1521-0111/91/1/49–57$25.00 http://dx.doi.org/10.1124/mol.116.106054MOLECULAR PHARMACOLOGY Mol Pharmacol 91:49–57, January 2017Copyright ª 2016 The Author(s).This is an open access article distributed under the CC BY Attribution 4.0 International license.

Myeloperoxidase Enhances Etoposide andMitoxantrone-Mediated DNA Damage: A Targetfor Myeloprotection in Cancer Chemotherapy s

Mandeep Atwal, Emma L. Lishman, Caroline A. Austin, and Ian G. CowellInstitute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne. United Kingdom

Received July 8, 2016; accepted November 8, 2016

ABSTRACTMyeloperoxidase is expressed exclusively in granulocytes andimmature myeloid cells and transforms the topoisomerase II(TOP2) poisons etoposide and mitoxantrone to chemical formsthat have altered DNA damaging properties. TOP2 poisons arevaluable andwidely used anticancer drugs, but they are associatedwith the occurrence of secondary acute myeloid leukemias. Thesefactors have led to the hypothesis that myeloperoxidase inhibitioncould protect hematopoietic cells from TOP2 poison-mediatedgenotoxic damage and, therefore, reduce the rate of therapy-related leukemia.We showhere thatmyeloperoxidase activity leadsto elevated accumulation of etoposide- and mitoxantrone-inducedTOP2A and TOP2B-DNA covalent complexes in cells, which areconverted to DNA double-strand breaks. For both drugs, the effect

ofmyeloperoxidase activity was greater for TOP2B than for TOP2A.This is a significant finding because TOP2B has been linked togenetic damage associatedwith leukemic transformation, includingetoposide-induced chromosomal breaks at the MLL and RUNX1loci. Glutathione depletion, mimicking in vivo conditions experi-enced during chemotherapy treatment, elicited further MPO-dependent increase in TOP2A and especially TOP2B-DNAcomplexes and DNA double-strand break formation. Togetherthese results support targeting myeloperoxidase activity to reducegenetic damage leading to therapy-related leukemia, a possibilitythat is enhanced by the recent development of novel specificmyeloperoxidase inhibitors for use in inflammatory diseases in-volving neutrophil infiltration.

IntroductionDrugs targeting DNA topoisomerase II (TOP2 poisons) are

important, effective, and widely used anticancer agents, butthey are associated with short- and long-term toxic sideeffects, including neutropenia and rare but life-threateningtherapy-related acute myeloid leukemia (t-AML) (Allan andTravis, 2005; Leone et al., 2010; Cowell and Austin, 2012). Ascancer survival rates have increased, t-AML has become amore important clinical problem, and it is estimated that upto 15% of all acute myeloid leukemia cases can be classifiedas t-AML (Mauritzson et al., 2002). Therapy-related acuteleukemias occur after a wide range of primary neoplasias, butprior treatment of breast cancer accounts for about 50% ofcases, while hematological malignancies account for approx-imately 30% (Kayser et al., 2011). In its normal cellular role,TOP2 facilitates changes to DNA topology by allowing onedouble stranded segment to pass through another via anenzyme-bridged DNA double-strand break (DSB) (Austin and

Marsh, 1998; Vos et al., 2011; Cowell andAustin, 2012). In thisconfiguration, each protomer of the homodimeric TOP2 en-zyme is covalently coupled to a cleaved DNA strand via a 59-phosphotyrosine linkage. TOP2 poisons such as etoposide andmitoxantrone exert their tumoricidal effect by stabilizing thisnormally transient enzyme-bridged break, resulting in theaccumulation of cytotoxic covalently linked TOP2 protein-DNA complexes, which can be processed in the cell to DNAdouble-strand breaks (Burma et al., 2001; Cowell and Austin,2012; Lee et al., 2012, 2016). Therapy related leukemias,especially those appearing after exposure to TOP2 poisonsoften contain recurrent chromosome translocations, includ-ing t(15,17)(PML-RARA), t(8,21)(AML-ETO), and 11q23 re-arrangements involving the MLL gene (Rowley and Olney,2002; Cowell and Austin, 2012). These genetic lesions disruptblood cell development and play a pivotal role in the develop-ment of the disease. Such t-AML cases arise as a result ofTOP2 poison-mediated DNA damage in bone marrow bloodprecursor cells. There are two vertebrate TOP2 paralogues,TOP2A and TOP2B; TOP2 poisons such as etoposide affectboth paralogues, but recent evidence points to a greater rolefor TOP2B in generating the genotoxic damage associatedwith TOP2 poisons (Azarova et al., 2007; Cowell et al., 2012;Smith et al., 2014a).

This work was supported by Breast Cancer Now [Grant 2012Novem-berPhD11] and Bloodwise [Programme Grant 12031].

dx.doi.org/10.1124/mol.116.106054.s This article has supplemental material available at molpharm.

aspetjournals.org.

ABBREVIATIONS: AML, acute myeloid leukemia; BSO, buthionine sulfoximine; DAPI, 4’,6-diamidino-2-phenylindole; DSB, DNA double-strandbreak; GSH, reduced glutathione; H2AX, histone H2A.X; gH2AX, S-139 phospho histone H2A.X; MPO, myeloperoxidase; MPOi-II, 4-(5-fluoro-1H-indol-3-yl)butanamide; PF-1355, 2-(6-(2,5-dimethoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide; SA, succinylacetone; TAR-DIS, trapped in agarose DNA immunostaining assay; t-AML, therapy-related acute myeloid leukemia; TOP2, topoisomerase II; TOP2A, DNAtopoisomerase IIb; TOP2B, DNA topoisomerase IIb; TOP2-CC, topoisomerase II covalent complex.

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We are interested in why cells of the myeloid hematopoieticlineage are sensitive to TOP2 poison-mediated genotoxicdamage, which leads to t-AML, and how this sensitivity couldbe reduced. Myeloperoxidase is expressed exclusively incells of the myeloid lineage; it is present at high levels inneutrophils where it exerts its antimicrobial action but is alsoexpressed in myeloid precursor/progenitor cells, includinghuman and mouse common myeloid progenitor and granulo-cyte/macrophage progenitor cells (Strobl et al., 1993; Moriet al., 2009; Goardon et al., 2011) and is readily detectable inex vivo normal human bone marrow CD341 cells (Supple-mental Fig. 1) (Strobl et al., 1993; Vlasova et al., 2011). Thus,MPO is likely to be present in the cells in which t-AML arises.In its physiologic role MPO generates hypochlorous acid fromhydrogen peroxide and chloride ions to kill pathogenic micro-organisms. However, MPO activity also leads to the oxidativeactivation of etoposide. This occurs by one-electron oxidationof the etoposide E-ring, yielding a phenoxy radical species andbyO-demethylation to the reactive orthoquinone (Supplemen-tal Fig. 2A) (Haim et al., 1986; Kalyanaraman et al., 1989;Kagan et al., 2001; Fan et al., 2006; Jacob et al., 2011; Vlasovaet al., 2011). Additionally, CYP3A4 and/or CYP3A5 can oxidizeetoposide to etoposide catechol, a metabolite found in patientplasma (Zheng et al., 2004; Zhuo et al., 2004), and oxidation ofetoposide by CYP3A4 has been implicated in the incidence oft-AMLs (Felix et al., 1998). Etoposide catechol can be oxidized toetoposide quinone by MPO via a semiquinone species (Supple-mental Fig. 2A). Etoposide quinone is more effective at inducingTOP2-mediatedDNAbreaks than etoposide in vitro (Jacob et al.,2011) and this is particularly true for TOP2B (Smith et al.,2014b). This increased potency of etoposide quinone as a TOP2poison has been attributed to covalent modification of the en-zyme by the quinone (Jacob et al., 2011; Smith et al., 2014b).Furthermore, etoposide metabolites have the potential to formadducts with DNA and protein (Haim et al., 1987; Lovett et al.,2001), which may also impact on genotoxicity.Oxidative activation of TOP2 poisons is not limited to

