Quantitation and localization of protein kinase C Beta II isozyme in colon

Abstract

The maintenance of colonic crypt PKC Beta 11 isozyme phics. levels may sustain the homeostatic balance between cell proliferation and apostasis. To define the role of PKC isozymes with respect to colonocyte phenotype, we determined the subcellular localization and crypt expression of PKC Beta 11 in relation to in situ cell proliferation and apostasis following carcinogen and diet manipulation. In a 2X2X2 factorial design, rats were fed diets containing either corn oil (containing n-6 PUFA) or fish oil (containing n-3 PUFA), cellulose (non-fermentable fiber) or pectin (highly fermentable fiber), and injected with azoxymethane (AOM) or saline. After 16 weeks, an intermediate time point when no macroscopic tumors are detected, colonic mucosa was processed for Western immunoblot and immunohistochemistry-morphodensitometric image analysis. In vivo cell proliferation was measured by incorporation of Brdu into DNA, and apostasis by TUNEL assay. Western blot analysis revealed that n-3 PUFA treatment blocked carcinogen induced overexpression of PKC Beta 11 (80 koa) in the distal colonic membrane (DM) fraction (p<0.05). Additionally, the n-3 Pl-TA/pectin groups (p<0.05). The 80 lea PKC Beta 11 isozyme was not detected in either the proximal cytologic (PC) or the distal cytologic (DC) fractions. Interestingly, a 50 koa immunoreactive band was observed in select samples (DM: 17/54, DC: 7/1 1, PM: 8/24, and PC: 8/8). Carcinogen significantly (p<0.05) enhanced PKC Beta 11 expression in all regions of the crypt (upper, middle, and lower (el-tiles). The kinetics of colonic cell proliferation (proliferative zone, and number of proliferative cells in the middle 1/3 of the crypt) paralleled the increase in PKC Beta 11 in carcinogen treated animals. There was also a significant (p<0.05) interaction between dietary fat and fiber on PKC Beta 11 expression. The kinetics of programmed cell death (apoptosis at the luminal surface) was inversely proportional to the expression of PKC Beta 11 in the upper fertile/apical region. These results suggest that an elevation in PKC Beta 11 expression along the crypt axis is linked to the enhancement of cell proliferation and suppression of apostasis, predictive biomarkers of tumor development.