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A Two-Mechanism Model for the Interaction of Etoposide Quinone with Topoisomerase II# Elizabeth G. Gibson, McKenzie M. King, Susan L Mercer, and Joseph E. Deweese Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00209 • Publication Date (Web): 17 Aug 2016 Downloaded from http://pubs.acs.org on August 23, 2016

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Chemical Research in Toxicology

A Two-Mechanism Model for the Interaction of Etoposide Quinone with Topoisomerase IIα α

Elizabeth G. Gibson,†‡ McKenzie M. King,† Susan L. Mercer,†‡ and Joseph E. Deweese†¶*

Department of Pharmaceutical Sciences, Lipscomb University College of Pharmacy and Health Sciences, Nashville, Tennessee 37204-3951 and Departments of Pharmacology and Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

* To whom correspondence should be addressed: Address: Dept. of Pharmaceutical Science,One University Park Drive, Nashville, TN 37204-3951; Phone: 615-966-7101; Fax: 615-966-7163; E-mail, [email protected] †

Lipscomb University College of Pharmacy and Health Sciences, Department of Pharmaceutical Sciences. ‡ Vanderbilt University School of Medicine, Department of Pharmacology. ¶ Vanderbilt University School of Medicine, Department of Biochemistry.

Running title: Etoposide Quinone Uses Two Mechanisms to Impact Topoisomerase IIα

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ABSTRACT

Topoisomerase II is an essential nuclear enzyme involved in regulating DNA topology to facilitate replication and cell division. Disruption of topoisomerase II function by chemotherapeutic agents is in use as an effective strategy to fight cancer. Etoposide is an anticancer therapeutic that disrupts the catalytic cycle of topoisomerase II and stabilizes enzyme-bound DNA strand breaks. Etoposide is metabolized into several species including active quinone and catechol metabolites. Our previous studies have explored some of the details of how these compounds act against topoisomerase II. In our present study, we extend those analyses by examining several effects of etoposide quinone on topoisomerase IIα including whether the quinone impacts ATP hydrolysis, DNA ligation, cleavage complex persistence, and enzyme:DNA binding. Our results demonstrate that the quinone inhibits relaxation at 100-fold lower levels of drug when compared to etoposide. Further, the quinone inhibits ATP hydrolysis by topoisomerase IIα. The quinone does appear to stabilize single-strand breaks similar to etoposide suggesting a traditional poisoning mechanism. However, there is minimal difference in cleavage complex persistence in the presence of etoposide or etoposide quinone. In contrast to etoposide, we find that etoposide quinone blocks enzyme:DNA binding, which provides an explanation for previous data showing the ability of the quinone to inactivate the enzyme over time. Finally, etoposide quinone is able to stabilize the Nterminal protein clamp implying an interaction between the compound and this portion of the enzyme. Taken together, the evidence supports a two-mechanism model for the effect of the quinone on topoisomerase II: 1) interfacial poison and 2) covalent poison that interacts with the N-terminal clamp and impacts the binding of DNA.

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INTRODUCTION Replication, transcription, and even mitosis are dependent upon regulation of DNA topology.1-3 This essential task is assigned to a class of enzymes known as DNA topoisomerases. These enzymes generate transient DNA strand breaks to alleviate topological impediments. There are two types of topoisomerases: Type I, which create single-stranded DNA brakes that allow for alleviation of torsional strain, and Type II, which create double-stranded DNA breaks that facilitate relaxation, unknotting, and decatenation.1, 2 Mammals have two isoforms of type II topoisomerases: topoisomerase IIα and IIβ. Topoisomerase IIα (TopoIIα) is up-regulated in response to cell growth and peaks during mitosis, making it an ideal cancer therapy target.3 TopoIIβ appears to function during transcription and does not fluctuate as widely through the cell cycle.3 TopoIIα is the focus of our current study because of its central role as an anti-cancer drug target. There are broadly two classes of compounds that impact TopoII: catalytic inhibitors and interfacial poisons.3-5 Inhibitors effect the catalytic cycle of the topoisomerase enzyme and decrease cleavage complexes leading to slow cell growth causing quiescence and mitotic failure. Poisons lead to stabilization of TopoII:DNA complexes (known as cleavage complexes) that results in strand breaks and cell death or repair of the damage in sub-lethal circumstances. In addition, some compounds poison TopoII in a non-traditional manner and are known as covalent poisons or redoxdependent poisons.6 These compounds often share various characteristics including covalent binding to the enzyme, poisoning of DNA cleavage, and sensitivity to reducing agents.

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Etoposide is an anti-cancer therapeutic that targets TopoII and is used to treat a variety of cancer types including both solid tumors and hematologic malignancies.4,

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Etoposide acts as an interfacial poison of TopoII, which lead to strand breaks as noted above.4,

5, 7

Around 2-3% of patients treated with this agent develop a secondary

leukemia associated with specific chromosomal translocations.8-13 The mechanism for leukemogenesis resulting from this therapy has not been fully clarified.14 Etoposide is metabolized by CYP3A4 to generate quinone and catechol metabolites, which may contribute to leukemogenic translocations.15-18 Both of these metabolites have activity against TopoII.19-21 Previous studies with TopoIIα have shown that the quinone metabolite displays characteristics of a covalent poison, including 5fold higher levels of DNA cleavage and producing a higher ratio of double-stranded to single-stranded break ratio than etoposide.20 Conversely, the catechol metabolite works similarly to the parent compound but can also be oxidized to the quinone, which makes this form less stable and potentially more toxic than etoposide.19,

20

Furthermore,

etoposide quinone induced high levels of DNA cleavage with TopoIIβ at half of the drug concentration needed with TopoIIα and reacted 2-4 times faster with the β isoform.21 ATP stimulates DNA cleavage with the β isoform in the presence of etoposide but not in the presence of etoposide quinone.21 The increased activity of the quinone against both isoforms of TopoII has led us to further explore the differences in the mechanism of action of etoposide and the quinone metabolite on the TopoIIα isoform. It is unclear if the quinone only exerts its action using an interfacial poison or if it is also acting outside of the active site. Using previous data as a guide, we performed studies to further clarify a hypothesized dual mechanism of the drug working both inside and outside the active site. Using purified TopoIIα, we investigated the ability of the 5



