Involvement of endonuclease G in nucleosomal DNA fragmentation under sustained endogenous oxidative stress
Involvement of endonuclease G in nucleosomal DNA fragmentation under sustained endogenous oxidative stress
复制标题
DOI：
10.1074/jbc.m510382200
复制
发表时间：
2006-03-10
影响因子：
4.8
通讯作者：
Shimamoto, N
中科院分区：
文献类型：
--
作者：
Ishihara, Y;Shimamoto, N
我们以前已经表明，3-氨基-1，2，4-三唑（ATZ）和巯基琥珀酸（MS），分别抑制过氧化氢酶和谷胱甘肽过氧化物酶的活性，在大鼠原代肝细胞引起持续的内源性氧化应激和凋亡细胞死亡没有半胱天冬酶-3激活。在这项研究中，我们研究了这种凋亡性细胞死亡的核小体DNA片段化的机制。ATZ + MS处理时间依赖性地增加了脱氧核苷酸转移酶介导的缺口末端标记（TUNEL）阳性细胞核的数量，从12小时开始，导致在24小时出现清晰的DNA梯状。脱氧核糖核酸酶（DNase）抑制剂金精三羧酸（ATA）完全抑制核小体DNA断裂，而泛半胱天冬酶抑制剂z-VAD-favorite则无此作用;此外，未检测到半胱天冬酶激活的DNase抑制剂的断裂，表明除半胱天冬酶激活的DNase外，还涉及DNase。考虑到内切核酸酶G（EndoG）以不依赖半胱天冬酶的方式起作用，我们首次克隆了大鼠EndoG cDNA。重组EndoG单独消化质粒DNA并诱导分离的肝细胞核中的核小体DNA片段化。重组EndoG活性抑制ATA，但不是过氧化氢，即使在10 mM。ATZ + MS刺激引起线粒体膜电位和EndoG从线粒体到细胞核的易位降低。通过应用RNA干扰，EndoG的mRNA水平几乎被完全抑制，并且EndoG蛋白的量降低到未处理细胞水平的大约一半。在这些条件下，TUNEL阳性细胞核的减少被显著抑制。这些结果表明，EndoG至少部分负责ATZ + MS诱导的内源性氧化应激条件下的核小体DNA片段化。
We have previously shown that inhibition of catalase and glutathione peroxidase activities by 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid (MS), respectively, in rat primary hepatocytes caused sustained endogenous oxidative stress and apoptotic cell death without caspase-3 activation. In this study, we investigated the mechanism of this apoptotic cell death in terms of nucleosomal DNA fragmentation. Treatment with ATZ + MS time-dependently increased the number of deoxynucleotidyl transferase-mediated nickend-labeling (TUNEL)-positive nuclei from 12 h, resulting in clear DNA laddering at 24 h. The deoxyribonuclease ( DNase) inhibitor, aurintricarboxylic acid (ATA), completely inhibited nucleosomal DNA fragmentation but the pan-caspase inhibitor, z-VAD-fmk was without effects; furthermore, the cleavage of inhibitor of caspase-activated DNase was not detected, indicating the involvement of DNase(s) other than caspase-activated DNase. Considering that endonuclease G (EndoG) reportedly acts in a caspase-independent manner, we cloned rat EndoG cDNA for the first time. Recombinant EndoG alone digested plasmid DNA and induced nucleosomal DNA fragmentation in isolated hepatocyte nuclei. Recombinant EndoG activity was inhibited by ATA but not by hydrogen peroxide, even at 10 mM. ATZ + MS stimulation elicited decreases in mitochondrial membrane potential and EndoG translocation from mitochondria to nuclei. By applying RNA interference, the mRNA levels of EndoG were almost completely suppressed and the amount of EndoG protein was decreased to approximately half the level of untreated cells. Under these conditions, decreases in TUNEL-positive nuclei were significantly suppressed. These results indicate that EndoG is responsible, at least in part, for nucleosomal DNA fragmentation under endogenous oxidative stress conditions induced by ATZ + MS.