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Originally published In Press as doi:10.1074/jbc.M104383200 on September 27, 2001

J. Biol. Chem., Vol. 276, Issue 48, 44488-44494, November 30, 2001
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Topoisomerase II Poisoning by ICRF-193*

Kuan-Chun HuangDagger §, Hanlin GaoDagger §, Edith F. YamasakiDagger , Dale R. Grabowski||, Shujun LiuDagger , Linus L. Shen**, Kenneth K. ChanDagger Dagger , Ram Ganapathi||, and Robert M. SnapkaDagger §§§

From the Departments of Dagger  Radiology and § Molecular Virology, Immunology, and Medical Genetics, The Ohio State University College of Medicine, Columbus Ohio, 43210, the || Taussig Cancer Center, Cleveland Clinic Foundation, Cleveland, Ohio 44195, the Dagger Dagger  College of Medicine and College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, and ** Abbott Laboratories, Abbott Park, Illinois 60064

Received for publication, May 14, 2001, and in revised form, September 11, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Antineoplastic bis(dioxopiperazine)s, such as meso-2,3-bis(2,6-dioxopiperazin-4-yl)butane (ICRF-193), are widely believed to be only catalytic inhibitors of topoisomerase II. However, topoisomerase inhibitors have little or no antineoplastic activity unless they are topoisomerase poisons, a special subclass of topoisomerase-targeting drugs that stabilize topoisomerase-DNA strand passing intermediates and thus cause the topoisomerase to become a cytotoxic DNA-damaging agent. Here we report that ICRF-193 is a very significant topoisomerase II poison. Detection of topoisomerase II poisoning by ICRF-193 required the use of a chaotropic protein denaturant in the topoisomerase poisoning assays. ICRF-193 caused dose-dependent cross-linking of human topoisomerase IIbeta to DNA and stimulated topoisomerase IIbeta -mediated DNA cleavage at specific sites on 32P-end-labeled DNA. Human topoisomerase IIalpha -mediated DNA cleavage was stimulated to a lesser extent by ICRF-193. In vivo experiments with MCF-7 cells also showed the requirement of a chaotropic protein denaturant in the assays and selectivity for the beta -isozyme of human topoisomerase II. Studies with two topoisomerase IIbeta -negative cell model systems confirmed significant topoisomerase II poisoning by ICRF-193 in the wild type cells and were consistent with beta -isozyme selectivity. Common use of only the detergent, SDS, in assays may have led to failure to detect topoisomerase II poisoning by ICRF-193 in earlier studies.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The bis(2,6-dioxopiperazine)s were originally synthesized as membrane permeant analogs of the metal chelator, EDTA, based on the hypothesis that metal chelation was involved in the activity of anticancer drugs (1). As predicted, the bis(2,6-dioxopiperazine)s did have anticancer activity (1-4) and were shown to selectively inhibit DNA synthesis in cultured cells while having little effect on RNA or protein synthesis (5). However, there is also evidence that metal chelation plays little, if any, role in their anticancer activity (2). Numerous bis(dioxopiperazine)s have been studied for anticancer activity and for interactions with other anticancer drugs and radiation (1-4). (I)-4,4'-(1,2-propanediyl)-bis-(4-piperazine-2,6-dione) (ICRF·159, NSC 129943) has pronounced effects on tumor vasculature, and it has been claimed that this is the basis of its antimetastatic activity (3, 6). Bis(2,6-dioxopiperazine)s were reported to be strong catalytic inhibitors of DNA topoisomerase II (7), and ICRF-1931 is the most potent of the analogs studied (8). The meso configuration of the 2,3-butanediyl linker has been shown to be critical for topoisomerase II inhibition (9). ICRF-193 was shown to inhibit topoisomerase II-dependent steps in SV40 DNA replication in intact mammalian cells (10), and topoisomerase II was shown to be the cytotoxic target of bis(dioxopiperazine)s in yeast (11).

The term "catalytic inhibitor," when applied to topoisomerases, is used to distinguish them from topoisomerase poisons that stabilize topoisomerase-DNA strand passing reaction intermediates in which the topoisomerases are covalently attached to the DNA at DNA strand breaks (12). This distinction is important because topoisomerase poisons are much more cytotoxic than topoisomerase catalytic inhibitors. Topoisomerase poisons, in contrast to topoisomerase catalytic inhibitors, often have good anticancer activity. The well known topoisomerase II poisons m-AMSA, VM-26, and VP-16 are in widespread clinical use, and topoisomerase I poisons are the focus of numerous studies and clinical trials. The term "topoisomerase poison" was coined to express the idea that this special class of topoisomerase-targeting drugs convert normal enzymes into cellular poisons. These drug-stabilized "cleavable complexes" or "DNA cleavage complexes" are thought to be converted to lethal DNA lesions, such as double strand DNA breaks, when they interact with DNA replication or transcription machinery (12, 13). Assays for topoisomerase poisoning use protein denaturants to inactivate the topoisomerases trapped in cleavage complexes by topoisomerase poisons, thus converting the cleavage complex into an irreversible protein-linked DNA strand break. SDS, a strong detergent, is used almost universally as the protein denaturant in these assays. Various topoisomerase poisoning assays then measure either the DNA strand breaks or the protein-DNA cross-links.

