Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line
Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line
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DOI：
10.1038/sj.cdd.4400959
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发表时间：
2002-03-01
影响因子：
12.4
通讯作者：
Knox, SJ
中科院分区：
文献类型：
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作者：
Armstrong, JS;Steinauer, KK;Knox, SJ
本研究的主要目的是确定在B细胞淋巴瘤系PW中细胞氧化还原状态调节后发生的生化信号事件的顺序，重点是线粒体信号的作用。L-丁硫酰亚胺磺酰亚胺（BSO），抑制γ谷氨酰半胱氨酸合成酶（gammaGCS），用于调节细胞的氧化还原状态。线粒体事件和下游凋亡信号和介质的顺序和作用进行了研究。BSO处理后，细胞谷胱甘肽（GSH）早期下降，随后活性氧（ROS）产生增加，在没有任何外部凋亡刺激的情况下诱导各种凋亡信号（在不同时间点可检测）。伴随细胞凋亡的生化事件序列包括总GSH减少95%和线粒体GSH部分（25%）保留，24 h时ROS产生无显著增加。在BSO处理后约3 h观察到核因子κ B亚基RelA的早期活化和核转位。BSO处理24 h后，细胞色素c释放到胞质溶胶中。在氧化还原调节长达72小时后，p53蛋白表达没有变化，并且观察到细胞增殖的p21（waf 1）非依赖性损失。令人惊讶的是，截短形式的p53以时间依赖性的方式表达，在BSO孵育后24小时开始。BSO处理后48和72小时之间发生不可逆的承诺凋亡时，线粒体GSH耗尽，并有增加ROS的生产。Procaspase 3蛋白水平显示与BSO孵育后的时间依赖性降低，特别是在48小时后，这与增加的ROS水平相对应。在96 h时，可检测到caspase 3裂解产物。pan-半胱天冬酶抑制剂zVADf在48 h部分阻断了凋亡诱导，72 h后无效。PW细胞可以从凋亡中获救，通过将它们从BSO孵育后长达48小时，但不是72小时与BSO。线粒体跨膜电位（DeltaPsi（m））在72 h观察期内在大多数细胞中保持完整，表明DeltaPsi（m）耗散不是PW细胞中诱导氧化还原依赖性凋亡的早期信号。这些数据表明，GSH单独减少可以作为凋亡信号传导的有效早期激活剂。线粒体GSH耗竭后ROS产生增加是一个重要事件，它不可逆地使PW细胞凋亡。
The primary objective of this study was to determine the sequence of biochemical signaling events that occur after modulation of the cellular redox state in the B cell lymphoma line, PW, with emphasis on the role of mitochondrial signaling. L-Buthionine sulphoximine (BSO), which inhibits gamma glutamyl cysteine synthetase (gammaGCS), was used to modulate the cellular redox status. The sequence and role of mitochondrial events and downstream apoptotic signals and mediators was studied. After BSO treatment, there was an early decline in cellular glutathione (GSH), followed by an increase in reactive oxygen species (ROS) production, which induced a variety of apoptotic signals (detectable at different time points) in the absence of any external apoptotic stimuli. The sequence of biochemical events accompanying apoptosis included a 95% decrease in total GSH and a partial (25%) preservation of mitochondrial GSH, without a significant increase in ROS production at 24 h. Early activation and nuclear translocation of the nuclear factor kappa B subunit RelA was observed at approximately 3 h after BSO treatment. Cytochrome c release into the cytosol was also seen after 24 h of BSO treatment. p53 protein expression was unchanged after redox modulation for up to 72 h, and p21(waf1) independent loss of cellular proliferation was observed. Surprisingly, a truncated form of p53 was expressed in a time-dependent manner, beginning at 24 h after BSO incubation. Irreversible commitment to apoptosis occurred between 48 and 72 h after BSO treatment when mitochondrial GSH was depleted, and there was an increase in ROS production. Procaspase 3 protein levels showed a time-dependent reduction following incubation with BSO, notably after 48 h, that corresponded with increasing ROS levels. At 96 h, caspase 3 cleavage products were detectable. The pan-caspase inhibitor zVADfmk, partially blocked the induction of apoptosis at 48 h, and was ineffective after 72 h. PW cells could be rescued from apoptosis by removing them from BSO after up to 48, but not 72 h incubation with BSO. Mitochondrial transmembrane potential (DeltaPsi(m)) remained intact in most of the cells during the 72 h observation period, indicating that DeltaPsi(m) dissipation is not an early signal for the induction of redox dependent apoptosis In PW cells. These data suggest that a decrease in GSH alone can act as a potent early activator of apoptotic signaling. Increased ROS production following mitochondrial GSH depletion, represents a crucial event, which irreversibly commits PW cells to apoptosis.