FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2031372644> ?p ?o ?g. }

• W2031372644 endingPage "23208" @default.
• W2031372644 startingPage "23200" @default.
• W2031372644 abstract "Topoisomerase I (Top1) is known to relax DNA supercoiling generated by transcription, replication, and chromatin remodeling. However, it can be trapped on DNA as cleavage complexes (Top1cc) by oxidative and carcinogenic DNA lesions, base damage, and camptothecin treatment. We show here that Top1 is also functionally involved in death receptor-induced programmed cell death. In cells exposed to TRAIL or Fas ligand, Top1cc form at the onset of apoptosis. Those apoptotic Top1cc are prevented by caspase inhibition and Bax inactivation, indicating that both caspases and the mitochondrial death pathway are required for their formation. Accordingly, direct activation of the mitochondrial pathway by BH3 mimetic molecules induces apoptotic Top1cc. We also show that TRAIL-induced apoptotic Top1cc are preferentially formed by caspase-3-cleaved Top1 at sites of oxidative DNA lesions with an average of one apoptotic Top1cc/100 kbp. Examination of Top1 knock-down cells treated with TRAIL revealed similar DNA fragmentation but a marked decrease in apoptotic nuclear fission with reduced formation of nuclear bodies. Thus, we propose that Top1 contributes to the full apoptotic responses induced by TRAIL. Topoisomerase I (Top1) is known to relax DNA supercoiling generated by transcription, replication, and chromatin remodeling. However, it can be trapped on DNA as cleavage complexes (Top1cc) by oxidative and carcinogenic DNA lesions, base damage, and camptothecin treatment. We show here that Top1 is also functionally involved in death receptor-induced programmed cell death. In cells exposed to TRAIL or Fas ligand, Top1cc form at the onset of apoptosis. Those apoptotic Top1cc are prevented by caspase inhibition and Bax inactivation, indicating that both caspases and the mitochondrial death pathway are required for their formation. Accordingly, direct activation of the mitochondrial pathway by BH3 mimetic molecules induces apoptotic Top1cc. We also show that TRAIL-induced apoptotic Top1cc are preferentially formed by caspase-3-cleaved Top1 at sites of oxidative DNA lesions with an average of one apoptotic Top1cc/100 kbp. Examination of Top1 knock-down cells treated with TRAIL revealed similar DNA fragmentation but a marked decrease in apoptotic nuclear fission with reduced formation of nuclear bodies. Thus, we propose that Top1 contributes to the full apoptotic responses induced by TRAIL. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) 2The abbreviations used are:TRAILTNF-related apoptosis-inducing ligandTNFtumor necrosis factorBH3Bcl-2 homology domain 3CPTcamptothecinNACN-acetyl-l-cysteinePARPpoly(ADP-ribose)-polymeraseROSreactive oxygen speciesTop1topoisomerase ITop1cctopoisomerase I cleavage complexesZbenzyloxycarbonylfmkfluoromethylketonesiRNAsmall interfering RNAAbantibody8-oxoG8-oxoguanine. is a promising therapeutic agent because it induces apoptosis in a wide variety of cancer cells without affecting normal tissues (1Ashkenazi A. Pai R.C. Fong S. Leung S. Lawrence D.A. Marsters S.A. Blackie C. Chang L. McMurtrey A.E. Hebert A. DeForge L. Koumenis I.L. Lewis D. Harris L. Bussiere J. Koeppen H. Shahrokh Z. Schwall R.H. J. Clin. Investig. 1999; 104: 155-162Crossref PubMed Scopus (2008) Google Scholar, 2Walczak H. Miller R.E. Ariail K. Gliniak B. Griffith T.S. Kubin M. Chin W. Jones J. Woodward A. Le T. Smith C. Smolak P. Goodwin R.G. Rauch C.T. Schuh J.C. Lynch D.H. Nat. Med. 1999; 5: 157-163Crossref PubMed Scopus (2236) Google Scholar). TRAIL belongs to the TNF family of cytokines, including TNFα and Fas ligand, which induce apoptosis by binding to their cognate plasma membrane receptors (3Ashkenazi A. Nat. Rev. Cancer. 2002; 2: 420-430Crossref PubMed Scopus (1111) Google Scholar). The binding of TRAIL to the DR4 or DR5 receptors and the binding of Fas ligand to Fas receptor cause the intracellular death domains of those receptors to trimerize, which leads to the recruitment of FADD and the activation of caspase-8. Caspase-8 then cleaves and thereby activates caspase-3 either directly (type I cells) or/and indirectly (type II cells) (4Scaffidi C. Fulda S. Srinivasan A. Friesen C. Li F. Tomaselli K.J. Debatin K.M. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 1675-1687Crossref PubMed Scopus (2633) Google Scholar) by activating the mitochondrial death pathway through the cleavage of Bid (5Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3085) Google Scholar). Cleaved Bid binds to and activates the pro-apoptotic Bcl-2 relatives Bax and Bak proteins, causing the release of mitochondrial cytochrome c and the activation of caspase-9 and caspase-3 (6Wei M.C. Lindsten T. Mootha V.K. Weiler S. Gross A. Ashiya M. Thompson C.B. Korsmeyer S.J. Genes Dev. 2000; 14: 2060-2071Crossref PubMed Google Scholar). Activated caspase-3 (and other downstream caspases) cleaves a broad array of intracellular targets including DNA topoisomerase I (Top1) (7Samejima K. Svingen P.A. Basi G.S. Kottke T. Mesner Jr., P.W. Stewart L. Durrieu F. Poirier G.G. Alnemri E.S. Champoux J.J. Kaufmann S.H. Earnshaw W.C. J. Biol. Chem. 1999; 274: 4335-4340Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). It is also required to induce the controlled rearrangement and degradation of nuclear structures with chromatin condensation, DNA fragmentation, nuclear fission, and release of apoptotic nuclear bodies in the extracellular space (8Samejima K. Earnshaw W.C. Nat. Rev. Mol. Cell Biol. 2005; 6: 677-688Crossref PubMed Scopus (237) Google Scholar). TRAIL-induced apoptosis also involves an accumulation of intracellular reactive oxygen species (ROS) (9Lee M.W. Park S.C. Yang Y.G. Yim S.O. Chae H.S. Bach J.H. Lee H.J. Kim K.Y. Lee W.B. Kim S.S. FEBS Lett. 2002; 512: 313-318Crossref PubMed Scopus (88) Google Scholar). TNF-related apoptosis-inducing ligand tumor necrosis factor Bcl-2 homology domain 3 camptothecin N-acetyl-l-cysteine poly(ADP-ribose)-polymerase reactive oxygen species topoisomerase I topoisomerase I cleavage complexes benzyloxycarbonyl fluoromethylketone small interfering RNA antibody 8-oxoguanine. Top1 removes DNA superhelical tensions generated during transcription, replication, and chromatin remodeling (10Wang J.C. Nat. Rev. Mol. Cell Biol. 2002; 3: 430-440Crossref PubMed Scopus (1916) Google Scholar) and is essential in higher eukaryotes (11Morham S.G. Kluckman K.D. Voulomanos N. Smithies O. Mol. Cell Biol. 1996; 16: 6804-6809Crossref PubMed Scopus (156) Google Scholar). It relaxes DNA by forming transient DNA single-strand breaks that are produced as Top1 forms a covalent bond between its active site tyrosine (Tyr723) and a 3′-DNA phosphate. These Top1 cleavage complexes (Top1cc) allow controlled rotation of the broken DNA around the intact strand (10Wang J.C. Nat. Rev. Mol. Cell Biol. 2002; 3: 430-440Crossref PubMed Scopus (1916) Google Scholar). Immediately after DNA relaxation, Top1 religates the break in the absence of added cofactor, such as ATP. Under normal conditions, the Top1cc are constitutively transient and almost undetectable because the DNA religation (closing) step is much faster than the DNA cleavage (nicking) step. The rapid resealing of Top1cc is inhibited by many common DNA base alterations (oxidation, alkylation, base mismatch, base loss), carcinogenic DNA adducts, and DNA backbone nicks (12Pourquier P. Pommier Y. Adv. Cancer Res. 2001; 80: 189-216Crossref PubMed Google Scholar). Top1cc can also be trapped with exquisite selectivity by camptothecin (CPT), a plant alkaloid (13Sirikantaramas S. Yamazaki M. Saito K. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 6782-6786Crossref PubMed Scopus (86) Google Scholar) whose semi-synthetic derivatives topotecan and irinotecan are used to treat human cancers (14Pommier Y. Nat. Rev. Cancer. 2006; 6: 789-802Crossref PubMed Scopus (1650) Google Scholar). Top1cc have also been detected in cells undergoing apoptosis in response to a wide range of stimuli, including arsenic trioxide (15Sordet O. Liao Z. Liu H. Antony S. Stevens E.V. Kohlhagen G. Fu H. Pommier Y. J. Biol. Chem. 2004; 279: 33968-33975Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), staurosporine (16Sordet O. Khan Q.A. Plo I. Pourquier P. Urasaki Y. Yoshida A. Antony S. Kohlhagen G. Solary E. Saparbaev M. Laval J. Pommier Y. J. Biol. Chem. 2004; 279: 50499-50504Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), tubulin and topoisomerase II (Top2) inhibitors (17Rockstroh A. Kleinert A. Kramer M. Grosse F. Soe K. Oncogene. 