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J. Biol. Chem., Vol. 277, Issue 24, 21458-21467, June 14, 2002

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The Role of Topoisomerase II in the Excision of DNA Loop Domains during Apoptosis^*

Victor T. Solovyan^§¶, Zinayida A. Bezvenyuk, Antero Salminen, Caroline A. Austin^**, and Michael J. Courtney

From the  A. I.Virtanen Institute for Molecular Sciences, University of Kuopio, P. O. Box 1627, FIN-70211 Kuopio, Finland, ^§ Institute of Molecular Biology and Genetics, Kiev-252627, Ukraine,  Kuopio University Hospital, University of Kuopio, Kuopio 70211, Finland, and ^** School of Biochemistry and Genetics, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, United Kingdom

Received for publication, November 5, 2001, and in revised form, February 25, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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 (1, 2). The domain level of chromatin organization is supported by the interaction of specific DNA sequences, matrix/scaffold attachment regions, with nuclear matrix proteins (3). The chromatin loops represent the basic structural components of higher-order chromatin folding, which is maintained during the cell cycle and in differentiated cells (3-5).

Disintegration of nuclear DNA into nucleosome-sized fragments represents a classical manifestation of apoptosis (6). 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 (7). 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 (8, 9) and is considered to be an initial step in DNA disintegration during apoptosis (7, 10-12).

The discovery of caspase-activated DNase (CAD/DFF40/CPAN; hereafter designated CAD) (13-15) 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 (13-15). At the same time, increasing evidence indicates that the formation of the HMW DNA fragments during apoptosis can proceed without internucleosomal DNA cleavage (7, 12, 16). 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.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Culture and Induction of 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).

Analysis of Nuclear Morphology-- 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).

Caspase 3 (DEVDase) Activity Assay-- 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 mM MgCl₂, 0.1 mM EDTA, 0.1 mM EGTA, and 0.1 mM phenylmethylsulfonyl fluoride (PMSF)) as described previously (12). 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.

Cell-free Apoptosis Assay-- Nuclei and cytosolic extracts for cell-free apoptosis assay were prepared as described previously (12). 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 mM sucrose, 5 mM EGTA, 1 mM dithiothreitol, 0.5 mM spermidine, 0.2 mM spermine, 1 mM PMSF, and 50% glycerol) at 1 × 10⁸ nuclei/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 II-specific (catalogue no. 2011-1; TopoGEN) or JB-1 topo II-specific antibodies (kindly provided by Dr. D. Sullivan, University of South Florida) at a dilution of 1:20.

Analysis of DNA Integrity-- 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 (12).

Western Blot-- 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 II or 18513 anti-topo II antibodies (36) (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.

Preparation of Nuclear Halo Structures and Induction of the Excision of DNA Loop Domains-- 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 mM Tris-HCl, pH 7.5, 20 mM EDTA, and 0.5% SDS) and subjected to fractionation by FIGE.

Exonuclease Protection Assay-- 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 mM Tris-HCl, pH 8.0, 50 mM EDTA, and 1% SDS), loaded into wells of a 1% agarose gel, and fractionated by conventional gel electrophoresis.

Isolation of DNA-associated Proteins-- 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 mM dithiothreitol, and 1 mM PMSF). DNase-treated samples were resolved in 7% SDS-PAGE, blotted onto nitrocellulose membrane, and probed with anti-topo II-specific antibody.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Etoposide and Serum Withdrawal Induce Apoptosis in NB-2a Cells with Distinct Patterns of DNA Fragmentation-- 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.

View larger version (70K): [in this window] [in a new window]   Fig. 1.   Apoptosis in NB-2a cells induced by serum withdrawal and by etoposide is accompanied by distinct patterns of DNA disintegration. Exponentially growing cells were incubated either without serum or in the presence of the indicated concentrations of etoposide for 0-3 days. At different time points, cells were collected and analyzed for nuclear morphology, DNA integrity, and caspase 3 (DEVDase) activity. A, nuclear morphology (Hoechst 33258 staining) of the control (con), serum-deprived (ser), and etoposide-treated cells (eto) after 48 h of apoptotic challenge. B and C, pattern of DNA disintegration in serum-deprived cells and etoposide-treated cells, respectively, revealed either by FIGE (top panels) or by conventional gel electrophoresis (bottom panels). Lanes C, control (nontreated) cells; lanes M and m, molecular weight standards; Midrange II PFG molecular weight markers (New England Biolabs) and a 1-kb DNA ladder, respectively. D, time course of caspase 3 activation during apoptosis induced by serum deprivation and by 10 µM etoposide. Caspase 3 activity is presented as nmol cleaved AMC/(s × g protein). Each time point contains the mean ± S.D. of three observations.