Topoisomerase catalytic inhibitors inhibit topoisomerase reactions at other steps of the topoisomerase reaction cycle and are much less cytotoxic because no DNA lesion is formed. For many topoisomerase II catalytic inhibitors, such as proflavine (14-16), the step inhibited appears to be DNA binding or DNA cleavage site recognition by the topoisomerase. ICRF-193 inhibits topoisomerase II by a unique mechanism in which the topoisomerase becomes locked in a "closed clamp" and is unable to hydrolyze ATP, as required to regenerate the active form of the enzyme (17).

Until recently, bis(dioxopiperazine)s were thought to be pure catalytic inhibitors of topoisomerase II that do not stabilize the covalent topoisomerase II-DNA strand passing intermediates that are the basis of topoisomerase poisoning. However, Jensen, et al. reported evidence for very weak topoisomerase II poisoning by ICRF-193 but found it far too weak to account for the cytotoxicity of the drug and suggested that it has a novel cytotoxic mechanism (18). Here we report evidence that ICRF-193 is a very significant topoisomerase II poison and that a chaotropic protein denaturant must be used to efficiently detect the topoisomerase poisoning. Studies with purified topoisomerases and with intact cells both indicate that ICRF-193 also has a preference for the beta -isozyme of human topoisomerase II. However, given the predominance of topoisomerase IIalpha in actively dividing cells, we feel that topoisomerase IIalpha poisoning probably contributes significantly to ICRF-193 cytotoxicity. Earlier studies of ICRF-193 may have failed to detect significant topoisomerase II poisoning because of the common use of SDS in topoisomerase poisoning assays.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Lines, Cell Culture Conditions, and Reagents-- MCF-7 human breast adenocarcinoma cells were from the American Type Culture Collection and were grown in Dulbecco's modified Eagle's medium with 10% fetal calf serum and a 5% CO2 atmosphere at 37 °C. CV-1 African green monkey kidney cells were from the American Type Culture Collection and were maintained in Eagle's minimal essential medium (Life Technologies, Inc.) supplemented with 10% calf serum. AMCV1 cells are a CV-1 cell subclone maintained in 3 µM m-AMSA (NSC 249992) (19). HL-60/AMSA cells are human myelogenous leukemia cells selected for resistance to m-AMSA, and HL-60/S cells are the parental drug-sensitive cells (20). HL-60/AMSA and HL-60/S cells were obtained from Drs. Leonard Zwelling and Miloslav Beran and were maintained in Iscove's modified Dulbecco's medium with 10% fetal calf serum at 37 °C in a 5% CO2 atmosphere. ICRF-193 was a gift of Dr. Donald Witiak and Dr. Andrei V. Blokhin. Topoisomerase IIalpha was from TopoGen (Columbus, OH) and Abbott Laboratories (Abbott Park, IL). Topoisomerase IIbeta was provided by Dr. Anni H. Andersen (University of Aarhus, Aarhus, Denmark) and Dr. Caroline Austin (University of Newcastle, Newcastle-upon-Tyne, UK). Topoisomerase IIalpha -specific antibody was from TopoGen, and topoisomerase IIbeta -specific antibody was from Dr. Daniel M. Sullivan (H. Lee Moffitt Hospital, Tampa, FL). The enzymes and antibodies used in these studies were fully active as shown by studies done concurrently with this work (19, 21).

GF/C Assay for Topoisomerase-DNA Cross-links-- GF/C glass fiber filter assays measure protein-DNA cross-links and are based on the selective binding of proteins to glass fiber filters in 0.4 M GuHCl. The assay is used to measure topoisomerase poisoning in intact cells and in in vitro assays involving purified enzymes and DNA substrates (19, 21). For in vitro assays, purified topoisomerases were incubated with [3H]dT-labeled SV40 DNA (12,000 dpm) in reaction buffer (10 mM Tris-HCl, pH 7.5, 50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 15 µg/ml bovine serum albumin, 1 mM ATP) for 15 min at 37 °C (22). The reactions were stopped by the addition of protein denaturants (SDS or GuHCl), and aliquots were removed and added to either 0.4 or 4.0 M GuHCl solutions for filtration through GF/C glass fiber filters. In 4 M GuHCl, all nucleic acids bind the filter, and scintillation counting of the dried filter gives a measure of total labeled DNA in the aliquot. In 0.4 M GuHCl, only protein binds to the glass fiber filter, and DNA is not retained unless it is cross-linked to protein. Comparison of the radioactivity retained on the filter in 0.4 M GuHCl to the radioactivity retained on the filter in 4 M GuHCl gives the fraction of DNA molecules cross-linked to protein. The topoisomerase subunits trapped in topoisomerase-DNA strand passing intermediates at the time of denaturation are covalently cross-linked to the substrate DNA and cause its retention on the glass fiber filter in 0.4 M GuHCl. Thus, the assay can be used to measure topoisomerase poisoning. For in vivo assays with intact cells, cellular DNA is prelabeled with [3H]dT (1.0 µCi/ml, 85 Ci/mmol; Amersham Pharmacia Biotech), and the cells are exposed to suspected topoisomerase poisons before lysis with protein denaturants (SDS or GuHCl). The protein denaturants trap drug-stabilized topoisomerase-DNA strand passing intermediates (cleavable complexes) as irreversible protein-DNA cross-links. The cellular DNA is sheared by vortexing, and duplicate aliquots of each lysate are added to 0.4 and 4 M GuHCl solutions for the GF/C filter assay.

