HIGH-DOSE SELECTION WITH MAFOSFAMIDE RESULTS IN SENSITIVITY TO DNA CROSS-LINKING AGENTS - CHARACTERIZATION OF HYPERSENSITIVE CELL-LINES

One of the most frequently used alkylating drugs in the therapy of a b road spectrum of tumors is cyclophosphamide. To elucidate the mechanis ms by which tumor cells acquire resistance to this agent, Chinese hams ter ovary cells (CHO-K1) were treated with a high dose of the cyclopho sphamide analogue mafosfamide, and survivors were analyzed as to their cell killing response, chromosomal aberrations, and DNA repair capaci ty, None of the surviving clones tested were mafosfamide resistant. Su rprisingly, some of the isolated cell lines exhibited a mafosfamide-hy persensitive phenotype. Two of these cell variants (designated as CHO- K1-4 and CHO-K1-12) were analyzed in more detail and proved to be cros s-sensitive to other DNA cross-linking antineoplastic drugs such as N- hydroxyethyl-N-chloroethylnitrosourea, treo-sulfan, melphalan, cisplat in, and mitomycin C. The hypersensitivity to the cytotoxic effect of m afosfamide was accompanied by a 2-3-fold increase in the frequency of chromosomal aberrations. The intracellular levels of glutathione and g lutathione S-transferase activity of the hypersensitive variants as we ll 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 p 53 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 phen otype is not due to a defect in nucleotide excision repair. The induct ion of DNA single-strand breaks upon mafosfamide treatment was very si milar 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 blo ck to DNA replication. From the data obtained, we conclude that hypers ensitivity to cross-linking agents upon mafosfamide selection is due t o changes in cell cycle progression of drug-treated cells.