Temporal relationship of CDK1 activation and mitotic arrest to cytosolic accumulation of cytochrome C and caspase-3 activity during Taxol-induced apoptosis of human AML HL-60 cells
Temporal relationship of CDK1 activation and mitotic arrest to cytosolic accumulation of cytochrome C and caspase-3 activity during Taxol-induced apoptosis of human AML HL-60 cells
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DOI：
10.1038/sj.leu.2401218
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发表时间：
1998-12-01
期刊：
影响因子：
11.4
通讯作者：
Bhalla, K
中科院分区：
文献类型：
--
作者：
Ibrado, AM;Kim, CN;Bhalla, K
抗微管抗癌药物紫杉醇抑制微管动力学，引起有丝分裂阻滞，诱导caspase-3切割和活性，导致人AML HL-60细胞凋亡。Caspase-3的切割是由线粒体释放和细胞内积聚的电子转移蛋白细胞色素c （cyt c）触发的。紫杉醇诱导的G2/M转变是由p34(cdc-2) （CDK1）介导的，如果过早激活，也可能引发细胞凋亡。在本研究中，在s期同步和释放后，将强制表达bcl-x(L) （HL-60/ bcl-x(L)）和/或新霉素耐药基因（HL-60/neo）的HL-60细胞暴露于紫杉醇中，以检测cdk1相关的细胞周期事件和cyt -c触发的细胞凋亡的分子级联。在紫杉醇治疗后的不同时间间隔，进行细胞周期蛋白B1和CDK1水平的免疫印迹分析。此外，测定免疫沉淀CDK1的体外组蛋白H1激酶活性及其酪氨酸磷酸化状态（通过抗磷酸酪氨酸免疫印迹分析）。这里的数据显示，虽然紫杉醇诱导的CDK1激酶活性峰值在HL-60/neo细胞中出现得更早，但紫杉醇处理的HL-60/neo细胞在细胞周期蛋白B1积累、CDK1酪氨酸去磷酸化和有丝分裂阻滞方面与HL-60/Bcl-x(L)细胞没有显著差异。紫杉醇诱导的CDK1激活和有丝分裂先于胞质积累（类似于cyt c的六倍）。后一事件被Bcl-x(L)过表达阻断，但不被caspase-3抑制剂阻断。尽管caspase抑制剂和高Bcl-x(L)水平抑制caspase-3的切割和活性，但它们对紫杉醇诱导的CDK1激活或有丝分裂停止没有显著影响。这些发现表明，Bcl-x(L)过表达不影响紫杉醇诱导的CDK1活性，导致G2/M过渡，这暂时早于胞质细胞c介导的分裂、caspase-3活性和凋亡。
The antimicrotubule anticancer drug, Taxol, suppresses microtubule dynamics, causes mitotic arrest, and induces caspase-3 cleavage and activity resulting in apoptosis of human AML HL-60 cells. Caspase-3 cleavage is triggered by the mitochondrial release and cytosolic accumulation of the electron transfer protein, cytochrome c (cyt c). Taxol-induced G2/M transition is mediated by p34(cdc-2) (CDK1) which, if prematurely activated, may also trigger apoptosis. In the present studies following S-phase synchronization and release, HL-60 cells with enforced expression of the bcl-x(L) (HL-60/Bcl-x(L)) and/or neomycin resistance gene (HL-60/neo) were exposed to Taxol to examine CDK1-related cell-cycle events and the cyt c-triggered molecular cascade of apoptosis. At various time-intervals after Taxol treatment, immunoblot analyses of cyclin B1 and CDK1 levels were performed. In addition, the in vitro histone H1 kinase activity of immunoprecipitated CDK1 and its tyrosine phosphorylation status (by anti-phosphotyrosine immunoblot analysis) were determined. Data presented here show that, while Taxol induced peak CDK1 kinase activity occurs earlier in HL-60/neo cells, there are no significant differences in cyclin B1 accumulation, tyrosine dephosphorylation of CDK1, and mitotic arrest of Taxol-treated HL-60/neo vs HL-60/Bcl-x(L) cells. Taxol-induced CDK1 activation and mitosis preceded the cytosolic accumulation (similar to six-fold) of cyt c. The latter event was blocked by Bcl-x(L) overexpression but not by inhibitors of caspase-3. Although the caspase inhibitors and high Bcl-x(L) levels inhibited caspase-3 cleavage and activity, they did not significantly affect Taxol-induced CDK1 activation or mitotic arrest. These findings indicate that Bcl-x(L) overexpression does not affect Taxol-induced CDK1 activity leading to G2/M transition, which temporally precedes the cytosolic cyt c-mediated cleavage and activity of caspase-3 and apoptosis.