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.

View larger version (54K): [in this window] [in a new window]   Fig. 2.   HMW DNA fragmentation and internucleosomal DNA fragmentation during apoptosis are mediated by separate mechanisms. A, nuclei isolated from nonapoptotic NB-2a cells were incubated for 1 h at 37 °C with cytosolic extracts prepared from the control (nonapoptotic) cells (con, lane 1) or from cells induced to undergo apoptosis either by etoposide (10 µM for 48 h) (eto, lane 2) or by serum withdrawal (48 h of deprivation) (ser, lane 3). Cytosolic extract of serum-deprived cells was incubated without nuclei (lane 4). B, nonapoptotic nuclei were incubated with cytosolic extract of serum-deprived cells (48 h of deprivation) alone (lane 1) or in the presence of 100 µM suramin (lane 2). C is as described in B, except that 0.1 mM Zn2+ was added to the cytosolic extract instead of suramin. Herein and in other experimental settings, all cytosolic extracts were diluted with cytosol-preparing buffer to give a standard concentration of protein (5 mg/ml). After incubation, nuclei were embedded in low-melting point agarose, lysed, and fractionated either by FIGE (top panels) or by conventional gel electrophoresis (bottom panels). The positions of the molecular weight markers are shown on the left. Note that the distinct patterns of DNA disintegration that occurred in apoptotic cells were reproducible in a cell-free apoptotic system and that the HMW and internucleosomal DNA fragmentation activities could be distinguished by pharmacological means.

The Caspase/CAD Pathway Is Not Involved in the Formation of HMW DNA Fragments during Etoposide-induced Apoptosis-- 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.

View larger version (48K): [in this window] [in a new window]   Fig. 3.   Analysis of DNA fragmentation activities during apoptosis in NB-2a cells. A, nuclei isolated from the nonapoptotic NB-2a cells were incubated with cytosolic extract of the nonapoptotic cells (N.cytosol) or with cytosolic extracts of etoposide-treated cells (Eto cytosol) prepared at 24, 36, and 48 h of etoposide treatment (10 µM). Before the addition of substrate nuclei, cytosolic extracts were preincubated for 30 min at 37 °C with (+) or without () recombinant caspase 3. B is as described in A, except that cytosolic extracts were prepared from the serum-deprived cells at different stages of serum deprivation, as indicated. After incubation, nuclei were embedded in low-melting point agarose, lysed, and fractionated either by FIGE (top panels) or by conventional gel electrophoresis (bottom panels). C shows the level of CAD in apoptotic cells revealed with anti-CPAN antibodies at different stages of apoptosis.

Because caspases play a pivotal role in the activation of the CAD-dependent pathway of DNA disintegration (37), 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.

View larger version (46K): [in this window] [in a new window]   Fig. 4.   The role of caspases in the regulation of HMW DNA cleavage during apoptosis in NB-2a cells. A, cells were incubated in serum-deficient medium for 24-48 h alone (lanes 1-3) or in the presence of 100 µM z-VAD-fmk (lanes 4-6). At different time points, cells were collected, embedded in agarose, lysed, and fractionated either by FIGE (top panel) or by conventional gel electrophoresis (bottom panel). B is as described in A, except that 10 µM etoposide was used instead of serum withdrawal to induce apoptosis. The bottom panel shows cleavage of poly(ADP-ribose) polymerase in etoposide-treated cells with or without z-VAD-fmk, as indicated. Lane C, control (nontreated cells).

NB-2a Cells Possess Nuclear Matrix-associated HMW DNA Fragmentation Activity with the Properties of Topo II-- 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 (17, 18). Data presented in Fig. 5B demonstrate 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 (19, 20) (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 II, but not topo II, was reduced to a negligible level (Ref. 21; results not shown). This suggests that topo II may be involved in inducible excision of DNA loop domains in high salt-extracted nuclei.