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quinone to effect DNA relaxation, DNA ligation, stability of cleavage complexes, and the ATPase activity of the enzyme. Furthermore, we studied the effect of the metabolite on enzyme:DNA binding by using a mobility shift assay, fluorescence anisotropy, and a clamp closing assay. Taken together, our data outlined below provide evidence that etoposide quinone utilizes at least two distinct mechanisms against TopoII: 1) inhibition of religation (interfacial poisoning) and 2) interaction with the N-terminal clamp (stabilization of the clamp and blocking of DNA binding). We propose a two-mechanism model for the action of etoposide quinone. EXPERIMENTAL PROCEDURES Enzymes and Materials. Wild-type TopoIIα was expressed in Saccharomyces cerevisiae JEL1∆top1 cells and purified as described previously.22 The enzyme was stored at 80ºC as a 1.5 mg/mL (4 µM) stock in 50 mM Tris-HCl, pH 7.7, 0.1 mM EDTA, 750 mM KCl, 5% glycerol, and 40 µM DTT (carried from the enzyme preparation). Negatively supercoiled pBR322 DNA was prepared using a Plasmid Mega Kit (Qiagen) as described by the manufacturer. Etoposide and 1,4-benzoquinone were obtained from Sigma. Etoposide quinone was synthesized as previously described.20 Drugs were stored at 4°C as 20 mM stock solutions in 100% DMSO, except 1,4benzoquinone which was stored as a 20 mM stock in H2O. Topoisomerase II-mediated Relaxation of Plasmid DNA. Reaction mixtures contained 4.4 nM wild-type human TopoIIα, 5 nM negatively supercoiled pBR322 DNA, and 1 mM ATP in 20 µL of 10 mM Tris-HCl, pH 7.9, 175 mM KCl, 0.1 mM NaEDTA, 5 mM MgCl2, and 2.5% glycerol. Assays were started by the addition of enzyme, and DNA relaxation mixtures were incubated for 15 min at 37°C. DNA relaxation reactions were carried out in the presence of 0−200 µM etoposide or etoposide quinone. DNA relaxation was 6 ACS Paragon Plus Environment

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stopped by the addition of 3 µL of stop solution (77.5 mM Na2EDTA, 0.77% SDS). Samples were mixed with 2 µL of agarose gel loading buffer, heated for 2 min at 45°C, and subjected to gel electrophoresis in 1% agarose gels. The agarose gel was then stained in ethidium bromide for 30 min. DNA bands were visualized by UV light and quantified using a Bio-Rad ChemiDoc MP Imaging System and Image Lab Software (Hercules, CA). Results were plotted using GraphPad Prism 6 (La Jolla, CA). DNA relaxation was monitored by the conversion of supercoiled plasmid DNA to relaxed topoisomers. Thin-Layer Chromatography-Based ATPase Assay. ATP hydrolysis was monitored using thin-layer chromatography (TLC) on a polyethylenimine (PEI) matrix (Merck KGaA, Darmstadt, Germany). Reaction mixtures contained 140 nM of wild-type of human topoisomerase IIα, 5 nM negatively supercoiled pBR322 DNA, and 1 mM ATP in 20 µL of 10 mM Tris-HCl, pH 7.9, 100 mM KCl, 1 mM EDTA, 5 mM MgCl2, and 2.5% glycerol. Reactions were incubated at 37oC and 4 µL samples were taken out at increasing time points (0-30 min) and spotted on the TLC plate. Reactions were run in the absence (1% DMSO as a control) or presence of etoposide, etoposide quinone, or etoposide catechol. The plate was then placed in 400 mM ammonium carbonate inside the TLC chamber and resolved. Separation of ADP from ATP was imaged using an AlphaImager system (Santa Clara, CA) and quantified using AlphaImager software. Data were calculated as the percent of ATP converted to ADP in each reaction. Topoisomerase II-mediated Cleavage of Plasmid DNA. Plasmid DNA cleavage reactions were performed using the procedure of Fortune and Osheroff.23 Reaction mixtures contained 220 nM of wild-type human TopoIIα and 5 nM negatively supercoiled pBR322 DNA in 20 µL of 10 mM Tris-HCl, pH 7.9, 100 mM KCl, 1 mM EDTA, 5 mM 7



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MgCl2, and 2.5% glycerol. Final reaction mixtures contained ~1 µM DTT, which represents the residual DTT carried along from the enzyme preparation. Unless stated otherwise, assays were started by the addition of enzyme, and DNA cleavage mixtures were incubated for 6 min at 37°C. DNA cleavage reactions were carried out in the absence or presence of 0−200 µM etoposide and etoposide quinone. DNA cleavage complexes were trapped by the addition of 2 µL of 5% SDS followed by 2 µL of 250 mM NaEDTA, pH 8.0. Proteinase K was added (2 µL of a 0.8 mg/mL solution), and reaction mixtures were incubated for 30 min at 37°C to digest TopoIIα. Samples were mixed with 2 µL of agarose gel loading buffer (60% sucrose in 10 mM Tris-HCl, pH 7.9), heated for 2 min at 45°C, and subjected to electrophoresis in 1% agarose gels in 40 mM Trisacetate, pH 8.3, and 2 mM EDTA containing 0.5 µg/mL ethidium bromide. Doublestranded DNA cleavage was monitored by the conversion of negatively supercoiled plasmid DNA to linear molecules. DNA bands were visualized by UV light and quantified using a Bio-Rad ChemiDoc MP Imaging System and Image Lab Software (Hercules, CA). Results were plotted using GraphPad Prism 6 (La Jolla, CA). Topoisomerase II-mediated Ligation of Plasmid DNA. Ligation assays were performed using chemical means to induce ligation. DNA cleavage/ligation equilibria were established with 220 nM wild-type TopoIIα for 6 min at 37°C using the same protocol above for plasmid-mediated DNA cleavage. In addition to stopping a control reaction with SDS, ligation reactions were treated with either 2 µL of 250 mM EDTA or 2 µL of 5 M NaCl prior to 2 µL of 5% SDS. Addition of EDTA or NaCl to the reaction induces ligation through either metal ion chelation or changing the ionic strength, respectively. Linear DNA product was used to quantify double-strand breaks (DSB), while nicked plasmid was used to quantify single8


