It has been proposed that O6-methylguanine DNA methyltransferase (MGMT) gene silencing in premalignant lesions and cancers of the lung might result in the acquisition of a 'mutator' phenotype. Previously, however, we found that Mgmt-/- mouse DNA failed to show an increase in spontaneous mutations. We thus hypothesized that only during exposure to specific environmental carcinogens would the consequences of MGMT deficiency become evident. Metabolism of the tobacco-derived nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) generates alkylating species that can react with the O6 position of deoxyguanine, thereby yielding substrates for MGMT-mediated repair. To investigate how MGMT might regulate the mutational effects of NNK, Mgmt-/- mice were crossed with a lacI-based transgenic reporter line (Big Blue™) thus enabling an assessment of the in vivo mutagenic effects of this agent. We observed the induction of a complex spectrum of NNK-dependent lacI mutations in both control and Mgmt-/- tissues, but only a trend in the mutant frequency increases that could be attributed to MGMT deficiency. The mutational spectra of NNK-treated Mgmt-/- lungs revealed an increase in the absolute number of G:C to A:T changes accompanied by a shift in these from CpG to GpG sites, consistent with an SN1 alkylation mechanism. In keeping with the high levels of MGMT expressed in the liver, more pronounced mutagenic effects and greater differences in O6 position of deoxyguanosine adduct levels following NNK were observed in Mgmt-/- versus wild-type mice. Extrapolating to humans, MGMT-deficient cells would likely exhibit an increased mutational burden, but only following exposures to specific environmental mutagens such as NNK.
Bibliographical noteFunding Information:
National Cancer Institute of Canada with funds from the Canadian Cancer Society (to F.R.J.), and also by grant R01-CA115309 from the National Cancer Institute (to L.A.P.). Alberta Heritage Foundation for Medical Research post-doctoral fellowships (to L.E.S. and H.A.L.).
We are indebted to Dr L. Samson for providing us with the Mgmt-deficient mouse strain, to M. Villemaire and L. Forster for their excellent technical assistance and to L. Zimmerman for maintenance of the animal colony. J.N.H. held a Natural Sciences and Engineering Research Council studentship, and F.R.J. was the recipient of a Canada Research Chair award.