In Vivo Topoisomerase Cleavage Complex Assay and Western Blotting of Nuclear Topoisomerase II-- The in vivo topoisomerase cleavage complex assay, as modified from Subramanin et al. (23), has been described (19). Briefly, cultured MCF-7 cells were treated with drugs or the drug solvent, Me2SO, and then lysed with either SDS buffer (10 mM Tris-HCl, pH 7.4, 10 mM EDTA, 0.6% SDS) or 6 M GuHCl. The DNA from the lysates was banded by ultracentrifugation in a CsCl step gradient and was eluted from the bottom. Free proteins are left at the top of the gradient, and proteins that are covalently attached to DNA band with the DNA. The fractions containing DNA were pooled and dialyzed to remove CsCl, MgCl2 was added to the dialyzed sample to a final concentration of 5 mM, and the sample was treated with DNase I (Roche Molecular Biochemicals) 0.1 unit/µl at 37 °C for 1 h. The DNase I-digested sample was then applied to a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech) with a slot blot device. The DNase treatment reduces the viscosity of the sample and greatly facilitates its application to the membrane. Proteins adhere to the membrane tightly. The membrane was subsequently blocked with 10% nonfat milk in TBS buffer (10 mM Tris-HCl, pH 8.0, 100 mM NaCl), followed by probing with either topoisomerase IIalpha - or topoisomerase IIbeta -specific antibody. The primary antibody was recognized by peroxidase-conjugated anti-rabbit IgG antibody. Finally, the chemiluminescent substrate (SuperSignal; Pierce) was added, and the signal detected by a Lumi-Imager (Roche Molecular Biochemicals) was analyzed by LumiAnalyst 3.0 software. Nuclear extracts of late log MCF-7 cells were prepared as described (24) and analyzed by Western blotting with topoisomerase IIalpha - and IIbeta -specific antibodies, as described (19).

Mapping Topoisomerase II Cleavage Sites-- Topoisomerase IIalpha - and IIbeta -mediated DNA cleavage sites were mapped on a 516-base pair DNA substrate, consisting of an EcoRI-ScaI fragment of pBR322 (residues 3846-4362). The DNA fragment was purified by agarose gel electrophoresis, and the excised band was isolated with a gel extraction kit (Qiagen, Valentia, CA). The overhanging EcoRI end was labeled with 32P in a 40-µl reaction containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, 50 µg/ml acetylated bovine serum albumin, 0.25 mM of each deoxynucleotide (dGTP, dCTP, and dTTP), 60 µCi of [alpha -32P]dATP (Amersham Pharmacia Biotech; 3000 Ci/mmol) and Klenow fragment, 5 units (USB Corp., Cleveland, OH). After a 15-min incubation at 25 °C, unlabeled dCTP, dGTP, dTTP, and dATP were added (10 nmol of each), and the incubation was continued for an additional 15 min before termination by heating at 70 °C for 10 min. The end-labeled DNA fragment was then purified with a mini-Quick Spin DNA column (Roche Molecular Biochemicals). Assays for topoisomerase IIalpha /beta -dependent DNA cleavage contained end-labeled DNA fragments (1-2 × 105 dpm/reaction), 10 mM HEPES-HCl, pH 7.9, 50 mM KCl, 5 mM MgCl2, 50 mM NaCl, 0.1 mM Na2EDTA, 1 mM ATP, and the drug being tested. After a 5-min preincubation at 37 °C, the reaction (total reaction volume, 20 µl) was started by the addition of 0.8 µg of purified human topoisomerase IIalpha or 1.2 µg of purified topoisomerase IIbeta . These amounts gave approximately equal topoisomerase II-mediated DNA cleavage with the isozyme-nonspecific topoisomerase II poison VM-26. The reaction mix was incubated at 37 °C for 30 min before termination by the addition of 2 µl of 4 M GuHCl. The DNA was purified by ethanol precipitation and then resuspended in 28 µl of proteinase K solution (0.2 mg/ml, 2 h, 45 °C). The DNA was then repurified by ethanol precipitation before resuspension in 4 µl of loading buffer (80% formamide, 10 mM NaOH, 1 mM EDTA, 0.1% xylene cyanol, and 0.1% bromphenol blue). The samples were heated to 70 °C for 2 min, cooled to room temperature, and then loaded onto a DNA sequencing gel (8% polyacrylamide, 19:1 acrylamide/bisacrylamide) containing 7 M urea in 1× Tris borate buffer (25). Electrophoresis was performed at 1800 V for 2 or 6 h. The gel was then transferred to Whatman 3MM paper, (Whatman, Clifton, NJ) and exposed to Hyperfilm-MP (Amersham Pharmacia Biotech).