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strand breaks (SSB). Samples were processed, resolved, and analyzed as described under the plasmid DNA cleavage method above. Results are shown relative to the level of DNA cleavage in the absence of compound, which was set to a value of 1 (not shown on figure). Persistence of Topoisomerase IIα-DNA Cleavage Complexes. The persistence of TopoIIα-DNA cleavage complexes established in the presence of drugs was determined using the procedure of Gentry, et al.24 Initial reactions contained 550 nM wild-type human TopoIIα enzyme, 50 nM DNA, and 25 µM etoposide or 25 µM etoposide quinone in a total of 20 µL of human cleavage buffer. Reactions were incubated at 37°C for 6 min and then diluted 25-fold with human cleavage buffer warmed to 37°C. Samples (20 µL) were removed at times ranging from 0-240 min, and DNA cleavage was stopped with 2 µL of 5% SDS followed by 2 µL of 250 mM EDTA (pH 8.0). Samples were digested with proteinase K and processed as described above for cleavage assays. Levels of DNA cleavage were set to 100% at time zero, and the persistence of cleavage complexes was determined by the decay of the linear reaction product over time. Electrophoretic Mobility Shift Assay to Assess Enzyme:DNA Binding. The ability of DNA to bind to TopoIIα was measured using an EMSA. Reactions consisting of 0-330 nM TopoIIα, DNA, 50 mM Tris, pH 7.9, 150 mM KCl, 0.5 mM NaEDTA, and 12.5% glycerol were incubated at 37°C and carried out in the presence of 10% DMSO or 50 µM etoposide quinone or 1,4-benzoquinone. Reactions were run: 1) with no drug (DMSO); 2) with enzyme and DNA reacting prior to the addition of compound; or 3) with enzyme and compound reacting prior to the addition of DNA. Reactions were processed by adding sample loading buffer and immediately subjected to gel electrophoresis in a 1% TBE gel stained with ethidium bromide. Gels were imaged using BioRad ChemiDoc MP 9



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Imaging system (Hercules, CA). Binding was qualitatively analyzed by DNA migration through the gel. Fluorescence anisotropy to monitor DNA binding to Topoisomerase IIα. A 40-mer sequence has been labeled on the top strand with 6-FAM (6-carboxyfluorescein) based upon a previously published sequence.25 Sequences for the strands are as follows: ‘top’ strand,

5’-

CGCAATCTGACAATGCGCTCATCGTCATCCTCGCGACGCG-3’

and

‘bottom’ strand, 5’-CGCGTGCCGAGGATGACGATGAGCGCATTGTCAGATTGCG-3’. Reactions were carried out in an 80 µL reaction mixture with an enzyme titration from 0150 nM human TopoIIα, 1 nM DNA, 50 mM KCl, +/-10 mM MgCl2, 50 mM Tris, pH 8.5, 5% glycerol, 10 µg/µL BSA. Reactions were run in the presence of 10% DMSO (no drug) or 50 µM etoposide or etoposide quinone, which was added to the enzyme and incubated for 5 minutes at 37°C. Enzyme was titrated into the reaction and successive fluorescence readings were measured on a Cytation3 imaging plate reader from BioTek (Winooski, VT) with the appropriate filter sets and anisotropy values were calculated using BioTek’s Gen5 software. The reactions are run in quadruplicate and fluorescent anisotropies calculated for each titration point were read ~10 times and averaged together. Data were analyzed using GraphPad Prism 6 (La Jolla, CA) and fitted to a one-site specific binding with Hill slope curve. Statistical analysis was performed within Prism 6 using a one-way ANOVA followed by a Tukey’s Post-Test Analysis. Protein N-terminal clamp closing assay. The stabilization of the N-terminal protein clamp was measured using a modified version of a previously described protocol.26-28 Briefly, 88 nM wild-type human TopoIIα and 2 nM pBR322 were incubated for 10 min at 37°C in a total of 50 µL of clamp closing buffer (50 mM Tris-HCl, pH 8.0, 100 mM KCl, 1 10


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mM EDTA, and 8 mM MgCl2) in the absence or presence of 100 µM etoposide, etoposide, quinone, or 1,4-benzoquinone. Control reactions including DNA only and etoposide quinone in the presence of 100 µM dithiothreitol (DTT) were also performed. After 10 min incubation, 2 mM ATP was added and an addition 10 min incubated was carried out at 37°C. Binding mixtures were then loaded into filter baskets containing glass fiber filters (Millipore) that were pre-equilibrated using clamp closing buffer. Filters were spun at low speed (~1 krpm) for 5-10 s. Reactions were then washed in 50 µL clamp closing buffer (low salt), 100 µL of high salt wash (1 M NaCl), and 100 µL of SDS wash (10 mM TrisHCl, pH 8.0, 1 mM EDTA, and 0.5% SDS) heated to 65°C. Baskets were transferred to new tubes after each wash. Eluates were precipitated in isopropanol and dried. Samples were then resuspended in nucleic acid loading buffer (Bio-Rad) and electrophoresed in a 1% agarose TAE gel containing ethidium bromide. Gels were imaged using BioRad ChemiDoc MP Imaging system (Hercules, CA). Supercoiled DNA bands were quantified for low salt, high salt, and SDS wash eluates for each condition and DNA eluting after the SDS wash was calculated as a percentage of the total from all three washes. Data were analyzed used GraphPad Prism 6 (La Jolla, CA), and statistical analysis was performed using a one-way ANOVA followed by a Tukey’s PostTest Analysis.

