You are here:
TRANSFECTION OF A RAT CYTOCHROME P-450B CDNA INTO C3H10T1/2CL8 MOUSE EMBRYO FIBROBLASTS
Citation:
Hansen, S., J. Ross, J. Siegfried, S. Leavitt, K. Rudo, R. Langenbach, AND S. Nesnow. TRANSFECTION OF A RAT CYTOCHROME P-450B CDNA INTO C3H10T1/2CL8 MOUSE EMBRYO FIBROBLASTS. U.S. Environmental Protection Agency, Washington, D.C., EPA/600/J-89/358.
Description:
A cDNA clone of a rat cytochrome P450b gene was used to construct an expression vector driven by an SV40 promoter and containing a G418-resistance selectable marker. his bifunctional plasmid (pJRSL100) was transfected into the C3H10T1/2CL8 mouse embryo fibroblast cell line. 418-resistant clones were selected and tested for enhanced sensitivity to the cytotoxic effects of the carcinogens 2-acetylaminofluorene (2-AAF) and N,N-dimethyl- nitrosamine (DMN), compounds which normally do not induce cytotoxicity nor morphological transformation in these cells. NA analyses of one subclone, 19P450b-4, which exhibited increased cytotoxic responses to 2-AAF and DMN compared to the parental C3H10T1/2CL8 cells, demonstrated an increase in the number of copies of the cytochrome P450b and demonstrated the appearance of unique restriction fragment bands relative to parental and control transfected cells. his subclone also exhibited increased levels of mRNA complementary to the P450b cDHA. etabolism studies of 2-AAF in this subclone demonstrated an increase in the C-hydroxylated metabolites 1-, 3-, 5/9-, and 7-hydroxy-AAF compared to parental C3H10T1/2CL8 cells. he results indicate that C3H10T1/2CL8 cells can be transfected with gene/cDNAs to increase their metabolic competency and that such transfected cells may enhance the usefulness of the C3H10T1/2CL8 cells in studies on chemically-induced cytotoxicity and morphological transformation.