View larger version (59K): [in this window] [in a new window]   Fig. 5.   Characterization of HMW DNA fragmentation activity in nonapoptotic NB-2a nuclei. A, nuclei isolated from nonapoptotic NB-2a cells were incubated with cytosolic extract of the nonapoptotic cells (lane 1) or with cytosolic extract of serum-deprived cells immediately (lane 2) or after preheating at 65 °C for 20 min (lane 3), followed by the analysis of DNA integrity as described. Note that preheating of the nuclei completely abrogated the cytosol-dependent formation of HMW but not oligonucleosomal DNA cleavage. B, nonapoptotic nuclei were embedded in low-melting point agarose and extracted with the high salt extraction buffer (see "Materials and Methods") to obtain DNA loop halos. After washing, high salt-extracted nuclei were incubated for 20 min at 37 °C in DNA cleavage buffer (see "Materials and Methods") containing either 5 mM EDTA (lane 1) or 5 mM Mg2+ (lanes 2-12). Etoposide (Eto) at a final concentration of 0, 20, or 40 µM (lanes 2-4, respectively), suramin at a final concentration of 10, 100, or 500 µM in the presence of 40 µM etoposide (Eto/Sur, lanes 5-7, respectively), or suramin at the same concentrations without etoposide (Sur, lanes 8-10, respectively) was added to the DNA cleavage buffer. After incubation, the high salt-extracted nuclei were lysed and subjected to fractionation by FIGE. In an additional experimental setting (lanes 11 and 12), the high salt-extracted nuclei were incubated in DNA cleavage buffer for 20 min at 37 °C. After incubation, samples were either treated immediately with lysis buffer (lane 11) or additionally incubated in the presence of 1 M NaCl for 20 min (lane 12) followed by treatment with lysis buffer and analysis of DNA integrity by FIGE. The top panel shows the pattern of DNA cleavage revealed by FIGE; the bottom panel shows noncleaved DNA isolated from the starts after sample fractionation by FIGE. Lane m, Midrange II PFG molecular weight markers (New England Biolabs).

Topo II Is Involved in Excision of DNA Loop Domains during Apoptosis in NB-2a Cells-- 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).

View larger version (47K): [in this window] [in a new window]   Fig. 6.   Topo II contributes to the excision of DNA loop domains during apoptosis in NB-2a cells. A, protective effect of suramin against HMW DNA fragmentation in apoptotic cells. Cells were treated for 48 h with 10 µM etoposide alone (lane 1) or in the presence of suramin at a final concentration of 0.25, 0.5, and 1 mM (lanes 2-4, respectively). After incubation, both attached and detached cells were collected, embedded in agarose, lysed, and fractionated by FIGE. Top panel shows DNA integrity revealed by FIGE; bottom panel shows noncleaved DNA isolated from the wells after cell fractionation by FIGE. B, cells were incubated for 48 h with 10 µM etoposide or in serum-deficient medium followed by the addition of suramin at a final concentration of 0.25 (lanes 2 and 5) and 0.5 mM (lanes 3 and 6). After an additional incubation for 1 h, both attached and detached cells were collected and analyzed for DNA integrity as described in A. C, apoptotic HMW DNA fragments are protein-associated. Agarose plugs containing 50-100-kb DNA fragments derived from either etoposide-treated (10 µM etoposide for 48 h, top panel) or serum-deprived cells (48 h of deprivation, bottom panel) were excised from the low-melting point agarose gel and treated with either ExoIII exonuclease or lambda exonuclease or pretreated with proteinase K followed by lambda exonuclease treatment for 0-40 min, as indicated. After incubation, 20-µl aliquots were transferred to 10 µl of stop buffer (50 mM Tris-HCl, pH 8.0, 50 mM EDTA, and 1% SDS), loaded into wells of 1% agarose gel, and fractionated by conventional gel electrophoresis. D, topo II enzyme is associated with the apoptotic HMW DNA fragments. Agarose plugs containing 50-100-kb DNA fragments derived from either etoposide-treated or serum-deprived cells were excised, and DNA fragments were extracted from agarose as described under "Materials and Methods." Extracted DNA was treated with DNase I for 30 min at 37 °C; digest was resolved in 7% SDS-PAGE, blotted onto nitrocellulose membrane, and probed with anti-topo II or anti-topo II antibodies. Lane 1, total nuclear lysate; lane 2, DNase I digestion mixture without DNA; lane 3, DNase I digest of total DNA purified from control cells; lanes 4 and 5, DNase I digests of HMW DNA fragments derived from serum-deprived and etoposide-treated cells, respectively. Left panel shows Coomassie Blue-stained gel; middle and right panels show membrane after probing with anti-topo II and anti-topo II antibody, respectively. Arrow indicates the position of the full-length topo II enzyme. E, topo II enzyme is involved in the formation of HMW DNA fragments in a cell-free apoptotic assay. Nuclei isolated from the nonapoptotic cells were incubated with nonapoptotic cytosolic extract (lane 1), with cytosolic extract prepared from etoposide-treated cells (lanes 2-6), or with cytosolic extract prepared from serum-deprived cells (lanes 7 and 8). Before incubation, apoptotic cytosolic extracts were preincubated for 1 h at room temperature alone (lanes 2 and 4) or with anti-nuclear factor B antibody (lane 3), anti-topo II antibody (lane 5), or anti-topo II antibody (lanes 6 and 8). After incubation, substrate nuclei were embedded in agarose, lysed, and fractionated by FIGE (top panel) or by conventional gel electrophoresis (bottom panel).

