title: Effects of bryostatin 1 and other pharmacological activators of protein kinase C on 1-[beta-D-arabinofuranosyl]cytosine-induced apoptosis in HL-60 human promyelocytic leukemia cells.
abstract: We have demonstrated previously that bryostatin 1, a macrocylic lactone with putative protein kinase C (PKC)-activating properties, synergistically augments the antileukemic actions of the deoxycytidine analog 1-[beta-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 x 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.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Medicine, Medical College of Virginia, Richmond 23298-0230.
authors.forename: W D
authors.initials: WD
authors.lastname: Jarvis
authors.forename: L F
authors.initials: LF
authors.lastname: Povirk
authors.forename: A J
authors.initials: AJ
authors.lastname: Turner
authors.forename: R S
authors.initials: RS
authors.lastname: Traylor
authors.forename: D A
authors.initials: DA
authors.lastname: Gewirtz
authors.forename: G R
authors.initials: GR
authors.lastname: Pettit
authors.forename: S
authors.initials: S
authors.lastname: Grant
mesh_terms: D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D054713:Bryostatins; D003561:Cytarabine / Q000008:administration & dosage / Q000494:pharmacology; D004249:DNA Damage; D004273:DNA, Neoplasm / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D006801:Humans; D007783:Lactones / Q000008:administration & dosage / Q000494:pharmacology*; D007951:Leukemia, Myeloid / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D018942:Macrolides; D011493:Protein Kinase C / Q000032:analysis / Q000187:drug effects*; D014407:Tumor Cells, Cultured
chemical_list: D000970:Antineoplastic Agents; D054713:Bryostatins; D004273:DNA, Neoplasm; D007783:Lactones; D018942:Macrolides; D003561:Cytarabine; C046785:bryostatin 1; D011493:Protein Kinase C