Apoptotic Topoisomerase I-DNA Complexes Induced by Staurosporine-mediated Oxygen Radicals*

Topoisomerase I (Top1), an abundant nuclear enzyme expressed throughout the cell cycle, relaxes DNA supercoiling by forming transient covalent DNA cleavage complexes. We show here that staurosporine, a ubiquitous inducer of apoptosis in mammalian cells, stabilizes cellular Top1 cleavage complexes. These complexes are formed indirectly as staurosporine cannot induce Top1 cleavage complexes in normal DNA with recombinant Top1 or nuclear extract from normal cells. In treated cells, staurosporine produces oxidative DNA lesions and generates reactive oxygen species (ROS). Quenching of these ROS by the antioxidant N-acetyl-l-cysteine or inhibition of the mitochondrial dependent production of ROS by the caspase inhibitor benzyloxycarbonyl-VAD prevents staurosporine-induced Top1 cleavage complexes. Down-regulation of Top1 by small interfering RNA decreases staurosporine-induced apoptotic DNA fragmentation. We propose that Top1 cleavage complexes resulting from oxidative DNA lesions generated by ROS in staurosporine-treated cells contribute to the full apoptotic response.

We reported previously in a meeting abstract (1) that staurosporine induces Top1 1 cleavage complexes as mammalian cells undergo apoptosis. However, at that time, the mechanism of formation of these complexes and their role in apoptosis remained incompletely investigated. Apoptosis, the death program of the cell, plays a crucial role for development and for adult homeostasis. Staurosporine, an alkaloid kinase inhibitor, is widely used to study the mechanisms of cell death because of its unique ability to induce apoptosis in a wide variety of mammalian cells (2). Although the mechanism(s) by which staurosporine initiates apoptosis is still unclear, it is now apparent that staurosporine induces apoptosis through the mitochondrial pathway (3,4). Staurosporine increases the permeability of mitochondrial outer membrane, thereby allowing the release of proteins normally located in the space between the inner and the outer mitochondrial membranes (5,6). Some of these molecules are involved in the activation of caspases (cytochrome c, Smac/DIABLO, and HtrA2/Omi), and others in nuclear modifications (endonuclease G and apoptosis inducing factor) (7). After cellular exposure to staurosporine, activated caspase-3 feeds back on permeabilized mitochondria, which further dissipates the mitochondrial transmembrane potential (⌬ m ) and induces the accumulation of intracellular reactive oxygen species (ROS) (8).
DNA Top1 is an ubiquitous and essential enzyme as it relaxes DNA supercoiling ahead of replication and transcription complexes (9 -11). DNA relaxation is because of the induction of transient single-strand breaks, thereby allowing rotation of the DNA double helix around the intact phosphodiester bonds opposite to the enzyme-mediated DNA cleavages. Once the DNA is relaxed, Top1 readily religates the break and regenerates intact duplex DNA. Under normal conditions, the covalent Top1-cleaved DNA intermediates, referred to as "cleavage complexes," are transient and remain at very low levels; the DNA religation ("closing") step is much faster than the DNA cleavage ("nicking") step. Top1 cleavage complexes can be schematically produced by two main mechanisms. First, Top1 cleavage complexes can be trapped by specific inhibitors such as camptothecin and its chemotherapeutic derivatives. These anticancer drugs specifically bind at the Top1-DNA interface and trap the cleavage complexes by preventing the DNA religation step (12)(13)(14). The second mechanism is related to DNA lesions that interfere with DNA nicking-closing activities of Top1. These modifications include frequent lesions such as oxidized bases (e.g. 8-oxoguanine), abasic sites, mismatches, and strand breaks (15)(16)(17).
Recently, Top1 cleavage complexes have been observed in cells undergoing apoptosis following treatment with arsenic trioxide (18) or UV irradiation (19). In the present study, we describe the staurosporine-induced apoptotic Top1 cleavage complexes, and we report the mechanism of their production and functional relevance. We demonstrate that Top1 cleavage complexes are related to DNA modifications during staurosporine-induced apoptosis rather than resulting from a direct drug-Top1 interaction. Our results suggest that staurosporine-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
induced Top1 cleavage complexes form in response to oxidative DNA lesions generated by radical oxygen species (ROS) and caspase activation. Finally we provide evidence for the functional role of Top1-DNA complexes in chromatin fragmentation during staurosporine-induced apoptosis.