Sanger dideoxy DNA sequence ladders were generated by using the fmol cycle DNA sequencing system (Promega, Madison WI). The primer 5'-AAATTCTTGAAGACGAAAGGGCC-3', complementary to the EcoRI end of the 516-base pair ScaI/EcoRI pBR322 primer was labeled at the 5' end by T4 polynucleotide kinase with [gamma -32P]ATP. The labeled primer was used directly without further purification. For each set of sequencing reactions, the appropriate d/ddNTP mix was added, and polymerase chain reaction amplification was carried out for 30 cycles with Taq DNA polymerase. Following the thermal cycling program, the reactions were stopped by the addition of fmol sequencing reaction stop solution, and DNA was denatured at 70 °C immediately before gel loading. Because the sequenced strand was labeled on the 5' EcoRI end and was complementary to the strand on which topoisomerase-mediated DNA cleavages were mapped, it was necessary to translate the sequence to determine the cutting sites. Many key DNA cleavage sites were also confirmed by Maxam and Gilbert sequence ladders, involving direct sequencing of the strand on which topoisomerase-mediated DNA cleavages were mapped (26). The 32P-end labeling for the Maxam and Gilbert sequencing was done as described above for the topoisomerase substrate DNA. A + G and C + T sequencing ladders were used.

Cytotoxicity Assay-- Cytotoxicity induced by ICRF-193 was determined by a soft agar colony forming assay. Cells (1 × 106/ml) in Iscove's modified Dulbecco's medium supplemented with 25 mM HEPES, 2 mM L-glutamine, and 10% fetal bovine serum were treated with a range of drug concentrations for 3 h at 37 °C in a humidified 5% CO2 plus 95% air atmosphere. Following treatment, the cells were washed, and 1 × 104 cells were plated in triplicate in 35 × 10-mm Petri dishes using Iscove's modified Dulbecco's medium supplemented with 25 mM HEPES, 2 mM L-glutamine, and 20% fetal bovine serum. Colonies were counted following incubation of the Petri dishes for 6-7 days in a humidified 5% CO2 plus 95% air atmosphere (27). Colony forming efficiency of the HL-60/S and HL-60/AMSA cells was 29 and 14%, respectively.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We use GuHCl as a protein denaturant in both in vitro and in vivo topoisomerase poisoning assays, because our experience has suggested that it traps topoisomerase-DNA cleavage complexes more efficiently than the detergent, SDS, even in the absence of topoisomerase poisons. Routine use of GuHCl for topoisomerase poisoning assays led us to the observation that topoisomerase poisons vary with respect to the increase in efficiency of cleavage complex trapping in GuHCl as opposed to SDS. A remarkable example is chloroquinoxaline sulfonamide, whose topoisomerase II poisoning is detectable only when GuHCl or urea is used as the protein denaturant in the topoisomerase poisoning assay (21). To specifically test the effects of protein denaturant on topoisomerase II poisoning by ICRF-193, we carried out in vitro topoisomerase poisoning assays, using purified human topoisomerase IIalpha and topoisomerase IIbeta . Topoisomerase IIalpha poisoning was not detected in this in vitro assay when the in vitro reactions were stopped with either SDS or with GuHCl (data not shown). ICRF-193 induced dose-dependent topoisomerase IIbeta -DNA cross-links when GuHCl was used to terminate the reaction, but no significant increase in topoisomerase IIbeta -DNA cross-links was detected when SDS was used to terminate the reaction (Fig. 1). The background topoisomerase IIbeta -DNA cross-linking, in the absence of added drug, was higher when GuHCl was used as the protein denaturant than when SDS was used. This suggests that GuHCl is more efficient than SDS at trapping topoisomerase II-DNA cleavage complexes even in the absence of topoisomerase poisons. ICRF-193 did not cause any filter binding of the DNA substrate in the absence of topoisomerase II.


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  		Fig. 1.   The effect of protein denaturant on ICRF-193-induced topoisomerase IIbeta -DNA cross-links in vitro. In vitro topoisomerase IIbeta DNA cleavage reactions contained cleavage buffer, [3H]dT-labeled SV40 DNA, and sufficient topoisomerase IIbeta to give ~5% cross-linking in the absence of added drugs when SDS was used to terminate the reaction. The reactions were terminated with either <FR><NU>1</NU><DE>10</DE></FR> volume of 10% SDS or <FR><NU>1</NU><DE>10</DE></FR> volume of 4 M GuHCl. , reactions terminated by addition of GuHCl; open circle , reactions terminated by addition of SDS. Error bars (± S.D., n = 4) are shown where they are larger than the symbols.

Because topoisomerase poisoning involves drug stabilization of topoisomerase-DNA cleavage complexes, the covalent attachment of the topoisomerase subunits to the DNA occurs at the site of a DNA strand break. To further test topoisomerase poisoning by ICRF-193, we mapped ICRF-193-induced topoisomerase IIalpha - and IIbeta -mediated cleavages on a cloned fragment of pBR322 (Fig. 2), using GuHCl to trap the topoisomerase II-DNA cleavage complexes by denaturation and inactivation of the enzyme. Much more extensive DNA cleavage was seen with topoisomerase IIbeta than with topoisomerase IIalpha . The ICRF-193-induced topoisomerase II-DNA cleavages at nucleotides 343 and 346 were seen for both topoisomerase II isozymes, but unique, strong ICRF-193-induced topoisomerase IIbeta -DNA cleavages were seen at many other sites, such as nucleotides 329, 396, and 399. Most of the ICRF-193-induced topoisomerase IIbeta cleavages (nucleotides 203, 207, 295, 298, 304, 313, 343, 346, 357, 361, 396, and 399; Fig. 3) correspond to sites of VM-26-stimulated topoisomerase IIalpha cleavages that we have mapped in separate experiments (not shown).


