Involvement of Endonuclease G in Nucleosomal DNA Fragmentation under Sustained Endogenous Oxidative Stress
2006; Elsevier BV; Volume: 281; Issue: 10 Linguagem: Inglês
10.1074/jbc.m510382200
ISSN1083-351X
AutoresYasuhiro Ishihara, Norio Shimamoto,
Tópico(s)Phagocytosis and Immune Regulation
ResumoWe 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. 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. The generation and elimination of reactive oxygen species (ROS) 2The abbreviations used are: ROS, reactive oxygen species; ActD, actinomycin D; ATA, aurintricarboxylic acid; ATZ, 3-amino-1,2,4-triazole; CAD, caspase-activated deoxyribonuclease; DiOC6, 3,3′-dihexyloxacarbocyanine iodide; DNase, deoxyribonuclease; EndoG, endonuclease G; GPx, glutathione peroxidase; ICAD, inhibitor of caspaseactivated deoxyribonuclease; MS, mercaptosuccinic acid; RNAi, RNA interference; siRNA, small-interfering RNA; SKF, SKF-525A; TNFα, tumor necrosis factor α; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end-labeling; NTA, nitrilotriacetic acid; Z, benzyloxycarbonyl.2The abbreviations used are: ROS, reactive oxygen species; ActD, actinomycin D; ATA, aurintricarboxylic acid; ATZ, 3-amino-1,2,4-triazole; CAD, caspase-activated deoxyribonuclease; DiOC6, 3,3′-dihexyloxacarbocyanine iodide; DNase, deoxyribonuclease; EndoG, endonuclease G; GPx, glutathione peroxidase; ICAD, inhibitor of caspaseactivated deoxyribonuclease; MS, mercaptosuccinic acid; RNAi, RNA interference; siRNA, small-interfering RNA; SKF, SKF-525A; TNFα, tumor necrosis factor α; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end-labeling; NTA, nitrilotriacetic acid; Z, benzyloxycarbonyl. are well balanced in normal cells. However, increased ROS generation or decreased antioxidative capacity inside cells shifts this balance toward excess ROS production, leading to oxidative stress (1Droge W. Physiol. Rev. 2002; 82: 47-95Crossref PubMed Scopus (7493) Google Scholar, 2Djordjevic V.B. Int. Rev. Cytol. 2004; 237: 57-89Crossref PubMed Scopus (210) Google Scholar, 3Galli F. Piroddi M. Annetti C. Aisa C. Floridi E. Floridi A. Contrib. Nephrol. 2005; 149: 240-260Crossref PubMed Scopus (181) Google Scholar). Cells exhibit diverse responses against oxidative stress, including proliferation (4Brar S.S. Kennedy T.P. Sturrock A.B. Huecksteadt T.P. Quinn M.T. Whorton A.R. Hoidal J.R. Am. J. Physiol. Cell Physiol. 2002; 282: C1212-C1224Crossref PubMed Scopus (138) Google Scholar, 5Adachi T. Togashi H. Suzuki A. Kasai S. Ito J. Sugahara K. Kawata S. Hepatology. 2005; 41: 1272-1281Crossref PubMed Scopus (151) Google Scholar), differentiation (6Steinbeck M.J. Kim J.K. Trudeau M.J. Hauschka P.V. Karnovsky M.J. J. Cell. Physiol. 1998; 176: 574-587Crossref PubMed Scopus (57) Google Scholar, 7Katoh S. Mitsui Y. Kitani K. Suzuki T. Biochem. J. 1999; 338: 465-470Crossref PubMed Scopus (41) Google Scholar), and cell demise (apoptosis and/or necrosis) (8Gorman A. McGowan A. Cotter T.G. FEBS Lett. 1997; 404: 27-33Crossref PubMed Scopus (206) Google Scholar, 9Rollet-Labelle E. Grange M.J. Elbim C. Marquetty C. Gougerot-Pocidalo M.A. Pasquier C. Free Radic. Biol. Med. 1998; 24: 563-572Crossref PubMed Scopus (222) Google Scholar, 10Kanno S. Ishikawa M. Takayanagi M. Takayanagi Y. Sasaki K. Biol. Pharm. Bull. 2000; 23: 37-42Crossref PubMed Scopus (36) Google Scholar, 11Kazzaz J.A. Xu J. Palaia T.A. Mantell L. Fein A.M. Horowitz S. J. Biol. Chem. 1996; 271: 15182-15186Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 12Xu Y. Bradham C. Brenner D.A. Czaja M.J. Am. J. Physiol. 