APOPTOSIS AND EXPRESSION OF BAX, BCL-X, AND BCL-2 APOPTOTIC REGULATORY PROTEINS IN COLORECTAL CARCINOMAS, AND ASSOCIATIONS WITH P53 GENOTYPE PHENOTYPE/

Aims-Spontaneous apoptosis and expression of the apoptotic regulatory proteins Bar, Bcl-x, and Bcl-2 were investigated in 50 colorectal carc inomas. The p53 genotypes/phenotypes and BAX genotypes were also deter mined, and possible associations of these with apoptosis and/or with e xpression of the different apoptotic regulatory proteins were studied. Methods-Terminal deoxynucleotidyl transferase (TdT) mediated dUTP lab elling of DNA fragments was used to detect apoptotic tumour cells in s ections and peroxidase immunohistochemistry was used to assess protein expression, p53 genotype/phenotype was determined using constant dena turant gel electrophoresis/immunoblotting and bar genotype was determi ned using polymerase chain reaction based methods. Results The distrib ution of tumour apoptotic indices was bimodal with a natural cut off a t 1.0% (range, 0.0-5.4%); the median fraction of apoptotic tumour cell s was 0.8%. Tumour apoptosis was not associated significantly with tum our DNA ploidy status. Normal mucosal tissue had less than 0.1% apopto tic cells. Staining intensities for Bar, Bcl-x, and Bcl-2 were strong; that is, equivalent to or greater than positive normal mucosal cells, in 11 of 50, 20 of 49, and 20 of 48 carcinomas. Frameshift mutations in the bar gene were detected in three of 42 tumours analysed, all of which were DNA diploid, and Bar protein expression in these tumours wa s absent or very low. Bar, Bcl-x, and Bcl-2 protein expression were no t correlated with tumour apoptosis or tumour DNA ploidy status. p53 wa s expressed in 34 of 50 tumours and p53 gene mutations were detected i n 22 of 29 p53 positive tumours analysed. Apoptosis was significantly lower in a greater number of p53 positive tumours than p53 negative tu mours. In addition, Bcl-2 protein expression was significantly higher in a greater number of p53 positive tumours compared with p53 negative tumours. Bar and Bcl-x protein expression were not significantly asso ciated with p53 phenotype/genotype. Conclusions-The results indicate t hat acquisition of a p53 phenotype is associated with lower spontaneou s apoptois and higher expression of Bcl-2. The results also suggest th at p53 is not a major determinant for Bar expression in colorectal car cinomas in vivo.