Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death.
Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death.
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DOI：
10.1084/jem.182.2.367
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发表时间：
1995-08-01
期刊：
影响因子：
--
通讯作者：
Kroemer G
中科院分区：
文献类型：
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作者：
Zamzami N;Marchetti P;Castedo M;Decaudin D;Macho A;Hirsch T;Susin SA;Petit PX;Mignotte B;Kroemer G
程序性细胞死亡（PCD）是一种生理过程，通常定义为细胞溶解前发生的核形态（凋亡）和/或染色体DNA特征性逐步降解的改变。然而，确定的PCD特征，如线粒体还原酶活性或细胞溶解的损失，可以在去核细胞中诱导，表明细胞质PCD控制。在这里，我们报告了一个连续的线粒体功能失调，细胞收缩和核碎裂之前。进行中的PCD的第一个环孢菌素A可降解步骤的特征在于线粒体跨膜电位的降低，如通过特异性荧光染料（5，5 ′，6，6 ′-四氯-1，1 ′，3，3 ′-四乙基苯并咪唑碳菁+碘化物; 3，3 ′二己基氧碳菁碘化物）测定的。线粒体跨膜电位降低的细胞荧光纯化细胞最初不能将氢乙啶（HE）氧化成乙锭。在体外短期培养后，这些细胞获得HE氧化的能力，从而揭示了PCD的第二步，其特征在于线粒体产生活性氧（ROS）。该步骤可被鱼藤酮和钌红选择性抑制，但不受环孢菌素A的影响。最后，细胞减少其体积，这是一个被自由基清除剂延迟的步骤，表明ROS在凋亡过程中的作用。这种伴随早期PCD的改变序列在非常不同的细胞凋亡诱导模型中发现：糖皮质激素诱导的淋巴细胞死亡，T细胞杂交瘤的活化诱导的PCD，以及肿瘤坏死因子诱导的U937细胞死亡。抗凋亡原癌基因Bcl-2的转染同时抑制线粒体的改变和类固醇或神经酰胺引发的凋亡细胞死亡。体内注射荧光染料如5，5 '，6，6'-四氯-1，1 '，3，3'-四乙基苯并咪唑羰花青碘化物; 3，3 '二己基氧羰花青碘化物;或HE允许检测程序性死亡但仍缺乏核DNA片段化的细胞。特别是，线粒体活性氧产生的评估提供了PCD介导的淋巴细胞消耗的准确情况。总之，线粒体功能的改变构成了早期PCD的一个重要特征。
Programmed cell death (PCD) is a physiological process commonly defined by alterations in nuclear morphology (apoptosis) and/or characteristic stepwise degradation of chromosomal DNA occurring before cytolysis. However, determined characteristics of PCD such as loss in mitochondrial reductase activity or cytolysis can be induced in enucleated cells, indicating cytoplasmic PCD control. Here we report a sequential disregulation of mitochondrial function that precedes cell shrinkage and nuclear fragmentation. A first cyclosporin A-inhibitable step of ongoing PCD is characterized by a reduction of mitochondrial transmembrane potential, as determined by specific fluorochromes (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine++ + iodide; 3,3'dihexyloxacarbocyanine iodide). Cytofluorometrically purified cells with reduced mitochondrial transmembrane potential are initially incapable of oxidizing hydroethidine (HE) into ethidium. Upon short-term in vitro culture, such cells acquire the capacity of HE oxidation, thus revealing a second step of PCD marked by mitochondrial generation of reactive oxygen species (ROS). This step can be selectively inhibited by rotenone and ruthenium red yet is not affected by cyclosporin A. Finally, cells reduce their volume, a step that is delayed by radical scavengers, indicating the implication of ROS in the apoptotic process. This sequence of alterations accompanying early PCD is found in very different models of apoptosis induction: glucocorticoid-induced death of lymphocytes, activation-induced PCD of T cell hybridomas, and tumor necrosis factor-induced death of U937 cells. Transfection with the antiapoptotic protooncogene Bcl-2 simultaneously inhibits mitochondrial alterations and apoptotic cell death triggered by steroids or ceramide. In vivo injection of fluorochromes such as 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolcarbocyanine iodide; 3,3'dihexyloxacarbocyanine iodide; or HE allows for the detection of cells that are programmed for death but still lack nuclear DNA fragmentation. In particular, assessment of mitochondrial ROS generation provides an accurate picture of PCD-mediated lymphocyte depletion. In conclusion, alterations of mitochondrial function constitute an important feature of early PCD.