Protein kinase C-beta, fibronectin, alpha(5)beta(1)-integrin, and tumor necrosis factor-alpha are required for phorbol diester-induced apoptosis in human myeloid leukemia cells.

The human myeloid HL-60 cell line and its cell variant HL-525 were used to study signaling events leading to apoptosis induction by phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC) enzymes. Unlike parental cells, HL-525 cells are PKC-beta deficient and resistant to PMA-induced apoptosis. These cells regain susceptibility to apoptosis induction after transfection with a PKC-beta expression vector. By using this vector and specific neutralizing monoclonal antibodies (mAbs), it was established that PMA-induced apoptosis also called for an interaction between cell-surface alpha(5)beta(1)-integrin and its deposited ligand fibronectin (FN), which is downstream of PKC-beta activation. Experiments with mAbs, the PKC-beta vector, and exogenous FN revealed that the next step entailed an interaction between secreted tumor necrosis factor-alpha and its type I receptor. By using a sphingomyelinase inhibitor, it was concluded that the subsequent step involved ceramide production. Moreover, a permeable ceramide was effective in inducing apoptosis in both HL-60 and HL-525 cells, and this induction was caspase-1 and/or -4 dependent because an inhibitor of these caspases abrogated the induced apoptosis. Based on these and related differentiation studies, we conclude that the above signaling events, the early ones in particular, are shared with PMA-induced macrophage differentiation in the HL-60 cells. It is likely that once these cells acquire their macrophage phenotype and perform their tasks, they become superfluous and are eliminated from the body by a self-triggered apoptotic process that involves our proposed signaling scheme. Copyright 2001 Wiley-Liss, Inc.