Martins L M, Mesner P W, Kottke T J, Basi G S, Sinha S, Tung J S, Svingen P A, Madden B J, Takahashi A, McCormick D J, Earnshaw W C, Kaufmann S H
Institute of Cell & Molecular Biology, University of Edinburgh, Edinburgh, UK.
Blood. 1997 Dec 1;90(11):4283-96.
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 G2 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-Ile-Asp-aminomethylcoumarin (VEID-AMC) as well as an altered spectrum of active caspases that were affinity labeled with N-(Nalpha-benzyloxycarbonylglutamyl-Nepsilon-biotin yllysyl) 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.
以往研究表明，在经起始刺激处理4至24小时后检测时，K562慢性粒细胞白血病细胞对多种诱导凋亡的药物具有抗性，包括拓扑异构酶II（topo II）毒药依托泊苷。在本研究中，比较了K562细胞和凋亡能力正常的HL-60急性粒单核细胞白血病细胞对依托泊苷的反应，特别着重于确定细胞的长期命运。当用不同浓度的依托泊苷处理细胞1小时，随后接种于软琼脂中时，这两种细胞系表现出相似的敏感性，在5至10 μmol/L依托泊苷浓度下集落形成减少90%。用17 μmol/L依托泊苷处理1小时后，在不到6小时内，HL-60细胞中半胱天冬酶底物聚（ADP-核糖）聚合酶（PARP）的裂解、DNA片段化及凋亡形态学变化就很明显。经过相同处理后，K562细胞停滞在细胞周期的G2期，但在出现类似凋亡变化之前的3至4天内看起来正常。当依托泊苷剂量增加到68 μmol/L时，HL-60细胞在2至3小时后出现凋亡变化，而K562细胞在24至48小时后才出现相同变化。K562细胞凋亡变化发展的这种延迟伴随着细胞色素c向细胞质的延迟释放以及裂解荧光底物天冬氨酸-谷氨酸-缬氨酸-天冬氨酸-氨基三氟甲基香豆素（DEVD-AFC）和缬氨酸-谷氨酸-异亮氨酸-天冬氨酸-氨基甲基香豆素（VEID-AMC）的肽酶活性的延迟出现，以及与N-（Nα-苄氧羰基谷氨酰-Nε-生物素基赖氨酰）天冬氨酸[（2,6-二甲基苯甲酰）氧基]甲基酮[z-EK（bio）D-aomk]亲和标记的活性半胱天冬酶谱的改变。另一方面，在无细胞条件下，从这两种细胞系制备的细胞质中半胱天冬酶-3的激活以难以区分的动力学发生。总体而言，这些结果表明，细胞色素c释放和半胱天冬酶激活上游信号级联的延迟导致在依托泊苷处理的K562细胞中凋亡活跃期开始前有很长的潜伏期。一旦凋亡活跃期开始，HL-60和K562细胞中活性半胱天冬酶种类的谱和亚细胞分布不同，但最终在这两种细胞系中死亡的细胞比例相似。
