Related Experiment Video
Updated: Aug 20, 2026
Cell Population Analyses During Skin Carcinogenesis
Published on: August 21, 2013
Quantifying levels of p53 mutation in mouse skin tumors
Tracie L Verkler1, Letha H Couch, Paul C Howard
1Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079, USA.
Abstract:
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.
Insights
A new PCR method detects mouse p53 mutations, finding the codon 270 CGT-->TGT mutation in all simulated solar light-induced tumors. This mutation may serve as a biomarker for UV-induced mutagenesis.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- The p53 tumor suppressor gene is frequently mutated in various cancers.
- Understanding mutation patterns induced by environmental factors like simulated solar light (SSL) is crucial for cancer prevention.
- Developing sensitive methods to detect low-frequency mutations is essential for early cancer detection and risk assessment.
Purpose of the Study:
- To develop and validate an allele-specific competitive blocker PCR (ACB-PCR) method for quantifying specific p53 mutations.
- To investigate the mutational status of p53 codon 270 and codon 244/245 in mouse skin tumors induced by SSL.
- To assess the potential of p53 mutations as biomarkers for SSL- or UV-induced mutagenesis.
Main Methods:
- Development of allele-specific competitive blocker PCR (ACB-PCR) for mouse p53 mutations.
- Optimization of PCR assays for allele-specific detection of CGT-->TGT and AAC/CGC-->AAT/TGC mutations.
- Quantification of mutant fractions (MFs) using standards and analysis of eight SSL-induced mouse skin tumors.
Main Results:
- The ACB-PCR method successfully amplified and quantified p53 mutant and wild-type sequences.
- All eight SSL-induced tumors exhibited detectable levels of the p53 codon 270 CGT-->TGT mutation, with MFs ranging from 10^-4 to 10^-2.
- No measurable levels of the p53 codon 244/245 tandem mutation were detected in any of the analyzed tumors.
Conclusions:
- The frequent detection of the p53 codon 270 CGT-->TGT mutation suggests that Pyr(me)CG sites are susceptible targets for SSL-induced mutagenesis.
- The absence of the codon 244/245 mutation may be due to its specific p53 mutant phenotype or lack of methylation.
- The p53 codon 270 CGT-->TGT mutation shows promise as a sensitive biomarker for SSL- or UV-induced mutagenesis and for evaluating co-carcinogenicity.