1997; 273: G795-G803PubMed Google Scholar, 13Gardner A.M. Xu F.H. Fady C. Jacoby F.J. Duffey D.C. Tu Y. Lichtenstein A. Free Radic. Biol. Med. 1997; 22: 73-83Crossref PubMed Scopus (331) Google Scholar), depending on the cell types or levels of oxidative stress. Apoptosis is a major cellular response against oxidative stress. In fact, there have been numerous reports on apoptosis induced by oxidative stress, and its mechanisms have been extensively discussed (14Suzuki Y.J. Forman H.J. Sevanian A. Free Radic. Biol. Med. 1997; 22: 269-285Crossref PubMed Scopus (1259) Google Scholar, 15Carmody R.J. Cotter T.G. Redox Rep. 2001; 6: 77-90Crossref PubMed Scopus (286) Google Scholar, 16Ueda S. Masutani H. Nakamura H. Tanaka T. Ueno M. Yodoi J. Antioxid. Redox Signal. 2002; 4: 405-414Crossref PubMed Scopus (467) Google Scholar, 17Suzuki Y.J. Antioxid. Redox Signal. 2003; 5: 741-749Crossref PubMed Scopus (27) Google Scholar, 18Matsuzawa A. Ichijo H. Antioxid. Redox Signal. 2005; 7: 472-481Crossref PubMed Scopus (243) Google Scholar, 19Culmsee C. Mattson M.P. Biochem. Biophys. Res. Commun. 2005; 331: 761-777Crossref PubMed Scopus (354) Google Scholar). The caspase family, which comprises a series of cysteine proteases, plays an important role in apoptosis and many groups have reported the involvement of caspases in apoptosis (20Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4124) Google Scholar, 21Nunez G. Benedict M.A. Hu Y. Inohara N. Oncogene. 1998; 17: 3237-3245Crossref PubMed Scopus (946) Google Scholar, 22Wolf B.B. Green D.R. J. Biol. Chem. 1999; 274: 20049-20052Abstract Full Text Full Text PDF PubMed Scopus (863) Google Scholar). Nucleosomal DNA fragmentation (DNA ladder formation) is one of the hallmarks of apoptosis and is executed by deoxyribonuclease (DNase) activated by apoptotic stimuli. Of the DNases that are activated by apoptotic stimuli, caspaseactivated DNase (CAD), which is induced in a caspase-dependent manner, is mainly involved in apoptotic DNA fragmentation. In normal cells, CAD exists as a heterodimer complex with an inhibitor subunit termed inhibitor of CAD (ICAD), and the DNase activity of CAD is masked by the state of the complex. ICAD has two sites cleaved by caspases. Once the caspases are activated and ICAD is cleaved, CAD is released from the complex and subsequently degrades chromosomal DNA to nucleosomal fragments (23Enari M. Sakahira H. Yokoyama H. Okawa K. Iwamatsu A. Nagata S. Nature. 1998; 391: 43-50Crossref PubMed Scopus (2805) Google Scholar, 24Sakahira H. Enari M. Nagata S. Nature. 1998; 391: 96-99Crossref PubMed Scopus (1422) Google Scholar). Whereas cell death under conditions in which the caspase family cannot function has been reported to resemble necrosis rather than apoptosis in several studies (25Hampton M.B. Orrenius S. FEBS Lett. 1997; 414: 552-556Crossref PubMed Scopus (590) Google Scholar, 26Nobel C.S. Burgess D.H. Zhivotovsky B. Burkitt M.J. Orrenius S. Slater A.F. Chem. Res. Toxicol. 1997; 10: 636-643Crossref PubMed Scopus (126) Google Scholar, 27Ueda S. Nakamura H. Masutani H. Sasada T. Yonehara S. Takabayashi A. Yamaoka Y. Yodoi J. J. Immunol. 1998; 161: 6689-6695PubMed Google Scholar, 28Samali A. Nordgren H. Zhivotovsky B. Peterson E. Orrenius S. Biochem. Biophys. Res. Commun. 1999; 255: 6-11Crossref PubMed Scopus (178) Google Scholar), a growing body of evidence that apoptotic cell death induced by oxidative stress occurs independently of caspase activation has been accumulated (29Carmody R.J. Cotter T.G. Cell Death Diff. 2000; 7: 282-291Crossref PubMed Scopus (126) Google Scholar, 30Shih C.M. Ko W.C. Wu J.S. Wei Y.H. Wang L.F. Chang E.E. Lo T.Y. Cheng H.H. Chen C.T. J. Cell. Biochem. 2004; 91: 384-397Crossref PubMed Scopus (148) Google Scholar, 31Kang Y.H. Lee E. Choi M.K. Ku J.L. Kim S.H. Park Y.G. Lim S.J. Int. J. Cancer. 2004; 112: 385-392Crossref PubMed Scopus (86) Google Scholar, 32Zheng Y. Yamaguchi H. Tian C. Lee M.W. Tang H. Wang H.G. Chen Q. Oncogene. 