathione S-transferase Ya cDNA hybrid genes. Expression and conferred resistance to an alkylating molecule in mammalian cells. J Biol Chem 262:3739-45 |