2007; 26: 123-131Crossref PubMed Scopus (8) Google Scholar, 18Sordet O. Goldman A. Pommier Y. Mol. Cancer Ther. 2006; 5: 3139-3144Crossref PubMed Scopus (28) Google Scholar), TNFα (17Rockstroh A. Kleinert A. Kramer M. Grosse F. Soe K. Oncogene. 2007; 26: 123-131Crossref PubMed Scopus (8) Google Scholar), and UV-C radiation (19Soe K. Rockstroh A. Schache P. Grosse F. DNA Repair. (Amst.). 2004; 3: 387-393Crossref PubMed Scopus (22) Google Scholar). In the present study, we demonstrated that apoptotic Top1cc also form in response to the physiological ligands TRAIL and Fas. We also investigated the mechanism of their production and provide evidence for their functional relevance during TRAIL-induced apoptosis. Cells, Drugs, and Chemical Reagents—The human leukemia (Jurkat) and colon carcinoma (HCT116) cell lines (American Type Culture Collection, Manassas, VA) were cultured as described (16Sordet O. Khan Q.A. Plo I. Pourquier P. Urasaki Y. Yoshida A. Antony S. Kohlhagen G. Solary E. Saparbaev M. Laval J. Pommier Y. J. Biol. Chem. 2004; 279: 50499-50504Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). HCT116 cells of each genotype (Bax+/-, Bax-/-, p53+/+, and p53-/-) (20Bunz F. Dutriaux A. Lengauer C. Waldman T. Zhou S. Brown J.P. Sedivy J.M. Kinzler K.W. Vogelstein B. Science. 1998; 282: 1497-1501Crossref PubMed Scopus (2549) Google Scholar, 21Zhang L. Yu J. Park B.H. Kinzler K.W. Vogelstein B. Science. 2000; 290: 989-992Crossref PubMed Scopus (797) Google Scholar) were kind gifts from Dr. Bert Vogelstein (John Hopkins Oncology Center, Baltimore, MD). Recombinant human soluble TRAIL (KillerTRAIL™) was obtained from Alexis (Axxora, San Diego, CA), and agonistic anti-Fas receptor antibody (clone CH11) was from Upstate Biotechnology (Lake Placid, NY). Etoposide, antimycin A, and N-acetyl-l-cysteine (NAC) were obtained from Sigma. CPT was obtained from the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, NCI, National Institutes of Health. BH3I-2′ was purchased from Calbiochem (EMD Biosciences, La Jolla, CA), and the caspase peptide inhibitor benzyloxycarbonyl-Val-Ala-dl-Asp(OMe)-fluoromethylketone (Z-VAD-fmk) was from Bachem (Torrance, CA). Detection of Cellular Topoisomerase Cleavage Complexes—Topoisomerase cleavage complexes were detected using the in vivo complex of enzyme bioassay as described (22Subramanian D. Kraut E. Staubus A. Young D.C. Muller M.T. Cancer Res. 1995; 55: 2097-2103PubMed Google Scholar). Briefly, the cells were lysed in 1% Sarkosyl and homogenized. The cell lysates were centrifuged on cesium chloride step gradients at 165,000 × g for 20 h at 20 °C. Twenty 0.5-ml fractions were collected and diluted (v/v) into 25 mm potassium phosphate buffer, pH 6.6. The DNA-containing fractions (fractions 7–11) were pooled (except in Fig. 1A) and applied to polyvinylidene difluoride membranes (Immobilon-P, Millipore, MA) using a slot blot vacuum manifold. Topoisomerase cleavage complexes were detected by immunoblotting using the C21 Top1 mouse monoclonal antibody (1:1,000 dilution) (a kind gift from Dr. Yung-Chi Cheng, Yale University, New Haven, CT) or a Top2α mouse monoclonal antibody (clone Ki-S1, 1:1,000 dilution) from Chemicon International (Temecula, CA). To identify the covalent DNA binding of the 80-kDa cleaved form of Top1 (see Fig. 4D, bottom panel), the DNA-containing fractions isolated as described above were precipitated with isopropanol (v/v) following addition of 0.5 m ammonium and a 10-min incubation at -20 °C. After centrifugation (10,000 × g, 15 min, 4 °C), the pellets (DNA-protein complexes) were washed with 70% ethanol. Following resuspension in 5 mm CaCl2 buffer containing protease inhibitors (Complete; Roche Applied Science), the DNA was digested with 50 units of DNase I for 30 min at room temperature. The reactions were stopped by the addition of loading buffer (v/v) (125 mm Tris-HCl, pH 6.8, 10% β-mercaptoethanol, 4.6% SDS, 20% glycerol, and 0.003% bromphenol blue), boiled, and applied to 8% SDS-PAGE gels. Top1 was detected by immunoblotting with the C21 Top1 monoclonal antibody. Western Blotting—Western blotting analyses on whole cell extracts were performed as described (16Sordet O. Khan Q.A. Plo I. Pourquier