POSSIBLE INVOLVEMENT OF PROTEIN-KINASE C-EPSILON IN PHORBOL ESTER-INDUCED GROWTH-INHIBITION OF HUMAN LYMPHOBLASTIC CELLS

Sustained activation of members of the protein kinase C (PKC) family i s known to influence the growth and differentiation of various cell ty pes, however, the specific roles for individual isoforms mediating the se cellular events have yet to be elucidated. Activation of PKC by pho rbol esters leads to growth inhibition in certain cell lines. The HT58 human B lymphoblastic cell may serve as a cellular model system to in vestigate the participation of individual isoforms in the initial even ts of growth arrest induced by phorbol ester. Determination of cell cy cle and investigation of apoptosis were performed by bow cytometric me asurements. Phorbol ester-induced translocation and down-regulation of the conventional alpha, beta and the novel epsilon isoforms of PKC we re demonstrated by Western blot analysis. At lower concentrations (0.5 ng/ml) phorbol myristate acetate (PMA) stimulated a G1 arrest with re tention of viability in the human HT58 B lymphoblastic cell. The prote in kinase inhibitor staurosporine at a concentration of 25 nM did not significantly alter HT58 cell viability. However, staurosporine (25 nM ) induced apoptosis in cells preincubated for 4 hr with 0.5-1.0 ng/ml PMA. The translocation of PKC-epsilon was observed within 30 min expos ure to 0.5 ng/ml PMA. After a 4 hr treatment, evidence for down-regula tion and an altered phosphorylation state of PKC-epsilon was seen. In contrast, the conventional alpha and beta isoforms were practically un affected by this PMA treatment. At higher PMA concentrations (50 ng/ml ) the alpha and beta isoforms showed a significant down-regulation. Th e preferential alterations in PKC-epsilon observed under the condition s required for PMA to influence the growth and survival of HT58 cells suggest a role for the Ca2+-independent epsilon isoform in mediating t he initial events of the phorbol ester stimulated cellular responses. Copyright (C) 1996 Elsevier Science Ltd