Caspase inhibition blocks cell death and results in cell cycle arrest in cytokine-deprived hematopoietic cells
Caspase inhibition blocks cell death and results in cell cycle arrest in cytokine-deprived hematopoietic cells
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DOI：
10.1074/jbc.m607961200
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发表时间：
2007-01-26
影响因子：
4.8
通讯作者：
Knudson, C. Michael
中科院分区：
文献类型：
--
作者：
Brown, Nicholas M.;Martin, Sean M.;Knudson, C. Michael
细胞因子剥夺通常用于研究细胞凋亡的分子过程。在 FL5.12 细胞中白细胞介素 (IL)-3 撤除后，Bax 发生构象变化，导致其线粒体靶向、细胞色素 c 释放、caspase-9 激活和细胞凋亡。过表达 Casp9DN（显性阴性 caspase-9）或用 caspase 抑制剂 Q-VD-OPh 处理的细胞增加了活力，但未能增加克隆存活。我们发现半胱天冬酶抑制的细胞中有很大一部分活细胞（本文称为“拯救”细胞）在IL-3添加回来后未能启动细胞分裂。 “拯救”的细胞线粒体电位降低，活性 Bax 染色，二氢乙锭（一种对超氧化物水平敏感的试剂）染色减少。剥夺后重新添加 IL-3 表明 Bax 激活被逆转，而改变的 5,5', 6,6'-四氯-1,1', 3,3'-四乙基苯并咪唑基碳花青碘化物和二氢乙锭染色持续数天。此外，“被拯救”的细胞对鱼藤酮（一种线粒体呼吸抑制剂）有抵抗力。这些细胞对 2-脱氧葡萄糖高度敏感，2-脱氧葡萄糖是一种糖酵解抑制剂，也是拟议的抗癌剂。我们得出的结论是，抑制 caspase-9 可以使细胞保留活力，但细胞会出现长期线粒体功能障碍，并进入一种独特的非分裂状态，该状态与恶性细胞具有一些相同的特性。
Cytokine deprivation has been classically used to study molecular processes of apoptosis. Following interleukin (IL)-3 withdrawal in FL5.12 cells, Bax undergoes a conformational change that results in its mitochondria targeting, cytochrome c release, activation of caspase-9, and apoptosis. Cells overexpressing Casp9DN (dominant negative caspase-9) or treated with the caspase inhibitor Q-VD-OPh increased viability but failed to increase clonogenic survival. We find that caspase-inhibited cells had a significant fraction of viable cells (herein termed "rescued" cells) that failed to initiate cell division after IL-3 add back. The "rescued" cells had reduced mitochondrial potential, stained for active Bax, and had reduced staining with dihydroethidium, an agent sensitive to superoxide levels. Readdition of IL-3 after deprivation demonstrated that Bax activation was reversed, whereas altered 5,5', 6,6'-tetrachloro-1,1', 3,3'-tetraethylbenzimidazolylcarbocyanine iodide and dihydroethidium staining persisted for days. Furthermore, the "rescued" cells were resistant to rotenone, an inhibitor of mitochondrial respiration. The cells were highly sensitive to 2-deoxyglucose, an inhibitor of glycolysis and proposed anti-cancer agent. We conclude that the inhibition of caspase-9 allows cells to retain viability, but cells have prolonged mitochondrial dysfunction and enter a unique nondividing state that shares some properties with malignant cells.