P. Urasaki Y. Yoshida A. Antony S. Kohlhagen G. Solary E. Saparbaev M. Laval J. Pommier Y. J. Biol. Chem. 2004; 279: 50499-50504Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) using the C21 Top1 monoclonal antibody (1:1,000 dilution), the poly(ADP-ribose)-polymerase (PARP) polyclonal antibody (1:5,000 dilution) (Roche Applied Science), the γ-H2AX monoclonal antibody (1:2,000 dilution) (Upstate Biotechnology), and the tubulin monoclonal antibody (1:5,000 dilution) (clone Ab-4; NeoMarkers, Fremont, CA). Identification of Apoptosis—Global DNA fragmentation was quantified by filter elution assay as described (23Bertrand R. Kohn K.W. Solary E. Pommier Y. Drug Dev. 1995; 34: 138-144Crossref Scopus (35) Google Scholar) and expressed as a percentage of fragmented DNA relative to total DNA. Oligonucleosomal DNA fragmentation was visualized by agarose gel electrophoresis using the quick apoptotic ladder kit from Biovision (Mountain View, CA). For sub-G1 analysis, DNA content was assessed by staining ethanol-fixed cells with propidium iodide and monitoring by FACScan (BD Biosciences, San Jose, CA). The number of cells with sub-G1 DNA content was determined with the CellQuest program (BD Biosciences). For electron microscopy, the cells were fixed in 0.1 m cacodylate buffer (pH 7.4) containing 2% glutaraldehyde and post-fixed in 1% osmium in same buffer. Then the cells were stained with 0.5% uranyl acetate, dehydrated in graded ethanol and propylene oxide, and infiltrated in equal volume of epoxy resin and propylene oxide overnight. The sample was embedded in epoxy resin and cured in 55 °C oven for 48 h. The cured block was thin sectioned and stained in uranyl acetate and lead citrate and examined by electron microscopy (Hitachi H700 microscope, Tokyo, Japan). Caspase activities were measured as described (18Sordet O. Goldman A. Pommier Y. Mol. Cancer Ther. 2006; 5: 3139-3144Crossref PubMed Scopus (28) Google Scholar) using the fluorogenic peptide substrate Z-DEVD-AFC (caspase-3) and Z-IETD-AFC (caspase-8) from Calbiochem. Confocal Microscopy Analyses of 8-Oxoguanine—After cytospin, Jurkat cells were fixed with 2% paraformaldehyde for 20 min and post-fixed/permeabilized with ice-cold 70% ethanol for 20 min. The cells were incubated with 5% bovine serum albumin for 1 h to block nonspecific binding before incubation with both mouse anti-8-oxoguanine (clone 483.15; Chemicon International) (1:250 dilution) and rabbit antihuman Top1 antibodies (raised against the 68-kDa C-terminal Top1 fragment; local source) (1:500 dilution) for 1.5 h. After washes, the cells were incubated with fluorescent secondary antibodies (Alexa-488 and Alexa-568; Molecular Probes, Eugene, OR) for 45 min. The slides were mounted using Vectashield mounting liquid (Vector Labs, Burlingame, CA), and fluorescences were visualized using a Nikon Eclipse TE-300 confocal laser microscope. Quantification of fluorescence intensity was determined using Adobe Photoshop 7.0 software and expressed as relative fluorescence intensity/cell. Top1 Silencing by RNA Interference—Top1 expression was stably down-regulated in HCT116 cells by the transfection of U6 promoter-driven DNA vectors stably expressing small interfering RNA (siRNA) hairpins targeting human Top1 (HCT116siTop1; cDNA sequence: 5′-CTT GAC AGC CAA GGT ATT C-3′) or a negative control sequence (HCT116siCtrl; cDNA sequence, 5′-GCG TCC TTT CCA CAA GAT A-3′) as described (24Miao Z.H. Player A. Shankavaram U. Wang Y.H. Zimonjic D.B. Lorenzi P.L. Liao Z.Y. Liu H. Shimura T. Zhang H.L. Meng L.H. Zhang Y.W. Kawasaki E.S. Popescu N.C. Aladjem M.I. Goldstein D.J. Weinstein J.N. Pommier Y. Cancer Res. 2007; 67: 8752-8761Crossref PubMed Scopus (86) Google Scholar). For transient down-regulation of Top1 expression, the cells were transfected with an siRNA duplex (Qiagen) against the sequence GGA CTC CAT CAG ATA CTA T from the Top1 mRNA. Transfections were performed using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's protocol. A negative control siRNA duplex from Qiagen (target DNA sequence: TTC TCC GAA CGT GTC ACG T) was used. The Top1 knock-down experiments were carried out 72 h after transfection. TRAIL and Fas Induce Apoptotic Top1cc—Trapped Top1cc can be detected in genomic DNA after cesium chloride gradient