King Malcolm Anthony
Department of Clinical Immunology, Pacific Medicine Laboratory Services, Royal North Shore Hospital, St. Leonards, NSW 2065, Australia.
Cytometry A. 2005 Feb;63(2):69-76. doi: 10.1002/cyto.a.20107.
Antimycin A (AMA) inhibits mitochondrial electron transport, collapses the mitochondrial membrane potential, and causes the production of reactive oxygen species. Previous work by me and my colleagues has demonstrated that AMA causes an array of typical apoptotic phenomena in HL-60 cells. The hypothesis that AMA causes HL-60 apoptosis by the intrinsic apoptotic pathway has now been tested.
Z-LEHD-FMK and Z-IETD-FMK were used as specific inhibitors of the initiator caspases 9 and 8, respectively. Caspase 3 activation, DNA fragmentation, and cellular disintegration were measured by flow cytometry. Cytochrome c release, chromatin condensation, and nuclear fragmentation were measured by microscopy.
AMA caused mitochondrial cytochrome c release and neither Z-LEHD-FMK nor Z-IETD-FMK inhibited that. In the absence of caspase inhibition there was a very close correlation between cytochrome c release and caspase 3 activation. Z-LEHD-FMK blocked caspase 3 activation but enhanced DNA fragmentation and failed to stop nuclear or cellular disintegration. Z-IETD-FMK also blocked caspase 3 activation but, in contrast to Z-LEHD-FMK, delayed DNA fragmentation and disintegration of the nucleus and the cell.
The hypothesis to explain AMA-induced HL-60 apoptosis was clearly inadequate because: (a) caspase 9 inhibition did not prevent DNA fragmentation or cell death, (b) apoptosis proceeded in the absence of caspase-3 activation, (c) the main pathway leading to activation of the executioner caspases was by caspase-8 activation, but caspase 8 inhibition only delayed apoptosis, and (d) activation of caspases 8 and 9 may be necessary for caspase-3 activation. Thus, in this cell model, apoptosis triggered from within the mitochondria does not necessarily proceed by caspase 9, and caspase 3 is not critical to apoptosis. The results provide further evidence that, when parts of the apoptotic network are blocked, a cell is able to complete the program of cell death by alternate pathways.
抗霉素A（AMA）抑制线粒体电子传递，使线粒体膜电位崩溃，并导致活性氧的产生。我和我的同事之前的研究表明，AMA在HL-60细胞中会引发一系列典型的凋亡现象。现在已经对AMA通过内源性凋亡途径导致HL-60细胞凋亡的假说进行了验证。
Z-LEHD-FMK和Z-IETD-FMK分别用作起始半胱天冬酶9和8的特异性抑制剂。通过流式细胞术检测半胱天冬酶3的激活、DNA片段化和细胞解体。通过显微镜检测细胞色素c释放、染色质浓缩和核碎裂。
AMA导致线粒体细胞色素c释放，Z-LEHD-FMK和Z-IETD-FMK均未抑制这一过程。在没有半胱天冬酶抑制的情况下，细胞色素c释放与半胱天冬酶3激活之间存在非常密切的相关性。Z-LEHD-FMK阻断了半胱天冬酶3的激活，但增强了DNA片段化，并且未能阻止核或细胞解体。Z-IETD-FMK也阻断了半胱天冬酶3的激活，但与Z-LEHD-FMK相反，它延迟了DNA片段化以及细胞核和细胞的解体。
解释AMA诱导HL-60细胞凋亡的假说明显不充分，原因如下：（a）半胱天冬酶9的抑制并未阻止DNA片段化或细胞死亡；（b）凋亡在没有半胱天冬酶-3激活的情况下仍会发生；（c）导致执行性半胱天冬酶激活的主要途径是通过半胱天冬酶-8激活，但半胱天冬酶8的抑制仅延迟了凋亡；（d）半胱天冬酶8和9的激活可能是半胱天冬酶-3激活所必需的。因此，在这个细胞模型中，由线粒体内引发的凋亡不一定通过半胱天冬酶9进行，并且半胱天冬酶3对凋亡并不关键。这些结果进一步证明，当凋亡网络的部分环节被阻断时，细胞能够通过替代途径完成细胞死亡程序。
