The role of O⁶-alkylguanine in cell killing and mutagenesis in chinese hamster ovary cells

Chinese hamster ovary cells with no detectable (<200 molecules/cell) O⁶-methylguanine-DNA methyltransferase (EC 2.1.1.63) were transfected with human cell DNA and pSV2neo plasmid by electroporation. Two stable transformant clones, GC-1 and GC-2, containing 4 × 10⁴ and 4-6 × 10³ methyltransferase molecules/cell respectively were isolated by successive screening in the presence of G418 and 2-chloroethyl-N-nitrosourea (CNU). Only three or four copies of pSV2neo DNA and no repetitive human DNA sequence were detected in these isolates. Secondary transfection of parent cells with GC-1 DNA yielded several clones containing 2-10 × 10³ methyltransferase molecules/cell. The rate of removal of O⁶-methylguanine in GC-1, GC-2 and parent cells in vivo reflected their methyltransferase levels, while the N-methylpurines were removed at similar rates in all three cell lines. The differential sensitivity of these cells to several alkylating agents, namely CNU, N-methyl-N-nitrosourea, N-methyl-N′-nitro-N-nitrosoguanidine and methyl-methane sulfonate (MMS), known to yield different proportions of O⁶-alkylguanine among the alkyl adducts in DNA, varied widely. The largest and smallest differences in toxic response were observed with CNU and MMS respectively. These cell lines showed no difference in sensitivity to the DNA cross-linking agent psoralen. These data strongly suggest that alkylating agents produce two classes of lethal lesions, one of which os O⁶-alkylguanine. Induction of mutations at the hypoxanthine-phosphoribosyltransferase locus in these cell lines suggests that, regardless of its relative yield, O⁶-methyl-guanine is the major mutagenic lesion for all alkylating agents.