etoposide because MPO has been implicated in the activationof mitoxantrone either directly or via the production ofreactive aldehydes (Panousis et al., 1995, 1997; Hazen et al.,1998; Parker et al., 1999; Evison et al., 2016).Using pharmacological inhibition and genetic manipula-

tion we show that MPO activity increases etoposide andmitoxantrone-induced formation of TOP2A and TOP2B cova-lent enzyme-DNA complexes in cells of myeloid origin andsimilarly increases DSB formation. GSH depletion amplifiedthis effect. This is consistent with the hypothesis that MPOinhibition, in a clinical setting, especially where cellular thiollevels are suppressed, could protect against genotoxic damagein the myeloid compartment by TOP2 poisons.

Materials and MethodsReagents and Antibodies. Etoposide, mitoxantrone, succinylacetone

(SA), dimethyl sulfoxide, Tween 20, Triton X-100, paraformaldehyde,DL-buthionine sulfoximine and PF-1355 (2-(6-(2,5-dimethoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide) were purchasedfrom Sigma-Aldrich (Dorset, UK). MPOi-II (4-(5-fluoro-1H-indol-3-yl)butanamide) was purchased fromMerck-Millipore (Watford, UK). Anti-MPO ab9535 (immunofluorescence) and ab134132 (Western blotting) werefrom Abcam (Cambridge UK), anti-mouse gH2AX 05-636 was obtainedfrom Merck-Millipore.

Cell Culture. All cell lines were maintained in RPMI-1640medium supplemented with 10% fetal bovine serum and 1% penicillinand streptomycin (Thermo Fisher Scientific, Paisley, UK). Cells werecultured at 37°C in a humidified atmosphere containing 5% CO2.Experiments were conducted on cells growing in log phase (2–5 �105cells/ml). For succinylacetone treatment of cells to downregulateMPO, cells were treated with 200 mM SA for 48 hours before additionof TOP2 poison and downstream analysis. SA was retained duringTOP2 poison treatment.

Stable Transfection of K562 Cells with MPO cDNA. K562cells were transfected with plasmid RC2016029 containing the MPOcoding sequence with a C-terminal MYC-DDK tag in the vector pCMV6-Entry (Origene, Rockville, MD). Transfection was performed usingDharmafect Duo (Dharmacon, Amersham, UK) transfection reagent.Transfectionmixtures contained 1 mg/ml of MPO plasmid (Origene) orG418 control plasmid with 20 mg/ml of Dharmafect Duo (Dharmacon)and 80% v/v serum-free RPMI 1640 medium. After selection withG418 (750 mg/ml), clonal lines were isolated by limited dilution. MPO-expressing lines were continuously grown in 500 mg/ml G418, theselection antibiotic. MPO expression was assessed using immunoflu-orescence and MPO activity assays.

MPO and GSH Colorimetric Activity Assays. MPO activityassays were performed using an Abcam MPO Activity Assay kit(ab105136, Abcam). Cells were harvested by centrifugation at 1000 gfor 5 minutes and homogenized in 4 pellet volumes of lysis bufferprovided. Bradford assays were conducted to ensure an equalconcentration of protein was used for each assay. The MPO activityassay was conducted according to manufacturer’s instructions withabsorbance readings measured at 415 nm. GSH assays were per-formed using a GSH assay kit (KA0797, Abnova, Taipei City, Taiwan),according to the manufacturer’s instructions.

Immunoblotting for MPO. Whole cell lysates of cells wereprepared (Mirski et al., 1993) and samples were resolved on precast4–20% SDS-polyacrylamide gels (NuSep, Wasserburg, Germany).Western blotting was performed by standard methods using ECLdetection.

Trapped in Agarose DNA Immunostaining Assay. Trapped inagarose DNA immunostaining (TARDIS) assays to quantify TOP2covalent protein-DNA complexes were carried out essentially asdescribed previously (Willmore et al., 1998; Cowell et al., 2011b).Briefly cells were treated for 1 hour with the desired dose of etoposideor mitoxantrone. Cells were then pelleted and washed in ice-cold PBS.After being recentrifuged, cells were mixed with molten 2% LMPagarose (Lonza, Basel, Switzerland) in PBS at 37°C and spread evenlyonto agarose coated slides. Agarose-embedded cells were lysed in (1%w/v SDS, 20 mM sodium phosphate, 10 mM EDTA, pH 6.5) andnoncovalently DNA bound proteins were removed using 1 M NaCl.TOP2 covalent complexeswere detected by immunofluorescence usingrabbit anti-TOP2A (4566) or anti-TOP2B (4555) antibodies, raised tothe C-terminal domain of human TOP2A and TOP2B, respectively,and Alexa-488 coupled anti-rabbit secondary antibodies (ThermoFisher Scientific). Slides were counterstained with Hoechst 33258 tovisualize DNA. Quantitative immunofluorescence was performed bycapturing images for Hoechst and Alexa-488 using a epifluorescencemicroscope (Olympus IX-81, Olympus, Tokyo, Japan) fitted with anOrca-AG camera (Hamamatsu, Tokyo, Japan) and suitable narrowband filter sets. Images were analyzed using Volocity 6.3 (PerkinElmer, Waltham, MA), and data representation and statistics wereperformed using GraphPad Prism 4.0 and R (San Diego, CA).