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  		Fig. 2.   ICRF-193-induced topoisomerase IIalpha and IIbeta cleavage in a 516-base pair ScaI/EcoRI fragment of pBR322. The pBR322 sequence is numbered from the ScaI cut site (see Fig. 3). TIIbeta , topoisomerase IIbeta ; TIIalpha , human topoisomerase IIalpha ; G, A, and T, dideoxy DNA sequencing ladders for guanine, adenine, and thymine, respectively. Lane D, substrate DNA only (no topoisomerase or drug). Inclusion of ICRF-193 (1.0 mM) in the topoisomerase II cleavage reactions is indicated by +. This gel was run at 1800 V for 2 h; a duplicate was run for 6 h to obtain better resolution for the high molecular weight fragments (not shown).


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  		Fig. 3.   Map of ICRF-193-induced topoisomerase IIalpha and IIbeta cleavage sites in the pBR322 fragment. At the EcoRI end, the primer used for dideoxy DNA sequence ladders is indicated by underlining, and the 32P-labeled adenine residues incorporated into the strand used for mapping topoisomerase II cleavages and Maxam and Gilbert DNA sequence ladders are indicated by bold type and shading. ICRF-193-induced topoisomerase II cleavage sites are indicated in bold type in the sequence, and the residues are further identified above these residues as either topoisomerase IIalpha or IIbeta cleavage sites with the relative strength of the cleavage indicated by bold text (strong cleavages), normal text (moderate cleavage), and gray text (weak cleavages). The break at C413 is a weak DNA strand break that is always present in our preparations of this pBR322 fragment.

ICRF-193 also caused dose-dependent protein-DNA cross-links in MCF-7 cells (Fig. 4A). In cells prelabeled with [3H]dT for 2 days, ICRF-193-induced protein-DNA cross-links increased steeply with the concentration of ICRF-193 and reached a maximum by about 5 µM. ICRF-193 did not cause protein DNA cross-links when it was added after lysis of the cells, suggesting that active enzymes are required for ICRF-193-induced protein DNA cross-links (data not shown). A higher level of ICRF-193-induced protein-DNA cross-linking was reached when cells were pulse labeled with [3H]dT immediately before exposure to the drug (Fig. 4B). ICRF-193-induced protein-DNA cross-links were only seen when MCF-7 cells were lysed with GuHCl in the presence of the drug but not when the cells were lysed with SDS (Fig. 4B). ICRF-193-induced protein-DNA cross-links were not significantly higher than background (Me2SO solvent control) when the cells were lysed with SDS buffer. This SDS-based cell lysis efficiently traps topoisomerase-DNA cleavage complexes stabilized by topoisomerase poisons such as teniposide (VM-26), etoposide (VP-16), and m-AMSA (19). The background in this assay consists of a few percent of nonspecific DNA binding to the filters plus a few percent of background protein-DNA cross-linking (removable by proteinase predigestion) that may be due to trapping of intracellular topoisomerase-DNA cleavage complexes in the absence of topoisomerase poisons (21). As in the in vitro experiment with topoisomerase IIbeta , protein-DNA cross-linking in the absence of the drug (the solvent controls) was higher with the GuHCl lysis than with the SDS lysis. Again, this suggests that GuHCl is more efficient at trapping topoisomerase-DNA cleavage complexes even in the absence of added topoisomerase poisons. Levels of topoisomerase IIalpha and IIbeta in nuclear extracts of the MCF-7 cells are indicated by Western blotting with topoisomerase IIalpha -specific antibody and topoisomerase IIbeta -specific antibody (Fig. 4B, bottom panel).


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  		Fig. 4.   ICRF-193-induced protein-DNA cross-links in MCF-7 cells. A, dose response for ICRF-193-induced protein-DNA cross-links in MCF-7 cells. MCF-7 cells in late log phase were labeled with [3H]dT (2 days, 1 µCi/ml). The cells were rinsed with serum-free medium and then treated for 15 min with ICRF-193 at the concentrations indicated. The medium was then drawn off, and the cells were lysed with 0.5 ml of 6 M GuHCl. The cell lysates were sheared by vortexing and assayed by the GF/C filter assay for protein-DNA cross-links. The error bars are ± S.D. (n = 4). B, denaturant dependence of ICRF-193-induced protein-DNA cross-links in MCF-7 cells. MCF-7 cells in log phase were labeled with [3H]dT (250 µCi/ml, 30 min, 37 °C), and the cells were treated with either 30 µM ICRF-193 (gray bars) or with the solvent (dimethyl sulfoxide (DMSO), black bars) during the last 15 min of the radiolabeling. The radiolabel and ICRF-193 were then removed, and the cells were lysed with either SDS-containing Hirt buffer or 6 M GuHCl. The cell lysates were sheared by vortexing and assayed by the GF/C filter assay for protein-DNA cross-links. The error bars are ± S.D. (n = 4). Western blots for topoisomerase IIalpha and IIbeta (bottom panel) were each done in duplicate on nuclear extracts (5 µg of total protein/blot) from late log MCF-7 cells to confirm that topoisomerase IIalpha was not greatly down-regulated.

