FIGURE 6.

PKC agonist treatment induces cyclin D1 expression in human colon cancer cells. A, PMA induces cyclin D1 expression in multiple colon cancer cell lines. FET, GEO, and DLD1 cells were treated with PMA for the indicated times, and the expression of cyclin D1 was analyzed by Western blotting. Even loading on blots was confirmed by immunoblotting for β-actin and/or fast green staining. B, the synthetic diacylglycerol DiC₈ induces cyclin D1 expression in colon cancer cells. DLD1 cells were treated with DiC₈ (D) or vehicle (ethanol; C) for 4 or 6 h, and expression of cyclin D1 and β-actin was determined by Western blotting. DiC₈ was added repeatedly in fresh medium (every hour) to compensate for its rapid metabolism in cells. C, induction of cyclin D1 expression by PMA in DLD1 cells is PKC-dependent. DLD1 cells were treated with PMA (P) or vehicle (C) for 5 h in the presence of the general PKC inhibitor BIM or vehicle (DMSO), as indicated, and analyzed as in B. D, PMA induction of cyclin D1 in colon cancer cells is dependent on ERK activity; as in C except that 10 or 20 μm U0126 was used in place of BIM. E, DLD1 cells express PKCϵ and PKCβII as PKC agonist-responsive isozymes. IEC-18 and DLD1 cells were analyzed for the expression of the indicated PKC isozymes by Western blotting. All panels represent analysis of the same protein extracts from each cell line. F, PKCβII activity is not required for PMA induction of cyclin D1 expression in DLD1 cells. Cells were treated with PMA (P) or vehicle (C) in the presence of the indicated concentrations of the PKCβ inhibitor 3-(1-(3-imidazol-1-ylpropyl)-1H-indol-3-yl)-4-anilino-1H-pyrrole-2,5-dione and analyzed for cyclin D1, PKCβII, and β-actin expression by immunoblotting. Data are representative of at least three independent experiments.