Immunofluorescence Analysis of gH2AX and MPO. Afterdrug treatment cells were washed and pelleted in ice-cold PBS andspotted onto poly-L-lysine slides. Cells were fixed in 4% formaldehydein PBS and permeabilized using KCM1T buffer (120 mMKCl, 20 mMNaCl, 10 mMTris-HCl pH 8.0, 1 mMEDTA, 0.1% Triton X-100). Afterblocking in (KCM1T, 2% bovine serum albumin, 10% dry milkpowder) cells were probed with primary anti-MPO antibody or anti-gH2AX in blocking buffer and Alexa-488 anti-rabbit or Alexa-594 anti-mouse secondary antibodies (Thermo Fisher Scientific). Slides were

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counterstained with DAPI (4’,6-diamidino-2-phenylindole) (VectorLaboratories, Burlingame, CA) and viewed using an epifluorescencemicroscope (Olympus IX-81). For gH2AX quantification, images werecaptured for DAPI and Alexa-594 and quantitative analysis wasperformed using Volocity 6.3 (Perkin Elmer) with data representationperformed in GraphPad Prism 4.0 and R.

GSH Depletion. NB4 cells were treated with 150 mM buthioninesulfoximine (BSO) for 4.5 hours (Griffith, 1982; Gantchev and Hunting,1997) before addition of TOP2 poison. BSO was retained in the mediumduring TOP2 poison treatment.

Statistics. Data are presented as themean values6 S.E.M. valuesfor n $ 3 replicate experiments; the number of replicates involved foreach treatment is indicated within the figures. Statistical analysiswas performed by one-way ANOVA with post hoc Tukey’s multi-ple comparison test. For signifying P values, * refers to P , 0.05,** refers to P , 0.01, and *** refers to P , 0.001.

ResultsMPO Inhibition Suppresses Etoposide-Induced

TOP2A and TOP2B Covalent DNA Complex Formationin NB4 Cells. MPO activity can be efficiently reduced in cellculture systems by the heme biosynthesis inhibitor succiny-lacetone (SA) (Pinnix et al., 1994; Kagan et al., 2001; Vlasovaet al., 2011). By using NB4 cells, an acute promyleocyticleukemia line that express MPO at a high level (Hu et al.,1993), we found that incubation with 200 mM SA for 48 hoursreducedMPO enzymatic activity to below the detection level ofthe assay employed, and as reported previously (Pinnix et al.,1994), it significantly depleted mature MPO protein (Fig. 1, Aand B). Quantification of replicate blots indicated that matureMPO protein level was reduced to less than 25% of that inuntreated cells. Notably, SA treatment of up to 72 hours didnot affect TOP2A or TOP2B protein levels in NB4 cells,accelerate acidification of the medium, nor reduce cell via-bility or cell growth (Supplemental Fig. 2, B–E). Similarly,SA did not affect TOP2A or TOP2B enzymatic activity invitro (Supplemental Fig. 2, F–H). We used the TARDISassay (Willmore et al., 1998; Cowell et al., 2011b) to quan-tify stabilized TOP2-DNA covalent complexes in etoposide-treated cells. This assay employs sensitive quantitativeimmunofluorescence to analyze TOP2 content in agaroseembedded “nuclear ghosts” that remain after noncovalentlyattached proteins and other cellular constituents have beenremoved from nuclear DNA by SDS-salt extraction (Supple-mental Fig. 3, A and B). Pretreatment with SA significantlyreduced the levels of etoposide-induced TOP2A and TOP2B-DNA complexes. This was true for both 10 and 100 mMetoposide (Fig. 1, C and D; Supplemental Fig. 3, A and B). Atboth doses of etoposide, the magnitude of the affect wasgreater for TOP2B than for TOP2A. For TOP2B with100 mM etoposide SA pretreatment resulted in a 38% re-duction in TOP2-complex formation, whereas the reductionwas 12% for TOP2A. For 10 mM etoposide, the respectivefigures were 55% and 18% (Fig. 1, C and D; SupplementalTable 1). As expected, SA pretreatment did not affect stabi-lized TOP2A or TOP2B-DNA complexes in K562 cells, achronic myeloid leukemia-derived cell line that does notexpress MPO (Supplemental Fig. 3, C and D). Etoposidequinone displays enhanced TOP2-mediated DNA cleavageactivity in vitro compared with the parent compound (Jacobet al., 2011), and this is more pronounced for TOP2B (Smithet al., 2014b). The observation that SA pretreatment, which

reducesMPO-mediated etoposide phenoxy radical production,partially suppressed etoposide-induced TOP2-CC formationin MPO-expressing NB4 cells supports the conclusion thatoxidative metabolism of etoposide plays a role in TOP2-mediated DNA damage in vivo.

MPO Expression in K562 Cells Stimulates Etoposide-Induced TOP2-DNA Covalent Complex Formation.K562 is a chronic myeloid leukemia derived cell line thatdoes not express detectable MPO (Hu et al., 1993) (Supple-mental Fig. 1). We transfected K562 cells with a human MPOexpression construct and isolated clonal K562 lines expressingMPO. Two K562MPO cell lines (MPO line 4 and MPO line 5)exhibited MPO activity at 37 6 7.5% and 46 6 6.0% of thelevel of NB4 cells, respectively. Both cell lines expressed MPOin all cells by immunofluorescence (Fig. 2A). Parental K562cells and K562MPO cell lines 4 and 5 were treated with 10 or100 mM etoposide and stabilized TOP2-DNA complexes werequantified. The K562MPO cell lines displayed significantlyhigher levels of drug-stabilized complexes for both TOP2isoforms compared with K562 cells lacking MPO expressionat 100 mM etoposide. At 10 mM etoposide, raised levels ofTOP2A and TOP2B stabilized complexes were observed withMPO line 5, and for line 4 significantly raised complex levels

Fig. 1. MPO inhibition reduces the level of TOP2-DNA covalent com-plexes formed by etoposide in NB4 cells. (A and B) Succinylacetone (SA)abolishedMPO activity (A) and reduced mature protein levels in NB4 cells(B); NB4 cells were treated with 200 mM SA for 48 hours and assayed forMPO activity (A) and MPO protein level by Western blotting of whole cellextracts (B). Blots were quantified by densitometry. (C and D) TOP2-DNAcovalent complexes were quantified by TARDIS analysis using antibodiesspecific to TOP2A (C) or TOP2B (D). Integrated fluorescence values weredetermined per nucleus (at least 500 nuclei per treatment per replicateexperiment). From these, median values were obtained for each treatmentand means of the medians were calculated from replicate experiments (n =3). Data are expressed as a percentage of the mean value obtained with100 mM etoposide in the absence of SA, 6S.E.M. Integrated fluorescencedata corresponding to an individual experiment are also shown inSupplemental Fig. 3, A and B. *P , 0.05.

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were observed only for TOP2A (Fig. 2B). After subtractingbackground complex levels, K562MPO line 5 displayed a 1.6-and 2.0-fold increase in TOP2A- and TOP2B-DNA complexes,respectively, compared with K562 after 100 mM etoposidetreatment. For line 4, which expressedMPO at a lower level, asmaller fold increase in complexes was observed (Supplemen-tal Table 1). By comparison, K562 cells transfected with anempty expression vector did not display increased TOP2covalent complex formation (Supplemental Fig. 3, G and H).