RESULTS AND DISCUSSION Etoposide quinone is more potent than etoposide at inhibiting relaxation. As seen in Figure 1, etoposide and etoposide quinone both inhibit relaxation by TopoIIα. 11 ACS Paragon Plus Environment


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Interestingly, etoposide quinone inhibits relaxation with ~100-fold less compound than etoposide (Figure 1). While this complements previous data that shows etoposide quinone is very potent when studying DNA cleavage20, the current results do not clarify the mechanism by which relaxation is inhibited. Interfacial poisons, like etoposide, have the ability to inhibit relaxation through poisoning the DNA cleavage/ligation process. However, other mechanisms may also impair relaxation, such as inhibition of ATP hydrolysis by some catalytic inhibitors. The ability of etoposide quinone to impair relaxation at such low levels could be caused by a different mode of action than the interfacial poisons or by a combination of mechanisms. We set out to elucidate alternative mechanisms for etoposide quinone to act upon TopoIIα using a series of assays. Etoposide quinone inhibits ATP hydrolysis. Strand passage by TopoIIα is ATP dependent, and ATP hydrolysis is required for full catalytic activity. Some agents can block ATP hydrolysis either directly or as a consequence of disrupting the catalytic cycle. As seen in Figure 2, etoposide has a minor effect on ATP hydrolysis, while etoposide quinone strongly inhibits hydrolysis by TopoIIα. While this may simply reflect the ability of this metabolite to poison DNA cleavage and block the enzyme from ligating, it may also be due to other effects. To further assess ATPase inhibition, we found that similar to etoposide, etoposide catechol also does not inhibit ATP hydrolysis at concentrations up to 200 µM (Figure S1). It should be noted that etoposide does inhibit ATP hydrolysis by yeast TopoII29, but additional analysis indicates that this inhibition occurs after phosphate release.30 The disparity between our results with etoposide and TopoIIα and the results with yeast TopoII may reflect fundamental mechanistic distinctions between the enzymes and/or differences in the techniques 12


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used to measure ATP hydrolysis. Further analysis will be required to clarify this matter. Therefore, we set out to further explore how etoposide quinone is inhibiting ATP hydrolysis while also poisoning DNA cleavage. Etoposide quinone blocks ligation at one scissile bond. Previous results demonstrated that etoposide quinone does inhibit ligation.20 However, the results were focused on ligation of double-stranded DNA breaks without examining the singlestranded DNA breaks. Therefore, we monitored both double- and single-stranded DNA breaks formed by human TopoIIα under conditions that induce DNA ligation. As seen in Figure 3, etoposide quinone induces far higher levels of double-stranded DNA breaks (DSB) and a higher proportion of DSB to single-strand breaks (SSB) when the reactions are terminated by SDS, which traps the reaction and denatures the enzyme. The addition of EDTA prior to SDS allows for ligation of cleaved DNA. The results show that in the presence of EDTA DSBs with both etoposide and etoposide quinone decrease significantly, while the single-strand breaks increase. Notably, single-strand breaks with etoposide quinone increase to a significant degree above SSB in reactions terminated with SDS (~4-fold increase). Further, the addition of NaCl, which promotes dissociation of the DNA from the enzyme and thereby induces ligation, leads to a decrease in DSBs and SSBs with both etoposide and the quinone. Based upon the results discussed above, etoposide quinone inhibits ligation, similar to etoposide and interfacial poisons. However, this mechanism alone does not explain the high degree of double-stranded DNA breaks. Therefore, we hypothesized that a second mechanism may be involved. Since our previous results demonstrate that the quinone can inactivate DNA cleavage when incubated with the enzyme prior to

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DNA20, we used a series of experiments to explore the impact of the compound on the persistence of enzyme:DNA complexes and on the ability of the enzyme to bind to DNA. Etoposide and etoposide quinone have a similar effect on the persistence of DNA cleavage complexes. Since etoposide quinone strongly induces double strand breaks, the stability of the TopoII:DNA complex in the presence of etoposide quinone was examined in comparison with complexes formed in the presence of etoposide. In this experiment, DNA cleavage assays with human TopoIIα were run in the presence of either etoposide or etoposide quinone and then diluted 10-fold in reaction buffer. Samples were taken from the diluted reaction over time and stopped using SDS. Results seen in Figure 4 track the cleavage levels detected over time, which are indicative of TopoII:DNA complexes. Throughout the four-hour time course, there is no statistically significant trend or difference between complexes formed in the presence of etoposide versus those formed in the presence of the quinone. However, it does appear that complexes with etoposide quinone persist longer than those formed in the presence of etoposide. This result, however, does not measure whether etoposide quinone can impede the ability of the enzyme to bind to DNA. Therefore, we set out to examine DNA binding in the presence of the quinone. Etoposide quinone impairs DNA binding. As discussed above, etoposide quinone inhibits the ability of TopoII to ligate cleaved DNA. However, previous studies have also demonstrated the ability of etoposide quinone to inactivate TopoII activity when the compound incubated with the enzyme prior to adding DNA.20 While the inhibition of ligation may be a consequence of a traditional interfacial poisoning mechanism, the ability to inactivate TopoII activity reflects the mechanism seen with some redoxdependent or covalent poisons, such as 1,4-benzoquinone.6 We hypothesized that the 14


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ability to inactivate the enzyme may imply the ability of etoposide quinone to block DNA binding to the TopoII. In order to determine whether etoposide quinone is inhibiting enzyme:DNA binding, we performed electrophoretic mobility shift assays (EMSA) to observe the change in migration of DNA in the gel when bound to TopoIIα. As seen in Figure 5, the covalent poison 1,4-benzoquinone impairs DNA binding when present with the enzyme prior to the addition of DNA. A similar effect is seen to a lesser extent in the presence of etoposide quinone, which suggests that the metabolite may reduce enzyme:DNA binding. In order to quantitate DNA binding by TopoIIα in the presence of etoposide or etoposide quinone, we employed fluorescence anisotropy using a fluorescently-labeled, duplex oligonucleotide. TopoIIα binding decreases the rotation of the DNA substrate in solution, resulting in higher anisotropy, which is measured as a polarized emission signal. If etoposide quinone interferes with enzyme:DNA binding, then the anisotropy will be diminished in the presence of the compound relative to that observed in its absence. As seen in Figure 6, increasing concentrations of human TopoIIα bind to the oligonucleotide resulting in an increasing fluorescence anisotropy signal. The binding curve is increased in the presence of Mg2+, which is required for DNA cleavage by the enzyme. The presence of etoposide does not appear to significantly change binding with or without Mg2+ when compared to the absence of drug. However, etoposide quinone leads to a 3-4-fold decrease in DNA binding compared to etoposide or the no drug control, regardless of the presence of Mg2+. The effect is evident when comparing the calculated Bmax values for each set (Figure 7). The drop in Bmax in the presence of 50 µM etoposide quinone (with Mg2+) is significant (p < 0.05) when compared to no drug 15