centrifugation and immunoblotting with Top1 antibody (16Sordet O. Khan Q.A. Plo I. Pourquier P. Urasaki Y. Yoshida A. Antony S. Kohlhagen G. Solary E. Saparbaev M. Laval J. Pommier Y. J. Biol. Chem. 2004; 279: 50499-50504Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). In human leukemia Jurkat cells exposed to TRAIL or to an agonistic anti-Fas antibody (anti-Fas Ab) that mimics Fas ligand, we detected high levels of Top1cc (Fig. 1A). By contrast, topoisomerase IIα cleavage complexes (Top2αcc) were not detectable under conditions where etoposide (positive control) produced a clear signal (Fig. 1A). Similarly, Top2βcc were not induced by TRAIL (data not shown). Those experiments indicate that the binding of TRAIL and anti-Fas Ab to their plasma membrane receptors DR4/DR5 and Fas, respectively (3Ashkenazi A. Nat. Rev. Cancer. 2002; 2: 420-430Crossref PubMed Scopus (1111) Google Scholar), effectively induce the formation of Top1cc. TRAIL and Fas ligand are potent inducers of apoptosis in tumor cells (1Ashkenazi A. Pai R.C. Fong S. Leung S. Lawrence D.A. Marsters S.A. Blackie C. Chang L. McMurtrey A.E. Hebert A. DeForge L. Koumenis I.L. Lewis D. Harris L. Bussiere J. Koeppen H. Shahrokh Z. Schwall R.H. J. Clin. Investig. 1999; 104: 155-162Crossref PubMed Scopus (2008) Google Scholar, 2Walczak H. Miller R.E. Ariail K. Gliniak B. Griffith T.S. Kubin M. Chin W. Jones J. Woodward A. Le T. Smith C. Smolak P. Goodwin R.G. Rauch C.T. Schuh J.C. Lynch D.H. Nat. Med. 1999; 5: 157-163Crossref PubMed Scopus (2236) Google Scholar), suggesting that the observed Top1cc were apoptotic Top1cc resulting from the activation of cell death pathways. In line with this possibility, TRAIL and anti-Fas Ab induced Top1cc with kinetics that coincided with DNA fragmentation (Fig. 1, B and C). Top1cc and apoptotic DNA fragmentation were both detected after 1 h of exposure to TRAIL (or 3 h for anti-Fas Ab). Top1cc persisted throughout the apoptotic process as the DNA became extensively fragmented (Fig. 1, B and C). To demonstrate that those Top1cc were due to apoptosis, we tested whether inhibition of the apoptotic pathways induced by TRAIL and anti-Fas Ab would prevent the formation of Top1cc. Pretreatment of cells with the pancaspase inhibitor Z-VAD-fmk inhibited TRAIL- and anti-Fas Ab-induced apoptosis, as well as the formation of Top1cc (Fig. 1D). As expected, Z-VAD-fmk also inhibited the activities of caspases 8 and 3 and the cleavage of PARP (Fig. 1E). Because the formation of apoptotic Top1cc by UV-C radiation and colcemid was recently reported to involve p53 (17Rockstroh A. Kleinert A. Kramer M. Grosse F. Soe K. Oncogene. 2007; 26: 123-131Crossref PubMed Scopus (8) Google Scholar), we examined Top1cc in p53-/- HCT116 cells exposed to TRAIL. We found that p53+/+ and p53-/- HCT116 cells both displayed similar induction of Top1cc in response to TRAIL (Fig. 1F, bottom panel). Both cell lines also exhibited similar apoptotic response to TRAIL (Fig. 1F, top panel), which further demonstrates that TRAIL-induced apoptosis is p53-independent (25Ravi R. Bedi A. Cancer Res. 2002; 62: 1583-1587PubMed Google Scholar). Collectively, our experiments demonstrate a close association between the formation of Top1cc and the induction of p53-independent apoptosis in cells exposed to TRAIL. TRAIL-induced Top1cc Require Activation of the Mitochondrial Death Pathway—In most cancer cells, TRAIL induces apoptosis by increasing the permeability of mitochondrial membranes (3Ashkenazi A. Nat. Rev. Cancer. 2002; 2: 420-430Crossref PubMed Scopus (1111) Google Scholar). Schematically, the proapoptotic Bax protein induces the formation of mitochondrial pores in the outer membrane by antagonizing the antiapoptotic Bcl-xL protein. Because the loss of Bax has been shown to protect HCT116 cells against TRAIL-induced apoptosis (25Ravi R. Bedi A. Cancer Res. 2002; 62: 1583-1587PubMed Google Scholar), we examined the formation of Top1cc in Bax+/- and Bax-/- HCT116 cells (21Zhang L. Yu J. Park B.H. Kinzler K.W. Vogelstein B. Science. 2000; 290: 989-992Crossref PubMed Scopus (797) Google Scholar). Fig. 2A shows that Bax-/- cells failed to induce Top1cc (bottom panel). They were also defective in apoptosis (top panel). These experiments demonstrate that Bax plays a key role in TRAIL-induced apoptotic Top1cc. Next, we investigated whether direct activation of the apoptotic mitochondrial pathway (26Grad J.M. Cepero E. Boise L.H. Drug Resist. Updates. 