This further supports the notion that MPO-mediated activa-tion contributes to etoposide-mediated DNA damage in MPOexpressing cells.MPO Contributes to Etoposide-Induced Cellular

DNA Damage. TOP2 poison-induced covalent DNA complexesare processed to DSBs that result in histone H2AX phosphory-lation (Sunter et al., 2010; Cowell et al., 2011a, 2012) as part ofa well-established response involving activation of the DNAdamage kinase ataxia-telangiectasia mutated (Burma et al.,2001). Thus,gH2AXcanbeusedas ameasure ofDSBsgeneratedby etoposide treatment. The inhibition ofMPOusing SA resultedin a 40% reduction in gH2AX induced by 100 mM etoposide (Fig.3A; Supplemental Table 1). Notably, this is equivalent to thedifference in gH2AX signal observed between 10 and 100 mMetoposide in the absence of SA. SA did not significantly affect thelevel of gH2AX observed after exposure to 10 mM etoposide. SAhad no effect on etoposide-induced gH2AX in K562 cells, whichdo not express MPO (Supplemental Fig. 3E).Etoposide-induced gH2AX formation was also examined in

K562MPO cell lines. For 10 mM etoposide, MPO expressionresulted in a significant rise in gH2AX intensity (Fig. 3B),reaching a 1.8-fold increase over K562 cells for line 5 (Supple-mental Table 1). At 100 mM etoposide, neither cell linedisplayed a significant increase in gH2AX over K562. How-ever, the lack of significant effect of MPO expression whencells were treated with 100 mM compared with the loweretoposide dose can be explained by the observation thatgH2AX formation reaches saturation at 100 mM etoposide inK562 cells (Supplemental Fig. 3F).K562 cells transfected with empty vector were also tested

for induction of gH2AX formation in comparison with non-transfected K562 cells. The empty vector control did not differsignificantly from the nontransfected cells in gH2AX signalformation upon etoposide treatment (Supplemental Fig. 3I).Thus, MPO activity results in elevatedH2AX phosphorylationin etoposide-treated cells.MPO Stimulates Mitoxantrone-Induced Accumulation

of TOP2-DNA Complexes and H2AX Phosphorylation.The inhibitor SA, which effectively abolishes MPO activityin NB4 cells (Fig. 1), resulted in a substantial reduction instabilized TOP2-DNA complexes induced by mitoxantrone(Fig. 4A). After subtraction of signal detected in control cells(0 mM mitoxantrone), TOP2A and TOP2B complexes in-duced by 0.5 mM mitoxantrone were reduced by 59 and 88%,respectively, whereas at 1 mMmitoxantrone the figures were

Fig. 3. MPOactivity results in raised levels of etoposide-induced H2AX phosphorylation. (A) NB4 cells werepretreated for 48 hours with SA (200 mM) thenincubated with etoposide (10 or 100 mM). (B) K562and K562MPO cell lines were incubated with 10 and100 mM etoposide or dimethyl sulfoxide vehiclecontrol. For both (A) and (B), gH2AX was quantifiedby immunofluorescence. Analysis was carried out asdescribed for TARDIS analysis in Fig. 1. Data areexpressed relative to the mean values obtained with100 mM etoposide in the absence of SA (A) or in wild-type parental K562 cells (B). Numbers of replicatesare indicated. *P , 0.05; **P , 0.01; ***P , 0.001.

Fig. 2. Expression of active MPO in K562 cells increases the level ofetoposide-stabilized TOP2-DNA covalent complexes. (A) Comparison ofMPOactivity (left) andMPO immunofluorescence (right) in K562 cells, twoMPO-expressing K562-derived cell lines (K562MPO line4 K562MPO line5), andin NB4 cells. (B) K562 and K562MPO cell lines were incubated with 10 or100 mM Etoposide or a vehicle control for 1 hour. Etoposide-stabilizedTOP2-DNA complexes were quantified by TARDIS analysis using anti-bodies specific for TOP2A or TOP2B as described for Fig. 1. Numbers ofreplicates are indicated. *P , 0.05; **P , 0.01; ***P , 0.001.

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43% and 63% (Supplemental Table 1). Similarly, SA pre-treatment reduced the mitoxantrone induced gH2AX signalby 39% for 1 mM mitoxantrone (Fig. 4B).K562MPO cell line 2 expresses MPO activity at 20% of the

level observed in NB4 cells (Fig. 4C). Mitoxantrone (1 mM)induced 2.2- and 2.9-fold more stabilized TOP2A and TOP2Bcomplexes in K562MPO cell line 2 than in parental K562 cells(Fig. 4D; Supplemental Table 1).Like etoposide, mitoxantrone is prone to oxidation by

peroxidases such as MPO, and mitoxantrone can form DNAadducts via activation by formaldehyde (Panousis et al., 1995;Parker et al., 1999), but less is known about the impact of thison TOP2-DNA complex formation in cells or the downstreamaccumulation of DNA DSBs. The observed MPO activityrequirement for maximal TOP2 complex formation as wellas H2AX phosphorylation supports the idea that MPO-mediated oxidation directly or indirectly enhances the activityof mitoxantrone as a TOP2 poison.Glutathione Depletion Stimulated Etoposide- and

Mitoxantrone-Induced DNA Damage in an MPO-DependentManner. Etoposide and mitoxantrone metabolites reactwith the low molecular weight thiol GSH (Yalowich et al.,

1996; Panousis et al., 1997; Kagan et al., 2001; Fan et al.,2006). It follows that the effect of high MPO expression onTOP2 poison-mediated DNA damage in cells may thereforebe limited by cellular GSH. To test this we used buthioninesulfoximine (BSO), which inhibits g-glutamylcysteine syn-thetase and thus leads to GSH depletion in cells (Griffith,1982). The addition of BSO did not affect TOP2 activityin in vitro activity assays (Supplemental Fig. 2, F–H).Glutathione levels were reduced by more than 70% afterpretreatment with BSO (150 mM, 4.5 hours; Fig. 5A). BSO-treated NB4 cells exhibited elevated levels of TOP2A- andTOP2B-DNA complexes induced by 10 mM etoposide (1.9- and3.4-fold, respectively), and for 100 mM etoposide BSO in-creased TOP2B-DNA complexes (1.6 fold) but did not increaseTOP2A-DNA complex levels (Fig. 5, B and C; SupplementalTable 2). In addition, BSO pretreatment significantly in-creased etoposide-induced H2AX phosphorylation at bothdoses of etoposide (2.2- and 2.0-fold increase, respectively;Fig. 5D; Supplemental Table 2). Significantly, BSO had noeffect on TOP2 complexes or H2AX phosphorylation inducedby etoposide when cells were pretreated with the MPOinhibitor SA. This is consistent with the previously described

Fig. 4. MPO activity enhances mitoxantrone-induced TOP2-DNA covalent complex formation and H2AX phosphorylation. (A and B) NB4 cells werepretreated with 200 mM SA for 48 hours followed by a 1-hour incubation with 0.5 or 1 mM mitoxantrone, or a vehicle control. TOP2-DNA covalentcomplexes and gH2AX were quantified as in Figs. 1 and 3. Data are expressed relative to the mean values obtained with 1 mM mitoxantrone in theabsence of SA. (C) Quantification of MPO activity in K562MPO line 2 compared with NB4 and parental K562 cells. (D) MPO expression in K562 cellsresults in enhanced mitoxantrone-induced TOP2-DNA protein complex formation. Data are expressed relative to the mean value obtained for parentalK562 cells treated with 1 mM mitoxantrone. Numbers of replicates are indicated. *P , 0.05; **P , 0.01; ***P , 0.001.

