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• W1999854664 abstract "Disintegration of nuclear DNA into high molecular weight (HMW) and oligonucleosomal DNA fragments represents two major periodicities of DNA fragmentation during apoptosis. These are thought to originate from the excision of DNA loop domains and from the cleavage of nuclear DNA at the internucleosomal positions, respectively. In this report, we demonstrate that different apoptotic insults induced apoptosis in NB-2a neuroblastoma cells that was invariably accompanied by the formation of HMW DNA fragments of about 50–100 kb but proceeded either with or without oligonucleosomal DNA cleavage, depending on the type of apoptotic inducer. We demonstrate that differences in the pattern of DNA fragmentation were reproducible in a cell-free apoptotic system and develop conditions that allow in vitro separation of the HMW and oligonucleosomal DNA fragmentation activities. In contrast to apoptosis associated with oligonucleosomal DNA fragmentation, the HMW DNA cleavage in apoptotic cells was accompanied by down-regulation of caspase-activated DNase (CAD) and was not affected by z-VAD-fmk, suggesting that the caspase/CAD pathway is not involved in the excision of DNA loop domains. We further demonstrate that nonapoptotic NB-2a cells contain a constitutively present nuclease activity located in the nuclear matrix fraction that possessed the properties of topoisomerase (topo) II and was capable of reproducing the pattern of HMW DNA cleavage that occurred in apoptotic cells. We demonstrate that the early stages of apoptosis induced by different stimuli were accompanied by activation of topo II-mediated HMW DNA cleavage that was reversible after removal of apoptotic inducers, and we present evidence of the involvement of topo II in the formation of HMW DNA fragments at the advanced stages of apoptosis. The results suggest that topo II is involved in caspase-independent excision of DNA loop domains during apoptosis, and this represents an alternative pathway of apoptotic DNA disintegration from CAD-driven caspase-dependent oligonucleosomal DNA cleavage. Disintegration of nuclear DNA into high molecular weight (HMW) and oligonucleosomal DNA fragments represents two major periodicities of DNA fragmentation during apoptosis. These are thought to originate from the excision of DNA loop domains and from the cleavage of nuclear DNA at the internucleosomal positions, respectively. In this report, we demonstrate that different apoptotic insults induced apoptosis in NB-2a neuroblastoma cells that was invariably accompanied by the formation of HMW DNA fragments of about 50–100 kb but proceeded either with or without oligonucleosomal DNA cleavage, depending on the type of apoptotic inducer. We demonstrate that differences in the pattern of DNA fragmentation were reproducible in a cell-free apoptotic system and develop conditions that allow in vitro separation of the HMW and oligonucleosomal DNA fragmentation activities. In contrast to apoptosis associated with oligonucleosomal DNA fragmentation, the HMW DNA cleavage in apoptotic cells was accompanied by down-regulation of caspase-activated DNase (CAD) and was not affected by z-VAD-fmk, suggesting that the caspase/CAD pathway is not involved in the excision of DNA loop domains. We further demonstrate that nonapoptotic NB-2a cells contain a constitutively present nuclease activity located in the nuclear matrix fraction that possessed the properties of topoisomerase (topo) II and was capable of reproducing the pattern of HMW DNA cleavage that occurred in apoptotic cells. We demonstrate that the early stages of apoptosis induced by different stimuli were accompanied by activation of topo II-mediated HMW DNA cleavage that was