Cyclophosphamide resistance developed in a human melanoma cell line

The Mer- human melanoma cell line MM253c1 was treated four times with cyclophosphamide activated in situ with rat liver microsomes, the fourth cycle being preceded by treatment with the mutagenic methylating agent N-methyl-N1-nitro-N-nitrosoguanidine. The resulting subline (MM253c1-4CG) showed increased resistance to activated cyclophosphamide; the resistance of seven allogeneic human tumor lines and of a fibroblast strain spanned the two extremes represented by the autologous MM253c1 lines. MM253c1-4CG cells were highly resistant to killing by methylating agents, a property indicative of conversion to the Mer+ phenotype. Compared with the parent line, MM253c1-4CG cells were resistant to DNA cross-linking agents having different structures and transport mechanisms (melphalan, mechlorethamine, and mitomycin) and were slightly more resistant to doxorubicin, gamma rays, and hydroxyurea, but were not resistant to killing by acrolein, cytarabine, hydrogen peroxide, 254 nm UV, [3H]thymidine, or vincristine. No difference in the intracellular concentration of potential alkylation targets (RNA, protein, and SH groups) was found, and neither cell line appeared able to activate cyclophosphamide or detoxify its metabolites. Caffeine and 3-aminobenzamide had no synergistic effect upon cyclophosphamide toxicity in either cell line. 3-Aminobenzamide showed synergism with the methylating agent 5-(3-methyl-1-triazeno)imidazole-4-carboxamide, the effect being greater in MM253c1-4CG than in MM253c1 cells. These results suggest that in vitro activation of cyclophosphamide produces a metabolite similar in stability to phosphoramide mustard, resistance to such toxicity being associated not with conversion to the Mer+ phenotype but with some intracellular change with confers resistance to a variety of DNA cross-linking agents.