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MPO-dependent (and therefore SA-suppressed) generation ofetoposide quinone via the phenoxy-radical (Haim et al., 1986;Kagan et al., 2001), leading to elevated TOP2-DNA complexaccumulation and histone H2AX phosphorylation due toelevated TOP2 poison activity (Gantchev and Hunting,1998; Jacob et al., 2011). This activity is then enhancedunder conditions of depleted glutathione, which wouldotherwise reduce the phenoxy radical and/or combine withetoposide quinone. BSO pretreatment also resulted in elevatedmitoxantrone-induced stabilized TOP2-DNA complexes andH2AX phosphorylation (Fig. 6, A–C), although the magnitudeof the effect was smaller than for etoposide (Fig. 5; Supplemen-tal Table 3).

Direct Small Molecule Inhibitors of MPO SuppressEtoposide- and Mitoxantrone-Induced TOP2A andTOP2B Covalent DNA Complex Formation and H2AXPhosphorylation in NB4 Cells. The work described aboveemployed the heme synthesis inhibitor SA that indirectlyreduces cellular MPO activity. Recently a number of directspecific MPO inhibitors have been developed. These includePF-1355 (2-(6-(2,5-dimethoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide) (Zheng et al., 2015) andMPOi-II (4-(5-fluoro-1H-indol-3-yl)butanamide) (Soubhyeet al., 2013). As expected, both of these inhibitors dramaticallyreduced MPO activity in NB4 cells (Supplemental Fig. 4).After 4 hours, MPO activity was undetectable in PF-1355-treated cells and for MPOi-II activity was reduced by morethan 90%. Pretreatment with either inhibitor also reduced the

levels of TOP2A and TOP2B DNA complexes and H2AXphosphorylation induced by etoposide or mitoxantrone inNB4 cells, having a greater effect on TOP2B (Fig. 7). Themagnitudes of the effects were comparable to those observedwith SA pretreatment (Supplemental Table 2; Figs. 1, C andD, 3A, and 4, A and B). As was observed for SA, PF-1355 andMPOi-II did not affect induction of TOP2A or TOP2B com-plexes or H2AX phosphorylation induced by etoposide ormitoxantrone in MPO nonexpressing K562 cells (Supplemen-tal Fig. 4).

DiscussionTherapy-related acute leukemia is a rare, but life threaten-

ing, complication of prior treatment of a primary cancer.Therefore, interventions that specifically protect myeloid cellsfrom the unwanted effects of cytotoxic chemotherapies wouldbe very welcome. MPO is expressed exclusively in cells ofmyeloid origin and is capable of enzymatic conversion of TOP2poisons, including etoposide, mitoxantrone, and anthracy-clines, to species with greater DNA damaging activity. So, inprinciple, MPO inhibition should partially protect myeloidprecursors from TOP2 poison-mediated DNA damage, whilepreserving the desired cytotoxic effects in the target tumorcells. This idea is supported by the data reported here showingthat three chemically distinct MPO inhibitors each reduceetoposide and mitoxantrone-induced TOP2-DNA complexand gH2AX formation in NB4 cells, whereas conversely,

Fig. 5. Glutathione depletion increases etoposide-mediated TOP2-DNA covalent complex formation and H2AX phosphorylation. (A) BSO preincubation(150 mM, 4.5 hours) resulted in 70% reduction of total and reduced glutathione in NB4 cells. (B–D) NB4 cells were incubated in the presence or absence ofSA (200 mM) for 48 hours, BSO (150 mM) for 4.5 hours, with both or with neither, followed by addition of 10 or 100 mM etoposide for 1 hour. TARDIS andgH2AX assays were performed as described in Figs 1 and 3. Numbers of replicates are indicated. *P , 0.05; **P , 0.01; ***P , 0.001.

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exogenous MPO expression in K562 cells leads to increasedetoposide- and mitoxantrone-induced damage.Notably, the suppression of MPO activity had an approxi-

mately threefold greater effect on TOP2B complex formationthan on TOP2A. The greater effect for TOP2B is of interest,because this TOP2 isoform appears to be required for the

majority of etoposide-inducedMLL and RUNX1 chromosomalbreaks (Cowell et al., 2012; Smith et al., 2014a) in a humanlymphoblastoid cell line model and for etoposide-mediatedcarcinogenesis in a mouse model (Azarova et al., 2007). TheMPO-dependent enhancement of the TOP2 poisoning activityof etoposide is likely to be due to production of etoposidephenoxy radicals and etoposide ortho-quinone in MPO-expressing cells (see Supplemental Fig. 2A). Etoposide phe-noxy radicals have been observed in vitro and in cell culturesystems (Haim et al., 1986; Kalyanaraman et al., 1989;Yalowich et al., 1996; Kagan et al., 2001; Vlasova et al.,2011) and can be further oxidized to etoposide quinone (Haimet al., 1986; Fan et al., 2006), which exhibits enhanced TOP2-dependent DNA cleavage activity compared with etoposide,especially for TOP2B (Jacob et al., 2011; Smith et al., 2014b).Etoposide ortho-quinone reacts spontaneously with GSH insolution (Fan et al., 2006), and so the effect of MPO expressionon TOP2-DNA complex accumulation may be limited by GSHtitration of etoposide quinone within cells. Indeed, we foundthat when GSH was depleted by BSO, treatment of NB4 cellswith 10 mM etoposide resulted in significantly elevatedTOP2A- and TOP2B-DNA complex accumulation (1.9- and3.4-fold, respectively), and this effect was abolished by SApretreatment (Fig. 5). Notably, although glutathione deple-tion amplified the effect of SA on etoposide induced TOP2-DNA complexes, we were still able to measure suppressionof etoposide-induced TOP2A and TOP2B complexes in theabsence of BSO.Although these data were mostly obtained with NB4 cells,

which express MPO at a high level (Supplemental Fig. 1) (Huet al., 1993), enhanced etoposide-induced TOP2-DNA complexformation and H2AX phosphorylation were also observed inK562 cells exogenously expressing MPO activity at less than50% of the level of NB4 cells. For the analogous mitoxantroneexperiments, exogenously expressed MPO resulted in a 1.6-and 2.9-fold increase in TOP2A- and TOP2B-DNA complexes,respectively, even though MPO was only expressed at 20% ofthe NB4 level. Thus, it appears that MPO can stimulateTOP2-mediated DNA damage even when expressed at mod-erate levels more similar to those that exist in bone marrowmyeloid precursor cells.While our results with etoposide in NB4 cells can be