2005; 24: 3339-3347Crossref PubMed Scopus (62) Google Scholar). In fact, cells have shown apoptosis-like morphological changes, chromatin condensation and/or DNA fragmentation without caspase activation in these papers, suggesting that these distinctive characteristics of apoptosis take place in a caspase-independent manner. An argument about the definition of apoptosis or necrosis has emerged in recent years (33Blagosklonny M.V. Leukemia. 2000; 14: 1502-1508Crossref PubMed Scopus (120) Google Scholar, 34Sloviter R.S. Trends Pharmacol. Sci. 2002; 23: 19-24Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 35Liao D.J. Med. Hypotheses. 2005; 65: 23-28Crossref PubMed Scopus (24) Google Scholar). However, it is agreed that nucleosomal DNA fragmentation is a hallmark of apoptosis at this time. Thus, it is generally accepted that the presence or absence of nucleosomal DNA fragmentation distinguishes apoptosis from necrosis. We have previously shown that sustained oxidative stress is evoked and, subsequently, chromatin condensation and nucleosomal DNA fragmentation (which are typical features of apoptosis) are observed in rat primary hepatocytes when catalase and glutathione peroxidase (GPx) activities are inhibited for 24 h by 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid (MS), respectively (36Shiba D. Shimamoto N. Free Radic. Biol. Med. 1999; 27: 1019-1026Crossref PubMed Scopus (39) Google Scholar, 37Ishihara Y. Shiba D. Shimamoto N. Free Radic. Res. 2005; 39: 163-173Crossref PubMed Scopus (31) Google Scholar). In addition, caspase-3 is not activated during this apoptotic cell death, indicating that the apoptotic morphological changes are induced in a caspase-3-independent manner (37Ishihara Y. Shiba D. Shimamoto N. Free Radic. Res. 2005; 39: 163-173Crossref PubMed Scopus (31) Google Scholar). It should be noted that nucleosomal DNA fragmentation has been clearly observed in this apoptotic cell death without caspase-3 activation. However, considering that ICAD is reportedly processed by caspases other than caspase-3, we cannot exclude the possibility of the participation of CAD in nucleosomal DNA fragmentation (38Wolf B.B. Schuler M. Echeverri F. Green D.R. J. Biol. Chem. 1999; 274: 30651-30656Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar, 39Zhou X. Gordon S.A. Kim Y.M. Hoffman R.A. Chen Y. Zhang X.R. Simmons R.L. Ford H.R. J. Immunol. 2000; 165: 1252-1258Crossref PubMed Scopus (40) Google Scholar). Thus, here we examined whether CAD is involved in the nucleosomal DNA fragmentation induced by ATZ+MS by investigating ICAD processing and, if not so, this nucleosomal DNA fragmentation can be considered to take place caspase-independently. We focused on endonuclease G (EndoG) as an executor of DNA fragmentation, which has been shown to be caspase-independently translocated from mitochondria to nuclei in response to apoptotic stimuli, followed by the induction of nucleosomal DNA fragmentation (40Li L.Y. Luo X. Wang X. Nature. 2001; 412: 95-99Crossref PubMed Scopus (1395) Google Scholar, 41van Loo G. Schotte P. van Gurp M. Demol H. Hoorelbeke B. Gevaert K. Rodriguez I. Ruiz-Carrillo A. Vandekerckhove J. Declercq W. Beyaert R. Vandenabeele P. Cell Death Diff. 2001; 8: 1136-1142Crossref PubMed Scopus (277) Google Scholar). Materials—WILLIAMS E medium, ATZ, MS, aurintricarboxylic acid (ATA), SKF-525A (SKF), tumor necrosis factor α (TNFα), actinomycin D (ActD), and Hoechst 33342 were obtained from Sigma-Aldrich. l-Ascorbic acid sodium salt (vitamin C) was obtained from Wako Pure Industries, Ltd. (Osaka, Japan). z-VAD-fmk was obtained from BIOMOL (Plymouth Meeting, PA). Antibodies were purchased from the following sources: anti-CAD antibody, anti-ICAD antibody, antihistone H1 antibody, and anti-β-actin antibody were from Santa Cruz Biotechnology, Inc. (Beverly, MA); anti-His