of Etoposide Quinone with Topoisomerase II# A Two ...€¦ · 1 A Two-Mechanism Model for the Interaction of Etoposide Quinone with Topoisomerase IIαααα Elizabeth G. Gibson,†‡

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Article

A Two-Mechanism Model for the Interactionof Etoposide Quinone with Topoisomerase II#

Elizabeth G. Gibson, McKenzie M. King, Susan L Mercer, and Joseph E. DeweeseChem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00209 • Publication Date (Web): 17 Aug 2016

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1

A Two-Mechanism Model for the Interaction of

Etoposide Quinone with Topoisomerase IIαααα

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 N-

terminal 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 redox-

dependent 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, 5

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 5-

fold 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

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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 1,4-

benzoquinone 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

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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

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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 Tris-

acetate, pH 8.3, and 2 mM EDTA containing 0.5 µg/mL ethidium bromide. Double-

stranded 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 single-

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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

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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 0-

150 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 Bio-

Tek (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

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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 Tris-

HCl, 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 Post-

Test 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.

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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

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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 single-

stranded 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 redox-

dependent or covalent poisons, such as 1,4-benzoquinone.6 We hypothesized that the

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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

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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 N-

terminal 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, 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 salt-

stable 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

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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

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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 double-

stranded 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.

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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.

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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, single-

stranded DNA break; TopoII, topoisomerase II.

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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

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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 (Pre-

DNA). 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

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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

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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

DNA +TII 0.1 1 10 100 0.1 1 10 100

Etoposide Etoposide Quinone

µM

Rel

SC

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Figure 2

0 10 20 30

0

5

10

15

20

25

Time (Min)

ATP Hydrolysis (%)

ND

EQ 25 µM

Etop 200 µM

Etop 25 µM

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Figure 3

SDS

EDTANaCl

SDS

EDTANaCl

SDS

EDTANaCl

SDS

EDTANaCl

0

2

4

6

8

10

Relative DNA Cleavage

Etoposide Etoposide Quinone

SSBDSB SSBDSB

50µm 50µm

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Figure 4

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Figure 5

No Cpd DNA Control

Pre-DNA Pre-Cpd

330 220

1,4-Benzoquinone Etoposide Quinone

330 220 330 220 330 220 330 220 nM (hTIIα)

Pre-DNA Pre-Cpd

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Figure 6

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Figure 7

ND +Mg

Etop +Mg

EQ +Mg

ND -Mg

Etop -Mg

EQ -Mg

ND +Mg

Etop +Mg

EQ +Mg

ND -Mg

Etop -Mg

EQ -Mg

0

25

50

75

100

125

150

Bmax (FAU)

10 µM 50 µM

**

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Figure 8

DNA

ND

Etop

EQ

BQ

EQ +DTT

0

5

10

15

20

Salt Stable DNA (%)

**

***

L H S L H S L H S

Etop EQ BQ

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Figure 9

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Table 1

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Documents

of Etoposide Quinone with Topoisomerase II# A Two ...€¦ · 1 A Two-Mechanism Model for the Interaction of Etoposide Quinone with Topoisomerase IIαααα Elizabeth G. Gibson,†‡