Morphodensitometric analysis of protein kinase C βII expression in rat colon: modulation by diet and relation to in situ cell proliferation and apoptosis

Abstract

We have recently demonstrated that overexpression of PKC β II renders transgenic mice more susceptible to carcinogeninduced colonic hyperproliferation and aberrant crypt foci formation. In order to further investigate the ability of PKC β II to modulate colonocyte cytokinetics, we determined the localization of PKC β II with respect to cell proliferation and apoptosis along the entire colonic crypt axis following carcinogen and diet manipulation. Rats were provided diets containing either corn oil [containing n-6 polyunsaturated fatty acids (PUFA)] or fish oil (containing n-3 PUFA), cellulose (non-fermentable fiber) or pectin (fermentable fiber) and injected with azoxymethane (AOM) or saline. After 16 weeks, an intermediate time point when no macroscopic tumors are detected, colonic sections were utilized for immunohistochemical image analysis and immunoblotting. Cell proliferation was measured by incorporation of bromodeoxyuridine into DNA and apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling. In the distal colon, PKC β II staining was localized to the upper portion of the crypt. In comparison, proximal crypts had more (P < 0.05) staining in the lower tertile. AOM enhanced (P < 0.05) PKC β II expression in all regions of the distal colonic crypt (upper, middle and lower tertiles). There was also an interaction (P < 0.05) between dietary fat and fiber on PKC β II expression (corn/pectin > fish/cellulose, fish/pectin > corn/cellulose) in all regions of the distal colonic crypt. With respect to colonic cell kinetics, proliferation paralleled the increase in PKC β II expression in carcinogen-treated animals. In contrast, apoptosis at the lumenal surface was inversely proportional to PKC β II expression in the upper tertile. These results suggest that an elevation in PKC β II expression along the crypt axis in the distal colon is linked to enhancement of cell proliferation and suppression of apoptosis, predictive intermediate biomarkers of tumor development. Therefore, select dietary factors may confer protection against colon carcinogenesis in part by blocking carcinogen-induced PKC β II expression.Abbreviations: AOM, azoxymethane; BrdU, bromodeoxyuridine; BT, biotinyl tyramide/amplification diluent; DAG, diacylglycerol; PBS, phosphate-buffered saline; PKC, protein kinase C; PUFA, polyunsaturated fatty acids; SA-HRP, streptavidin-conjugated horseradish peroxidase.

Protein kinase C βII and TGFβRII in ω-3 fatty acid–mediated inhibition of colon carcinogenesis

“…PKCβII is a lipid-dependent protein kinase whose activity can be modulated in vitro by ω-3 fatty acids ( Holian and Nelson, 1992 ; Davidson et al, 2000 ; Seung Kim et al, 2001 ). Therefore, we assessed the relationship between colonic PKCβII activity and dietary ω-3 fatty acids during AOM-induced colon carcinogenesis in vivo.…”

“…Animals were terminated at 16 wk after AOM injection. Colonic epithelial cell extracts from the distal colon were fractionated into membrane and cytosolic fractions and subjected to immunoblot analysis for PKCβII expression ( Davidson et al, 2000 ). Band intensity was quantitated using the Bio-Rad Laboratories Fluor-s imaging system.…”

The Multifaceted Effects of Omega-3 Polyunsaturated Fatty Acids on the Hallmarks of Cancer

“…PKC β 2, which is induced early in colon carcinogenesis, leading to self-sufficiency, cancer promotion, and carcinogen induced epithelial hyper-proliferation [72–76], is significantly decreased in rats fed FO. This blocked PKC β 2 hyperproliferation [74, 77]. …”