antibody was from Amersham Biosciences; anti-EndoG antibody was from MoBiTec (Goettingen, Germany); anti-cytochrome c antibody was from BD Pharmingen (San Diego, CA); and anti-cytochrome c oxidase subunit IV (COX IV) antibody was from Molecular Probes (Eugene, OR). All other chemicals were obtained from Sigma-Aldrich or Wako and were of the highest quality commercially available. Preparation of Rat Primary Hepatocytes—Male Wistar rats (150-200 g of body weight) were used. After the rats were anesthetized by intraperitoneal administration of sodium pentobarbital at a dose of 50 mg/kg, hepatocytes were isolated by an in situ collagenase perfusion method, as previously described (37Ishihara Y. Shiba D. Shimamoto N. Free Radic. Res. 2005; 39: 163-173Crossref PubMed Scopus (31) Google Scholar). Cells were plated onto 6-well collagen type I-coated plates or 60-mm collagen type I-coated dishes (Sumitomo Bakelite Co., Ltd., Tokyo, Japan) in WILLIAMS E medium containing 10% fetal bovine serum, 300 nm insulin, and 100 nm dexamethasone. Cells were incubated in a 95% air, 5% CO2 atmosphere at 37 °C for 2 h, and the medium was changed before they were subjected to the experiments described below. The average cell viability was about 90% according to trypan blue extrusion. Determination of Apoptotic Morphological Features—Chromatin condensation was assessed using DNA-binding fluorochrome Hoechst 33342. Hepatocytes grown in 6-well collagen type I-coated plates were washed with ice-cold phosphate-buffered saline (PBS) and fixed with 4% formaldehyde in PBS. The cells were then stained with Hoechst 33342 (1 μg/ml) for 5 min at room temperature. The nuclei were visualized using a fluorescence microscope (Nikon, Tokyo, Japan) at an excitation wavelength of 365 nm. Data were captured by a digital CCD camera (Hamamatsu C4742-95-10, Hamamatsu Photonics, Hamamatsu, Japan). For the detection of DNA fragmentation, an Apoptosis DNA Ladder kit (Wako) was used. The extracted DNA was separated by 1.5% agarose gel electrophoresis, stained with SYBR Green I and then visualized under ultraviolet light. Fluorographs of the DNA ladder were stored on a computer-assisted image analyzer (Fluor-S MultiImager, Bio-Rad). Immunoblotting Analysis—Equal amounts of proteins were loaded and separated by SDS-PAGE using a 7.5-15% (w/v) gradient polyacrylamide gel, and then transferred onto a polyvinylidene difluoride membrane. The blocked membranes were incubated overnight at 4 °C with the following antibodies at the stated dilutions (depending on the specific experiment): anti-CAD (1:200), anti-ICAD (1:200), anti-cytochrome c (1:500), anti-EndoG (1:500), anti-COX IV (1:1000), anti-histone H1 (1:200), or anti-β-actin (1:200). The membranes were then incubated with alkaline phosphatase-conjugated secondary antibody (Bio-Rad) and visualized with alkaline phosphatase substrates. Band intensity was measured by Multianalyst (Bio-Rad). Cloning of Rat EndoG—Rat liver QuickClone cDNA (BD Biosciences Clontech, Mountain View, CA) was amplified with Platinum TaqDNA Polymerase (Invitrogen) and PCR × Enhancer system (Invitrogen) using the EndoG-specific primers ESP1 (TAGCGCGCTGGTCCTCCGGTA, forward primer) and ESP2 (GCTTGAGTGAGTCCTACTCCA, reverse primer). The reaction conditions recommended by the manufacturer were used with minor modifications. Briefly, 30 cycles of amplification were carried out at 95 °C for 30 s, at 55 °C for 30 s and at 68 °C for 1 min. One-tenth of the amplification product from the first 30 cycles was reamplified under the same conditions. The products of the second amplification were analyzed by agarose gel electrophoresis and cloned using the TOPO TA Cloning kit (Invitrogen) in pCR 2.1-TOPO vectors. Positive clones were selected, followed by purification and sequencing of