P53 functional assay in bladder cancer

Mutation of the p53 tumor suppressor gene is thought to correlate with clinical outcome in patients with invasive transitional carcinoma of the urinary bladder. Assessment of p53 status is routinely performed using immunohistochemical staining techniques. These techniques are variable and do not always correlate with the actual presence of a gene mutation. Also, a gene mutation do not necessarily correlate with the function of the gene product. To improve upon our ability to identify p53 gene mutation which are relevant for alteration of biological function, we applied a functional analysis of p53 gene product to human transitional cell carcinomas. Messenger RNA was isolated from fresh transitional cell carcinomas obtained at surgery. Reverse transcription to cDNA was performed. The p53 sequence was amplified with polymerase chain reaction (PCR). The PCR-product linked to an expression vector was cotransfected into host yeast strain ylG397. The yeast cells used in this assay contain reporter plasmid which in the presence of a normal p53 gene product causes white colony formation and in the presence of mutant p53 gene product causes red pigment formation due to interruption of the adenine biosyntetic pathway. Transitionell cell carcinomas assayed by this methode with wild type p53 gene resulted in the growth of white colonies, those with functional relevant p53 gene mutation resulted in red pigmented growth of the yeast assay colonies. The status of p53 tumor suppressor gene in transitional cell carcinoma may serve as a very important prognostic indicator. Assays for p53 status must therefore be highly reliable and biological relevant. The functional assay of p53 tumor suppressor gene status is easy to perform and reproductible. We suggest that this new assay may provide a useful adjunct in the assessment of p53 tumor suppressor gene status paticularly in whom immunohistichemistry is equivocal.