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with Mg2+. Comparisons of the Bmax in the absence of Mg2+ shows a change that is significant at the p < 0.1 level. Based upon binding curve analysis, there is no significant change in Kd under any of the conditions (Table 1). Therefore, etoposide quinone can impair binding of DNA to the enzyme when present with the enzyme prior to the addition of DNA. As discussed below, this result may clarify how etoposide quinone can inactivate DNA cleavage by the enzyme. Etoposide quinone stabilizes the N-terminal clamp of TopoIIα. The ability of etoposide quinone to block binding suggests a structural effect on the enzyme of some type. Previous research has shown that reactive quinones are able to block the Nterminal protein clamp of TopoII.28 Therefore, we tested whether etoposide quinone could stabilize the N-terminal protein clamp using an assay to measure the stability of the enzyme:DNA complex.26, 27 The protein clamp closing assay examines the stability of enzyme:DNA complexes by using successive washes of low salt, high salt, and SDS solutions. Stabilization of the N-terminal clamp is indicated by DNA that is retained in a glass fiber filter until the SDS wash. It should be noted that this assay does not measure the enzyme:DNA cleavage complexes. Instead, the complexes that elute are those where DNA is not cleaved by TopoII. As seen in figure 8, DNA alone and TopoIIα with DNA do not remain bound at significant levels to the glass fiber filters. Etoposide appears to cause a low level of DNA to remain bound, but this is not statistically significant. However, both etoposide quinone and 1,4-benzoquinone lead to higher levels of DNA in the SDS wash step. Interestingly, when etoposide quinone is reacted with DTT prior to the addition of enzyme and DNA, the ability to stabilize the N-terminal clamp is lost, which is consistent with our previous work on the redox-sensitive nature of the quinone.19, 16

20


By stabilizing the N-terminal

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clamp, ATP hydrolysis is also disrupted,26,

27

which is consistent with the results from

Figure 2. Taken together, etoposide quinone is able to induce the formation of a saltstable closed clamp with TopoII on DNA. The ability of etoposide quinone to stabilize the N-terminal clamp provides an explanation for how this metabolite inhibits ATP hydrolysis, blocks DNA binding and inactivates the enzyme. Taken together, these data provide evidence for action of etoposide quinone outside of the active site of TopoIIα. Conclusions The TopoII interfacial poison etoposide is metabolized into active species including a catechol and a quinone. Our previous studies have demonstrated that etoposide quinone displays characteristics of a redox-dependent covalent poison that reacts with TopoII. However, the mechanism has not been fully elucidated. Therefore, we set out to explore the mechanism of action of etoposide quinone against TopoIIα further. We found that the quinone inhibits plasmid DNA relaxation by TopoIIα at 100-fold lower concentration when compared to etoposide (1 µM vs 100 µM). While etoposide quinone does appear to strongly inhibit ATP hydrolysis, this is likely the effect of both interfacial poisoning and of stabilization of the N-terminal clamp, as discussed below. Relaxation is inhibited by interfacial poisons, so we examined the ability of the enzyme to ligate DNA under different conditions in the presence of etoposide and the quinone. Our results show that in the presence of EDTA, the DSBs formed by TopoIIα in the presence of the quinone become SSBs. Therefore, the quinone does appear to be acting similar to the parent compound and is likely blocking ligation on one strand. When ligation is induced by adding NaCl, both the DSBs and SSBs are decreased, which may reflect the fact that some of the action of the quinone is non-covalent in

17



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nature. However, the DSBs after NaCl treatment are still four times higher in the presence of the quinone when compared to etoposide. Since the DSBs appeared to maintain some stability, we employed a DNA cleavage persistence assay to study the comparative persistence of DNA cleavage over time. We found no significant difference in DNA cleavage persistence after a 4 h incubation in a dilution-based assay. Therefore, if the quinone complexes are more stable under some reaction conditions, the current assay was unable to detect that stability. As mentioned above, etoposide quinone displays the ability to inactivate TopoII activity when present with the enzyme prior to the addition of DNA. This is not seen in the presence of etoposide.20 We explored whether this result could be due to the ability of the quinone to inhibit TopoII:DNA binding. We employed EMSA to examine the ability of the quinone to block enzyme:DNA binding. Our results show that there is a decrease in binding, but this result was qualitative. In order to more fully assess the impact of the quinone on enzyme:DNA binding, we utilized a fluorescence anisotropy assay using a fluorescently-labeled oligonucleotide similar to previous studies.25, 31 By measuring the change in fluorescence anisotropy in the presence of increasing concentrations of TopoIIα, we were able to plot the binding of the enzyme to DNA. Our data shows that etoposide quinone, unlike etoposide, inhibits the ability of the enzyme to bind to DNA in a quantitative manner. While there is no change in Kd, there is a decrease in the Bmax at both 10 and 50 µM etoposide quinone. This is consistent with the quinone making the enzyme less available for binding to DNA. Finally, we performed a clamp-closing assay to measure the ability of etoposide quinone to stabilize the N-terminal protein clamp. Our results show that etoposide 18


