Involvement of protein kinase C-β and ceramide in tumor necrosis factor-α-induced but not Fas-induced apoptosis of human myeloid leukemia cells

The role of protein kinase C-β (PKC-β) in apoptosis induced by tumor necrosis factor (TNF)-α and anti-Fas monoclonal antibody (mAb) in the human myeloid HL-60 leukemia cell line was studied by using its variant HL-525, which is deficient in PKC-α. In contrast to the parental HL-60 cells, HL- 525 is resistant to TNF-α-induced apoptosis but sensitive to anti-Fas mAb- induced apoptosis. Both cell types expressed similar levels of the TNF- receptor I, whereas the Fas receptor was detected only in HL-525 cells. Transfecting the HL-525 cells with an expression vector containing PKC-β reestablished their susceptibility to TNF-α-induced apoptosis. The apoptotic effect of TNF-α in HL-60 and the transfectants was abrogated by fumonisin, an inhibitor of ceramide generation, and by the peptide Ac-YVAD-BoMK, an inhibitor of caspase-1 and -4. Supplementing HL-525 cells with exogenous ceramides bypassed the PKC-β deficiency and induced apoptosis, which was also restrained by the caspase-1 and -4 inhibitor. The apoptotic effect of anti-Fas mAb in HL-525 cells was abrogated by the antioxidants N- acetylcysteine and glutathione and by the peptide z-DEVD-FMK, an inhibitor of caspase-3 and -7. We suggest that TNF-α-induced apoptosis involves PKC-β and then ceramide and, in turn, caspase-1 and/or -4, whereas anti-Fas mAb- induced apoptosis utilizes reactive oxygen intermediates and, in turn, caspase-3 and/or -7.