2001; 4: 85-91Crossref PubMed Scopus (59) Google Scholar) was sufficient to induce apoptotic Top1cc. Both the Bcl-2 homology domain 3 (BH3) mimetics antimycin A (27Tzung S.P. Kim K.M. Basanez G. Giedt C.D. Simon J. Zimmerberg J. Zhang K.Y. Hockenbery D.M. Nat. Cell Biol. 2001; 3: 183-191Crossref PubMed Scopus (439) Google Scholar) (Fig. 2B) and BH3I-2′ (28Degterev A. Lugovskoy A. Cardone M. Mulley B. Wagner G. Mitchison T. Yuan J. Nat. Cell Biol. 2001; 3: 173-182Crossref PubMed Scopus (543) Google Scholar) (Fig. 2C), which bind to and inhibit Bcl-xL, led to the induction of apoptotic Top1cc. Altogether, our results indicate a pivotal role of the mitochondrial death pathway for the induction of apoptotic Top1cc by TRAIL. TRAIL-induced Apoptotic Top1cc Are Linked to the Generation of Oxidative DNA Lesions—Because Top1cc are readily induced by oxidative base damages (e.g. 8-oxoguanine (8-oxoG)), DNA strand breaks, and ROS (12Pourquier P. Pommier Y. Adv. Cancer Res. 2001; 80: 189-216Crossref PubMed Google Scholar, 29Daroui P. Desai S.D. Li T.K. Liu A.A. Liu L.F. J. Biol. Chem. 2004; 279: 14587-14594Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 30Pourquier P. Ueng L.-M. Fertala J. Wang D. Park H.-J. Essigman J.M. Bjornsti M.-A. Pommier Y. J. Biol. Chem. 1999; 274: 8516-8523Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), we searched for the presence of 8-oxoG in TRAIL-treated cells (9Lee M.W. Park S.C. Yang Y.G. Yim S.O. Chae H.S. Bach J.H. Lee H.J. Kim K.Y. Lee W.B. Kim S.S. FEBS Lett. 2002; 512: 313-318Crossref PubMed Scopus (88) Google Scholar). Immunofluorescence confocal microscopy (Fig. 3, A and B) showed 8-oxoG nuclear staining in TRAIL-treated cells, indicative of TRAIL-induced oxidative damage (3Ashkenazi A. Nat. Rev. Cancer. 2002; 2: 420-430Crossref PubMed Scopus (1111) Google Scholar, 31Izeradjene K. Douglas L. Tillman D.M. Delaney A.B. Houghton J.A. Cancer Res. 2005; 65: 7436-7445Crossref PubMed Scopus (92) Google Scholar, 32Mohr A. Buneker C. Gough R.P. Zwacka R.M. Oncogene. 2007; PubMed Google Scholar). Moreover, caspases inhibition by Z-VAD-fmk (Fig. 1E) prevented TRAIL-induced 8-oxoG (Fig. 3, A and B), which is in agreement with recent studies showing ROS production by caspase activation (33Ricci J.E. Gottlieb R.A. Green D.R. J. Cell Biol. 2003; 160: 65-75Crossref PubMed Scopus (421) Google Scholar, 34Ricci J.E. MunoZ-Pinedo C. Fitzgerald P. Bailly-Maitre B. Perkins G.A. Yadava N. Scheffler I.E. Ellisman M.H. Green D.R. Cell. 2004; 117: 773-786Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar). Our results and these publications suggest that TRAIL-induced caspase activation leads to the generation of oxidative DNA lesions and apoptotic Top1cc. To further demonstrate that TRAIL-induced apoptotic Top1cc were related to oxidative DNA lesions, we tested the effect of the potent antioxidant N-acetyl-l-cysteine (NAC). Fig. 3 shows that quenching ROS with NAC prevented both TRAIL-induced 8-oxoG (Fig. 3, A and B) and Top1cc (Fig. 3C). Fas-induced apoptosis has also been associated with the intracellular accumulation of ROS (35Shakibaei M. Schulze-Tanzil G. Takada Y. Aggarwal B.B. Antioxid. Redox. Signal. 2005; 7: 482-496Crossref PubMed Scopus (73) Google Scholar) and with the generation of oxidative DNA lesions (36Nathan I. Dizdaroglu M. Bernstein L. Junker U. Lee C. Muegge K. Durum S.K. Cytokine. 2000; 12: 881-887Crossref PubMed Scopus (16) Google Scholar). Accordingly, NAC prevented the induction of Top1cc by anti-Fas Ab (Fig. 3C). Altogether, our findings indicate that both caspase activation and oxidative DNA damage are involved in the production of apoptotic Top1cc induced by death receptor activation. Caspase-3-cleaved Top1 Is Preferentially Associated with the Apoptotic Top1cc Induced by TRAIL—We next investigated whether modifications of Top1 itself might also contribute to the formation of apoptotic Top1cc induced by TRAIL. The 100-kDa native Top1 protein is indeed a known substrate of caspase-3 (7Samejima K. Svingen P.A. Basi G.S. Kottke T. Mesner Jr., P.W. Stewart L. Durrieu F. Poirier G.G. Alnemri E.S. Champoux J.J. Kaufmann S.H. Earnshaw W.C. J. Biol. Chem. 1999; 274: 4335-4340Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Caspase-3 cleaves Top1 after aspartate residue 146 and generates an 80-kDa C-terminal fragment (Fig. 4A) that remains capable of forming Top1cc (7Samejima K. Svingen P.A. Basi G.S. Kottke T. Mesner Jr., P.W. Stewart L. Durrieu F. Poirier G.G. Alnemri E.S. Champoux J.J. Kaufmann S.H. Earnshaw W.C. J. Biol. Chem. 1999; 274: 4335-4340Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 37Pommier Y. Laco G.S. Kohlhagen G. Sayer J.M. Kroth H. Jerina D.