Activation of multiple interleukin-1 beta converting enzyme homologues in cytosol and nuclei of HL-60 cells during etoposide-induced apoptosis
Activation of multiple interleukin-1 beta converting enzyme homologues in cytosol and nuclei of HL-60 cells during etoposide-induced apoptosis
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DOI：
10.1074/jbc.272.11.7421
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发表时间：
1997-03-14
影响因子：
4.8
通讯作者：
Kaufmann, SH
中科院分区：
文献类型：
--
作者：
Martins, LM;Kottke, T;Kaufmann, SH
最近的遗传和生化研究表明，半胱氨酸依赖的天冬氨酸定向蛋白酶（半胱天冬氨酸酶）在细胞凋亡的活性阶段。在本研究中，利用三种互补技术跟踪依托泊苷诱导HL-60人白血病细胞凋亡过程中caspase的激活。免疫印迹显示，在依托泊苷诱导的细胞凋亡过程中，procaspase-2的水平没有变化，而在加入依托泊苷2-3小时后，procaspase-3的水平显著降低。同时，裂解devd -氨基三氟甲基香豆素和veid -氨基甲基香豆素的胞质肽酶活性分别提高了100倍和20倍；但yvad -氨基三氟甲基香豆素裂解活性仅增加1.5倍。与N-（N- α -苄基氧羰基谷氨酰基-N-epsilon-生物素酰酰）-天冬氨酸[（2,6-二甲基苯甲酰）氧]甲基酮的亲和标记表明，在加入依托泊苷后的前6小时内，细胞质中依次出现了多个活性的半胱天冬酶物种。对一维和二维凝胶的分析显示，两种酶与caspase-6结合，三种酶与活性caspase-3结合，表明这些酶的一些剪接或修饰变体在细胞凋亡过程中是活跃的。在HL-60凋亡细胞的细胞核中也发现了与胞质半胱天冬酶同源的多肽。这些结果不仅表明依托泊苷诱导的HL-60细胞凋亡伴随着胞浆和细胞核中多种caspase的选择性激活，而且表明其他caspase前体如procaspase-2在凋亡过程中存在但未被激活。
Recent genetic and biochemical studies have implicated cysteine-dependent aspartate-directed proteases (caspases) in the active phase of apoptosis. In the present study, three complementary techniques were utilized to follow caspase activation during the course of etoposide-induced apoptosis in HL-60 human leukemia cells. Immunoblotting revealed that levels of procaspase-2 did not change during etoposide-induced apoptosis, whereas levels of procaspase-3 diminished markedly 2-3 h after etoposide addition. At the same time, cytosolic peptidase activities that cleaved DEVD-aminotrifluoromethylcoumarin and VEID-aminomethylcoumarin increased 100- and 20-fold, respectively; but there was only a 1.5-fold increase in YVAD-aminotrifluoromethylcoumarin cleavage activity. Affinity labeling with N-(N-alpha-benzyloxycarbonylglutamyl-N-epsilon-biotinyllysyl)- aspartic acid [(2,6-dimethylbenzoyl)oxy] methyl ketone indicated that multiple active caspase species sequentially appeared in the cytosol during the first 6 h after the addition of etoposide. Analysis on one- and two-dimensional gels revealed that two species comigrated with caspase-6 and three comigrated with active caspase-3 species, suggesting that several splice or modification variants of these enzymes are active during apoptosis. Polypeptides that comigrate with the cytosolic caspases were also labeled in nuclei of apoptotic HL-60 cells. These results not only indicate that etoposide-induced apoptosis in HL-60 cells is accompanied by the selective activation of multiple caspases in cytosol and nuclei, but also suggest that other caspase precursors such as procaspase-2 are present but not activated during apoptosis.