Involvement of Endonuclease G in Nucleosomal DNA Fragmentation under Sustained Endogenous Oxidative Stress

Abstract

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 nick end-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.

4-hydroperoxy-cyclophosphamide mediates caspase-independent T-cell apoptosis involving oxidative stress-induced nuclear relocation of mitochondrial apoptogenic factors AIF and EndoG

“…13,35 Experiments using antioxidants further indicate that ROS acts upstream of Bax relocation and mitochondrial depolarization. 13 We observed that NAC substantially inhibited ROS production, upregulation of p53, conformational changes of Bax, disruption of the DC m , and cell death induction.…”

The Drosophila melanogaster Gene cg4930 Encodes a High Affinity Inhibitor for Endonuclease G

“…EndoG knock-out mice have no defect in apoptotic DNA degradation (16,17), but this might be due to redundancy with CAD and additional nucleases (1,2,18). RNA interference experiments and genetic data in Saccharomyces cerevisiae support a role of EndoG in apoptosis (8,19,20). EndoG is also thought to function in a non-apoptotic form of programmed cell death (21).…”

Endoplasmic Reticulum Stress-induced Apoptosis in Leishmania through Ca2+-dependent and Caspase-independent Mechanism

“…Endo G is a mitochondrial nuclease that has been suggested to play a role in mitochondrial DNA replication (38), and its role in nuclear DNA fragmentation during apoptosis has been evidenced from the studies on Caenorhabditis elegans (39,40). The release of Endo G from mitochondria and its translocation to nuclei have been documented in different cell lines during apoptotic stimuli (41)(42)(43). In Leishmania, both Cyt c and Endo G have been reported to play important roles in apoptosis (6).…”