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quinone and 1,4-benzoquinone are able to stabilize the closed clamp, while etoposide or etoposide quinone with DTT are both unable to stabilize the clamp. This result may provide a mechanism for how etoposide quinone is able to block DNA binding and inactivate the enzyme when the metabolite is present with TopoII prior to the addition of DNA. Further, this result may also explain the strong inhibition of ATP hydrolysis by etoposide quinone. Stabilization of the N-terminal protein clamp is expected to inhibit ATP hydrolysis by TopoII.26, 27 Taken together, we propose a two-mechanism model for the interaction of etoposide quinone with TopoII (Figure 9). First, etoposide quinone can act as an interfacial TopoII poison and inhibit ligation. Second, the quinone appears to be able to act, outside the active site, in a way that: A) blocks DNA binding when present before DNA, which inactivates the enzyme and likely involves protomer adduction19, 20, and B) promotes increased double-stranded DNA breaks and stabilization of the N-terminal clamp when the DNA is present before the compound. It is possible that some of the double-strand DNA breaks result from interfacial poisoning, but this mechanism likely cannot explain the full enhancement seen in the presence of the quinone. We hypothesize that the closing of the N-terminal clamp may lead to an increase doublestranded breaks by stabilizing the enzyme on DNA, but further work will be needed to explore this connection and determine whether this model holds true. Testing this model will require additional experimentation including the use of an active site mutant to determine whether some of the effects of etoposide quinone are dependent upon poisoning of DNA cleavage. It will also be of interest to explore whether these same effects are seen with TopoIIβ.

19



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The present work has examined the action of etoposide quinone on the function of TopoIIα and specifically examined the impact on DNA binding and enzyme function. In summary, etoposide quinone can block enzyme-DNA binding and inactivate the enzyme when present prior to DNA. When the enzyme binds to DNA, etoposide quinone can stabilize the enzyme:DNA complex and result in higher levels of double-stranded breaks.

ASSOCIATED CONTENT Supporting information Figure S1 with ATP hydrolysis by TopoIIα in the presence of etoposide catechol and 1,4-benzoquinone and Table S1 with Kd and Bmax Values for Fluorescence Anisotropy in the Presence of 10 µM Compound are available in Supporting Information. The Supporting Information is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Phone: 615-966-7101; fax: 615-966-7163; E-mail: [email protected].

Funding This work was funded in part by a New Investigator Award from the American Association of Colleges of Pharmacy and by support from Lipscomb University College of Pharmacy and Health Sciences. Notes The authors declare no competing financial interest.

20


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ACKNOWLEDGEMENTS We thank Dr. Anni Andersen for providing the expression vector for His-tagged human topoisomerase IIα. We would like to thank Dr. Steve Phipps for helpful discussions regarding

statistical

analysis.

E.G.G.

and

M.M.K.

were

participants

in

the

Pharmaceutical Sciences Summer Research Program of the Lipscomb University College of Pharmacy and Health Sciences.

ABBREVIATIONS Bmax, maximum binding; DTT, dithiothreitol; DSB, double-stranded DNA break; EDTA, ethylenediaminetetracetic acid; EMSA, electrophoretic mobility shift assay; FAU, fluorescence anisotropy units; Kd, dissociation constant; SC, supercoiled; SSB, singlestranded DNA break; TopoII, topoisomerase II.

21



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REFERENCES (1)

Wang, J. C. (2002) Cellular roles of DNA topoisomerases: a molecular perspective. Nat. Rev. Mol. Cell Biol. 3, 430-440.

(2)

Bates, A. D., and Maxwell, A. (2005) DNA Topology. Oxford University Press, New York.

(3)

Nitiss, J. L. (2009) DNA topoisomerase II and its growing repertoire of biological functions. Nat. Rev. Cancer 9, 327-337.

(4)

Deweese, J. E., and Osheroff, N. (2009) The DNA cleavage reaction of topoisomerase II: wolf in sheep's clothing. Nucleic Acids Res. 37, 738-749.

(5)

Pommier, Y., Leo, E., Zhang, H., and Marchand, C. (2010) DNA topoisomerases and their poisoning by anticancer and antibacterial drugs. Chem. Biol. 17, 421-433.

(6)

Gibson, E. G., and Deweese, J. E. (2013) Covalent poisons of topoisomerase II. Curr. Top. Pharm. 17, 1-12.

(7)

Wu, C. C., Li, T. K., Farh, L., Lin, L. Y., Lin, T. S., Yu, Y. J., Yen, T. J., Chiang, C. W., and Chan, N. L. (2011) Structural basis of type II topoisomerase inhibition by the anticancer drug etoposide. Science 333, 459-462.

(8)

Pui, C.-H., Ribeiro, R. C., Hancock, M. L., Rivera, G. K., Evans, W. E., Raimondi, S. C., Head, D. R., Behm, F. G., Mahmoud, M. H., Sandlund, J. T., and Crist, W. M. (1991) Acute myeloid leukemia in children treated with epipodophyllotoxins for acute lymphoblastic leukemia. N. Engl. J. Med. 325, 1682-1687.

(9)

Winick, N. J., McKenna, R. W., Shuster, J. J., Schneider, N. R., Borowitz, M. J., Bowman, W. P., Jacaruso, D., Kamen, B. A., and Buchanan, G. R. (1993) Secondary acute myeloid leukemia in children with acute lymphoblastic leukemia treated with etoposide J. Clin. Oncol. 11, 209-217. 22