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10739-10744Crossref PubMed Scopus (50) Google Scholar) and still possesses one functional nuclear localization signal (38Mo Y.Y. Wang C. Beck W.T. J. Biol. Chem. 2000; 275: 41107-41113Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) (Fig. 4A). Fig. 4B (upper panel) shows that TRAIL induced the appearance of the proteolytic Top1 form (80CL) (7Samejima K. Svingen P.A. Basi G.S. Kottke T. Mesner Jr., P.W. Stewart L. Durrieu F. Poirier G.G. Alnemri E.S. Champoux J.J. Kaufmann S.H. Earnshaw W.C. J. Biol. Chem. 1999; 274: 4335-4340Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Compared with the other caspase substrate, PARP (Fig. 4C), Top1 cleavage remained incomplete (Fig. 4B, upper panel) (7Samejima K. Svingen P.A. Basi G.S. Kottke T. Mesner Jr., P.W. Stewart L. Durrieu F. Poirier G.G. Alnemri E.S. Champoux J.J. Kaufmann S.H. Earnshaw W.C. J. Biol. Chem. 1999; 274: 4335-4340Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). This partial cleavage of Top1 coincided with the generation of apoptotic Top1cc in response to TRAIL (Fig. 4B, lower panel). We next investigated which of the two forms of Top1, the 100-kDa native Top1 or the 80-kDa caspase-processed Top1, formed the apoptotic Top1cc. To address this question, TRAIL-induced Top1cc were isolated from the DNA-containing cesium chloride fractions. Following DNA digestion with DNase I, Western blotting was performed to determine the molecular mass of the Top1 molecules involved in the TRAIL-induced Top1cc. Approximately 90% of the Top1cc consisted of the truncated 80-kDa Top1 fragment (Fig. 4, D, bottom panel, and E). By contrast, the 80-kDa Top1 form corresponded to only ∼40% of total Top1 in whole cell extracts (Fig. 4, D, top panel, and E). These experiments demonstrate that the apoptotic Top1cc generated during TRAIL-induced apoptosis correspond primarily to the 80-kDa C-terminal fragment of Top1 generated by caspase-3. Functional Relevance of TRAIL-induced Apoptotic Top1cc—First, to determine whether apoptotic Top1cc could directly contribute to the apoptotic DNA fragmentation, we determined the frequency of TRAIL-induced Top1cc. Using CPT as a calibrator (39Goldwasser F. Bae I. Valenti M. Torres K. Pommier Y. Cancer Res. 1995; 55: 2116-2121PubMed Google Scholar), we estimated that TRAIL induced approximately one Top1cc/100 kbp in cells treated with TRAIL for 4 h (Fig. 5). That number scores relatively low compared with the observed DNA double-strand break frequency produced by apoptotic endonucleases (8Samejima K. Earnshaw W.C. Nat. Rev. Mol. Cell Biol. 2005; 6: 677-688Crossref PubMed Scopus (237) Google Scholar) (Fig. 6, G and H). Thus, it is unlikely that apoptotic Top1cc contribute to the direct breakdown of genomic DNA.FIGURE 6Apoptotic Top1cc are involved in TRAIL-induced apoptotic nuclear modifications. A–C, Top1 was silenced in HCT116 cells using U6 promoter-driven DNA vectors stably expressing siRNA hairpins targeting human Top1 (HCT116siTop1) or a negative control sequence (HCT116siCtrl). A, Top1 expression was examined by Western blotting. Tubulin was used as a loading control. B, cells were treated with 0.1 μg/ml TRAIL for 18 h. Apoptotic sub-G1 DNA contents are indicated by the brackets. C, percentage of cells with sub-G1 DNA (means ± S.D. of three independent experiments). The cells were treated with the indicated concentration of TRAIL for 18 h. Asterisks denote statistically significant difference from the TRAIL-treated HCT116siCtr cells (*, p < 0.05; **," @default.
• W2031372644 created "2016-06-24" @default.
• W2031372644 creator A5006775012 @default.
• W2031372644 creator A5015342151 @default.
• W2031372644 creator A5018411232 @default.
