Quantifying levels of p53 mutation in mouse skin tumors.
Allele-specific competitive blocker PCR (ACB-PCR) amplification and quantification was developed for mouse p53 codon 270 CGT-->TGT base substitution and codon 244/245 AAC/CGC-->AAT/TGC tandem mutation. PCR products corresponding to p53 mutant and wild-type DNA sequences were generated. These DNAs were mixed in known proportions to construct samples with defined mutant fractions and the allele-specific detection of each mutation was systematically optimized. Each assay was used to analyze eight simulated solar light (SSL)-induced tumors. By analyzing mutant fraction (MF) standards in parallel with PCR products generated from tumor samples, p53 mutants could be quantified as subpopulations within the tumors. All eight tumors contained detectable levels of p53 codon 270 CGT-->TGT mutation. Three tumors had p53 MFs between 10(-4) and 10(-3). Five tumors had p53 MFs between 10(-3) and 10(-2). None of the eight mouse skin tumors had measurable levels of p53 codon 244/245 tandem mutation. Frequent detection of p53 codon 270 CGT-->TGT mutation provides additional evidence that a pyrimidine dinucleotide overlapping a methylated CpG site (Pyr(me)CG) is a susceptible target for SSL-induced mutagenesis. The absence of p53 codon 244/245 mutation in tumors may be explained by its mutant p53 phenotype and/or indicate that this site is not methylated. These initial results indicate that p53 codon 270 CGT-->TGT mutation may be a sensitive biomarker for SSL- or UV-induced mutagenesis. This mutational endpoint may be useful for evaluating the co-carcinogenicity of compounds administered in combination with UV or SSL.
Verkler
Tracie L
TL
Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079, USA.
Couch
Letha H
LH
Howard
Paul C
PC
Parsons
Barbara L
BL
D000818:Animals; D003062:Codon / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods; D017931:DNA Primers; D005453:Fluorescence; D016158:Genes, p53 / Q000235:genetics*; D005819:Genetic Markers / Q000235:genetics; D051379:Mice; D008817:Mice, Mutant Strains; D009154:Mutation / Q000235:genetics*; D009381:Neoplasms, Radiation-Induced / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011840:Radiation, Nonionizing / Q000009:adverse effects; D012878:Skin Neoplasms / Q000235:genetics*
D003062:Codon; D017931:DNA Primers; D005819:Genetic Markers