EXPERIMENTAL PROCEDURES
Drugs and Chemical Reagents-Staurosporine (STP) and N-acetyl-L-cysteine (NAC) were obtained from Sigma; camptothecin (CPT) was obtained from the Drug Synthesis and Chemistry Branch, NCI (National Institutes of Health, Bethesda); and the caspase peptide inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp(OMe)-fluoromethyl ketone (Z-VADfmk) was from Bachem (Torrance, CA). Stock solutions for STP, CPT, and Z-VAD-fmk were prepared in dimethyl sulfoxide (Me 2 SO) and stored at Ϫ20°C. Further dilutions were made in culture medium just before use. The final concentration of Me 2 SO in culture medium never exceeded 0.1% (v/v), which was nontoxic to the cells. NAC was diluted in culture medium prior to use. [2-14 C]Thymidine was obtained from PerkinElmer Life Sciences. Apurinic/apyrimidinic endonuclease-1 (Ape1) was obtained from Sigma, and Top1 and formamidopyrimidine-DNA glycosylase (Fpg) were prepared as described previously (17,20).
Detection of Cellular Top1-DNA Complexes-Top1-DNA complexes were detected using the in vivo complex of enzyme bioassay (22). Briefly, 10 6 cells were lysed in 1% Sarkosyl and homogenized with a Dounce homogenizer. Cell lysate was layered on cesium chloride step gradients and centrifuged at 165,000 ϫ g for 20 h at 20°C. Twenty fractions (0.5 ml each) were collected from the bottom, diluted into an equal volume of 25 mM potassium phosphate buffer, pH 6.6, and applied to polyvinylidene difluoride membrane (Immobilon-P, Millipore, MA) by using a slot-blot vacuum manifold. Top1-DNA complexes were detected by immunoblotting using the C21 Top1 mouse monoclonal antibody (a kind gift from Dr. Yung-Chi Cheng, Yale University, New Haven, CT).
Top1 Cleavage Complexes with Recombinant Top1 and Nuclear Extracts-3Ј-End-labeled DNA fragments were incubated with Top1 (17) or 0.35 M NaCl nuclear extracts (23). DNA fragments resulting from Top1 cleavage were separated by PAGE and visualized by PhosphorImager (Amersham Biosciences).
DNA Fragmentation Assays-DNA fragmentation-related apoptosis was quantified by using the previously reported filter elution assay (26). Cells were incubated with [2-14 C]thymidine (0.02 Ci/ml) for 2 days and chased overnight in radioisotope-free medium. After drug treatment, cells were loaded onto a protein-absorbing filter (Metricel® Membrane Filter, 0.8-m pore size, 25 mm diameter; Pall Corp.), washed with phosphate-buffered saline, and lysed in 0.2% sodium Sarkosyl, 2 M NaCl, 0.04 M EDTA, pH 10.0. The filters were then washed with 0.02 M EDTA, pH 10.0. DNA was depurinated by incubation of filters in 1 M HCl at 65°C and then released from the filters with 0.4 M NaOH at room temperature. Radioactivity was counted by liquid scintillation spectrometry in each fraction (wash, lysis, EDTA wash, and filter). DNA fragmentation was measured as the fraction of disintegrations/min in the lysis fraction plus EDTA wash relative to the total intracellular disinte-grations/min. For sub-G 1 analysis, DNA content was assessed by staining ethanol-fixed cells with propidium iodide and monitoring by FACScan (BD Biosciences). The number of cells with sub-G 1 DNA were determined with a CellQuest program (BD Biosciences).
ROS Detection-Cells were exposed to 0.5 M H 2 DCF-DA (Molecular Probes) for 5 min and then examined by phase contrast and fluorescence microscopy. All photomicrographs were taken at equal magnification (ϫ20) and exposure times. Quantification of DCF fluorescence was determined with the Adobe Photoshop program and expressed as relative fluorescence intensity/cell. Top1 Silencing by RNA Interference-Top1 was silenced in HCT116 cells by the transfection of U6 promoter-driven DNA vectors stably expressing small interfering RNA (siRNA) hairpins targeting human Top1 (cDNA sequence, 5Ј-CTT GAC AGC CAA GGT ATT C-3Ј) or a negative control sequence (cDNA sequence, 5Ј-GCG TCC TTT CCA CAA GAT A-3Ј) as described (18).