reversible after removal of apoptotic inducers, and we present evidence of the involvement of topo II in the formation of HMW DNA fragments at the advanced stages of apoptosis. The results suggest that topo II is involved in caspase-independent excision of DNA loop domains during apoptosis, and this represents an alternative pathway of apoptotic DNA disintegration from CAD-driven caspase-dependent oligonucleosomal DNA cleavage. high molecular weight caspase-activated DNase topoisomerase phenylmethylsulfonyl fluoride field inversion gel electrophoresis mouse embryonic fibroblast fluoromethylketone aminomethyl-coumarin arabinosylcytosine At the higher level of chromatin compaction, nuclear DNA is arranged into loop domains by periodical attachment of the chromatin fiber to the nuclear matrix (1Mirkovitch J. Mirault M.E. Laemmli U.K. Cell. 1984; 39: 223-232Abstract Full Text PDF PubMed Scopus (856) Google Scholar, 2Jackson D.A. Dolle A. Robertson G. Cook P.R. Cell Biol. Int. Rep. 1992; 16: 687-696Crossref PubMed Scopus (48) Google Scholar). The domain level of chromatin organization is supported by the interaction of specific DNA sequences, matrix/scaffold attachment regions, with nuclear matrix proteins (3Laemmli U.K. Kas E. Poljak L. Adachi Y. Curr. Opin. Genet. Dev. 1992; 2: 275-285Crossref PubMed Scopus (306) Google Scholar). The chromatin loops represent the basic structural components of higher-order chromatin folding, which is maintained during the cell cycle and in differentiated cells (3Laemmli U.K. Kas E. Poljak L. Adachi Y. Curr. Opin. Genet. Dev. 1992; 2: 275-285Crossref PubMed Scopus (306) Google Scholar, 4Nelson W.G. Pienta K.J. Barrack E.R. Coffey D.S. Annu. Rev. Biophys. Biophys. Chem. 1986; 15: 457-475Crossref PubMed Scopus (255) Google Scholar, 5Jackson D.A. Cook P.R. Int. Rev. Cytol. 1995; 162A: 125-149PubMed Google Scholar). Disintegration of nuclear DNA into nucleosome-sized fragments represents a classical manifestation of apoptosis (6Wyllie A.H. Kerr J.F. Currie A.R. Int. Rev. Cytol. 1980; 68: 251-306Crossref PubMed Scopus (6720) Google Scholar). In addition, another type of DNA cleavage during apoptosis has been reported to yield a set of the high molecular weight (HMW)1 DNA fragments of about 50–100 kb (7Oberhammer F. Wilson J.W. Dive C. Morris I.D. Hickman A. Wakeling A.E. Walker P.R. Sikorska M. EMBO J. 1993; 12: 3679-3684Crossref PubMed Scopus (1161) Google Scholar). The formation of HMW DNA fragments is widely thought to result from the excision of DNA loop domains at the positions of their attachment to the nuclear matrix (8Lagarkova M.A. Iarovaia O.V. Razin S.V. J. Biol. Chem. 1995; 270: 20239-20241Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 9Li T.K. Chen A.Y., Yu, C. Mao Y. Wang H. Liu L.F. Genes Dev. 1999; 13: 1553-1560Crossref PubMed Scopus (148) Google Scholar) and is considered to be an initial step in DNA disintegration during apoptosis (7Oberhammer F. Wilson J.W. Dive C. Morris I.D. Hickman A. Wakeling A.E. Walker P.R. Sikorska M. EMBO J. 1993; 12: 3679-3684Crossref PubMed Scopus (1161) Google Scholar, 10Brown D.G. Sun X.-M. Cohen G.M. J. Biol. Chem. 1993; 268: 3037-3039Abstract Full Text PDF PubMed Google Scholar, 11Walker P.R. Weaver V.M. Lach B. LeBlanc J. Sikorska M. Exp. Cell Res. 1994; 213: 100-106Crossref PubMed Scopus (181) Google Scholar, 12Solovyan V. Bezvenyuk Z. Huotari V. Tapiola T. Suuronen T. Salminen A. Mol. Brain Res. 1998; 62: 43-55Crossref PubMed Scopus (33) Google Scholar). The discovery of caspase-activated DNase (CAD/DFF40/CPAN; hereafter designated CAD) (13Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1646) Google Scholar, 14Enari M. Sakahira H. Yokayama H. Okawa K. Iwamatsu A. Nagata S. Nature. 