explained by etoposide redox activity in MPO expressing cellsand the finding that etoposide quinone is a more effectiveTOP2 poison than its parent compound in vitro (Jacob et al.,2011; Smith et al., 2014b), the situation is less clear formitoxantrone. Mitoxantrone can be oxidized by MPO, andproducts of this oxidation react covalently with DNA, both incell-free systems and in cells (Panousis et al., 1995, 1997).Furthermore,mitoxantrone is activated by formaldehyde, alsoresulting in covalent interaction with DNA (Parker et al.,1999). However, it is currently unclear whether the metabolicproducts of mitoxantrone function as TOP2 poisons, and if sowhat their relative activity is compared with the parentcompound. We show here that MPO downregulation signifi-cantly impairs mitoxantrone-induced TOP2-DNA complexaccumulation in NB4 cells. Again the effect is larger forTOP2B than TOP2A. For 0.5 mM mitoxantrone, SA preincu-bation decreased TOP2B-DNA complexes to a level that wasno longer significantly above the background level obtained inthe absence of mitoxantrone. Exogenous expression of MPO inK562 cells resulted in a more than doubling in TOP2A- and

Fig. 6. Glutathione depletion potentiates mitoxantrone-mediated TOP2covalent complex formation and H2AX phosphorylation. NB4 cells werepreincubated with SA, BSO, or both as described for Fig 4, followed byaddition of 1 mM mitoxantrone for 1 hour. TOP2A TARDIS (A), TOP2BTARDIS (B), and gH2AX assays (C) were performed as described in Figs 1and 3. *P , 0.05; **P , 0.01; ***P , 0.001.

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TOP2B-DNA complexes induced by mitoxantrone. Together,these data strongly suggest that oxidative metabolism ofmitoxantrone leads to increased TOP2A and TOP2B poison-ing. However, our data do not distinguish between directenhanced poisoning of TOP2 by mitoxantrone metabolites,and generation of DNA adducts or repair intermediates thatact as TOP2 poisons (Kingma et al., 1995; Sabourin andOsheroff, 2000).As was observed for etoposide, GSH depletion increased the

level of TOP2A- and TOP2B-DNA complexes induced bymitoxantrone consistent with the involvement of a metabolitethat reacts readily with free thiols. Furthermore, high in-tensity chemotherapy leads to depletion of thiols includingGSH (Jonas et al., 2000; Kasapovi�c et al., 2010; Kadam andAbhang, 2013), and so the high level of TOP2 poison-inducedDNA damage observed in BSO-treated cells is likely to reflectthe generation of genetic lesions in bone marrow cells duringcytotoxic chemotherapy regimens.MPO has become a target of interest for novel anti-

inflammatory drug development (Malle et al., 2007), and anumber of potent specific MPO inhibitors were recentlyreported (Tidén et al., 2011; Forbes et al., 2013; Soubhyeet al., 2013, 2016; Li et al., 2015; Zheng et al., 2015). We haveused two such inhibitors, PF1355 andMPOi-II, and show that

like SA, they reduce etoposide- and mitoxantrone-induceddamage in MPO-expressing cells. Thus, novel compoundsdeveloped for a different clinical need could, in principle, berepurposed to reduce unwanted and carcinogenic TOP2poison-induced genotoxic damage in critical bone marrowcells and could have a significant impact in the frequency oftherapy-induced secondary leukemias.

Authorship Contributions

Participated in research design: Atwal, Austin, Cowell.Conducted experiments: Atwal, Lishman, Austin.Performed data analysis: Atwal, Cowell.Wrote or contributed to the writing of the manuscript: Atwal,

Austin, Cowell.

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Atwal et al Molecular Pharmacology

Supplemental Data

Myeloperoxidase enhances etoposide and mitoxantrone mediated DNA

damage: a target for myeloprotection in cancer chemotherapy

Mandeep Atwal, Emma L. Lishman, Caroline A. Austin, Ian G. Cowell

Table of contents

Supplemental Figure Legends

Supplemental Figures 1-4

Supplemental References

Supplemental Tables 1-3

2

Supplemental Figure Legends

Supplemental Figure 1 . MPO is expressed in CD34+ bone marrow cells. CD34+ cells were isolated

from normal adult bone marrow MN cells using immunomagnetic separation and were stored

cryogenically. After thawing cells were cultured in StemSpan serum free medium for expansion of

hematopoietic cells supplemented with cytokine cocktail CC100 (StemCell technologies, Vancouver

Canada). After the number of days shown, cells were spotted onto Nunc Lab-Tek II CC2 chamber slides

and processed for immunofluorescence as described in Material and Methods using anti-MPO ab9535

(Abcam). Images were obtained using an Olympus IX81 microscope, with a 40X objective and

Hamamatsu Orca-AG camera tacking z-slices at 0.20μm intervals. Images shown are extended focus

projections (Volocity 6.3. Perkin Elmer). Bar = 10μm

Supplemental Figure 2. (A) Enzymatic oxidation of etoposide. Figure adapted from (Gantchev and

Hunting, 1998). (B) SA treatment does not affect TOP2A or TOP2B protein levels in NB4 cells. Whole

cell extracts were prepared from NB4 cells treated with 200µM SA for 48 or 72 hours and from

untreated cells. Extracts were run on 4-20% SDS PAGE gels and Western blotting was performed using

antibodies specific for TOP2A, TOP2B or actin. Three independent sets of extracts were prepared and

analysed by Western blotting. A representative gel (72 hr treatment) is shown on the left and

quantification from the triplicate experiments is shown on the right. (C-E) SA does not affect the

viability (C), or growth (D) of NB4 cells; nor does it result in a significant acceleration of acidification

of the cell culture medium up to 72 hours of culture (E). (F & G) Neither SA nor BSO affect TOP2 plasmid

relaxation activity in vitro. Assays were carried out as in (Fortune and Osheroff, 2001). Recombinant

TOP2A (F) or TOP2B (G) were incubated with 200μM SA or 150μM BSO on ice for 30 min. Reactions

were initiated by the addition of 1mM ATP and incubating for 30min at 37℃. Plasmid topoisomers

were resolved on a 0.8 % agarose gel (Sup=supercoiled plasmid DNA, Rel=Relaxed). (H) Neither SA nor

BSO affect TOP2 decatenation activity (Haldane and Sullivan, 2001). Recombinant TOP2A and TOP2B

were incubated with 200μM SA or 150μM BSO on ice for 30 min before initiating the reaction by

adding 1mM ATP and incubating at 37℃ for 30 minutes. Products were separated by agarose gel

electrophoresis (Cat=catenated kDNA, Decat=decatenated minicircles)

Supplemental Figure 3. (A& B) Example of a single replicate experiment of the TARDIS TOP2A (A) and

TOP2B (B) covalent complex quantification shown in Fig 1 C and D. Data are depicted as scattergrams,

in blue, where each dot represents the integrated fluorescence originating from a single nucleus, with

boxplots superimposed. Boxplots show the median and upper and lower quartiles. (C-E) SA does not

affect etoposide-induced TOP2 covalent complex formation or H2AX phosphorylation in K562 cells.