Page 22 of 39

Page 23 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(10) Smith, M. A., Rubinstein, L., and Ungerleider, R. S. (1994) Therapy-related acute myeloid leukemia following treatment with epipodophyllotoxins: estimating the risks. Med. Pediatr. Oncol. 23, 86-98. (11) Relling, M. V., Yanishevski, Y., Nemec, J., Evans, W. E., Boyett, J. M., Behm, F. G., and Pui, C. H. (1998) Etoposide and antimetabolite pharmacology in patients who develop secondary acute myeloid leukemia. Leukemia 12, 346-352. (12) Smith, M. A., Rubinstein, L., Anderson, J. R., Arthur, D., Catalano, P. J., Freidlin, B., Heyn, R., Khayat, A., Krailo, M., Land, V. J., Miser, J., Shuster, J., and Vena, D. (1999) Secondary leukemia or myelodysplastic syndrome after treatment with epipodophyllotoxins. J. Clin. Oncol. 17, 569-577. (13) Leone, G., Pagano, L., Ben-Yehuda, D., and Voso, M. T. (2007) Therapy-related leukemia and myelodysplasia: susceptibility and incidence. Haematologica 92, 1389-1398. (14) Cowell, I. G., and Austin, C. A. (2012) Mechanism of Generation of Therapy Related Leukemia in Response to Anti-Topoisomerase II Agents. Int. J. Environ. Res. Public Health 9, 2075-2091. (15) van Maanen, J. M., de Vries, J., Pappie, D., van den Akker, E., Lafleur, V. M., Retel, J., van der Greef, J., and Pinedo, H. M. (1987) Cytochrome P-450-mediated O-demethylation: a route in the metabolic activation of etoposide (VP-16-213). Cancer Res. 47, 4658-4662. (16) Relling, M. V., Evans, R., Dass, C., Desiderio, D. M., and Nemec, J. (1992) Human cytochrome P450 metabolism of teniposide and etoposide. J. Pharmacol. Exp. Ther. 261, 491-496.

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(17) Relling, M. V., Nemec, J., Schuetz, E. G., Schuetz, J. D., Gonzalez, F. J., and Korzekwa, K. R. (1994) O-demethylation of epipodophyllotoxins is catalyzed by human cytochrome P450 3A4. Mol. Pharmacol. 45, 352-358. (18) Zhuo, X., Zheng, N., Felix, C. A., and Blair, I. A. (2004) Kinetics and regulation of cytochrome P450-mediated etoposide metabolism. Drug Metab. Dispos. 32, 9931000. (19) Jacob, D. A., Gibson, E. G., Mercer, S. L., and Deweese, J. E. (2013) Etoposide Catechol Is an Oxidizable Topoisomerase II Poison. Chem. Res. Tox. 26, 11561158. (20) Jacob, D. A., Mercer, S. L., Osheroff, N., and Deweese, J. E. (2011) Etoposide quinone is a redox-dependent topoisomerase II poison. Biochemistry 50, 56605667. (21) Smith, N. A., Byl, J. A., Mercer, S. L., Deweese, J. E., and Osheroff, N. (2014) Etoposide quinone is a covalent poison of human topoisomerase IIβ. Biochemistry 53, 3229-3236. (22) Regal, K. M., Mercer, S. L., and Deweese, J. E. (2014) HU-331 is a catalytic inhibitor of topoisomerase IIα. Chem. Res. Toxicol. 27, 2044-2051. (23) Fortune, J. M., and Osheroff, N. (1998) Merbarone inhibits the catalytic activity of human topoisomerase IIα by blocking DNA cleavage. J. Biol. Chem. 273, 1764317650. (24) Gentry, A. C., Pitts, S. L., Jablonsky, M. J., Bailly, C., Graves, D. E., and Osheroff, N. (2011) Interactions between the etoposide derivative F14512 and human type II topoisomerases: implications for the C4 spermine moiety in promoting enzymemediated DNA cleavage. Biochemistry 50, 3240-3249. 24


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(25) Gilroy, K. L., and Austin, C. A. (2011) The impact of the C-terminal domain on the interaction of human DNA topoisomerase II alpha and beta with DNA. PLoS One 6, e14693. (26) Roca, J., and Wang, J. C. (1992) The capture of a DNA double helix by an ATPdependent protein clamp: a key step in DNA transport by type II DNA topoisomerases. Cell 71, 833-840. (27) Roca, J., Ishida, R., Berger, J. M., Andoh, T., and Wang, J. C. (1994) Antitumor bisdioxopiperazines inhibit yeast DNA topoisomerase II by trapping the enzyme in the form of a closed protein clamp. Proc. Natl. Acad. Sci. U. S. A. 91, 1781-1785. (28) Bender, R. P., and Osheroff, N. (2007) Mutation of cysteine residue 455 to alanine in human topoisomerase IIα confers hypersensitivity to quinones: enhancing DNA scission by closing the N-terminal protein gate. Chem. Res. Toxicol. 20, 975-981. (29) Morris, S. K., and Lindsley, J. E. (1999) Yeast topoisomerase II is inhibited by etoposide after hydrolyzing the first ATP and before releasing the second ADP. J. Biol. Chem. 274, 30690-30696. (30) Baird, C. L., Harkins, T. T., Morris, S. K., and Lindsley, J. E. (1999) Topoisomerase II drives DNA transport by hydrolyzing one ATP. Proc. Natl. Acad. Sci. U.S.A. 96, 13685-13690. (31) Gilroy, K. L., Leontiou, C., Padget, K., Lakey, J. H., and Austin, C. A. (2006) mAMSA resistant human topoisomerase IIβ mutation G465D has reduced ATP hydrolysis activity. Nucleic Acids Res. 34, 1597-1607.

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Figure Legends: Figure 1: Etoposide quinone inhibits plasmid DNA relaxation by TopoIIα. Structures of etoposide (left) and etoposide quinone (right) are shown above an ethidium bromide stained relaxation gel. Plasmid DNA relaxation by TopoIIα is monitored by gel electrophoresis in the absence (+TII) or presence of 0.1-100 µM etoposide or etoposide quinone. Positions of supercoiled (SC) and relaxed (Rel) plasmids are denoted at right. Supercoiled plasmid DNA standard is at left (DNA). Results are representative of four independent experiments.

Figure 2: Etoposide quinone inhibits TopoIIα-mediated ATP hydrolysis. TLC-based ATPase assays were performed with 1% DMSO (ND, black), 25 µM (blue) or 200 µM (green) etoposide (Etop), and 25 µM etoposide quinone (EQ, red). Time points were taken at 10, 20, and 30 min and percent ATP converted to ADP was quantified. Error bars represent the standard deviation of three or more independent experiments.