Staurosporine Induces Top1-DNA Complexes in Cells Undergoing Apoptosis-
The presence of Top1-DNA complexes in genomic DNA can be detected after cesium chloride gradient centrifugation and fractionation from tissue culture cells or tumor samples (22). In staurosporine-treated leukemia CEM cells, immunoblotting revealed the presence of Top1 in the DNA-containing fractions (fractions 7-10) (Fig. 1A). Unlike the Top1 inhibitor camptothecin, staurosporine was not able to generate Top1 cleavage complexes in normal DNA in the presence of recombinant human Top1 (Fig. 1B), indicating that staurosporine does not directly poison Top1. Cellular Top1-DNA complexes are therefore likely to be secondary to intracellular modifications induced by staurosporine.
Given that staurosporine is a potent inducer of apoptosis, we tested whether the cellular Top1-DNA complexes resulted from the engagement of apoptotic pathway(s). In CEM cells, staurosporine induces detectable Top1-DNA complexes after 3 h ( Fig.  2A), concomitantly with DNA fragmentation (Fig. 2B), and activation of caspases, which was demonstrated by the ability of cell lysates to cleave DEVD-AFC, a fluorogenic peptide substrate for caspase-3 and closely related caspases (Fig. 2C, top  panel). Caspase activation was further confirmed by the caspasedependent cleavage of PARP (Fig. 2C, bottom panel). Staurosporine-induced Top1-DNA complexes were also observed in the human colon carcinoma HCT116 cells undergoing apoptosis (Fig. 2, D-F) and in human leukemia HL-60 cells (supplemental Fig. 2). Thus, the induction of Top1-DNA complexes by staurosporine can be observed in different human cell types as they undergo apoptosis.
Staurosporine Induces Oxidative DNA Lesions-Because Top1 cleavage complexes can be produced by oxidative DNA lesions such as oxidized bases, abasic sites, and strand breaks (15)(16)(17), we investigated the generation of such lesions by staurosporine. Permeabilized CEM cells were exposed to purified formamidopyrimidine DNA glycosylase (Fpg), an enzyme that converts oxidized purines (e.g. 8-oxoguanine) into DNA singlestrand breaks (20). By using the alkaline elution assay (24), we observed that Fpg induced single-strand breaks in staurosporine-treated cells (Fig. 3A), indicating the presence of oxidative base damage. We also tested base damage in staurosporinetreated cells using recombinant apurinic endonuclease 1 (Ape1) that cleaves DNA adjacent to abasic sites (28). As visualized by agarose gel electrophoresis, Ape1 induced chromatin fragmentation in nuclei from staurosporine-treated cells (Fig. 3B). Nuclear extracts from staurosporine-treated cells failed to cleave a normal duplex oligonucleotide containing a canonical Top1 cleavage site (29) (Fig. 3C), indicating that the effect of staurosporine is not on Top1 itself.
Inhibition of ROS Prevents Apoptotic Top1-DNA Complexes-To further investigate whether apoptotic Top1-DNA complexes result from oxidative DNA lesions, we used 2Ј,7Јdichlorohydrofluorescein diacetate (H 2 DCF-DA) to detect intracellular H 2 O 2 and other peroxides (30). Fluorescence microcopy showed that staurosporine increased ROS levels in CEM cells (Fig. 4, A and B). Then we investigated whether inhibition of ROS levels would affect the generation of Top1-DNA complexes. Treatment of CEM cells with the antioxidant N-acetyl-L-cysteine (NAC) together with staurosporine prevented both staurosporine-induced ROS production (Fig. 4, A and B) and Top1-DNA complexes (Fig. 4C).
Caspases have been involved recently in the production of ROS by staurosporine (8). Consistently, inhibition of caspases by the peptide Z-VAD-fmk prevented staurosporine-induced ROS production (Fig. 4, A and B). Under these conditions, Z-VAD-fmk also prevented the formation of Top1-DNA complexes (Fig. 4C). Altogether, these findings support the hypothesis linking oxidative DNA damage with Top1-DNA complexes during apoptosis.
