Quantitative assessment of the role of O6-methylguanine in the initiation of carcinogenesis by methylating agents.

Induction of transformation, cell lethality, and DNA lesions were quantitatively compared in Syrian hamster embryo cells (HEC) treated with three different methylating agents: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N-nitrosourea (MNU), or methyl methanesulfonate (MMS). Each induced transformation in a dose-dependent manner. On a molar basis, MNNG was approximately equal to 100- and 500-fold more effective than MNU and MMS, respectively. For each carcinogen the induction and repair of O6- and N7-methylguanine (O6- and N7-MeGua) relative to total guanine content was compared. At concentrations that induced equivalent transformation frequencies, the induction of O6-MeGua was the same for all three carcinogens, but N7-MeGua induction was 30-fold higher with MMS than with MNNG or MNU. The capacity to repair methylation lesions in HEC is limited because only between 50% and 70% of both O6- and N7-MeGua lesions were removed from the DNA within 24 hr after treatment, independent of methylating carcinogen. No consistent effect on either the rate of DNA replication or the size distribution of nascent strands correlated with O6-MeGua induction. These data support the hypothesis that O6-MeGua is the critical lesion for initiation of carcinogenesis by methylating agents. The frequency of transformation relative to O6-MeGua induction is 40- to 750-fold more than that of mutation. Based on the quantitative data for induction of O6-MeGua and transformation, the target size for initiation of carcinogenesis was calculated as a minimum of 10(4) nucleotides. This suggests that one of many genes can initiate carcinogenesis or that initiation is not the result of a single base mutation.