Expression of a rat glutathione-S-transferase complementary DNA in rat mammary carcinoma cells: impact upon alkylator-induced toxicity.

The role of glutathione-S-transferase (GST) in alkylator drug resistance has been studied in MatB rat mammary carcinoma cells. A series of GST transfectant cell lines was established by using an expression vector containing the complementary DNA for the rat GST Yc gene under regulation of the SV40 early region promoter and the antibiotic resistance plasmid pSV2neo. Transfectant cell lines expressing up to 4-fold higher total GST activity than in the parental wild type cell line were identified. Southern blot analysis confirmed a DNA fragment corresponding in size to the transfected GST Yc complementary DNA. Wild type MatB cells contain very low levels of Yc protein, whereas the Yc+ clones showed greatly increased amounts of the Yc subunit. The effect of increased GST Yc activity on the sensitivity of the transfected clones to various cytotoxic agents was assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell survival assay. The clones expressing recombinant GST Yc were more resistant to melphalan (6- to 12-fold), mechlorethamine (10- to 16-fold), and chlorambucil (7- to 30-fold). In late passage populations of the GST Yc+ clones that had been grown over a period of 14 months under continuous selection in G418, GST activity was decreased and it was paralleled by a decrease in Yc protein. These late passage clones with diminished GST Yc content also demonstrate a partial reversion toward the wild type phenotype as determined by cytotoxicity assays using melphalan, mustargen, and chlorambucil. Interstrand DNA cross-links induced by mechlorethamine were significantly lower at 0, 2, and 20 h posttreatment in one of the GST Yc+ clones when compared to wild type MatB cells. These studies indicate that GST Yc overexpression can confer resistance to alkylating agents and that this correlates with inhibition of DNA cross-link formation.