the cDNA. Expression and Purification of Recombinant Rat EndoG—Rat EndoG cDNA with 6× His tag was subcloned into the pET 30 vector (Novagen, Madison, WI), and the construct was transformed into Escherichia coli BL21(DE3). The transformed cells were cultured and then stimulated in recombinant EndoG by the addition of isopropyl β-d-thiogalactopyranoside. The collected cells were resuspended in 5 volumes of lysis buffer (50 mm NaHPO4, pH 8.0, 300 mm NaCl, and 0.05% Tween 20) including 10 mm imidazole. All subsequent procedures were conducted at 4 °C. Lysozyme was added at a final concentration of 1 mg/ml. After 30 min incubation on ice, the cell suspension was sonicated and centrifuged at 10,000 × g for 30 min at 4 °C to remove the cellular debris. The supernatant was loaded onto a Ni-NTA agarose (Qiagen, Hilden, Germany) column. After the column was washed with lysis buffer containing 50 mm imidazole, the protein was eluted with lysis buffer containing 100 mm imidazole. The eluted protein was dialyzed against buffer A (20 mm HEPES, pH 7.0, 1.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, and 1 mm dithiothreitol) containing 10 mm KCl and then loaded onto SP-500C (TOSOH Corp., Tokyo, Japan) column. The column was washed with buffer A containing 50 mm KCl and EndoG was eluted with a linear gradient from 50 to 700 mm KCl in buffer A. DNase Assay—Recombinant rat EndoG activity was determined by the method of Widlak et al. (42Widlak P. Li L.Y. Wang X. Garrard W.T. J. Biol. Chem. 2001; 276: 48404-48409Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Recombinant rat EndoG (100 ng) was mixed with pUC18 plasmids (1 mg) or nuclei isolated from rat primary hepatocytes (2 × 105 nuclei) and the mixture was incubated in the presence or absence of ATA or hydrogen peroxide for 20 min at 37 °C followed by agarose gel electrophoresis. DNA fragmentation was analyzed by electrophoresis using 1.5% agarose gel. Measurement of Mitochondrial Membrane Potential—Hepatocytes were incubated with 200 nm DiOC6 (3Galli F. Piroddi M. Annetti C. Aisa C. Floridi E. Floridi A. Contrib. Nephrol. 2005; 149: 240-260Crossref PubMed Scopus (181) Google Scholar) for 15 min under the abovementioned culture conditions. The cells were then washed with ice-cold phosphate-buffered saline and detached from the plates by trypsinization. Fluorescence was determined using FACSCalibur™ (BD Bioscience, San Jose, CA) with an excitation wavelength at 488 nm and an emission wavelength at 530 nm. Generally, 10,000 events were monitored and data analysis was performed by Cell Quest software (BD Bioscience). Cell Fractionation—To separate the mitochondrial fraction from the cytosolic fraction, an ApoAlert® Cell Fractionation kit (BD Biosciences Clontech) was used according to the manufacturer's instructions. Hepatocytes were homogenized in fractionation buffer, and the homogenates were centrifuged at 700 × g for 10 min at 4 °C. Supernatants were further centrifuged at 10,000 × g for 25 min at 4 °C. Supernatants from the second centrifugation were designated as cytosolic fractions and pellets resuspended in fractionation buffer were used as mitochondrial fractions. Isolation of nuclei was carried out by the method described by Staal et al. (43Staal F.J. Anderson M.T. Herzenberg L.A. Methods Enzymol. 