1998; 391: 43-50Crossref PubMed Scopus (2805) Google Scholar, 15Halenbeck R. MacDonald H. Roulston A. Chen T.T. Conroy L. Williams L.T. Curr. Biol. 1998; 23: 537-540Abstract Full Text Full Text PDF Google Scholar) has made a significant contribution to the understanding of the mechanisms of DNA disintegration during apoptosis. After caspase 3-dependent inactivation of the CAD inhibitor (ICAD), active CAD initiates disintegration of nuclear DNA to oligonucleosomal DNA fragments (13Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1646) Google Scholar, 14Enari M. Sakahira H. Yokayama H. Okawa K. Iwamatsu A. Nagata S. Nature. 1998; 391: 43-50Crossref PubMed Scopus (2805) Google Scholar, 15Halenbeck R. MacDonald H. Roulston A. Chen T.T. Conroy L. Williams L.T. Curr. Biol. 1998; 23: 537-540Abstract Full Text Full Text PDF Google Scholar). At the same time, increasing evidence indicates that the formation of the HMW DNA fragments during apoptosis can proceed without internucleosomal DNA cleavage (7Oberhammer F. Wilson J.W. Dive C. Morris I.D. Hickman A. Wakeling A.E. Walker P.R. Sikorska M. EMBO J. 1993; 12: 3679-3684Crossref PubMed Scopus (1161) Google Scholar, 12Solovyan V. Bezvenyuk Z. Huotari V. Tapiola T. Suuronen T. Salminen A. Mol. Brain Res. 1998; 62: 43-55Crossref PubMed Scopus (33) Google Scholar,16Cohen G.M. Sun X.M. Snowden R.T. Dinsdale D. Killeter D.N. Biochem. J. 1992; 286: 331-334Crossref PubMed Scopus (671) Google Scholar). This implies that distinct pathways may be involved in the formation of HMW and oligonucleosomal DNA fragments during apoptosis. In this report, we describe apoptosis in NB-2a neuroblastoma cells that can proceed either with or without internucleosomal DNA fragmentation, depending on the type of apoptotic inducer. We demonstrate that HMW DNA cleavage and internucleosomal DNA cleavage represent separate programs of DNA disintegration and present evidence of the involvement of topo II in the formation of HMW DNA fragments during apoptosis. Mouse NB-2a cells obtained from American Type Culture Collection (CCL 131) were routinely cultured in an atmosphere of 10% CO2 in Dulbecco's modified Eagle's medium supplemented with 2 mm glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% fetal calf serum (Invitrogen). Apoptosis was induced in exponentially growing NB-2a cells either by serum withdrawal or by cell treatment with 10 μm etoposide (Calbiochem). Cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100, followed by staining with the nuclear dye Hoechst 33258 (0.1 μg/ml; Sigma). Cytosolic extracts were prepared by treatment of cells with 10 volumes of ice-cold cytosol-preparing buffer (10 mm PIPES, pH 7.5, 10 mm KCl, 1 mm dithiothreitol, 2 mmMgCl2, 0.1 mm EDTA, 0.1 mm EGTA, and 0.1 mm phenylmethylsulfonyl fluoride (PMSF)) as described previously (12Solovyan V. Bezvenyuk Z. Huotari V. Tapiola T. Suuronen T. Salminen A. Mol. Brain Res. 1998; 62: 43-55Crossref PubMed Scopus (33) Google Scholar). The activity of caspase 3-like proteases was assayed using the fluorogenic substrate Ac-DEVD-AMC (BD PharMingen) at a final concentration of 20 μm. Caspase assays were performed according to the manufacturer's protocol. Nuclei and cytosolic extracts for cell-free apoptosis assay were prepared as described previously (12Solovyan V. Bezvenyuk Z. Huotari V. Tapiola T. Suuronen T. Salminen A. Mol. Brain Res. 1998; 62: 43-55Crossref PubMed Scopus (33) Google Scholar). Briefly, cells were collected, resuspended in 1 volume of cytosol-preparing buffer, transferred to a 2-ml Dounce homogenizer, allowed to swell for 20 min on ice, and lysed with gentle strokes of a B-type pestle. After centrifugation of the cell lysate at 1000 ×g for 5 min, the crude nuclear pellet was used for the preparation of nuclei, whereas the supernatant, after an additional centrifugation