Protein kinase Cε mediates PMA-induced growth inhibition of low population density NIH 3T3 fibroblasts

Phorbol 12-myristate-13-acetate (PMA), a potent tumor promoter and activator of most protein kinase C (PKC) isotypes, was found to significantly inhibit the growth of low population density (1-5% confluency) NIH 3T3 cells. Higher cell population density (above 10% confluency) provided protection from this growth inhibitory effect of PMA. PMA-induced growth arrest is accompanied by an elevation in the level of p21^Cip1 protein, along with cell cycle arrest at the G1/S transition. Activation of PKC is required for this growth inhibitory response since the pan PKC inhibitor GF109203 blocked this effect of PMA. However, the classical PKC inhibitor Gö6976 had no effect, strongly suggesting the involvement of novel PKC isotypes (δ and/or ε). Overexpression of PKCε, but not PKCδ, was found to potentiate PMA-induced growth inhibition. Overexpression of a kinase-inactive dominant-negative mutant of PKCε (K437R) decreased the growth inhibitory effect of PMA and also blocked the PMA-induced increase in the level of p21^Cip1 protein. Taken together, these results indicate that PMA has a cell population density-dependent effect on the growth of NIH 3T3 cells and that the PMA growth inhibitory effect at low cell population density is mediated through activation of PKCε.