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J. Biol. Chem., Vol. 281, Issue 10, 6726-6733, March 10, 2006

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

Yasuhiro Ishihara and Norio Shimamoto¹

From the Department of Biology, Graduate School of Science, Osaka University, Osaka 532-8686, Japan and the Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, Kagawa 769-2193, Japan

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.

Received for publication, September 21, 2005 , and in revised form, December 28, 2005.

The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/Gen-Bank^TM/EBI Data Bank with accession number(s) AB221075 [GenBank] .

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, 1314-1, Shido, Sanuki, Kagawa 769-2193, Japan. Tel.: 81-87-894-5111 (ext. 6513); Fax: 81-87-894-0181; E-mail: n-shimamoto{at}kph.bunri-u.ac.jp.

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