at 16,000 × g for 30 min at 4 °C, was aliquoted and used as a cytosolic extract in the reconstituted apoptosis system. Nuclei were purified by centrifugation of a crude nuclear pellet at 1000 × g for 10 min through a layer of 1 m sucrose prepared in cytosol-preparing buffer, followed by washing and resuspension in nuclear storage buffer (10 mm PIPES, pH 7.4, 80 mm KCl, 20 mm NaCl, 250 mmsucrose, 5 mm EGTA, 1 mm dithiothreitol, 0.5 mm spermidine, 0.2 mm spermine, 1 mm PMSF, and 50% glycerol) at 1 × 108nuclei/ml. Prepared nuclei were either stored at −70 °C or used immediately in reconstitution experiments. In the reconstituted apoptosis system, 2 μl of nuclei (2 × 105) were incubated with 10 μl of cytosolic extracts (5 mg/ml protein) for 1 h at 37 °C, followed by embedding of the nuclei into low-melting point agarose and analysis of DNA integrity. In some experiments, cytosolic extracts were pretreated for 1 h at room temperature with anti-topo IIα-specific (catalogue no. 2011-1; TopoGEN) or JB-1 topo IIβ-specific antibodies (kindly provided by Dr. D. Sullivan, University of South Florida) at a dilution of 1:20. Intact cells or purified nuclei were embedded in low-melting point agarose drops (50 μl) and incubated with 10 volumes of lysis buffer (20 mm Tris-HCl, pH 7.5, 20 mm EDTA, 0.5% sodium sarkosyl (Sigma), and 0.5% SDS) containing 100 μg/ml proteinase K and 40 μg/ml RNase A for 1 h at 37 °C. Agarose drops containing deproteinized DNA samples were washed three times with washing buffer (lysis buffer without protein denaturants), loaded quantitatively in the wells of a 1% agarose gel, and subjected to either conventional or field inversion gel electrophoresis (FIGE) as described earlier (12Solovyan V. Bezvenyuk Z. Huotari V. Tapiola T. Suuronen T. Salminen A. Mol. Brain Res. 1998; 62: 43-55Crossref PubMed Scopus (33) Google Scholar). Samples were solubilized in 1× Laemmli SDS-PAGE sample buffer and boiled for 3 min. Extracted polypeptides (30 μg) were resolved at 200 V on 10% SDS-PAGE gels and electrophoretically transferred to ECL-nitrocellulose membrane (0.45 μm; Amersham Biosciences) for 2 h at 100 V. Membranes were blocked for 1 h at room temperature in phosphate-buffered saline containing 1% bovine serum albumin, 1% nonfat dried milk, and 0.05% Tween 20. Membranes were then incubated in the same solution for 1 h at room temperature with anti-CPAN (dilution, 1:250) and anti-poly(ADP-ribose) polymerase (dilution, 1:1000; Roche Molecular Biochemicals) with 18211 anti-topo IIα or 18513 anti-topo IIβ antibodies (36Willmore E. Frank A.J. Padget K. Tilby M.J. Austin C.A. Mol. Pharmacol. 1998; 54: 78-85Crossref PubMed Scopus (131) Google Scholar) (dilution, 1:500) followed by incubation with horseradish peroxidase-conjugated secondary IgG (1:4000) in an identical solution for 1 h at room temperature and then detected by enhanced chemiluminescence (Pierce) according to the manufacturer's instructions. Intact cells were embedded in low-melting point agarose, extracted once with high salt extraction buffer (20 mm Tris-HCl, pH 7.5, 1 mm EDTA, 1 mm PMSF, and 2 m NaCl) for 1 h at 4 °C, washed three times for 30 min at 4 °C with washing buffer (high salt extraction buffer without NaCl), and incubated for 20 min at 37 °C in DNA cleavage buffer (washing buffer supplemented with 5 mm MgCl2). After incubation, high salt-extracted cells were treated with lysis buffer (20 mmTris-HCl, pH 7.5, 20 mm EDTA, and 0.5% SDS) and subjected to fractionation by FIGE. Cells were induced to undergo apoptosis by etoposide treatment as described above. Cells were collected, embedded in agarose, lysed, and fractionated by FIGE in low-melting point agarose gel. Agarose plugs containing 50–100-kb DNA fragments derived either from etoposide-treated