Detection of p53 mutation in human and rat tumor cells by yeast functional assay

Yeast functional assay is a simple and sensitive screening method for detection of p53 mutaion in human tumor tissue. The RT-PCR products are directly assayed according to its DNA binding capacity in yeast. Relative population of cells carrying mutant p53 allele is ascertained by counting red colonies. We analyzed the sensitivity and the clinical application of this method. Mutant p53 alleles are detectable from samples containing at least 6% of mutant p53 cDNA. It is disclosed that more than 20% of red colonies suggests the existence of clonal expansion of cells carrying mutant p53 allele. We reconstructed this method to establish a yeast assay for rat p53 gene. By this method, several p53 mutations were newly identified in rat tumor cell lines. Hepatocellular carcinoma and cholangiofibrosis tissues in LEG rat liver were then assayed with this method. Ratios of red colony were significantly elevated in these tissues, but were not large enough to suggest the existence of clonal p53 mutation. This result suggests that p53 mutaion is not a primary event for the development of hepatocellular carcinoma in LEG rat and that increased red colonies may indicate a possible genomic instability in LEC rat liver. In conclusion, the yeast functional assay is sensitive and useful method for the detection of clinical mutation. This assay for rat p53 gene provides a new monitor system of p53 status in various experiment.