Effects of bryostatin 1 and other pharmacological activators of protein kinase C on 1-[β- d-arabinofuranosyl]cytosine-induced apoptosis in HL-60 human promyelocytic leukemia cells

Abstract

We have demonstrated previously that bryostatin 1, a macrocyclic lactone with putative protein kinase C (PKC)-activating properties, synergistically augments the antileukemic actions of the deoxycytidine analog 1-[β- d-arabinofuranosyl]cytosine (ara-C) in HL-60 human promyelocytic leukemia cells (Grant et al., Biochem Pharmacol 42: 853–867, 1991), and that this effect appears to be related to sensitization to ara-C-induced apoptosis (Grant et al., Cancer Res 52: 6270–6278, 1992). In the present studies, we have assessed the extent of this damage by quantitative spectrofluorophotometry of small molecular weight, double-stranded DNA fragments in order to provide: (a) a more complete characterization of the interaction between ara-C and bryostatin 1, and (b) a direct comparison of the relative effects of bryostatin 1 treatment with other pharmacological manipulations known to modulate protein kinase C activity. Exposure of cells to ara-C (10 −9 to 10 −4 M; 1–24 hr) induced time- and concentration-related increases in the extent of DNA fragmentation. Treatment with bryostatin 1 (10 −11 to 10 −7 M; 1–24 hr) alone failed to induce DNA damage, but promoted substantial time- and concentration-related increases in the extent of fragmentation induced by a subsequent 6-hr exposure to ara-C. Maximal potentiation of fragmentation (e.g. 2- to 3-fold greater than that obtained with ara-C alone) was observed following a 24-hr pretreatment with 10 −8 M or 10 −7 M bryostatin 1, and correlated closely with enhanced inhibition of HL-60 cell clonogenicity. The stage-1 tumor-promoter phorbol dibutyrate potentiated the effects of ara-C in a biphasic manner, maximally augmenting the response at 2.5 × 10 −8 M, but exerting no effect at 10 −7 M, whereas the stage-2 tumor-promoter mezerein failed to augment ara-C-related DNA fragmentation at low concentrations, and antagonized ara-C action at high concentrations. In contrast, ara-C-related DNA fragmentation was attenuated or abolished either by continual preexposure to synthetic diglyceride or by pretreatment with exogenous phospholipase C at all concentrations tested. Increased DNA fragmentation was not specifically related to recruitment of cells into S-phase or enhancement of ara-C-related cellular differentiation. Finally, concentrations of bryostatin 1 that maximally potentiated ara-C-related DNA fragmentation were associated with virtually complete down-regulation of total cellular PKC activity, whereas diglyceride and phospholipase C, which suppressed the response to ara-C, moderately increased total PKC activity. Taken together, these findings provide a quantitative basis for assessing the ability of bryostatin 1 and related compounds to potentiate ara-C-induced damage to DNA, and underscore the complex and pleiotropic effects that modulation of the PKC family of isoenzymes may exert on ara-C-related apoptosis in human leukemia cells.