or serum-deprived cells were excised from 1% low-melting point agarose gel, washed three times with STE buffer (10 mm Tris-HCl, pH 8.0, 0.1 mm EDTA, and 20 mm NaCl), and melted at 65 °C. ExoIII exonuclease buffer (supplied by manufacturers) was added to a final concentration of 1×, and 50–100-kb DNA fragments were treated with ExoIII exonuclease (Roche Molecular Biochemicals) for 0–40 min. For lambda exonuclease assay, 50–100-kb DNA fragments in 1× lambda exonuclease buffer were incubated for 30 min at 37 °C either with or without 0.1 mg/ml proteinase K, and then the samples were incubated for 15 min at 70 °C in the presence of 1 mm PMSF followed by treatment with lambda exonuclease (New England Biolabs) for 0–40 min. After incubation, 20 μm aliquots were transferred to 10 μm stop buffer (50 mmTris-HCl, pH 8.0, 50 mm EDTA, and 1% SDS), loaded into wells of a 1% agarose gel, and fractionated by conventional gel electrophoresis. Apoptosis in NB-2a cells was induced either by serum deprivation or by etoposide treatment as described above. Apoptotic cells were embedded in agarose, lysed, and fractionated by FIGE in the presence of 0.1% SDS. Agarose plugs containing 50–100-kb DNA fragments were excised from the gel, and DNA was extracted from the agarose using a Qiagen DNA purification kit. Extracted DNA was treated with 10 units of DNase I (Promega) for 30 min at 37 °C in DNase digestion buffer (10 mm Tris-HCl, pH 7.5, 5 mm MgCl2, 1 mmdithiothreitol, and 1 mm PMSF). DNase-treated samples were resolved in 7% SDS-PAGE, blotted onto nitrocellulose membrane, and probed with anti-topo IIβ-specific antibody. The treatment of NB-2a neuroblastoma cells with a genotoxic agent, etoposide, and withdrawal of growth factors both induced cell death, associated with the caspase 3 activation and chromatin condensation typical of apoptosis (Fig.1). Analysis of DNA integrity revealed that apoptosis induced by serum deprivation and apoptosis induced by etoposide were associated with distinct patterns of DNA disintegration (Fig. 1). Whereas serum withdrawal induced disintegration of nuclear DNA into HMW fragments of about 50–100 kb with concomitant development of an oligonucleosomal DNA ladder (Fig. 1B), etoposide induced the formation of HMW but not oligonucleosomal DNA fragments over the entire range of concentrations tested (Fig. 1C). No DNA laddering was observed in the floating etoposide-treated cells, in contrast to that seen in the serum-deprived cells (results not shown). The distinct patterns of DNA fragmentation caused by serum withdrawal and etoposide were accompanied by an increase in the activity of caspase 3-like proteases (Fig. 1D), thus indicating that activation of caspases is invariably associated with apoptotic DNA disintegration but does not necessarily lead to the formation of an oligonucleosomal DNA ladder in NB-2a cells. Data presented in Fig. 2 demonstrate that the distinct patterns of DNA disintegration induced by serum withdrawal and etoposide in NB-2a cells were reproducible in a reconstituted cell-free apoptotic system, in which nuclei isolated from nonapoptotic NB-2a cells were treated with cytosolic extracts prepared from apoptotic cells. Whereas cytosolic extract prepared from etoposide-treated cells induced the formation of 50–100-kb DNA fragments without production of an oligonucleosomal DNA ladder, the cytosolic extract of serum-deprived cells induced both HMW and internucleosomal DNA fragmentation in substrate nuclei (Fig.2A). Furthermore, the formation of HMW DNA fragments induced by cytosolic extract of serum-deprived cells was almost completely abolished by suramin, without affecting the internucleosomal DNA cleavage (Fig. 2B), whereas the presence of Zn2+ions in the cytosolic extract inhibited the