[Evaluation of yeast functional assay to detect p53 gene mutation in minute endoscopic samples of colonic mucosal neoplasia].

Mutation in the p53 tumor suppressor gene is known to play a critical role in the carcinogenesis of many types of human cancers, especially in colon carcinogenesis. To investigate the early stage of neoplastic processes, a simple and reliable method is needed to evaluate the state of p53 mutation. For this purpose, we tested the availability of the recently developed yeast functional assay to minute endoscopic samples. The assay consisted of 3 steps: 1) extraction of mRNA from the minute samples, 2) reverse transcriptional polymerase chain reaction (RT-PCR) amplification of the mRNA, and 3) transformation of yeasts by means of crude PCR products. This was an effective way to analyze many samples within a short period of time (2 days). The results were visible either as white colonies (wild type) or red colonies (mutant type). At first, we tested whether the quantity of mutants was measurable or not by analyzing a calibrated mixture of the cells known to harbor mutant and wild-type. The percentage of the red colonies that showed mutant p53 alleles was in proportion to the initial input percentage of the mutant p53 cells. Secondly, in clinical cases, it was found that minute samples (10-20 mg) were sufficient both for the analysis of yeast functional assays and pathologic examinations. The samples taken from normal mucosa and adenomas revealed fewer red colonies than the background value of this assay (10%). All the samples from adenocarcinomas yielded red colonies over 20%. The percentage of red colonies was well consistent with the ratio of cancer cell numbers to total cells. However, the percentage was not always correlated with the number of immunohistochemically positive cells stained with monoclonal antibody to detect abnormal p53. These results indicate that this assay combined with histological examination may provide useful information on determining carcinogenesis in endoscopic samples.