K562 cells were treated for 48 hours with 200µM SA before addition of etoposide. Samples were

analysed by TARDIS as described in Fig. 1 (A & B) or for γH2AX immunofluorescence (C). (F) Dose

response curve in K562 cells for γH2AX immunofluorescent signal at different doses of etoposide (0-

200µM) for 1 hour. Data is shown as mean ±S.E.M for three separate experiments. (G-I) G418 does

not affect the level of etoposide stabilised TOP2-DNA covalent complexes in K562 cells. K562 and K562

stably transfected with the empty vector used to construct the MPO overexpressing cell lines in Figs 2

& 4 and cultured in G418-containing medium were incubated with etoposide (10µM or 100µM) or

DMSO (0.02%v/v) as a vehicle control for 1 hour. TOP2-DNA covalent complexes were quantified using

the TARDIS assay (G & H) and γH2AX was quantified by immunofluorescence (I). Data are shown as

mean ±S.E.M for three separate experiments. (J-L) SA does not affect mitoxantrone-induced TOP2

covalent complex formation or H2AX phosphorylation in K562 cells. K562 cells were treated for 48

3

hours with 200µM SA before addition of etoposide. Samples were analysed by TARDIS as described in

Fig. 1 or for γH2AX immunofluorescence.

Supplemental Figure 4. (A) Inhibition of MPO activity in NB4 cells by PF-1355 and MPOi-II. NB4 cells

were incubated with PF-1355 (10 μM, 4 hr) or MPOi-II (5μM, 4hr) before assaying for myeloperoxidase

activity. (B-C) MPO inhibitors PF-1355 and MPOi-II do not affect the level of TOP2-DNA covalent

complex formation or H2AX phosphorylation induced by etoposide in K562 cells. (D-E) MPO inhibitors

PF-1355 and MPOi-II do not affect the level of TOP2-DNA covalent complex formation or H2AX

phosphorylation induced by mitoxantrone in K562 cells. For B-E cells were pre-treated with MPO

inhibitors PF-1355 (10 μM, 4hr) or MPOi-II (5 μM, 4 hr) before adding etoposide, mitoxantrone, or a

vehicle control as indicated for one hour.

Supplemental References

Fortune JM, and Osheroff N (2001) Topoisomerase II-catalyzed relaxation and catenation of plasmid DNA. Methods Mol Biol Clifton NJ 95:275–281.

Gantchev TG, and Hunting DJ (1998) The ortho-quinone metabolite of the anticancer drug etoposide (VP-16) is a potent inhibitor of the topoisomerase II/DNA cleavable complex. Mol Pharmacol 53:422–428.

Haldane A, and Sullivan DM (2001) DNA topoisomerase II-catalyzed DNA decatenation. Methods Mol Biol Clifton NJ 95:13–23.

4

Supplemental Figures

Fig 1

5

Fig 2

6

Fig 3

7

Fig 4

8

Cell Line Assay Pre-Treatment Etoposide % +ve control* SEM* p <0.05** % +ve control % reduction

/ µM corrected*** induced by SA****

NB4 TOP2A CCs None 10 39.4 2.3 39.3

NB4 TOP2A CCs SA 10 32.3 3.9 Y 32.2 18.1

NB4 TOP2A CCs None 100 100.0 1.4 100.0

NB4 TOP2A CCs SA 100 87.7 0.2 Y 87.6 12.4

NB4 TOP2B CCS None 10 50.5 4.9 50.5

NB4 TOP2B CCS SA 10 22.6 6.7 Y 22.6 55.3

NB4 TOP2B CCS None 100 100.0 11.1 100.0

NB4 TOP2B CCS SA 100 61.7 7.9 Y 61.7 38.3

NB4 gH2AX None 10 48.8 5.4 43.5

NB4 gH2AX SA 10 37.9 1.6 N 32.4 25.5

NB4 gH2AX None 100 100.0 4.9 94.7

NB4 gH2AX SA 100 59.6 2.8 Y 54.1 42.9

Cell Line Assay Pre-Treatment Mitoxantrone/ % +ve control* SEM* Sig (P -value)** % +ve control % reduction

/ µM corrected*** induced by SA****

NB4 TOP2A CCs None 0.5 62.1 4.3 59.7

NB4 TOP2A CCs SA 0.5 28.9 4.3 Y 24.2 59.4

NB4 TOP2A CCs None 1 100.0 6.1 95.4

NB4 TOP2A CCs SA 1 55.5 5.0 Y 54.1 43.3

NB4 TOP2B CCS None 0.5 67.6 13.1 41.0

NB4 TOP2B CCS SA 0.5 29.5 5.1 Y 4.9 88.0

NB4 TOP2B CCS None 1 100.0 7.2 76.1

NB4 TOP2B CCS SA 1 54.9 4.5 Y 27.9 63.3

NB4 gH2AX None 0.5 58.9 1.8 39.9

NB4 gH2AX SA 0.5 43.6 2.7 N 27.1 31.9

NB4 gH2AX None 1 100.0 5.2 81.0

NB4 gH2AX SA 1 61.0 3.7 Y 49.8 38.5

* Mean value relative to mean of value obtained with 100 mM etoposide / 1mM mitoxantrone, without SA pretreatment, ± SEM

** Significance of difference between control and SA pretreatment

*** Mean value relative to mean of value obtained with 100 mM etoposide / 1mM mitoxantrone,

without SA pretreatment with the value thus obtained for 0µM Etoposide/Mitoxantrone subtracted

**** Applies to values after subtraction of mean 0µM Etoposide/Mitoxantrone values

9

K562 TOP2A CCs 10 38.6 1.2 37.7

K562-line4 TOP2A CCs 10 59.8 2.4 Y 58.9 56.3 1.563009

K562-line5 TOP2A CCs 10 75.2 0.5 Y 74.4 97.5 1.974904

K562 TOP2A CCs 100 100.0 3.4 98.3

K562-line4 TOP2A CCS 100 134.3 12.9 Y 125.7 27.9 1.279367

K562-line5 TOP2A CCS 100 163.9 15.7 Y 157.1 59.9 1.598552

K562 TOP2B CCS 10 14.7 1.1 5.8

K562-line4 TOP2B CCS 10 24.8 2.8 N 19.9 242.1 3.420875

K562-line5 TOP2B CCS 10 30.0 4.4 Y 26.0 346.0 4.460311

K562 TOP2B CCS 100 100.0 8.6 92.1

K562-line4 TOP2B CCS 100 135.4 6.7 Y 133.5 45.0 1.449709

K562-line5 TOP2B CCS 100 191.8 8.0 Y 188.1 104.3 2.042724

K562 gH2AX 10 29.0 1.5 29.0

K562-line4 gH2AX 10 44.6 3.2 Y 44.6 53.7 1.537452

K562-line5 gH2AX 10 52.7 5.0 Y 52.7 81.6 1.815669

K562 gH2AX 100 100.0 2.4 100.0

K562-line4 gH2AX 100 113.6 2.8 N 113.6 13.6 1.136158

K562-line5 gH2AX 100 133.9 18.1 N 133.9

Cell Line Assay Mitox % +ve control* SEM* p <0.05** % +ve control % Increase Fold increase/ µM corrected*** compared to K562**** Compare to K562****