1995; 252: 168-174Crossref PubMed Scopus (42) Google Scholar) with slight modifications. Cells were suspended in buffer B (10 mm HEPES, pH 7.8, 10 mm KCl, 2 mm MgCl2, 0.1 mm EDTA, 0.5 mm dithiothreitol, 1 μg/ml leupeptin, 1 μg/ml pepstatin, and 20 μm APMSF) and incubated on ice for 15 min. Nonident-40 at a final concentration of 0.6% was added to the cell suspension, which was immediately vortexed followed by centrifugation. A white pellet was washed with buffer B and used as a nuclear fraction. RNA Interference (RNAi)—Silencing of EndoG gene expression in primary rat hepatocytes was achieved by the siRNA technique. Duplexes of 21 nucleotides with two 3′-overhanging TT against rat EndoG were designed and synthesized by Proligo (Kyoto, Japan). The sense strand of the siRNA against EndoG was GUA CAG CCG CAG CUU GAC UTT. Scrambled nonspecific siRNA was used as a negative control (the sense strand was CGU GCA GUC GUC UCG CAA ATT). Transfection of rat primary hepatocytes with synthesized siRNA was carried out by electroporation using the Nucleofection® system (Amaxa, Koln, Germany), according to the protocols provided by the manufacturer. Briefly, 1.3 × 106 cells were resuspended in 100 μl of nucleofector solution (Human T Cell Nucleofector kit) containing 150 pmol of double-stranded siRNAs. After electroporation, transfected cell suspensions were transferred into 6-well collagen type I-coated plates with 1.2 ml of prewarmed cultured medium. After 2 h of incubation, the medium was changed, and the cells were further incubated for 12 h. Subsequently, the medium was changed, and the cells were further incubated with ATZ+MS in the presence or absence of inhibitors for 24 h. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis—Total RNA extraction from hepatocytes was performed using an RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. First-strand cDNA was synthesized from 1 μg of total RNA using a TaKaRa RNA PCR kit (AMV) Ver. 2.1 (TaKaRa, Otsu, Japan). For PCR analysis, cDNA was amplified by Platinum TaqDNA polymerase (Invitrogen). β-Actin was used as the endogenous expression standard. Each PCR program consisted of a 2-min initial denaturation step at 94 °C, followed by 25 cycles (for EndoG) or 20 cycles (for β-actin) at 94 °C for 30 s, at 55 °C for 30 s and at 72 °C for 1 min, on a PCR Thermal Cycler Personal (TaKaRa). The primer sequences were as follows: EndoG forward primer, CCG CGA GTC CTA CGT GCT GA; EndoG reverse primer, ATC ACA TAG GAA CGC AGC TCG AC; β-actin forward primer, TTC AAC ACC CCA GCC ATG TA; and β-actin reverse primer, TGA TCC ACA TCT GCT GGA AG. The amplified products were separated by electrophoresis on agarose gels. The band intensity was measured using a Multianalyst (Bio-Rad). Terminal Deoxynucleotidyl Transferase-mediated Nick End-labeling (TUNEL) Assay—The TUNEL assay was performed using the Dead-End™ Fluorometric TUNEL System (Promega) according to the manufacturer's instructions. After the hepatocytes were fixed, fluorescein-12-dUTP was catalytically incorporated into the fragmented DNA at the 3′-OH by terminal deoxynucleotidyl transferase. The fluorescein-12-dUTP-labeled DNA was visualized directly by fluorescence microscopy using an excitation wavelength at 488 nm. TUNEL-positive cells were determined from fluorographs for at least 300 nuclei. Determination of Protein Content—The protein content was determined according to the method of Bradford (44Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215632) Google Scholar) using bovine serum albumin as a standard. Statistical Analysis—Data for each variable are expressed as the means ± S.E. Data obtained from the two groups were compared using the Student's t test with Holm's corrections for multiple comparisons. Probability (p) values of less than 0.05 were considered significant. Time Course for Increases in TUNEL-positive Cells Induced by ATZ+MS Treatments—TUNEL-positive nuclei were not observed for up to 24 h in untreated hepatocytes. Treatment with ATZ+MS for 9 h had no effect on DNA fragmentation. However, treatment periods longer than 12 h induced a time-dependent increase in the number of TUNEL-positive nuclei and almost all nuclei were TUNEL-positive after 24 h of incubation (Fig. 1A). The inset in Fig. 1A showed morphological