Isolation and characterization of a Chinese hamster ovary cell line resistant to bifunctional nitrogen mustards

A drug-resistant derivative of a Chinese hamster ovary cell line has been generated by chronic exposure to progressively higher concentrations of chlorambucil. The cells exhibit greater than 20-fold resistance to the cytotoxic effects of chlorambucil and comparable levels of cross-resistance to mechlorethamine and melphalan. These drugs all belong to a class of bifunctional alkylating agents which generate DNA cross-links by reaction at the N-7 position of guanine. However, no resistance is observed to several other drugs which possess a similar mechanism of action, to cis-platinum diammine dichloride or to bischloroethylnitrosourea and mitomycin C, which cross-link DNA via the O6 position of guanine. Lack of resistance to vincristine, colchicine, or Adriamycin coupled with the failure of the calcium channel blocker verapamil to reverse the phenotype, indicates that the mechanism of resistance is distinct from that characterized by the multidrug-resistant phenotype. Support for this view comes from the finding that no significant alteration in melphalan uptake could be demonstrated. The phenotype is very stable and has been maintained during 12 months of continuous culture without further selection. A slightly elevated basal level of glutathione is present in the resistant cells, but resistance is not overcome by depletion of intracellular glutathione with buthionine sulfoximine. A cytosolic protein with a molecular weight of approximately 25,000 is constitutively overexpressed in the resistant cells. Although these cells have an abnormal karyotype, no conclusive evidence for any cytogenetic indicator of gene amplification could be detected.