Protein kinase CβII activation by 1-β-D-arabinofuranosylcytosine is antagonistic to stimulation of apoptosis and Bcl-2α down-regulation

Abstract

1-β-D-Arabinofuranosylcytosine (ara-C) stimulates the formation of both diglyceride and ceramide in the acute myelogenous leukemia cell line HL-60 (Strum, J. C., Small, G. W., Pauig, S. B., and Daniel, L. W. (1994) J. Biol. Chem 269, 15493-15497). ara-C also causes apoptosis in HL-60 cells which can be mimicked by exogenous ceramide. However, the signaling role for ara-C- induced diacylglycerol (DAG) is not defined. We found that Bcl-2 levels were increased by treatment of HL-60 cells with exogenous DAG or 12-O- tetradecanoylphorbol-13-acetate (TPA). In contrast, exogenous ceramide treatment caused a decrease in cellular Bcl-2 levels. Thus, ara-C stimulates the synthesis of two second messengers with opposing effects on Bcl-2. Since the effects of ara-C-induced DAG could be due to protein kinase C (PKC) activation, we determined the effects of ara-C on PKC isozymes. ara-C caused an increase in membrane-bound PKCβII (but not PKCα or PKCδ). ara-C or TPA- iuduced translocation of PKCβII was inhibited by 1-O-octadecyl-2-O-methyl- rac-glycero-3-phosphocholine (ET-18-OCH3), and ara-C-induced apoptosis was stimulated by pre-treatment of the cells with ET-18-OCHs. ET-18-OCH3 also inhibited stimulation of Bcl-2 by TPA and enhanced the decrease in Bcl-2 observed in ara-C-treated cells. These data indicate that ara-C-induced apoptosis is limited by ara-C-stimulated PKCβII through effects on Bcl-2. To further determine the role of PKC, we used antisense oligonucleotides directed toward PKCβII. The antisense, but not the sense, oligonucleotide inhibited PKCβII activation and enhanced ara-C-induced apoptosis. These data demonstrate that the stimulation of apoptosis by ara-C is self-limiting and can be enhanced by inhibition of PKC.