PKC-δ inhibits anchorage-dependent and -independent growth, enhances differentiation, and increases apoptosis in CaCo-2 cells

Background & Aims: Previous studies showed decreased protein kinase C (PKC)-δ expression in azoxymethane-induced rat and sporadic human colonic tumors. To elucidate the role of PKC-δ on the neoplastic phenotype of human colon cancer cells, we established stable transfectants of this isoenzyme in CaCo-2 cells. Methods: Human PKC-δ complementary DNA was subcloned into 2 distinct metallothionein-regulated expression vectors. Polyclonal populations of PKC-δ transfectants were characterized by Western blotting. PKC-δ activity was measured in situ using a PKC-δ–specific substrate. Proliferation was determined by Coulter counter, and cell cycle distribution was analyzed by flow cytometry. In vitro transformation was assessed by growth in soft agar and differentiation by changes in alkaline phosphatase and sucrase isomaltase. Apoptosis was evaluated by 4',6-diamidino-2-phenylindole dihydrochloride and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling staining. Results: In the presence of Zn2+, PKC-δ transfectants expressed a 4-fold increase in the protein and a 2-fold increase in activity of PKC-δ. PKC-δ transfectants exhibited a 30% decrease (P < 0.05) in cell growth and an enhanced differentiation phenotype. Increased PKC-δ expression induced a significant G0/G1 arrest, inhibited anchorage-independent growth (50%, P < 0.05), and caused a 2-fold increase in apoptosis (P < 0.05). Conclusions: Our studies show that increased expression of PKC-δ inhibits anchorage-dependent and -independent growth, while inducing cellular differentiation and limiting survival of this human colon cancer cell line.GASTROENTEROLOGY 2001;120:1700-1712