Because topo II enzyme is known to remain covalently attached to the 5' ends of broken DNA during topo II-mediated DNA cleavage (19, 20), 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' exonuclease ExoIII 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 II 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 II 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 II 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 B antibody had no obvious effect on the formation of HMW DNA fragments, both anti-topo II and anti-topo II antibodies inhibited cytosol-dependent HMW DNA cleavage in substrate nuclei, with the protective effect of the anti-topo II antibody being more evident. In the cell-free apoptotic system, anti-topo II 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). The results suggest the involvement of topo II enzyme in the formation of HMW DNA fragments in the cell-free apoptotic system.

Activation of Topo II-mediated Excision of DNA Loop Domains Accompanies the Early Stages of Apoptosis in NB-2a Cells-- We further examined at what stage of apoptosis the activation of topo II-mediated HMW DNA cleavage takes place by using in vitro conditions that activate the excision of DNA loop domains in high salt-extracted nuclei, as in Fig. 5.

Data presented in Fig 7 demonstrate that whereas treatment of the cells with etoposide for 0-24 h still did not promote an obvious disintegration of nuclear DNA in vivo (Fig. 7A, lanes 1-4), a subsequent incubation of these cells in DNA cleavage buffer resulted in an increasing accumulation of HMW DNA fragments (Fig. 7A, lanes 5-8). In vitro induced HMW DNA cleavage subsequent to in vivo etoposide treatment was inhibited by suramin (Fig. 7A, lanes 9-12) and reversible under conditions of salt-induced religation (Fig. 7A, lanes 13-16). This suggests the involvement of topo II in the induction of HMW DNA cleavage after incubation of etoposide-treated cells in DNA cleavage buffer. An essentially similar activation of HMW DNA cleavage, inhibitable by suramin and reversible under salt-dependent religation conditions, was also observed in the serum-deprived cells after their incubation in DNA cleavage buffer (Fig. 7B). In vitro activation of HMW DNA cleavage that occurred in both etoposide-treated and in serum-deprived cells was observed in the cells at the early stages of apoptosis, well before the beginning of apoptotic DNA disintegration (Fig. 7, A and B) and, more interesting, was rapidly reversible by removal of etoposide from the culture medium or by readdition of serum to the serum-deprived cells (Fig. 7C). The results suggest an early engagement of topo II in HMW DNA cleavage that occurs at the reversible stages of apoptosis.