• W2031372644 creator A5021361253 @default.
• W2031372644 creator A5042114293 @default.
• W2031372644 creator A5071424632 @default.
• W2031372644 date "2008-08-01" @default.
• W2031372644 modified "2023-09-30" @default.
• W2031372644 title "Topoisomerase I Requirement for Death Receptor-induced Apoptotic Nuclear Fission" @default.
• W2031372644 cites W1484817497 @default.
• W2031372644 cites W1496626589 @default.
• W2031372644 cites W1499746632 @default.
• W2031372644 cites W1562326085 @default.
• W2031372644 cites W1567114372 @default.
• W2031372644 cites W1798491560 @default.
• W2031372644 cites W1913661233 @default.
• W2031372644 cites W1965037168 @default.
• W2031372644 cites W1965239599 @default.
• W2031372644 cites W1971840717 @default.
• W2031372644 cites W1973109581 @default.
• W2031372644 cites W1975426967 @default.
• W2031372644 cites W1980730914 @default.
• W2031372644 cites W1990098732 @default.
• W2031372644 cites W2012173926 @default.
• W2031372644 cites W2013222252 @default.
• W2031372644 cites W2032781906 @default.
• W2031372644 cites W2033307305 @default.
• W2031372644 cites W2033376776 @default.
• W2031372644 cites W2035768602 @default.
• W2031372644 cites W2036878177 @default.
• W2031372644 cites W2038118195 @default.
• W2031372644 cites W2040829831 @default.
• W2031372644 cites W2044418958 @default.
• W2031372644 cites W2044989903 @default.
• W2031372644 cites W2049855638 @default.
• W2031372644 cites W2056731952 @default.
• W2031372644 cites W2063732648 @default.
• W2031372644 cites W2064218445 @default.
• W2031372644 cites W2065634344 @default.
• W2031372644 cites W2069704309 @default.
• W2031372644 cites W2070498380 @default.
• W2031372644 cites W2080086497 @default.
• W2031372644 cites W2080125477 @default.
• W2031372644 cites W2081374600 @default.
• W2031372644 cites W2084772268 @default.
• W2031372644 cites W2084969545 @default.
• W2031372644 cites W2085712777 @default.
• W2031372644 cites W2085755885 @default.
• W2031372644 cites W2089521856 @default.
• W2031372644 cites W2091129135 @default.
• W2031372644 cites W2095646094 @default.
• W2031372644 cites W2105634121 @default.
• W2031372644 cites W2106284224 @default.
• W2031372644 cites W2114351091 @default.
• W2031372644 cites W2115511665 @default.
• W2031372644 cites W2124624490 @default.
• W2031372644 cites W2129493754 @default.
• W2031372644 cites W2136007486 @default.
• W2031372644 cites W2146274229 @default.
• W2031372644 cites W2154329375 @default.
• W2031372644 cites W2155292823 @default.
• W2031372644 cites W2158720649 @default.
• W2031372644 cites W2166931288 @default.
• W2031372644 cites W2168026882 @default.
• W2031372644 doi "https://doi.org/10.1074/jbc.m801146200" @default.
• W2031372644 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2516995" @default.
• W2031372644 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18556653" @default.
• W2031372644 hasPublicationYear "2008" @default.
• W2031372644 type Work @default.
• W2031372644 sameAs 2031372644 @default.
• W2031372644 citedByCount "16" @default.
• W2031372644 countsByYear W20313726442013 @default.
• W2031372644 countsByYear W20313726442014 @default.
• W2031372644 countsByYear W20313726442016 @default.
• W2031372644 countsByYear W20313726442017 @default.
• W2031372644 countsByYear W20313726442018 @default.
• W2031372644 countsByYear W20313726442023 @default.
• W2031372644 crossrefType "journal-article" @default.
• W2031372644 hasAuthorship W2031372644A5006775012 @default.
• W2031372644 hasAuthorship W2031372644A5015342151 @default.
• W2031372644 hasAuthorship W2031372644A5018411232 @default.
• W2031372644 hasAuthorship W2031372644A5021361253 @default.
• W2031372644 hasAuthorship W2031372644A5042114293 @default.
• W2031372644 hasAuthorship W2031372644A5071424632 @default.
• W2031372644 hasBestOaLocation W20313726441 @default.
• W2031372644 hasConcept C147897179 @default.
• W2031372644 hasConcept C185592680 @default.
• W2031372644 hasConcept C190283241 @default.
• W2031372644 hasConcept C552990157 @default.
• W2031372644 hasConcept C55493867 @default.
• W2031372644 hasConcept C86803240 @default.
• W2031372644 hasConcept C95444343 @default.
• W2031372644 hasConcept C98274493 @default.
• W2031372644 hasConceptScore W2031372644C147897179 @default.
• W2031372644 hasConceptScore W2031372644C185592680 @default.
• W2031372644 hasConceptScore W2031372644C190283241 @default.

• next