DNA damage in cyclophosphamide-resistant tumor cells: the role of glutathione

The relationship between DNA damage removal and cellular sensitivity to the alkylating agent cyclophosphamide (CP), currently one of the most effective drugs in the treatment of human tumors, has not been well established. In the present study, we have analyzed drug-induced DNA cross-link persistence in the CP-resistant subline KHT-rep/iv and its sensitive counterpart KHT-iv. The alkaline elution technique was used to detect both DNA-DNA and DNA-protein cross-links formed following exposure to clinically achievable CP concentrations. In addition, the role of glutathione (GSH) in the cross-link persistence was analyzed. The KHT-rep/iv cell line was chosen for study because compared to the parent KHT-iv line, this cell line demonstrates 2-3-fold increased GSH levels, and resistance to CP is known to be mediated by thiols. Following exposure to 4-hydroperoxy-cyclophosphamide (4-OOHCP), an active form of CP, the KHT-rep/iv cells exhibited fewer total cross-links and a shift in the time of maximal cross-link formation (6.2 at 2 h versus 2.9 at 4 h for the parent and resistant lines, respectively). When the GSH content of the resistant cells was reduced to equal that of the parent cell line prior to the alkaline elution assay, both the total number and time course of development of cross-links were similar between the two lines. Also, when the cells were exposed to mechlorethamine, an alkylating agent which forms cross-links at a much faster rate than 4-OOHCP, the time course of cross-link formation after mechlorethamine exposure was similar between the two sublines, with maximal cross-link formation occurring after a 1-h incubation. The present findings suggest that (a) the increased GSH levels in KHT-rcp/iv cells may be responsible for the delay in maximal cross-link development after treatment with 4-OOHCP, possibly by binding with the monoadduct produced by the drug; and (b) the faster reaction rate of mechlorethamine cross-link formation may prevent the tripeptide from binding to the monoadduct. These results are consistent with the hypothesis that GSH may "quench" the formation of cross-links following 4-OOHCP exposure.