Nitric oxide as a carcinogen: analysis by yeast functional assay of inactivating p53 mutations induced by nitric oxide

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We have used a yeast p53 functional assay to study induction of mutations in the p53 tumor suppressor gene by nitric oxide and cytosine methylation. The yeast assay identifies only biologically important p53 mutations. p53 cDNA was treated with the nitric oxide donor sydnonimine, PCR-amplified and transfected into yeast. Sydnonimine produced a significant, dose-dependent increase in C:G→A:T transversions. Many important p53 mutational hotspots are postulated to arise by deamination of methylCpG in tumors. We therefore examined nitric oxide induction of mutations in p53 cDNA methylated by PCR-mediated substitution of 5-methylcytosine for cytosine or by treatment with the SssI CpG methylase. Both methylation procedures increased the baseline mutation rate, and nitric oxide treatment produced a further increase in mutation yield. Sequence analysis showed that methylation alone led to C:G→T:A transitions, whereas nitric oxide treatment simply produced more C:G→A:T transversions. Thus the most important factor in C:G→T:A transition at CpG sites identified in this experimental system is cytosine methylation, consistent with spontaneous conversion of 5-methylcytosine to thymine by deamination.