High-dose selection with mafosfamide results in sensitivity to DNA cross-linking agents: characterization of...

One of the most frequently used alkylating drugs in the therapy of a broad spectrum of tumors is cyclophosphamide. To elucidate the mechanisms by which tumor cells acquire resistance to this agent, Chinese hamster ovary cells (CHO-K1) were treated with a high dose of the cyclophosphamide analogue mafosfamide, and survivors were analyzed as to their cell killing response, chromosomal aberrations, and DNA repair capacity. None of the surviving clones tested were mafosfamide resistant. Surprisingly, some of the isolated cell lines exhibited a mafosfamide-hypersensitive phenotype. Two of these cell variants (designated as CHO-K1-4 and CHO-K1-12) were analyzed in more detail and proved to be cross-sensitive to other DNA cross-linking antineoplastic drugs such as N-hydroxyethyl-N-chloroethylnitrosourea, treosulfan, melphalan, cisplatin, and mitomycin C. The hypersensitivity to the cytotoxic effect of mafosfamide was accompanied by a 2-3-fold increase in the frequency of chromosomal aberrations. The intracellular levels of glutathione and glutathione S-transferase activity of the hypersensitive variants as well as growth rate were comparable to wild-type cells. Both the variant and the parental cells did not exhibit an increase in the amount of p53 upon UV irradiation. Furthermore, sensitive cells displayed similar UV-induced unscheduled DNA synthesis and showed identical amounts of ERCC1 mRNA as wild-type cells, indicating that the hypersensitive phenotype is not due to a defect in nucleotide excision repair. The induction of DNA single-strand breaks upon mafosfamide treatment was very similar in wild-type and mutants, and the removal of mafosfamide-induced DNA cross-links was not reduced in hypersensitive cells. However, the hypersensitive cell variants exhibited a less severe drug-induced block to DNA replication. From the data obtained, we conclude that hypersensitivity to cross-linking agents upon mafosfamide selection is due to changes in cell cycle progression of drug-treated cells.