INVERSE CORRELATION BETWEEN RER(-CANCER CELL-LINES() STATUS AND P53 MUTATION IN COLORECTAL)

The high point mutation rate of replication error-prone (RER(+)) cells could theoretically lead to inactivation of the p53 gene by polyclona l mutations, which might explain the conflicting results that have bee n published on the p53 status of RER(+) colon cancers, To address this issue, we tested the p53 status of 21 human colorectal cancer cell li nes, including four showing microsatellite instability (RER(+) phenoty pe). Denaturing gradient gel electrophoresis (DGGE) followed by sequen cing showed that all four RER(+) cell lines were mild type for p53 whi le 15 of the 17 RER(-) cell lines contained p53 mutations (P=0.001), E ight cell lines (four RER(+) and four RER(-)) were analysed using thre e complementary methods to test more rigorously the polyclonal mutatio n hypothesis, (i) Of 87 single-cell clones (seven to 14 per cell line) examined by DGGE, only those derived from known p53 mutant cell lines showed altered profiles, (ii) Antibody DO-7 stained more than 80% of nuclei from the p53 mutant cell lines, but only 15% of nuclei from the RER(+) cell lines, (iii) A yeast functional assay which can simultane ously detect polyclonal mutations at over 500 different sites in the p 53 cDNA scored all four RER(+) cell lines as containing only transcrip tionally active p53, These data thus do not support the polyclonal mut ation hypothesis and instead suggest that mismatch repair deficiency p rovides a p53-independent pathway for development of colorectal cancer s.