The high levels of ICRF-193-induced protein-DNA cross-links achieved in MCF-7 cells (Fig. 4) suggested the possibility of testing for topoisomerase poisoning in vivo and for determining the in vivo topoisomerase II isozyme preference of ICRF-193. We used the IVCT assay and antibodies specific for topoisomerase IIalpha and IIbeta to test which topoisomerase II isozyme is covalently linked to DNA by ICRF-193 (see "Experimental Procedures"). VM-26 and XK469 were included as controls (Fig. 5). The results indicated ICRF-193 selectivity for topoisomerase IIbeta , although this selectivity was not as great as that of XK469. As previously reported (19), XK469 was highly selective for topoisomerase IIbeta and VM-26 was less selective for topoisomerase II isozyme. When SDS was used for lysis of ICRF-193-treated cells, neither topoisomerase IIalpha nor topoisomerase IIbeta was detectable in the IVCT assay (not shown).


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  		Fig. 5.   Topoisomerase II isozyme specificity of ICRF-193. Subconfluent MCF-7 cells were treated with solvent (dimethyl sulfoxide (DMSO)), 0.18 mM ICRF-193, 1 mM XK469, or 0.1 mM VM-26 for 10 min at 37 °C, and the cells were lysed by the addition of 6 M GuHCl. The relative amounts of topoisomerase IIalpha and topoisomerase IIbeta covalently linked to the DNA were then determined by the in vivo topoisomerase cleavage complex assay.

To further characterize topoisomerase poisoning by ICRF-193 in vivo, we studied two topoisomerase IIbeta -negative cell lines. AMCV1 cells are highly resistant to m-AMSA, have no topoisomerase IIbeta , have significantly reduced topoisomerase IIalpha , and have normal levels of topoisomerase I (19). As shown in Fig. 6A, ICRF-193 and m-AMSA induce high levels of protein-DNA cross-links in drug-sensitive parental CV-1 cells, but neither ICRF-193 nor m-AMSA induced a significant increase in cross-links above background (solvent control) in the AMCV1 cells. This result shows that m-AMSA and ICRF-193 are both topoisomerase II poisons of similar strength in the parental CV-1 cells. To further characterize in vivo topoisomerase poisoning by ICRF-193, we studied m-AMSA-resistant HL-60/AMSA cells and their drug-sensitive parental cells, HL-60/S. HL-60/AMSA cells have normal levels of a mutant topoisomerase IIalpha , which is sensitive to non-DNA intercalating topoisomerase II poisons but resistant to DNA intercalating topoisomerase II poisons (28). In addition, HL-60/AMSA does not express topoisomerase IIbeta (20). Topoisomerase II poisoning by m-AMSA was greatly reduced in the HL-60/AMSA line in comparison with the drug-sensitive parental HL-60/S line (Fig. 6B). This is in agreement with previous results because m-AMSA is a DNA intercalating topoisomerase II poison (28). ICRF-193-induced topoisomerase II poisoning was also significantly reduced in HL-60/AMSA cells as compared with HL-60/S cells at 50 µM (p = 0.00014), 100 µM (p = 0.0006), and 200 µM (p = 0.00025) ICRF-193. Because ICRF-193 is not a DNA intercalating drug, this reduction in topoisomerase II poisoning is consistent with beta -isozyme selectivity. Survival of HL-60/S cells, in comparison with survival of HL-60/AMSA cells, in a clonogenic assay following a 3-h exposure to 50 µM ICRF-193 showed a decrease of 30% percent (p = 0.035), also consistent with beta -isozyme selectivity and the decreased levels of ICRF-193-induced protein-DNA cross-linking (Fig. 6).


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  		Fig. 6.   ICRF-193-induced protein-DNA cross-links in topoisomerase IIbeta -negative cell lines. A, ICRF-193- and m-AMSAinduced protein-DNA cross-links in m-AMSA-resistant AMCV1 cells (gray bars) and parental drug-sensitive CV-1 cells (black bars). The error bars are ± S.D. (n = 4). B, ICRF-193 and m-AMSA-induced protein-DNA cross-links in m-AMSA-resistant HL-60/AMSA cells (gray bars) and drug-sensitive parental HL-60/S cells (black bars). The error bars are ± S.D. (n = 4). The results of clonogenic survival assays (± S.E.) of HL-60/S and HL-60/AMSA cells following 3-h exposures to 50 and 100 µM ICRF-193 are shown. The cytotoxicity was essentially flat in this ICRF-193 dose range, consistent with the cross-linking that reaches a plateau at about 5 µM ICRF-193 in MCF-7 cells (Fig. 4). DMSO, dimethyl sulfoxide.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our in vitro experiments with purified topoisomerases and DNA substrates have shown that ICRF-193 causes both topoisomerase II-DNA cross-links and specific topoisomerase II-mediated DNA cleavages, many of which correspond to sites of topoisomerase II poisoning by VM-26. The in vitro data also indicates that detection of topoisomerase II poisoning by ICRF-193 requires a chaotropic protein denaturant, as is the case with the quinoxaline topoisomerase II poison, chloroquinoxaline sulfonamide (21). In agreement with the in vitro results, our in vivo experiments also indicate a requirement for the use of a chaotropic protein denaturant. The higher levels of ICRF-193-induced protein-DNA cross-links in pulse-labeled cells than in prelabeled cells may reflect preferential trapping of topoisomerase II on newly replicated DNA. Both our in vitro and in vivo experiments also indicate that ICRF-193 targets topoisomerase IIbeta to a greater extent than it targets topoisomerase IIalpha . Our in vitro experiments with purified topoisomerases suggest much higher beta -isozyme selectivity than the in vivo experiments. This may be due to the fact that the in vitro reactions do not precisely mimic conditions in the nucleus of intact cells (ions, dielectric constant, etc.). The in vivo experiments may reflect the true level of isozyme selectivity in intact cells.