View larger version (47K): [in this window] [in a new window]   Fig. 7.   Activation of topo II-mediated HMW DNA cleavage in NB-2a cells at the early stages of apoptosis. A, cells were incubated with 10 µM etoposide (Eto) for 0-24 h. At different time points (indicated at the top of the panel), cells were collected and embedded in agarose, followed by an additional incubation for 20 min at 37 °C in the DNA cleavage buffer (CB) (see "Materials and Methods") containing either 5 mM EDTA (lanes 1-4) or 5 mM Mg2+ alone (lanes 5-8) or in the presence of 0.5 mM suramin (CB/Sur; lanes 9-12). In an additional experimental setting, samples were incubated in the DNA cleavage buffer and transferred to ice, NaCl was added to the DNA cleavage buffer at a final concentration of 1 M, and samples were incubated for an additional 20 min at 0 °C to induce a topo II-mediated religation reaction (lanes 13-16). After incubation, samples were treated with lysis buffer and fractionated by FIGE. B is as described in A, except that serum withdrawal (ser) was used as an apoptosis inducer instead of etoposide. C, cells were incubated in serum-deficient medium for 18 h (lane 1). After readdition of serum, cells continued to be incubated in serum-containing medium for 2-6 h (lanes 2-4). At different time points after the addition of serum (indicated at the top of the panel), cells were collected, embedded in low-melting point agarose, and additionally incubated in the DNA cleavage buffer as described above. After incubation, cells were treated with lysis buffer and analyzed for DNA integrity by FIGE. Top panels show DNA integrity in cell samples revealed by FIGE. Bottom panels show noncleaved DNA isolated from wells after cell fractionation by FIGE. Note that sensitivity to in vitro "inducible" conditions that activate HMW DNA cleavage in apoptotic cells occurs before overt apoptotic DNA disintegration in vivo.

Topo II-deficient Fibroblasts Are Resistant to Apoptosis Induced by Oxidative Stress-- To further elucidate the role of topo II in the formation of HMW DNA fragments during apoptosis, we examined the apoptotic response in wild-type and topo II knockout mouse embryonic fibroblasts (MEFs). The data presented in Fig. 8A demonstrate that serum deprivation induced cell death in MEFs in a manner associated with definite activation of caspase 3 that was comparable in both wild-type and topo II/ cells and induced disintegration of nuclear DNA into HMW DNA fragments that was attenuated in the topo II-deficient cells. Because serum deprivation induced oligonucleosomal DNA cleavage at the advanced stages of apoptosis (data not shown), we chose to examine a different apoptotic response in MEFs that proceeds without oligonucleosomal DNA fragmentation to exclude a possible involvement of CAD in disintegration of nuclear DNA. The data presented in Fig. 8B demonstrate that hydrogen peroxide induced an apoptotic-like chromatin shrinkage in MEFs that was associated with only a weak activation of caspase 3 but with clear disintegration of nuclear DNA, predominantly into HMW DNA fragments, without obvious formation of low molecular weight DNA tail. Both the number of shrunken nuclei and the level of HMW DNA fragmentation were markedly reduced in topo II-deficient fibroblasts as compared with the wild-type cells, indicating the involvement of topo II in HMW DNA cleavage during H₂O₂-induced cell death. Furthermore, in vitro incubation of H₂O₂-treated fibroblasts under conditions that activate the excision of DNA loop domains resulted in a massive accumulation of 50-100-kb DNA fragments that coincided with the beginning of apoptotic DNA disintegration in vivo (Fig. 8C). Again, in vitro activation of HMW DNA cleavage in the early apoptotic fibroblasts was markedly reduced in cells lacking topo II (Fig. 8C).

View larger version (49K): [in this window] [in a new window]   Fig. 8.   Analysis of apoptotic response in topo II-deficient MEFs. A, wild-type (wt) and topo II-deficient cells (/) were incubated in serum-free medium for 0-48 h. At different time points, cells were collected and analyzed for DEVDase activity (top panel) and HMW DNA cleavage (bottom panel), as indicated. B, cells were exposed to 0.5 mM H2O2 for 0-24 h. At different time points (as indicated in the panel), cells were collected and analyzed for DEVDase activity (top panel) and HMW DNA cleavage (middle panel). The nuclear morphology of Hoechst 33342-stained cells is shown in the bottom panel. The proportion of shrunken (apoptotic) nuclei calculated as an average of five independent fields is shown in parentheses. C, peroxide-induced sensitivity to in vitro activation of HMW DNA cleavage. Cells were exposed to 0.5 mM H2O2 for 0-8 h. At different time points (indicated at the top of the panel), cells were collected and embedded in agarose, followed by an additional incubation for 20 min at 37 °C in DNA cleavage buffer (CB) (see "Materials and Methods") containing either 5 mM EDTA (lanes 1-10) or 5 mM Mg2+ (lanes 11-20). After incubation, cells were lysed and analyzed for DNA integrity by FIGE.