features of hepatocytes treated with ATZ+MS at 12 h. By phase contrast microscopy (Eclipse TE300, Nikon), two hepatocytes in the figure were observed to be apart from interconnected hepatocytes and rounded up. However, they adhered to the surface of the culture dish. The sizes of ATZ+MS treated hepatocytes including rounded ones were smaller than those of untreated hepatocytes, showing that cellular shrinkage occurred. Nuclear condensation was noted in the rounded hepatocytes. In addition, electrophoretic analysis with extracted DNA at 24 h showed clear DNA laddering when cells were treated with ATZ+MS, confirming our previous reports (Fig. 1B) (36Shiba D. Shimamoto N. Free Radic. Biol. Med. 1999; 27: 1019-1026Crossref PubMed Scopus (39) Google Scholar, 37Ishihara Y. Shiba D. Shimamoto N. Free Radic. Res. 2005; 39: 163-173Crossref PubMed Scopus (31) Google Scholar). Pretreatment with SKF (a cytochrome P450 inhibitor) or vitamin C (an antioxidant) almost completely inhibited both the increase in the number of TUNEL-positive nuclei and the appearance of DNA laddering induced by ATZ+MS (Fig. 1, A and B). The onset and time course for the increase in TUNEL-positive nuclei were similar to those for the increase in chromatin condensation (37Ishihara Y. Shiba D. Shimamoto N. Free Radic. Res. 2005; 39: 163-173Crossref PubMed Scopus (31) Google Scholar). Involvement of DNases Other Than CAD in ATZ+MS-induced DNA Fragmentation—As described above, clear nucleosomal DNA fragmentation was observed when hepatocytes were treated with ATZ+MS. Thus, to ensure whether DNase(s) were responsible for this fragmentation, ATA, which is a DNase inhibitor, was used (45Hallick R.B. Chelm B.K. Gray P.W. Orozco Jr., E.M. Nucleic Acids Res. 1977; 4: 3055-3064Crossref PubMed Scopus (227) Google Scholar). Pretreatment with 30 μm ATA had no effect on DNA laddering induced by ATZ+MS. However, pretreatment with 100 μm ATA completely suppressed DNA laddering, suggesting that DNase(s) were involved in DNA fragmentation induced by ATZ+MS (Fig. 2A). We next investigated the potential for CAD, which is well characterized as an apoptotic DNase, to induce nucleosomal DNA fragmentation (23Enari M. Sakahira H. Yokoyama H. Okawa K. Iwamatsu A. Nagata S. Nature. 1998; 391: 43-50Crossref PubMed Scopus (2805) Google Scholar, 24Sakahira H. Enari M. Nagata S. Nature. 1998; 391: 96-99Crossref PubMed Scopus (1422) Google Scholar). First, we investigated effects of a pan-caspase inhibitor, z-VAD-fmk, on DNA laddering. The addition of the z-VAD-fmk at a concentration of 10, 50, or 250 μm neither affected TUNEL-positive nuclei (Fig. 2B) nor nucleosomal DNA fragmentation (Fig. 2C) induced by ATZ+MS. Secondly, we examined whether ICAD was cleaved in hepatocytes treated with ATZ+MS because CAD is released and activated by the processing of ICAD. After 24 h of incubation, few ICAD-cleaved products were observed in untreated hepatocytes. Treatment with ATZ+MS showed similar quantities of ICAD-cleaved products to those in untreated cells (Fig. 2D). Stimulation by TNFα+ActD is well known to induce caspase-dependent apoptosis in many types of cell, including hepatocytes (46Xu Y. Bialik S. Jones B.E. Iimuro Y. Kitsis R.N. Srinivasan A. Brenner D.A. Czaja M.J. Am. J. Physiol. 1998; 275: C1058-C1066Crossref PubMed Google Scholar, 47Kim Y.M. Kim T.H. Chung H.T. Talanian R.V. Yin X.M. Billiar T.R. Hepatology. 2000; 32: 770-778Crossref PubMed Scopus (210) Google Scholar, 48Kim H. Song Y. Biochem. Biophys. Res. Commun. 2002; 295: 937-944Crossref PubMed Scopus (22) Google Scholar). In fact, treatment with TNFα+ActD induced cell death in rat primary hepatocytes accompanied by caspase activation, chromatin condensation and nucleosomal DNA fragmentati
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