Properties of Human Melanoma Cells Resistant to 5-(3′,3′-Dimethyl-1-triazeno)imidazole-4-carboxamide and Other Methylating Agents¹

In a cloned line of human melanoma cells (MM253c1), the dose of 5-(3′,3′-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC) giving 37% survival was lowered by a factor of 6.8 when mouse liver microsomes were present. MM253c1 sublines resistant to microsome-activated DTIC, 5-(3′-methyl-1-triazeno)imidazole-4-carboxamide (MTIC), or N-methyl-N′-nitro-N-nitrosoguanidine were derived separately by one treatment of the parent line with a highly toxic level of the particular agent. Compared with MM253c1, the sublines had a higher chromosome and DNA content and a high degree of crossresistance to all of these agents, to N-methyl-N-nitrosourea, and to ethyl methanesulfonate, but less resistance to methyl methanesulfonate and dimethyl sulfate and no resistance to killing by melphalan, ultraviolet light, γ-rays, or light-activated DTIC and its photoproducts. Later passages of MM253c1 exhibited a spontaneous increase in chromosome and DNA content without affecting drug resistance. MTIC-induced DNA damage and repair were compared in late-passage MM253c1 and MM253c1-3D, a resistant subline obtained after three cycles of treatment with microsome-activated DTIC. Protein synthesis and, after allowance for pool size effects, nucleotide synthesis were not inhibited by MTIC during the first 12 hr. After 12 hr, inhibition of DNA synthesis occurred and correlated with cell death. The level of DNA repair synthesis induced by MTIC was the same in each line and was much less than that induced by equitoxic ultraviolet light. Sedimentation of DNA in alkaline sucrose (pH 13) revealed one to three breaks/10⁸ daltons in both lines during the first 24 hr after a lethal dose of MTIC (0.3 mm). MM253c1-3D showed fewer breaks than MM253c1 in the alkaline elution method (pH 12.5 and pH 12.7) 4 hr after treatment with 0.06 mm but not with 0.3 mm MTIC. The nucleoid (pH 8) and DNA-unwinding rate (pH 11.7) methods, detecting a much lower level of spontaneous or enzymically induced breaks, showed immediate dose-dependent formation of breaks, followed by substantial resealing within 1 to 2 hr and complete recovery after 24 hr; no major difference was found between the two lines. Reproductive death in both cell lines therefore follows replication of DNA carrying alkalilabile sites, possibly phosphate triesters or apurinic sites. MM253c1-3D and the other resistant lines may be mutants having an enhanced O-methyl repair system not readily detected by methods specifice for excision repair.