K562 TOP2A CCs 1 100.0 9.755053 97.8

K562-line2 TOP2A CCs 1 214.9 4.310977 Y 157.5 61.1 1.610827

K562 TOP2B CCS 1 100.0 37.78822 79.9

K562-line2 TOP2B CCS 1 247.6 13.76075 Y 231.1 189.2 2.891881

* Mean value relative to mean of value obtained for K562 cells and 100µM etoposide / 1mM mitoxantrone, ± SEM

** Significance of difference between K562 cells and MPO-expressing clones

*** Mean value relative to mean of value obtained with 100 µM etoposde / 1mM mitoxantrone, for 0µM Etoposide/Mitoxantrone subtracted

**** Applies to values after subtraction of mean 0µMEtoposide/Mitoxantrone values

Table 1 MPO activity affects etoposide and mitoxantrone-induced TOP2 CC and γH2AX levels and associated statistics

10

Cell Line Assay Pre-Treatment Etoposide % +ve control* SEM* p <0.05** % +ve control Fold increase induced

/ µM corrected*** by SA/BSO/SA+BSO****

NB4 TOP2A CCs None 10 48.8 8.4 47.3

NB4 TOP2A CCs SA 10 21.5 2.2 Y 20.2 0.43

NB4 TOP2A CCs BSO 10 90.9 9.4 Y 88.8 1.88

NB4 TOP2A CCs BSO+SA 10 23.6 4.0 Y 22.3 0.47

NB4 TOP2A CCs None 100 100.0 3.6 98.6

NB4 TOP2A CCs SA 100 75.4 4.1 Y 74.1 0.75

NB4 TOP2A CCs BSO 100 100.8 5.5 N 98.7 1.00

NB4 TOP2A CCs BSO+SA 100 73.8 3.9 Y 72.5 0.74

NB4 TOP2B CCS None 10 33.0 2.9 24.3

NB4 TOP2B CCS SA 10 18.2 4.7 Y 14.5 0.60

NB4 TOP2B CCS BSO 10 89.2 5.3 Y 82.9 3.42

NB4 TOP2B CCS BSO+SA 10 16.1 2.6 Y 10.3 0.43

NB4 TOP2B CCS None 100 100.0 1.9 91.3

NB4 TOP2B CCS SA 100 63.5 9.4 Y 59.8 0.65

NB4 TOP2B CCS BSO 100 154.1 9.3 Y 147.8 1.62

NB4 TOP2B CCS BSO+SA 100 87.8 8.5 N 82.0 0.90

NB4 gH2AX None 10 53.8 8.3 45.5

NB4 gH2AX SA 10 39.7 2.2 N 31.5 0.69

NB4 gH2AX BSO 10 110.7 9.5 Y 102.0 2.24

NB4 gH2AX BSO+SA 10 47.3 10.3 Y 40.2 0.88

NB4 gH2AX None 100 100.0 6.9 91.7

NB4 gH2AX SA 100 59.1 3.5 Y 50.9 0.55

NB4 gH2AX BSO 100 189.9 26.2 Y 181.2 1.98

NB4 gH2AX BSO+SA 100 49.9 2.0 Y 42.8 0.47

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Cell Line Assay Pre-Treatment Mitoxantrone/ % +ve control* SEM* p <0.05** % +ve control Fold increase induced

/ µM corrected*** by SA/BSO/SA+BSO****

NB4 TOP2A CCs None 1 100.0 9.3 93.5

NB4 TOP2A CCs SA 1 48.0 4.8 Y 41.2 0.44

NB4 TOP2A CCs BSO 1 140.5 12.0 Y 130.3 1.39

NB4 TOP2A CCs BSO+SA 1 62.9 7.4 Y 56.1 0.60

NB4 TOP2B CCS None 1 100.0 11.5 65.7

NB4 TOP2B CCS SA 1 51.3 6.4 Y 21.7 0.33

NB4 TOP2B CCS BSO 1 142.8 6.2 Y 106.0 1.61

NB4 TOP2B CCS BSO+SA 1 45.5 1.6 Y 15.9 0.24

NB4 gH2AX None 1 100.0 5.9 80.5

NB4 gH2AX SA 1 55.7 3.4 Y 38.4 0.48

NB4 gH2AX BSO 1 147.1 8.0 Y 128.7 1.60

NB4 gH2AX BSO+SA 1 63.7 5.4 Y 44.8 0.56

* Mean value relative to mean of value obtained with 100 µM etoposide / 1mM mitoxantrone, without SA pretreatment, ± SEM

** Significance of difference between control (No SA or BSO) and SA/BSO/SA+BSO pretreatment

*** Mean value relative to mean of value obtained with 100 mM etoposide / 1mM mitoxantrone, without SA pretreatment with the value thus obtained for 0µM Etop/Mtx subtracted

Table 2 Effect of MPO inhibition and GSH depletion on etoposide and mitoxantrone-induced TOP2 CC and γH2AX levels and associated statistics

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Assay TOP2 poison % reduction in mean values

SA PF-1355 MPOI-II

TOP2A CC 10µM Etop 18 s 40 s 40 s TOP2B CC 10µM Etop 55 s 65 s 81 s

γH2AX 10µM Etop 26 8 15

TOP2A CC 100µM Etop 12 s 22 s 19 s TOP2B CC 100µM Etop 38 s 54 s 66 s

γH2AX 100µM Etop 43 s 48 s 54 s

TOP2A CC 0.5µM Mitox 59 s 46 s 52 s TOP2B CC 0.5µM Mitox 88 s 59 s 57 s

γH2AX 0.5µM Mitox 32 s 38 s 30 s

TOP2A CC 1µM Mitox 43 s 48 s 53 s TOP2B CC 1µM Mitox 63 s 62 s 66 s

γH2AX 1µM Mitox 39 s 41 s 39 s

Table 3. Comparison of the effects of succinylacetone and direct MPO inhibitors on etoposide and mitoxantrone-induced TOP2-DNA covalent complexes and H2AX

phosphorylation. Figures were calculated as the % reduction in TARDIS or γH2AX quantitative fluorescence signals measured in TOP2 poison treated cells, after subtracting

the no-TOP2-poison background signal. s designates where the minus inhibitor versus plus inhibitor values differed significantly (p< 0.05)