A number of studies support the idea that bis(2,6-dioxopiperazine)s act as topoisomerase II catalytic inhibitors in vivo. Decatenation of newly replicated simian virus 40 daughter chromosomes is mediated by topoisomerase II, and this step is rate-limiting in SV40 DNA replication (29, 30). Classical topoisomerase II catalytic inhibitors such as proflavine and 9-aminoacridine block the decatenation step in SV40 replication, causing dramatic accumulations of catenated daughter chromosomes (16, 31). In just the same way, ICRF-193 causes pronounced, dose-dependent blockage of SV40 decatenation (10). Consistent with this, bis(2,6-dioxopiperazine)s interfere with mitosis and cause pronounced polyploidy in replicating cultured mammalian cells (32). In yeast, resistance to ICRF-193 has been found to result from overexpression of topoisomerase II, whereas depletion of topoisomerase II activity leads to increased toxicity (11). These observations are also consistent with topoisomerase II catalytic inhibition as a mechanism of cytotoxicity. However, many potent topoisomerase II catalytic inhibitors have no significant anticancer activity (proflavine and 9-aminoacridine being two examples), whereas a large number of structurally diverse topoisomerase II poisons do have clear anticancer activity. Thus, pure catalytic inhibition of topoisomerase II remains questionable as a mechanism of anticancer activity.

Several lines of evidence support the idea that there is some mechanism of bis(2,6-dioxopiperazine) cytotoxicity that is not due simply to topoisomerase II catalytic inhibition. First, one of the earliest studies of bis(dioxopiperazine) analogs found that they cause DNA strand breaks in cultured cells (2). In more recent studies, Chinese hamster ovary cells selected for resistance to the bis(2,6-dioxopiperazine) ICRF-187, and cross-resistant to other bis(2,6-dioxopiperazine)s were also resistant to well known topoisomerase II poisons, such as anthracyclines and the epipodophyllotoxin, VP-16 (33). The level of topoisomerase II protein in these bis(2,6-dioxopiperazine)-resistant cells was reduced by half, as was topoisomerase II poisoning by VP-16. Reduced levels of topoisomerase II and decreased topoisomerase II poisoning are commonly seen in cells selected for resistance to topoisomerase II poisons because these drugs convert the normal enzyme into a toxin. One would also expect an increase in topoisomerase II levels to give resistance to a topoisomerase II catalytic inhibitor, the opposite of what is observed in this case. Recently, it has been shown that stabilization of topoisomerase I-DNA cleavage complexes by camptothecins and stabilization of topoisomerase II-DNA cleavage complexes by topoisomerase II poisons cause extensive sumoylation of the topoisomerases (34, 35). However, treatment with ICRF-193 also caused sumoylation of topoisomerase II (35). This was interpreted as evidence that drug binding alone might cause sumoylation of topoisomerases and that stabilization of topoisomerase-DNA cleavage complexes might not be required for the sumoylation. However, this result can also be interpreted as additional evidence that ICRF-193 is able to stabilize topoisomerase II-DNA cleavage complexes.

The cytotoxicity of topoisomerase II poisons, such as VP-16 and m-AMSA is very dependent on RAD52 function, with loss of RAD52 greatly increasing the toxicity of these drugs. The cytotoxicity of bis(2,6-dioxopiperazine)s was found to be partially dependent on RAD52, suggesting that the bis(2,6-dioxopiperazine)s generate DNA lesions of some type (36). Ku-deficient cells were found to be hypersensitive to ICRF-193 (37). This is consistent with topoisomerase poisoning, and the authors indicated that they could not rule out ICRF-193-induced DNA damage. However, they interpreted their results based on the assumption that ICRF-193 is only a topoisomerase catalytic inhibitor. More recent studies have found detectable levels of ICRF-193-induced topoisomerase II-DNA cross-links but concluded that the levels of ICRF-193-induced cleavable complexes were far too low to account for the cytotoxicity of the drug (18). We find in the studies described here that ICRF-193 is a significant topoisomerase II poison but that its topoisomerase II poisoning requires a chaotropic protein denaturant in the assays for efficient detection and that it is selective for the beta -isozyme of topoisomerase II.

