Boulares A H, Zoltoski A J, Yakovlev A, Xu M, Smulson M E
Department of Biochemistry and Molecular Biology, Georgetown University School of Medicine, Washington, DC 20007, USA.
J Biol Chem. 2001 Oct 12;276(41):38185-92. doi: 10.1074/jbc.M100629200. Epub 2001 Jul 18.
During apoptosis, endonucleases cleave DNA into 50-300-kb fragments and subsequently into internucleosomal fragments. DNA fragmentation factor (DFF) is implicated in apoptotic DNA cleavage; this factor comprises DFF45 and DFF40 subunits, the former of which acts as a chaperone and inhibitor of the catalytic subunit and whose cleavage by caspase-3 results in DFF activation. Disruption of the DFF45 gene blocks internucleosomal DNA fragmentation and confers resistance to apoptosis in primary thymocytes. The role of DFF-mediated DNA fragmentation in apoptosis was investigated in primary fibroblasts from DFF45(-/-) and control (DFF45(+/+)) mice. DFF45 deficiency rendered fibroblasts resistant to apoptosis induced by tumor necrosis factor (TNF). TNF induced rapid cleavage of DNA into approximately 50-kb fragments in DFF45(+/+) fibroblasts but not in DFF45(-/-) cells, indicating that DFF mediates this initial step in DNA processing. The TNF-induced activation of poly(ADP-ribose) polymerase (PARP), which requires PARP binding to DNA strand breaks, and the consequent depletion of the PARP substrate NAD were markedly delayed in DFF45(-/-) cells, suggesting a role for DFF in PARP activation. The activation of caspase-3 and mitochondrial events important in apoptotic signaling, including the loss of mitochondrial membrane potential and the release of cytochrome c, induced by TNF were similarly delayed in DFF45(-/-) fibroblasts. DFF45(-/-) and DFF45(+/+) cells were equally sensitive to the DNA-damaging agent and PARP activator N-methyl-N'-nitro-N-nitrosoguanidine. Inhibition of PARP by 3-aminobenzamide partially protected DFF45(+/+) cells against TNF-induced death and inhibited the associated release of cytochrome c and activation of caspase-3. These results suggest that the generation of 50-kb DNA fragments by DFF, together with the activation of PARP, mitochondrial dysfunction, and caspase-3 activation, contributes to an amplification loop in the death process.
在细胞凋亡过程中，核酸内切酶将DNA切割成50 - 300 kb的片段，随后再切割成核小体间的片段。DNA片段化因子（DFF）与凋亡性DNA切割有关；该因子由DFF45和DFF40亚基组成，前者作为催化亚基的伴侣蛋白和抑制剂，其被半胱天冬酶-3切割会导致DFF激活。DFF45基因的破坏会阻止核小体间DNA片段化，并使原代胸腺细胞对细胞凋亡产生抗性。在来自DFF45(-/-)小鼠和对照（DFF45(+/+)）小鼠的原代成纤维细胞中研究了DFF介导的DNA片段化在细胞凋亡中的作用。DFF45缺陷使成纤维细胞对肿瘤坏死因子（TNF）诱导的细胞凋亡产生抗性。TNF可诱导DFF45(+/+)成纤维细胞中的DNA快速切割成约50 kb的片段，但在DFF45(-/-)细胞中则不会，这表明DFF介导了DNA加工的这一初始步骤。TNF诱导的聚（ADP - 核糖）聚合酶（PARP）激活（这需要PARP与DNA链断裂结合）以及随后PARP底物NAD的消耗在DFF45(-/-)细胞中明显延迟，这表明DFF在PARP激活中发挥作用。TNF诱导的半胱天冬酶-3激活以及凋亡信号传导中重要的线粒体事件，包括线粒体膜电位丧失和细胞色素c释放，在DFF45(-/-)成纤维细胞中同样延迟。DFF45(-/-)和DFF45(+/+)细胞对DNA损伤剂和PARP激活剂N - 甲基 - N'-硝基 - N - 亚硝基胍同样敏感。用3 - 氨基苯甲酰胺抑制PARP可部分保护DFF45(+/+)细胞免受TNF诱导的死亡，并抑制相关的细胞色素c释放和半胱天冬酶-3激活。这些结果表明，DFF产生50 kb的DNA片段，连同PARP激活、线粒体功能障碍和半胱天冬酶-3激活，在死亡过程中促成了一个放大循环。
