Glutathione depletion as a determinant of sensitivity of human leukemia cells to cyclophosphamide.

The role of glutathione (GSH) as a determinant of cellular sensitivity to the cytotoxic and DNA-damaging effects of cyclophosphamide (CP) was studied in a dual culture system of rat hepatocytes and K562 human chronic myeloid leukemia cells, which have elevated aldehyde dehydrogenase activity with a corresponding insensitivity to activated CP. Exposure of K562 cells to 50 microM DL-buthionine-S,R-sulfoximine for 24 h resulted in a depletion of cellular GSH content to 10% of control values without toxicity. Subsequent 1-h exposure of GSH-depleted cells to activated cyclophosphamide, obtained by incubation of CP with suspension cultures of rat hepatocytes, resulted in a 5-fold potentiation of the cytotoxicity of CP. Alkaline elution analysis of cellular DNA demonstrated that the level of apparent interstrand cross-linking was 3 to 4 times higher in GSH-depleted cells than in nondepleted cells. GSH-depleted cells were, in addition, more sensitive to induction of DNA single strand breaks than nondepleted cells. Depletion of GSH content did not increase cellular sensitivity to the cytotoxicity of phosphoramide mustard. Preincubation of K562 cells with 1 mM cysteine for 4 h resulted in an approximately 60% increase in cellular GSH content, which was accompanied by decreased sensitivity to the cytotoxicity of hepatocyte-activated CP. Exposure of nondepleted cells to clinically relevant concentrations of hepatocyte-activated CP resulted in depletion of cellular GSH content. Replenishment of GSH content in these cells was relatively slow following CP exposure. Acrolein was highly effective at depleting cellular GSH content, whereas phosphoramide mustard had no effect on cellular GSH content. The depletion of GSH by intracellularly released acrolein may be important in the mechanism of cytotoxicity of CP.