	DISCUSSION

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The HMW DNA Cleavage and Internucleosomal DNA Cleavage Represent Separate Programs of Apoptotic DNA Disintegration in NB-2a Cells-- Disintegration of nuclear DNA into nucleosome-sized DNA fragments represents the most typical feature of the apoptotic process in a variety of cellular models. Here we demonstrate that apoptosis, even within the same cell type, can be either associated with or proceed without the formation of oligonucleosomal DNA fragments, depending on the type of apoptotic inducer. In contrast to oligonucleosomal DNA fragmentation, HMW DNA cleavage invariably accompanies apoptosis induced by a variety of stimuli in NB-2a cells (22). The formation of HMW DNA fragments during apoptosis seems to originate from the excision of DNA loop domains at the positions of their attachment to the nuclear matrix, inasmuch as the pattern of HMW DNA fragmentation in apoptotic cells was essentially the same as the profile of HMW DNA cleavage induced in high salt-extracted nuclei. The distinct patterns of DNA fragmentation that accompany apoptosis induced by different stimuli in NB-2a cells raise the question of whether these two major periodicities of DNA cleavage, i.e. the excision of DNA loop domains and internucleosomal DNA fragmentation, are mediated by a common mechanism during apoptotic execution.

Previously, we demonstrated that the patterns of DNA disintegration were additive when NB-2a cells were challenged simultaneously with different apoptotic stimuli, each of which induced a distinct pattern of DNA disintegration (12). On the other hand, Zn²⁺ ions, a well-known inhibitor of both caspases (23) and DFF40/CAD (24), abrogated internucleosomal DNA cleavage but led to the accumulation of HMW DNA fragments in serum-deprived NB-2a cells (22). The results presented in this report demonstrate that the HMW and internucleosomal DNA fragmentation activities can also be separated in a cell-free apoptotic system, further supporting the idea that HMW DNA cleavage and internucleosomal DNA cleavage represent separate programs of DNA disintegration in apoptotic cells.

The Caspase/CAD Pathway Is Not Essential for the Excision of DNA Loop Domains in Apoptotic Cells-- Our data demonstrate that the pattern of DNA fragmentation induced by different apoptotic stimuli in NB-2a cells was reproducible in a cell-free apoptotic system. Thus, cytosolic extract prepared from etoposide-treated cells potentiated the formation of HMW DNA fragments in substrate nuclei, whereas cytosolic extract of serum-deprived cells induced the formation of both HMW and oligonucleosomal DNA fragments. The difference in the pattern of cytosol-dependent DNA fragmentation was also observed when substrate nuclei were incubated with cytosolic extracts prepared from okadaic acid- or AraC-treated cells that underwent apoptosis associated either with or without internucleosomal DNA fragmentation, respectively (22). Apoptotic cell death that proceeds without internucleosomal DNA cleavage is not restricted to the genotoxic apoptotic inducers (e.g. etoposide or AraC), inasmuch as staurosporine, a protein kinase inhibitor, also induced apoptosis in NB-2a cells accompanied by HMW but not internucleosomal DNA cleavage, with the pattern of fragmentation being reproducible in a cell-free system (results not shown).

The lack of an internucleosomal DNA cleavage in NB-2a cells during apoptosis induced by several stimuli, as well as the inability of a cytosolic extract from these cells to induce the formation of oligonucleosomal DNA fragments in a cell-free system, suggests that apoptosis that proceeds without internucleosomal DNA fragmentation is associated with activation of a domain nuclease, a nuclease that can excise DNA loop domains but is unable to perform internucleosomal DNA cleavage. At present, a number of nucleases implicated in apoptotic DNA disintegration have been described (reviewed in Ref. 25). The common feature of all these nucleases is that they induce oligonucleosomal DNA fragmentation during apoptosis. Increasing evidence suggests that CAD, whose activation is critically dependent on caspase activity, appears to be a pivotal nuclease implicated in oligonucleosomal DNA fragmentation induced by diverse apoptotic stimuli (26-30). Although CAD is able to induce HMW DNA fragmentation per se (31, 32), CAD-mediated HMW DNA cleavage is considered to be an initial step of DNA disintegration, which was always accompanied by the formation of oligonucleosomal DNA ladder in a variety of apoptotic models. The inability of a domain nuclease to induce the formation of oligonucleosomal DNA fragments and the down-regulation of CAD during etoposide-induced apoptosis suggest that HMW DNA cleavage in the absence of oligonucleosomal DNA fragmentation is mediated by a nuclease distinct from CAD.

