Nitrogen mustard-DNA interaction in melphalan-resistant mammary carcinoma cells with elevated intracellular glutathione and glutathione-S-transferase activity

Abstract

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 microM 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 microM 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 microM), the numbers of HN2-induced cross-links were significantly lower in MLNr cells than in WT cells. However, treatment of MLNr cells with 60 microM HN2 resulted in cross-link levels similar to those obtained using 30 microM HN2 in WT cells. The 35% decrease in MLN accumulation observed in MLNr cells could not entirely explain the absence of cross-links, 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 microM [14C]-MLN. Intracellular depletion of GSH by a nontoxic concentration of L-buthionine-(S, R)-sulfoximine (BSO, 100 microM; about 70% GSH depletion) significantly sensitized MLNr cells to MLN and increased cross-link formation. A nontoxic concentration (50 microM) 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of the Yc1 glutathioneS-Transferase mRNA as the overexpressed species in a nitrogen mustard-resistant rat mammary carcinoma cell line

“…To measure cytotoxicity of melphalan in the WT and MLNr cells, we used the MTT assay as previously described (16). Cells were exposed to various concentrations of BSO for 20 hours and then divided for analysis of GSH concentration and for subsequent exposure to melphalan.…”

Isolated limb infusion as a model to test new agents to treat metastatic melanoma

“…In efforts to identify mechanisms of drug resistance, glutathione (GSH) has emerged as a major protein associated with the melphalan resistance pathway . Depletion of GSH has been shown to reverse resistance to this alkylating agent .…”

Covalent Protein Adduction of Nitrogen Mustards and Related Compounds

“…39 As glutathione conjugation is a major pathway for detoxification of electrophiles in the body, 44 it would also be expected that glutathione conjugates would be observed for HN and CVAA. 45,46 In theory, protein adducts may also be utilized for blister agent detection, once adduction sites have been properly characterized. Detection methods for such adducts involve isolation of a relevant macromolecule, digestion via one or more proteases, and peptide analysis via LC-MS. 47 Protein adduction will be covered in greater depth in Section 2.4, and protein adduct analysis will be covered in Section 2.5.…”

“…Nitrogen mustard therapeutic compounds have demonstrated their ability to form DNA adducts and DNA cross-links at a variety of nucleophilic sites on DNA. 24,46,114 In addition to binding to DNA, these compounds may also bind to the DNA repair protein O 6 -alkyguanine DNA alkyltransferase (AGT) and form DNA-protein cross-links, hindering AGT function and thus representing an alternative mechanism for these compounds. 115 Resistance to these drugs is typically correlated with increased GSH and glutathione-S-transferase (GST) levels.…”

“…As detailed in the methods section, a SIM method was utilized for this experiment. While it is known that these therapeutic compounds are capable of adducting to nitrogen in DNA (as this is their mode of action as antineoplastic drugs), 24,46,114 the focus here is to ensure that protein adduction by these drugs would not produce the same adduct structures as previously identified for HN-2 and HN-3. Therefore, SIM methods were utilized as a rapid comparison for adducts of interest, as the molecular weights and RT of HN-adducted peptides were identified and characterized in previous studies.…”