von Haefen Clarissa, Wieder Thomas, Essmann Frank, Schulze-Osthoff Klaus, Dörken Bernd, Daniel Peter T
Department of Hematology, Oncology and Tumor Immunology, University Medical Center Charité, University of Berlin, Germany.
Oncogene. 2003 Apr 17;22(15):2236-47. doi: 10.1038/sj.onc.1206280.
Caspase-8 is a key effector of death-receptor-triggered apoptosis. In a previous study, we demonstrated, however, that caspase-8 can also be activated in a death receptor-independent manner via the mitochondrial apoptosis pathway, downstream of caspase-3. Here, we show that caspases-3 and -8 mediate a mitochondrial amplification loop that is required for the optimal release of cytochrome c, mitochondrial permeability shift transition, and cell death during apoptosis induced by treatment with the microtubule-damaging agent paclitaxel (Taxol). In contrast, Smac release from mitochondria followed a different pattern, and therefore seems to be regulated independently from cytochrome c release. Taxol-induced cell death was inhibited by the use of synthetic, cell-permeable caspase-3- (zDEVD-fmk) or caspase-8-specific (zIETD-fmk) inhibitors. Apoptosis signaling was not affected by a dominant-negative FADD mutant (FADD-DN), thereby excluding a role of death receptor signaling in the amplification loop and drug-induced apoptosis. The inhibitor experiments were corroborated by the use of BJAB cells overexpressing the natural serpin protease inhibitor, cytokine response modifier A. These data demonstrate that the complete activation of mitochondria, release of cytochrome c, and execution of drug-induced apoptosis require a mitochondrial amplification loop that depends on caspases-3 and -8 activation. In addition, this is the first report to demonstrate death receptor-independent caspase-8 autoprocessing in vivo.
半胱天冬酶-8是死亡受体触发的细胞凋亡的关键效应因子。然而，在先前的一项研究中，我们证明半胱天冬酶-8也可通过线粒体凋亡途径在不依赖死亡受体的情况下被激活，该途径位于半胱天冬酶-3的下游。在此，我们表明半胱天冬酶-3和-8介导了一个线粒体放大环，在用微管破坏剂紫杉醇（泰素）处理诱导的细胞凋亡过程中，该放大环是细胞色素c最佳释放、线粒体通透性转换孔开放和细胞死亡所必需的。相比之下，线粒体中Smac的释放遵循不同的模式，因此似乎是独立于细胞色素c的释放而被调控的。使用合成的、可穿透细胞的半胱天冬酶-3特异性抑制剂（zDEVD-fmk）或半胱天冬酶-8特异性抑制剂（zIETD-fmk）可抑制紫杉醇诱导的细胞死亡。凋亡信号传导不受显性负性FADD突变体（FADD-DN）的影响，从而排除了死亡受体信号传导在放大环和药物诱导的细胞凋亡中的作用。通过使用过表达天然丝氨酸蛋白酶抑制剂细胞因子反应调节因子A的BJAB细胞，证实了抑制剂实验的结果。这些数据表明，线粒体的完全激活、细胞色素c的释放以及药物诱导的细胞凋亡的执行需要一个依赖于半胱天冬酶-3和-8激活的线粒体放大环。此外，这是首次在体内证明不依赖死亡受体的半胱天冬酶-8自身加工的报告。
