TY - JOUR
T1 - 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
AU - David Jarvis, W.
AU - Povirk, Lawrence F.
AU - Turner, Amy J.
AU - Traylor, Rebecca S.
AU - Gewirtz, David A.
AU - Pettit, George
AU - Grant, Steven
PY - 1994/3/2
Y1 - 1994/3/2
N2 - 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.
AB - 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.
KW - PKC activators
KW - apoptosis
KW - ara-C
KW - bryostatin 1
KW - leukemia
KW - protein kinase C
UR - http://www.scopus.com/inward/record.url?scp=0028181584&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0028181584&partnerID=8YFLogxK
U2 - 10.1016/0006-2952(94)90484-7
DO - 10.1016/0006-2952(94)90484-7
M3 - Article
C2 - 8135859
AN - SCOPUS:0028181584
SN - 0006-2952
VL - 47
SP - 839
EP - 852
JO - Biochemical Pharmacology
JF - Biochemical Pharmacology
IS - 5
ER -