It is not clear why topoisomerase poisoning is difficult to detect with SDS-based assays of drugs such as ICRF-193 and chloroquinoxaline sulfonamide. It is possible that detergents like SDS may not denature topoisomerases instantly, allowing the release of certain drugs from the ternary topoisomerase-drug-DNA complex before the enzyme is completely inactivated. This might allow the still active enzyme to religate the DNA strand breaks before being completely denatured. Release of the drug from the ternary complex upon the binding of the first detergent molecules would be very dependent on the individual topoisomerase poisons and their specific interactions with the enzyme and/or the DNA. For instance, DNA-intercalating topoisomerase II poisons, such as m-AMSA, may be less likely to be released from the ternary complex by SDS binding to the enzyme, allowing complete inactivation of the enzyme without loss of the drug from the complex. On the other hand, drugs bound primarily by hydrophobic interactions with the topoisomerase might be easily released by SDS binding. Chaotropic protein-denaturants may inactivate topoisomerases before dissociation of topoisomerase poisons from the ternary complex. Alternately, the topoisomerase-DNA cleavage complexes stabilized by chloroquinoxaline sulfonamide and ICRF-193 may differ in some fundamental way from those stabilized by drugs whose topoisomerase-DNA cleavage complexes are detectable with SDS-based assays. Consistent with this is the observation that measurements of GuHCl-denatured topoisomerase-DNA cleavage complexes indicate that topoisomerase II poisoning by ICRF-193 is comparable with that of m-AMSA (Fig. 6). The fact that ICRF-193 is much less cytotoxic than m-AMSA, given comparable levels of topoisomerase-DNA cleavage complexes, again suggests that there may be some important difference in the structure of the cleavage complexes or their interactions with cellular machinery such as DNA replication forks or DNA damage signaling pathways.

The success of topoisomerase poisons such as m-AMSA, adriamycin, VM-26, and VP-16 in the treatment of cancer has been the rationale for academic, governmental, and pharmaceutical industry drug discovery efforts designed to discover new and more effective topoisomerase poisons. Many of these drug discovery programs have either searched directly for topoisomerase poisons or have used assays for topoisomerase poisoning to test for mechanisms of compounds found to be cytotoxic to tumor cell lines. It is likely that these topoisomerase poisoning assays have tended to use the much more available topoisomerase IIalpha and the commonly used detergent, SDS. The findings that drugs may be selective for topoisomerase IIbeta (Ref. 19 and this report), that topoisomerase IIbeta may be a significant cytotoxic target (24), and that detection of topoisomerase poisoning by some drugs requires a chaotropic protein denaturant (Ref. 21 and this report) suggest that some topoisomerase poisons may have been overlooked. In particular, cytotoxic compounds found to be topoisomerase catalytic inhibitors but not topoisomerase poisons may be worth revisiting with assays using chaotropic protein denaturants and/or topoisomerase IIbeta .

    ACKNOWLEDGEMENTS

We thank TopoGen (Columbus, OH) and Abbott Laboratories (Abbott Park, IL) for purified human topoisomerase IIalpha and Dr. Caroline Austin (University of Newcastle, UK) and Dr. Anni H. Andersen (University of Aarhus, Denmark) for purified human topoisomerase IIbeta . We also thank TopoGen for anti-human topoisomerase IIalpha antibody and Dr. Daniel M. Sullivan (H. Lee Moffitt Hospital, Tampa, FL) for anti-topoisomerase IIbeta antibody. We thank Dr. Andrei V. Blokhin and the late Dr. Donald T. Witiak for ICRF-193 and Dr. Leonard Zwelling and Dr. Miloslav Beran for HL-60/AMSA and parental HL-60/S cells. We also thank Dr. Russ Hille for useful suggestions on the manuscript.

    FOOTNOTES

* This work was supported by Grant NCI RO1 CA80961 (to R. M. S.), Contract NO1-CM-57201 (to K. K. C.), Grant U01CA63185 (to K. K. C. and R. M. S.), Grants DK56917 and CA74939 (to R. G.), and Grant P30 CA16058 (to the Ohio State University Comprehensive Cancer Center) from the Public Health Service.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.

These authors contributed equally to this work.

§§ To whom correspondence should be addressed: Ohio State University, Dept. of Radiology, 103 Wiseman Hall, 400 West 12th Ave., Columbus, OH 43210. Tel.: 614-292-9375; Fax: 614-292-7237; E-mail: snapka.1@osu.edu.

Published, JBC Papers in Press, September 27, 2001, DOI 10.1074/jbc.M104383200

    ABBREVIATIONS

The abbreviations used are: ICRF-193, meso-2,3-bis(2,6-dioxopiperazin-4-yl)butane; GuHCl, guanidinium chloride; m-AMSA, 4'-(9-acridinylamino)methanesulfon-m-aniside; VM-26 (teniposide), 4'-dimethylepipodophyllotoxin 9-[4,6-O-2-thenylidene-beta -D-glycopyranoside]; VP-16 (etoposide), 4'-dimethylepipodophyllotoxin 9-[4,6-O-ethylidene-beta -D-glucopyranoside].

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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