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.
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.
复制标题
苔藓抑素 1 和蛋白激酶 C 的其他药理学激活剂对 1-β-D-阿拉伯呋喃糖基]胞嘧啶诱导的 HL-60 人早幼粒细胞白血病细胞凋亡的影响。
DOI：
10.1016/0006-2952(94)90484-7
复制
发表时间：
1994
影响因子：
5.8
通讯作者：
Grant,S
中科院分区：
文献类型：
--
作者：
Jarvis,WD;Povirk,LF;Turner,AJ;Traylor,RS;Gewirtz,DA;Pettit,GR;Grant,S
我们先前已经证明苔藓抑素1，一种具有推定的蛋白激酶C（PKC）-活化特性的大环内酯，协同增强脱氧胞苷类似物1-[β-d-阿拉伯呋喃糖基]胞嘧啶（ara-C）在HL-60人早幼粒细胞白血病细胞中的抗白血病作用（Grantet al.，Biochem Pharmacol 42：853-867，1991），并且该作用似乎与对阿糖胞苷诱导的细胞凋亡的敏化有关（Grantet等人，Cancer Res52：6270-6278，1992）。在目前的研究中，我们已经评估了这种损害的程度，通过定量荧光分光光度法的小分子量，双链DNA片段，以提供：（a）阿糖胞苷和苔藓抑素1之间的相互作用的一个更完整的表征，和（B）苔藓抑素1治疗的相对影响与其他药理学操作的直接比较已知的调节蛋白激酶C的活性。将细胞暴露于阿糖胞苷（10− 9至10− 4 M; 1-24小时）可诱导DNA片段化程度的时间和浓度相关性增加。单独用苔藓抑素1（10− 11至10− 7 M; 1-24小时）处理未能诱导DNA损伤，但促进了随后6小时暴露于阿糖胞苷诱导的片段化程度的时间和浓度相关性增加。在用10− 8 M或10− 7 M苔藓抑素1预处理24小时后，观察到最大的片段化增强（例如，比单独使用ara-C获得的片段化增强2- 3倍），并且与HL-60细胞克隆形成的增强抑制密切相关。第1阶段肿瘤促进剂佛波醇二丁酸酯以双相方式增强阿糖胞苷的作用，在2.5 × 10− 8 M时最大程度地增强反应，但在10− 7 M时没有作用，而第2阶段肿瘤促进剂美泽瑞因在低浓度时不能增强阿糖胞苷相关的DNA片段化，在高浓度时拮抗阿糖胞苷的作用。与此相反，阿糖胞苷相关的DNA片段被衰减或废除，无论是连续预暴露于合成甘油二酯或预处理与外源性磷脂酶C在所有浓度的测试。DNA片段化的增加与S期细胞的募集或阿糖胞苷相关细胞分化的增强没有特异性关系。最后，苔藓抑素1的浓度，最大限度地增强阿糖胞苷相关的DNA片段与几乎完全下调总细胞PKC活性，而甘油二酯和磷脂酶C，抑制阿糖胞苷的反应，适度增加总PKC活性。两者合计，这些发现提供了定量的基础评估苔藓抑素1和相关化合物的能力，以加强阿糖胞苷诱导的DNA损伤，并强调了复杂的和多效性的影响，PKC家族的同工酶的调制可能会对阿糖胞苷相关的细胞凋亡在人类白血病细胞。
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 (Grantet al., Biochem Pharmacol42: 853–867, 1991), and that this effect appears to be related to sensitization to ara-C-induced apoptosis (Grantet al., Cancer Res52: 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 (10−9to 10−4M; 1–24 hr) induced time- and concentration-related increases in the extent of DNA fragmentation. Treatment with bryostatin 1 (10−11to 10−7M; 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 10−8M or 10−7M 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 × 10−8M, but exerting no effect at 10−7M, 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.