Mechanistic aspects of the cytotoxic activity of glufosfamide, a new tumour therapeutic agent.

Beta-D-glucosyl-ifosfamide mustard (D 19575, glc-IPM, INN = glufosfamide) is a new agent for cancer chemotherapy. Its mode of action, which is only partly understood, was investigated at the DNA level. In the breast carcinoma cell line MCF7 glufosfamide inhibited both the synthesis of DNA and protein in a dose-dependent manner, as shown by the decreased incorporation of [3H-methyl]-thymidine into DNA and [14C]-methionine into protein of these cells. Treatment of MCF7 cells with 50 microM glufosfamide was sufficient to trigger poly(ADP-ribose) polymerase (PARP) activation, as revealed by immunofluorescence analysis. Both CHO-9 cells, which are O6-methylguanine-DNA methyltransferase (MGMT)-deficient, and an isogenic derivative, which has a high level of MGMT, showed the same cytotoxic response to beta-D-glc-IPM, indicating that the O6 position of guanine is not the critical target for cytotoxicity. By contrast, a sharp decrease in survival of cross-link repair deficient CL-V5 B cells was observed already at concentrations of 0.1 mM beta-D-glc-IPM, whereas the wild-type V79 cells showed a 90% reduction in survival only after treatment with 0.5 mM of this compound. The therapeutically inactive beta-L-enantiomer of glufosfamide also showed genotoxic effects in the same assays but at much higher doses. This was probably due to small amounts of ifosfamide mustard formed under the conditions of incubation. The results indicate that the DNA crosslinks are the most critical cytotoxic lesions induced by beta-D-glc-IPM.