Molecular Characterization of Cellular Proteins from Colorectal Tumors

Abstract

Aims and background Recent evidence has suggested that progressive stages of colorectal tumorigenesis can be defined by a sequence of genetic events characterized by altered expression of certain genes and the appearance of cancer-specific proteins. Although the significance of these events is still not clear, expression of cancer-specific protein components may be directly involved in the neoplastic transformation. The purpose of the present study was to compare molecular characteristics of cellular proteins from human colorectal tumors and normal colonic mucosa. Methods Normal mucosa and colorectal tumors from 18 patients were fractionated by a differential centrifugation scheme into four cellular fractions, i.e., nuclear, mitochondrial (10P), microsomal (100P) and cytosolic (100S). The proteins of these fractions from normal and tumorigenic mucosa were analyzed by one-dimensional polyacrylamide gel electrophoresis followed by Coomassie brilliant blue R-250 and silver nitrate staining. Nuclear proteins from normal and neoplastic tissues which had revealed the most significant diversities were further characterized by two-dimensional gel electrophoresis. Electrophoretically cancer-specific nuclear proteins in the molecular mass zone 35-40 kDa were used as immunogen to produce rabbit polyclonal antibodies. Results Electrophoretic analysis by one-dimensional gel electrophoresis showed clear differences in molecular characteristics of cellular proteins between normal and tumorigenic mucosa, especially among nuclear fractions. The latter were also confirmed by their two-dimensional electrophoresis results. Rabbit antibodies raised against electrophoretically specific nuclear proteins characterized by molecular mass of 35-40 kDa cross-reacted with 36 kDa polypeptide in 15 of 18 (83.3%) studied nuclear fractions of colorectal tumors but not with any normal mucosa. In some cases, nuclear cancer-associated components of 38 and 40 kDa were also recognized by these antibodies. Conclusions During colorectal carcinogenesis, specific expression of several nuclear proteins takes place. One of them, the polypeptide of 36 kDa not found in normal colonic epithelium, was shared by over 83% of the studied carcinomas despite variations in detailed cancer properties. This particular nuclear protein may be considered as a potential marker for the colon malignancy.