Apoptotic Top1-DNA Complexes Contribute to DNA Fragmentation-As shown in Fig. 5A, NAC prevents apoptotic DNA fragmentation in staurosporine-treated CEM cells. To evaluate further its involvement in chromatin fragmentation during apoptosis, Top1 was down-regulated by siRNA hairpins targeting human Top1 (Fig. 5B). Top1 silencing reduced staurosporine-induced DNA fragmentation in HCT116 cells (Fig. 5C). Similar results were observed with another siRNA hairpin sequence targeting Top1 (supplemental Fig. 5). Apoptotic DNA fragmentation (Fig. 5D) and internucleosomal DNA laddering (Fig. 5E) were also reduced in P388/CPT45 cells deficient for Top1 (31). Together, these findings suggest the contribution of Top1 cleavage complexes to apoptotic DNA fragmentation.

DISCUSSION
This study reports the formation of Top1 cleavage complexes by staurosporine, a ubiquitous inducer of apoptosis in mammalian cells. These apoptotic Top1 cleavage complexes were observed in three human cell lines (two leukemia cell lines, CEM and HL-60, and one colon carcinoma cell line, HCT116) undergoing apoptosis. Staurosporine is a protein kinase inhibitor (32) that cannot induce Top1 cleavage complexes in normal DNA in the presence of recombinant Top1 or nuclear extracts from normal cells (Figs. 1 and 3). Thus, the mechanism of formation of the apoptotic Top1 complexes induced by staurosporine is indirect rather than resulting from drug-Top1 interaction.
We have observed that oxidative DNA lesions occur during staurosporine-induced apoptosis likely as a result of ROS production (Figs. 3 and 4), and we propose that staurosporineinduced apoptotic Top1 cleavage complexes result from these DNA modifications. Consistently, quenching of ROS with the antioxidant NAC prevents staurosporine-induced Top1 cleavage complexes (Fig. 4). Similarly, arsenic trioxide induces oxidative DNA lesions (33) and apoptotic Top1 cleavage complexes (18), suggesting that the ROS-dependent formation of oxidative DNA lesions is a common mechanism for the trapping of Top1 cleavage complexes during apoptosis (34). In fact, cellular exposure to hydrogen peroxide (H 2 O 2 ) was recently shown to induce Top1 cleavage complexes (35). Thus we propose that staurosporine induces the generation of ROS that damage DNA (oxidized bases and abasic sites), thereby generating Top1 cleavage complexes in apoptotic cells (Fig. 6). It is also possible that some of the DNA breaks produced by apoptotic nucleases such as DFF40/CAD (36), endonuclease G (37), or Ape1 (38) contribute to the trapping of Top1 cleavage complexes (15). Cellular exposure to staurosporine induces mitochondrial outer membrane permeabilization (3), which is followed by the release of cytochrome c and the downstream activation of caspase-9 and caspase-3 (6). Activated caspase-3 was recently shown to feed back on permeabilized mitochondria and cleave the 75-kDa subunit (NDUFS1) of complex I, which leads to a disruption of the mitochondrial transmembrane potential (⌬ m ) and the production of ROS (8). Mitochondrial production of ROS is likely to contribute to Top1 cleavage complex formation because caspase inhibition by Z-VAD-fmk inhibits staurosporine-induced ROS and Top1 cleavage complexes (Fig. 4). Activation of caspases could therefore serve to generate ROS that lead to Top1 cleavage complexes during staurosporineinduced apoptosis (Fig. 6).
In conclusion, our findings raise the possibility that Top1, which is abundant and ubiquitous in mammalian cells, could participate in apoptosis by directly generating DNA strand breaks. Like apoptotic endonucleases, Top1 is nonessential for apoptosis because Top1 silencing reduces but does not abrogate chromatin DNA fragmentation (Fig. 5C). However, the redundancy and the nonlinearity of endonuclease activation during apoptosis might have evolved to confer a robust pathway ensuring that deficiency in one of the apoptotic nuclease is not sufficient to abrogate DNA fragmentation. Top1-DNA complexes could also engage the apoptotic machinery in trans as trapping of Top1 by camptothecins is among the most efficient inducers of apoptosis (40). Apoptotic Top1-DNA complexes could therefore serve to amplify the apoptotic process engaged by staurosporine (see Fig. 6), as well as other agents including arsenic trioxide (18) and UV irradiation (19). FIG. 6. Proposed mechanism for the induction of Top1-DNA complexes during staurosporine-induced apoptosis. Staurosporine induces the caspase-dependent generation of ROS that produce DNA lesions, which in turn generate Top1-DNA complexes. Top1-DNA complexes contribute to chromatin degradation by generating DNA strand breaks and by activating caspases.