cytosol-dependent formation of oligonucleosomal but not HMW DNA fragments in substrate nuclei (Fig. 2C). These data indicate that the lack of internucleosomal DNA fragmentation is a characteristic feature of etoposide-induced apoptosis in NB-2a cells and that the formation of HMW and oligonucleosomal DNA fragments, at least in a cell-free system, is mediated by separate nuclease activities. Data presented in Fig. 3 demonstrate that the capacity of cytosolic extracts to initiate disintegration of DNA in substrate nuclei was progressively increased during apoptosis induced by either etoposide or serum deprivation in NB-2a cells. Pretreatment of cytosolic extracts of etoposide-treated cells with recombinant caspase 3 potentiated cytosol-dependent formation of HMW DNA fragments without producing an oligonucleosomal DNA ladder (Fig.3A), thus suggesting that caspase 3 may be involved in the activation of HMW DNA fragmentation activity. In contrast to the cytosolic extract prepared from etoposide-treated cells, the cytosolic extract of serum-deprived cells induced both HMW DNA cleavage and internucleosomal DNA cleavage in substrate nuclei, but only when the cytosolic extract was prepared from the cells at the late stage of apoptosis (i.e. after 48 h of serum deprivation; Fig.3B). Cytosolic extract prepared from the cells at an advanced stage of apoptosis (after 36 h of serum deprivation) possessed a weak DNA fragmentation capacity; however, this extract potentiated the formation of both HMW and oligonucleosomal DNA fragments in substrate nuclei after pretreatment with recombinant caspase 3 (Fig. 3B). In contrast, cytosolic extract of the early apoptotic cells (after 24 h of serum deprivation) induced disintegration of DNA in substrate nuclei mainly to HMW DNA fragments without obvious production of an oligonucleosomal DNA ladder, even after pretreatment with recombinant caspase 3 (Fig. 3B). Only HMW DNA fragmentation without any sign of internucleosomal DNA cleavage was observed when the cytosolic extract of nonapoptotic cells was pretreated with recombinant caspase 3 (results not shown). The capacity of the late apoptotic extract but not the early apoptotic extract to induce internucleosomal DNA fragmentation in substrate nuclei was consistent with up-regulation of CAD protein observed during apoptosis induced by serum deprivation in NB-2a cells (Fig.3C). At the same time, the progressive increase in the capacity of cytosolic extracts of etoposide-treated cells to induce HMW DNA fragmentation was accompanied by down-regulation of CAD in etoposide-treated cells (Fig. 3C). These results indicate that CAD is selectively induced during apoptosis associated with oligonucleosomal but not HMW DNA fragmentation, thus supporting the hypothesis that CAD has no role in the formation of HMW DNA fragments. Because caspases play a pivotal role in the activation of the CAD-dependent pathway of DNA disintegration (37Wolf 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), we investigated the pattern of DNA fragmentation in apoptotic cells in the presence of a broad range caspase inhibitor, z-VAD-fmk. Data presented in Fig. 4A demonstrate that z-VAD-fmk effectively suppressed internucleosomal DNA cleavage but only partially inhibited the formation of HMW DNA fragments in serum-deprived cells. In contrast, z-VAD-fmk, although effectively suppressing the cleavage of caspase-targeted poly(ADP-ribose) polymerase, possessed only a slight inhibitory effect on the formation of HMW DNA fragments during etoposide-induced apoptosis (Fig.4B). The results suggest that caspases are not essential in the induction of HMW DNA fragmentation in NB-2a cells during etoposide-induced apoptosis. Data presented in Fig 5A demonstrate that heating of substrate nuclei