We examined the relationship between intracellular levels of glutathione (GSH), glutathione-S-transferase (GST) activity, and the kinetics of DNA cross-links induced by the bifunctional alkylating drugs melphalan (MLN), chlorambucil (CLB), and mechlorethamine (HN2) in a rat mammary carcinoma cell line (WT) and in a subline selected in vitro for primary resistance to MLN (MLNr, 16-fold resistance). MLNr cells exhibit a 2-fold increase in intracellular GSH concentration and an approximately 5-fold increase in GST activity as compared with the parent cells. They are cross-resistant to a variety of drugs, including CLB (6-fold) and HN2 (14-fold). Treatment of WT cells with 30 μm MLN or CLB induced a significant accumulation of DNA-DNA cross-links for up to 8 h, which decreased over a 24-h period. In MLNr cells, no significant cross-link formation was induced by either MLN of CLB at any time between 0 and 24 h. Doses of up to 100 μm MLN failed to induce cross-links in MLNr cells. Formation of cross-links was observed immediately after treatment with HN2 in both cell lines and was followed by a subsequent decrease during a 24-h incubation in drug-free medium. At an equimolar concentration (30 μm), the mumbers of HN2-induced cross-links were significantly lower in MLNr cells than in WT cells. However, treatment of MLNr cells with 60 μm HN2 resulted in cross-link levels similar to those obtained using 30 μm HN2 in WT cells. The 35% decrease in MLN accumulation observed in MLNr cells could not entirely explain the absence of crosslinks, since thin-layer chromatographic analysis demonstrated that both cell lines accumulate a significant amount of MLN and metabolize it to the same extent. Significant amounts of MLN were also detected in nuclei isolated from WT and MLNr cells that had been treated with 30 μm [14C]-MLN. Intracellular depletion of GSH by a nontoxic concentration of l-buthionine-(S, R)-sulfoximine (BSO, 100 μm; about 70% GSH depletion) significantly sentisized MLNr cells to MLN and increased cross-link formation. A nontoxic concentration (50 μm) of ethacrynic acid (EA, an inhibitor of GST showing some specificity for Yc/Yp subunits) also sensitized MLNr cells to MLN and increased cross-link formation. Our data demonstrate that both EA and BSO are effective modulators of nitrogen mustard cytotoxicity in tumor cells resistant to alkylating drugs. The limited number of cross-links formed in MLNr cells after treatment with MLN or even CLB suggests that efficient repair of drug-DNA monoadducts is operative in these cells and that the increases obtained in the presence of BSO and EA may be related to the involvement of both GSH and GST in drug-DNA interactions such as monoadduct repair.
ASJC Scopus subject areas
- Cancer Research