The characteristic feature of CAD is that caspases play a pivotal role in its activation in a variety of apoptotic models (37). Our cell-free system experiments demonstrate that cytosol-dependent formation of HMW DNA fragments in substrate nuclei was potentiated when early apoptotic cytosolic extracts were pretreated with recombinant caspase 3. This suggests that caspases can activate the pathway leading to the excision of DNA loop domains, at least in a cell-free apoptotic system. At the same time, in apoptotic cells a pan-caspase inhibitor, z-VAD-fmk, suppressed oligonucleosomal but not HMW DNA fragmentation, despite completely suppressing the caspase-dependent cleavage of poly(ADP-ribose) polymerase (Fig. 4) or DEVDase activity in apoptotic cytosolic extracts that reached 5- to 10-fold of the activity in nonapoptotic cytosolic extract (results not shown). In MEFs, oxidative stress induced HMW but not oligonucleosomal DNA fragmentation, accompanied by a weak activation of DEVDase that reached no more than 1.5- to 2-fold as compared with the control cytosolic extract. Finally, as we demonstrated previously (33), the excitatory neurotransmitter glutamate induced massive HMW DNA cleavage in cultured neurons that was neither accompanied by activation of caspase 1, 2, 3, 5, 8, or 9 nor inhibitable by z-VAD-fmk or Boc-D-fmk. All these data suggest that caspases can activate but are not essential for the induction of HMW DNA cleavage during apoptosis, thus implying that the caspase/CAD pathway does not play a significant role in the induction of the excision of DNA loop domains in apoptotic cells.

Recently, a mitochondrially located apoptosis-inducing factor, AIF, has been described, which induced disintegration of nuclear DNA into HMW but not oligonucleosomal DNA fragments in a cell-free apoptotic system via a caspase-independent mechanism (34). In a cell-free apoptotic system, it has been shown that AIF-modulated HMW DNA cleavage and caspase/CAD-dependent internucleosomal DNA fragmentation represent two parallel pathways of apoptotic DNA disintegration (35). In our cell-free system, experiments with anti-AIF antibody did not neutralize the cytosol-dependent HMW DNA cleavage (results not shown). However, in accord with the above-mentioned report (35), our results demonstrate that caspase-dependent oligonucleosomal DNA fragmentation and caspase-independent HMW DNA cleavage do co-exist in intact cells and can be differentially triggered, depending on the type of apoptotic inducers.

Topo II Is a Domain Nuclease That Contributes to the Formation of HMW DNA Fragments during Apoptosis-- The absence of internucleosomal DNA cleavage during apoptosis suggests that apoptotic formation of HMW DNA fragments is mediated by a nuclease activity (domain nuclease) that is constrained to the chromosomal DNA at specific positions and is unable to induce the formation of oligonucleosomal DNA fragments. These positions could coincide with the position of the attachment of nuclear DNA to the nuclear matrix, consistent with the widely accepted belief that apoptotic HMW DNA fragments represent excised DNA loop domains.

Our data demonstrate that nonapoptotic NB-2a cells contain a HMW DNA fragmentation activity located in the high salt-insoluble nuclear fraction, thus supporting the suggestion that it is a component of the nuclear matrix. Upon induction, this activity initiates a highly organized cleavage of DNA in high salt-extracted nuclei into 50-100-kb DNA fragments, indicating excision of DNA loop domains. The sensitivity of HMW DNA cleavage to both etoposide and suramin, as well as the reversibility of the excision of DNA loop domains under conditions that promote topo II-dependent religation, adds an essential credence to the idea that the nuclease activity responsible for HMW DNA cleavage in high-salt extracted nuclei (i.e. the domain nuclease) possesses the properties of topoisomerase II.