Figure 3: Etoposide quinone poisons TopoIIα by inhibiting ligation. Both double-strand and single-strand breaks were tracked during plasmid DNA cleavage reactions with human TopoIIα in the presence of 50 µM etoposide or etoposide quinone. Reactions were run for 6 min and then were treated with SDS (to stop the reaction), EDTA (to induce ligation) then SDS after 5 min, or NaCl (to induce ligation) then SDS after 5 min. Error bars represent the standard deviation of three or more independent experiments.

Figure 4: Persistence of TopoIIα−DNA cleavage complexes does not vary significantly in the presence of etoposide or etoposide quinone. Assays were conducted in the 26


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presence of 25 µM etoposide (Etop, blue) or 25 µM etoposide quinone (EQ, red). For these reactions, DNA cleavage levels at time zero were set to 100% to allow a direct comparison and plotted on a logarithmic scale. Error bars represent the standard deviation of at least three independent experiments.

Figure 5: Etoposide quinone impairs binding of human TopoIIα to DNA. TopoIIα at 220 and 330 nM binds to plasmid DNA in the absence of compound (No Cpd) causing a slower migration of the DNA through a gel compared with the DNA control lane with plasmid only. In contrast, 50 µM of either 1,4-benzoquinone or etoposide quinone impede DNA binding. There is a greater effect when the compound is added to the enzyme prior to DNA (Pre-Cpd) than when the DNA is present before compound (PreDNA). Gels are representative of three independent experiments.

Figure 6: Etopoisde quinone impairs DNA binding by TopoIIα. Incubation of a 40-mer HEX labeled oligonucleotide duplex with increasing concentrations of human topoisomerase IIα were performed in the presence or absence of Mg2+ as denoted and were treated with 10% DMSO (ND), etoposide (Etop), or etoposide quinone (EQ). Left panel shows compounds at 10 µM, while the right panel shows compounds at 50 µM. Curves were fit with a Hill slope using Graphpad Prism. Error bars represent the standard deviation of three independent experiments.

Figure 7: Etoposide quinone reduces the ability of TopoIIα to be available for binding. Results are shown for 10% DMSO control (ND), etoposide (Etop), and etoposide quinone (EQ) with or without Mg2+ with compounds at 10 or 50 µM. Statistically 27



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Page 28 of 39

significant difference (p < 0.05) is denoted by ** and represents the comparison of ND +Mg vs 50 µM EQ +Mg. Bmax is plotted as fluorescence anisotropy units (FAU). Results are plotted as the mean and SD of Bmax values calculated by Graphpad Prism.

Figure 8: Etoposide quinone stabilizes the N-terminal clamp of TopoIIα similar to benzoquinone. Gel image shows a representative gel image for low salt (L), high salt (H), and SDS (S) washes for etoposide (Etop), etoposide quinone (EQ), and benzoquinone (BQ). Bar graph depicts the percent of plasmid DNA recovered from glass fiber filters after washing with an SDS solution by using a total DNA flow through from low salt, high salt, and SDS washes. DNA without enzyme (DNA, grey) and enzyme without compound (ND, black) are shown along with enzyme plus DNA in the presence of 100 µM etoposide (Etop, blue), etoposide quinone (EQ, red), benzoquinone (BQ, orange), or etoposide quinone with 100 µM DTT (EQ+DTT). Reactions were incubated for 10 min prior to the addition of ATP followed by an additional incubation before applying samples to the filters. Statistically significant differences (based upon one-way ANOVA with Tukey’s multiple comparisons post-test) are shown for Etop vs EQ (**p < 0.05) and for BQ vs EQ (***P < 0.001). Error bars represent the standard deviation of four or more independent experiments.

Figure 9: Etoposide quinone appears to use a two-mechanism model to impact TopoII. First, the metabolite acts in the same manner as etoposide by blocking ligation as an interfacial poison (blue). Second, the quinone appears to also act as a covalent poison (red) possibly somewhere around the N-terminal clamp (potentially at more than one site). This mechanism involves covalent adduction of the protomers, which can lead to 28


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several effects: inactivation of the enzyme likely through the blocking of DNA binding to the enzyme and can also lead to a stabilized closed-clamp form when DNA is present before the compound adducts. Further, the high proportion of DSB induced by the quinone may involve the combination of both mechanisms (purple arrows), but is likely primarily due to the trapping of the cleaved strand of DNA in the closed clamp (larger purple arrow). However, it must be noted that for there to be interfacial poisoning and high levels of DSB, the DNA must be present and bound to the enzyme prior to the quinone. Image generated using Pymol from PDB ID 4GFH.

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Figure 1

Etoposide DNA +TII

0.1

1

10

Etoposide Quinone 100

0.1

1

10

100 µM Rel

SC

30


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Figure 2

25

20

ATP Hydrolysis (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


15 ND 10

Etop 200 µM

5

Etop 25 µM EQ 25 µM

0 0

10

20

30

Time (Min)

31



Figure 3

10

8

6

4

2

DSB SSB Etoposide 50µm

SD ED S TA N aC l

SD ED S TA N aC l

SD ED S TA N aC l

0

SD ED S TA N aC l

Relative DNA Cleavage

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

DSB SSB Etoposide Quinone 50µm

32


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Figure 4

33



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Figure 5

DNA No Cpd Control 330 220

Pre-DNA Pre-Cpd Pre-DNA Pre-Cpd 330 220

330 220

1,4-Benzoquinone

34

330 220 330 220 nM (hTIIα)

Etoposide Quinone


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Figure 6

35



Figure 7 150

** 125

100

75

50

25

50 µM

10 µM

36


EQ -Mg

Etop -Mg

ND -Mg

EQ +Mg

Etop +Mg

ND +Mg

EQ -Mg

Etop -Mg

ND -Mg

EQ +Mg

Etop +Mg

0

ND +Mg

Bmax (FAU)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 8

Etop L

H

EQ S

L

H

BQ S L

H

S

*** 20

15

**

10

EQ +DTT

BQ

Etop

ND

0

EQ

5

DNA

Salt Stable DNA (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


37



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Figure 9

38


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Table 1

39


Two-Mechanism Model for the Interaction of ... - ACS Publications

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