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Nitric oxide as a carcinogen: analysis by yeast functional assay of inactivating p53 mutations induced by nitric
1Department of Neurosurgery, Hokkaido University School of Medicine, Sapporo, Japan.
Abstract:
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.
Insights
Nitric oxide and cytosine methylation induce mutations in the p53 tumor suppressor gene. Cytosine methylation is the primary driver of C:G-->T:A transitions at CpG sites, mimicking in vivo deamination.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- The p53 tumor suppressor gene is crucial for preventing cancer.
- Mutations in p53 are common in many human cancers.
- Nitric oxide and DNA methylation are implicated in carcinogenesis.
Purpose of the Study:
- To investigate the mutagenic effects of nitric oxide and cytosine methylation on the p53 gene.
- To elucidate the role of cytosine methylation in p53 mutations at CpG sites.
Main Methods:
- Utilized a yeast functional assay to detect biologically relevant p53 mutations.
- Treated p53 cDNA with a nitric oxide donor (sydnonimine).
- Introduced cytosine methylation into p53 cDNA using PCR-mediated substitution or SssI CpG methylase.
Main Results:
- Nitric oxide significantly increased C:G-->A:T transversions in a dose-dependent manner.
- Cytosine methylation alone induced C:G-->T:A transitions.
- Combined treatment with nitric oxide and methylation further increased mutation yield.
Conclusions:
- Cytosine methylation is a key factor in C:G-->T:A transitions at CpG sites, supporting the deamination of 5-methylcytosine to thymine.
- Nitric oxide primarily induces C:G-->A:T transversions.
- These findings provide insights into p53 mutational mechanisms in cancer.