Here we presented several independent lines of evidence supporting the involvement of the topo II enzyme, in particular, topo II, in the excision of DNA loop domains during apoptosis: (i) HMW DNA cleavage in apoptotic cells was sensitive to the catalytic inhibitor of topo II, suramin; (ii) HMW DNA fragments isolated from apoptotic cells were associated with the topo II enzyme; (iii) anti-topo II antibody suppressed the cytosol-dependent formation of HMW DNA fragments in a cell-free apoptotic system; and (iv) topo II-deficient cells were resistant to apoptosis associated with the HMW DNA cleavage. Furthermore, by using our system for the in vitro induction of the excision of DNA loop domains, we demonstrated the activation of topo II-mediated HMW DNA cleavage in cells at early stages of apoptosis induced by etoposide and by serum-deprivation in NB-2a cells or by oxidative stress in MEFs. In vitro induced HMW DNA cleavage was observed in apoptotic cells before or at the beginning of apoptotic DNA disintegration and was reversible after the removal of apoptotic inducers.

Unlike the majority of nucleases, topo II-mediated DNA cleavage proceeds through the formation of a cleavable complex between topo II enzyme and the substrate DNA (19, 20), in which DNA is cleaved, but double-stranded DNA breaks are clamped by the topo II enzyme and are transient due to the capacity of topo II to religate the cleaved DNA. The in vitro induction of HMW DNA cleavage in etoposide-treated cells is not unexpected, inasmuch as etoposide is a topo II-specific drug that promotes the formation of a cleavable complex between topo II enzyme and substrate DNA (19). However, it is interesting that in vitro activation of HMW DNA cleavage was also observed in serum-deprived cells and in H₂O₂-treated cells. Serum deprivation and oxidative stress both mimicked the effect of etoposide on the induction of HMW DNA cleavage in vitro, thus suggesting that apoptotic conditions provoked the formation of a cleavable complex between topo II and DNA loop domains. Thus, the engagement of topo II in the formation of the cleavable complex with DNA loop domains may represent an early and reversible step in the pathway leading to the excision of DNA loops during apoptosis. Our results support the recent findings of Li et al. (9) in that they demonstrated a rapid and reversible activation of topo II-dependent excision of DNA loop domains during apoptosis in human monoblastic leukemia cells.

To summarize (Fig. 9), our results demonstrate that apoptosis in NB-2a cells can proceed either with or without internucleosomal DNA cleavage, depending on the type of apoptotic inducer, but is invariably accompanied by the formation of HMW DNA fragments of about 50-100 kb. Formation of HMW DNA fragments, which seems to result from the excision of DNA loop domains, represents a separate program of apoptotic DNA disintegration that neither requires CAD nor is critically dependent on the caspase activity. Topoisomerase II, which is engaged in HMW DNA cleavage at the early stage of apoptosis and contributes to HMW DNA fragmentation at the advanced stages of apoptosis, may thus be considered as at least one of the domain nucleases responsible for the excision of DNA loop domains during apoptosis.

View larger version (30K): [in this window] [in a new window]   Fig. 9.   Multiple pathways of apoptotic DNA fragmentation. The hypothetical scheme of higher-level chromatin folding and patterns of apoptotic DNA disintegration are shown in the middle and at the bottom of the figure, respectively. Apoptotic signals activate at least two different pathways of DNA disintegration: one is accomplished by the formation of an oligonucleosomal DNA ladder and can be mediated by CAD, whereas the other involves topo II-dependent HMW DNA cleavage in the absence of oligonucleosomal DNA fragmentation. Whereas caspases can activate both pathways, only the pathway associated with oligonucleosomal DNA fragmentation is critically dependent on caspase activity. The scheme demonstrates the principal pathways of apoptotic execution only; the possibility that topo II-mediated DNA damage per se can trigger activation of caspases or the caspase/CAD pathway is not shown.

	ACKNOWLEDGEMENT

We thank Dr. D. Sullivan for kindly providing anti-topo II antibody.

	FOOTNOTES

^* This work was supported by Academy of Finland Grant 44190, the University of Kuopio, and the Ella and Georg Ehrnroothin Fund.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 358-17-163659; Fax: 358-17-163030; E-mail: victor.solovyan@uku.fi.

Published, JBC Papers in Press, April 8, 2002, DOI 10.1074/jbc.M110621200

	ABBREVIATIONS

The abbreviations used are: HMW, high molecular weight; CAD, caspase-activated DNase; topo, topoisomerase; PMSF, phenylmethylsulfonyl fluoride; FIGE, field inversion gel electrophoresis; MEF, mouse embryonic fibroblast; fmk, fluoromethylketone; AMC, aminomethyl-coumarin; AraC, arabinosylcytosine.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES