Quantification and characterization of total cellular p53 protein in colorectal cancer

Immunochemical methods were developed for the optimal detection and characterization of total cellular p53 protein expression, both in the nuclear-attached and soluble fractions of colorectal cancers, in order to improve the correlation between protein deregulation and gene status. Seventy colorectal carcinomas were studied using 3 monoclonal antibodies in a sensitive analyzing system combining flow cytometry (nuclear-bound fraction) and enzyme-linked immunosorbent assay (ELISA; soluble fraction). DNA indices were calculated on the DNA histograms and mutations of the p53 gene were searched for in a subset of 41 cases. Three p53 expression patterns were found: 35 tumors were classified as pattern "A," characterized by high p53 expression including "mutant" conformation and missense mutations of the gene (16/17 cases tested), pattern "B" consisted of 15 tumors with total absence of p53 expression corresponding to nonsense mutations of the gene (8/9 cases tested), and pattern "C" of 20 tumors presenting low or undetectable nuclear-bound p53 but intermediate p53 protein content (pAb (1801+) in the soluble fraction. The latter pattern was associated with wild-type genes (14/15 cases tested), and with tumors that were often localized in the right colon compared to pattern "A" and "B" tumors (45% versus 8%, P < 0.009) and were frequently near-diploid (80% versus 29%, P < 0.0002). No correlation was found between tumor stage and the patterns of p53 expression. The results indicate that both flow cytometry (FCM) and ELISA seem necessary for the proper characterization of the p53 expression pattern, thus achieving a high degree of concordance with molecular analysis of gene mutations.