selectively abrogates the cytosol-dependent formation of HMW DNA fragments but not oligonucleosomal DNA fragments, suggesting that a heat-labile component of HMW DNA fragmentation activity pre-exists in nuclei prepared from nonapoptotic NB-2a cells. Because the formation of HMW DNA fragments is widely believed to originate from the excision of DNA loop domains at the positions of their attachment to the nuclear matrix, we analyzed DNA fragmentation activity in nonapoptotic NB-2a nuclei extracted with a high concentration of salt (a procedure commonly used for the preparation of histone-depleted DNA loop domains attached to the insoluble nuclear matrix (17Paulson J.R. Laemmli U.K. Cell. 1977; 12: 817-828Abstract Full Text PDF PubMed Scopus (782) Google Scholar, 18Cook P.R. Brazell I.A. J. Cell Sci. 1976; 22: 287-302PubMed Google Scholar). Data presented in Fig. 5Bdemonstrate that incubation of the high salt-extracted nuclei in DNA cleavage buffer induced cleavage of nuclear DNA into 50–100-kb DNA fragments, with a pattern of fragmentation similar to that found in apoptotic cells. The observation that an ordered cleavage of DNA into the HMW DNA fragments is retained in the high salt-extracted nuclei strongly supports the idea that HMW DNA fragments represent DNA loop domains excised by a nuclear matrix-associated domain nuclease. The excision of DNA loop domains in the high salt-extracted nuclei proceeded in a highly efficient manner, was slightly potentiated by etoposide (Fig. 5B, lanes 2–4), and was inhibited by a catalytic inhibitor of topo II, suramin (Fig.5B, lanes 5–10). Furthermore, the inhibitory effect of suramin was markedly suppressed in the presence of etoposide, a drug that potentiates topo II-dependent DNA cleavage (19Burden D.A. Osheroff N. Biochim. Biophys. Acta. 1998; 1400: 139-154Crossref PubMed Scopus (497) Google Scholar,20Liu L.F. CRC Crit. Rev. Biochem. 1983; 15: 1-24Crossref PubMed Scopus (170) Google Scholar) (Fig. 5B, lanes 5–7). Also, conditions that favor topo II-dependent rejoining reaction lead to almost complete religation of the cleaved HMW DNA fragments into noncleaved DNA (Fig. 5B, lane 12). The biochemical properties of an inducible HMW DNA cleavage in high salt-extracted nuclei observed and demonstrated here add credence to the suggestion that the domain nuclease possesses the properties of topo II. An efficient HMW DNA cleavage (>90% of the total DNA was cleaved into 50–100-kb fragments under inducible conditions), which was inhibitable by suramin and reversible under salt-dependent religation conditions, was also observed by us in postmitotic cerebellar granule neurons, in which the level of topo IIα, but not topo IIβ, was reduced to a negligible level (Ref.21Tsutsui K. Sano K. Kikuchi A. Tokunaga A. J. Biol. Chem. 2001; 276: 5769-5778Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar; results not shown). This suggests that topo IIβ may be involved in inducible excision of DNA loop domains in high salt-extracted nuclei. To evaluate the role of topo II in degradation of nuclear DNA during apoptosis, we first analyzed the effect of suramin on the pattern of DNA fragmentation in apoptotic NB-2a cells. As in the in vitro model of inducible excision of DNA loop domains (Fig. 5), suramin effectively suppressed the formation of HMW DNA fragments during apoptosis induced by etoposide in NB-2a cells (Fig.6A). In contrast to serum deprivation, in which suramin at these concentrations suppressed all features of apoptosis (i.e. DNA fragmentation and caspase activation), the protective effect of suramin against HMW DNA fragmentation in etoposide-treated cells occurred despite the persistence of high caspase 3 activity (results not shown), thus suggesting that the protective effect of suramin is downstream of caspase activation in this apoptotic pathway and may be caused by the direct inhibition of topo II. The addition of suramin to the cells at advanced stages of apoptosis, when activation of caspases had already occurred, also resulted in suppression of HMW DNA fragmentation in both etoposide-treated and serum-deprived cells, thus suggesting a reversible feature of HMW DNA cleavage during apoptosis (Fig.6B). Because topo II enzyme is known to remain covalently attached to the 5′ ends of broken DNA during topo II-mediated DNA cleavage (19Burden D.A. Osheroff N. Biochim. Biophys. Acta. 1998; 1400: 139-154Crossref PubMed Scopus (497) Google Scholar, 20Liu L.F. CRC Crit. Rev. Biochem. 1983; 15: 1-24Crossref PubMed Scopus (170) Google Scholar), we further investigated the role of topo II in apoptosis by analyzing whether HMW DNA fragments isolated from apoptotic cells are topo II-associated. Data presented in Fig. 6C demonstrate that the HMW DNA fragments fractionated from etoposide-treated cells under denaturing conditions were sensitive to the 3′-5′ exonucleaseExoIII but exhibited a marked resistance to the 5′-3′ exonuclease lambda. Pretreatment of isolated HMW DNA fragments with proteinase K abolished this resistance (Fig. 6C), suggesting that protection against lambda exonuclease was caused by protein(s) associated with the 5′ termini of the HMW DNA fragments. HMW DNA fragments isolated from serum-deprived cells possessed similar properties (Fig. 6C), although the resistance to the lambda exonuclease was not so evident as in the case of DNA fragments isolated from etoposide-treated cells. Treatment of the isolated HMW DNA fragments with DNase I followed by analysis of the digest with SDS-PAGE revealed no visible polypeptides associated with the HMW DNA fragments after Coomassie Blue staining, except a ∼35-kDa protein seen in apoptotic but not control preparations (Fig. 6D). Probing of the same digest with anti-topo IIα antibody revealed immunoreactive bands associated with HMW DNA fragments, but no obvious band corresponding to the full-length 170-kDa protein was detected (Fig.6D). In contrast, anti-topo IIβ antibody revealed a clear ∼180-kDa band associated with DNA fragments derived from either serum-deprived or etoposide-treated cells (Fig. 6D). The data indicate that topo IIβ is at least one of the enzymes associated with apoptotic HMW DNA fragments. Cell-free system experiments (Fig.6E) further revealed that antibody raised against full-length topo II protein possessed a protective effect against HMW DNA cleavage induced by apoptotic cytosolic extract in substrate nuclei. Whereas anti-nuclear factor κB antibody had no obvious effect on the formation of HMW DNA fragments, both anti-topo IIα and anti-topo IIβ antibodies inhibited cytosol-dependent HMW DNA cleavage in substrate nuclei, with the protective effect of the anti-topo IIβ antibody being more evident. In the cell-free apoptotic system, anti-topo IIβ antibody almost completely suppressed the formation of HMW DNA fragments in substrate nuclei induced by cytosolic extracts of etoposide-treated cells, and it markedly suppressed the HMW DNA cleavage without affecting the oligonucleosomal DNA fragmentation induced by cytosolic extract of serum-deprived cells (Fig.6E)" @default.
• W1999854664 created "2016-06-24" @default.
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• W1999854664 date "2002-06-01" @default.
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• W1999854664 title "The Role of Topoisomerase II in the Excision of DNA Loop Domains during Apoptosis" @default.
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• W1999854664 doi "https://doi.org/10.1074/jbc.m110621200" @default.
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