Protein kinase C activation modulates pro- and anti-apoptotic signaling pathways.
Activation of protein kinase C (PKC) by TPA in human U937 myeloid leukemia cells is associated with induction of adherence, differentiation, and G0/G1 cell cycle arrest. In this study, we demonstrate that in addition to these differentiating cells about 25% of U937 cells accumulated in the subG1 phase after TPA treatment. This effect proved to be phorbol ester-specific, since other compounds such as retinoic acid or vitamin D3 failed to induce apoptosis in conjunction with differentiation. Only a specific inhibitor of PKC, GF109203X, but not the broad-spectrum kinase inhibitor staurosporine or a tyrosine kinase inhibitor genistein could reverse the induction of apoptosis. Bryostatin-1, another specific PKC activator with distinct biochemical activity failed to induce apoptosis. Moreover, bryostatin-1 completely abolished the induction of apoptosis in U937 cells even if added 8 hours after TPA treatment. Apart from apoptosis induced by various chemotherapeutic drugs, TPA-related cell death is not mediated by an autocrine Fas-FasL loop and could not be prevented by a blocking antibody to the Fas receptor. However, a 75% reduction in the number of apoptotic cells after TPA stimulation was achieved by preincubation with a blocking antibody to the TNFalpha receptor. Tetrapeptide cleavage assays revealed a four-fold increase in the DEVD-cleavage activity in U937 cells compared to a three-fold increase in TUR cells. Immunoblotting demonstrated that TUR cells did not activate significant levels of caspase-3 or -7, whereas in U937 cells a 20-kDa cleavage product corresponding to activated caspase-3 was detectable after 3 d TPA exposure. Moreover, immunoblots revealed a strongly reduced expression of the adaptor molecule APAF-1, which is required for cytochrome c-dependent activation of caspase-9 and subsequently caspase-3. APAF-1 proved to be inducible after PKC activation with phorbol ester in U937, but not in TUR cells. Thus, APAF-1 expression may, at least in part, be regulated by PKC activity and reduced APAF-1 levels are associated with resistance to various inducers of apoptosis. Furthermore, TPA exposure of U937 cells is associated with increased levels of the pro-apoptotic proteins Bak and Bcl-xs, whereas simultaneously a decline in the Bcl-2 expression was noticable.
Meinhardt
G
G
Department of Hematology/Oncology, Medizinische Klinik Innenstadt of the Ludwig-Maximilians-University, Munich, Germany. geroldmeinhardt@yahoo.de
Roth
J
J
Totok
G
G
D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D053477:Apoptotic Protease-Activating Factor 1; D015153:Blotting, Western; D054713:Bryostatins; D002117:Calcitriol / Q000494:pharmacology; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D053222:Fas Ligand Protein; D005434:Flow Cytometry; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D007783:Lactones / Q000494:pharmacology; D018942:Macrolides; D008301:Maleimides / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014212:Tretinoin / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D020298:U937 Cells; D019014:fas Receptor / Q000378:metabolism
C506592:APAF1 protein, human; D000970:Antineoplastic Agents; D053477:Apoptotic Protease-Activating Factor 1; D054713:Bryostatins; D004791:Enzyme Inhibitors; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D007211:Indoles; D007527:Isoenzymes; D007783:Lactones; D018942:Macrolides; D008301:Maleimides; D008562:Membrane Glycoproteins; D011506:Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha; D019014:fas Receptor; C046785:bryostatin 1; D014212:Tretinoin; D011493:Protein Kinase C; D020169:Caspases; D002117:Calcitriol; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate