Comparison of caspase activation and subcellular localization in HL-60 and K562 cells undergoing etoposide-induced apoptosis
Comparison of caspase activation and subcellular localization in HL-60 and K562 cells undergoing etoposide-induced apoptosis
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DOI：
10.1182/blood.v90.11.4283.4283_4283_4296
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发表时间：
1997-12-01
期刊：
影响因子：
20.3
通讯作者：
Kaufmann, SH
中科院分区：
文献类型：
--
作者：
Martins, LM;Mesner, PW;Kaufmann, SH
先前的研究表明，在初始刺激治疗后4至24小时检测K562慢性髓性白血病细胞对多种药物诱导的凋亡具有抗性，包括拓扑异构酶II （topo II）毒性etopo苷。在本研究中，我们比较了K562细胞和凋亡：精通HL-60急性髓单细胞白血病细胞对依托泊苷的反应，特别强调了确定细胞的长期命运。当细胞被不同浓度的。两株细胞株在5 ~ 10 μ mol/L依托泊苷的作用下，菌落形成减少90%，表现出相似的敏感性。17 μ mol/L依托波苷作用1小时后，HL-60细胞在不到6小时内出现caspase底物聚（adp -核糖）聚合酶（PARP）的断裂、DNA断裂和凋亡形态学改变。同样处理后，K562细胞停留在细胞周期的G(2)期，但在3 - 4天后出现类似的凋亡变化。当剂量增加到68 μ mol/L时，HL-60细胞在2 ~ 3 h后出现明显的凋亡变化，K562细胞在24 ~ 48 h后出现相同的凋亡变化。K562细胞凋亡变化发展的延迟伴随着细胞色素c向细胞质的延迟释放和肽酶活性的延迟出现，肽酶活性可裂解荧光底物asp - glu - val - asp -氨基三氟甲基香豆素（DEVD-AFC）和val - glu - ii -氨基三氟甲基香豆素（VEID-AMC），以及活性半胱天冬酶谱的改变，这些半胱天冬酶与N-（N- α -苄氧羰基谷氨酰基-N-epsilon-生物素酰酰）天冬氨酸密切相关[（2,6-二甲基苯甲酰基）氧]甲基酮[z-EK（生物）D-aomk]。另一方面，在无细胞条件下，caspase-3的激活在两种细胞系制备的细胞质中以难以区分的动力学发生。综上所述，这些结果表明，在依托泊苷处理的K562细胞中，细胞色素c释放和caspase激活上游信号级联的延迟导致细胞凋亡活动性阶段启动前有很长的潜伏期。在HL-60和K562细胞中，一旦开始凋亡的活跃阶段，活性caspase种类的光谱和亚细胞分布不同，但在这两种细胞系中最终被杀死的细胞比例相似。(C) 1997年由美国血液病学会出版。
Previous studies have shown that K562 chronic myelogenous leukemia cells are resistant to induction of apoptosis by a variety of agents, including the topoisomerase II (topo II) poison etoposide, when examined 4 to 24 hours after treatment with an initiating stimulus. In the present study, the responses of K562 cells and apoptosis:proficient HL-60 acute myelomonocytic leukemia cells to etoposide were compared, with particular emphasis on determining the long-term fate of the cells. When cells were treated with varying concentrations of. etoposide for 1 hour and subsequently plated in soft agar, the two cell lines displayed similar sensitivities, with a 90% reduction in colony formation at 5 to 10 mu mol/L etoposide. After treatment with 17 mu mol/L etoposide for 1 hour, cleavage of the caspase substrate poly(ADP-ribose) polymerase (PARP), DNA fragmentation, and apoptotic morphological changes were evident in HL-60 cells in less than 6 hours. After the same treatment, K562 cells arrested in G(2) phase of the cell cycle but otherwise appeared normal for 3 to 4 days before developing similar apoptotic changes. When the etoposide dose was increased to 68 mu mol/L, apoptotic changes were evident in HL-60 cells after 2 to 3 hours, whereas the same changes were observed in K562 cells after 24 to 48 hours. This delay in the development of apoptotic changes in K562 cells was accompanied by delayed release of cytochrome c to the cytosol and delayed appearance of peptidase activity that cleaved the fluorogenic substrates Asp-Glu-Val-Asp-aminotrifluoromethylcoumarin (DEVD-AFC) and Val-Glu-IIe-Aspaminomethylcoumarin (VEID-AMC) as well as an altered spectrum of active caspases that were affinity labeled with N-(N-alpha-benzyloxycarbonylglutamyl-N-epsilon-biotinyllysyl) aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone [z-EK(bio)D-aomk]. On the other hand, the activation of caspase-3 under cell-free conditions occurred with indistinguishable kinetics in cytosol prepared from the two cell lines. collectively, these results suggest that a delay in the signaling cascade upstream of cytochrome c release and caspase activation leads to a long latent period before the active phase of apoptosis is initiated In etoposide-treated K562 cells. Once the active phase of apoptosis is initiated, the spectrum and subcellular distribution of active caspase species differ between HL-60 and K562 cells, but a similar proportion of cells are ultimately killed in both cell lines. (C) 1997 by The American Society of Hematology.