Intercalating TOP2 Poisons Attenuate Topoisomerase Action at Higher Concentrations

Decatenation Assays.

Two Hundred nanograms of kDNA was incubated in reaction buffer [50 mM Tris-HCl (pH 7.5), 125 mM NaCl, 10 mM MgCl₂, 5 mM Dithiothreitol, and 100 μg/ml albumin], with or without drug, as indicated in the figure legends; the reaction volume was 20 µl, and reactions were initiated by the addition of TOP2A or TOP2B and incubated for 30 minutes at 37°C. Loading buffer was added and samples were run on a 0.8% gel in TBE buffer [100 mM Tris-borate (pH 8.3), 2 mM EDTA]. Gels were stained with ethidium bromide after electrophoresis and visualized on a Bio-Rad EZ gel doc.

DNA Cleavage Assays.

pTCS1 DNA substrate (3.9 µg) was incubated in reaction buffer [10 mM Tris-HCl (pH 8.0), 10 mM MgCl₂, 50 mM KCl, 150 µg/ml bovine serum albumin, and 1 mM ATP] in the presence or absence of mitoxantrone, doxorubicin, or epirubicin with or without etoposide for 30 minutes at 37°C. The reactions were initiated by the addition of 1.2 µg of TOP2A or TOP2B, and the reaction volume was 100 µl. Reactions were terminated by addition of SDS to 1%, followed by addition of EDTA to 25 mM and proteinase K to 0.5 mg/ml, and incubated for 60 minutes at 45°C. The DNA was precipitated overnight at −20°C following the addition of sodium acetate to 300 mM and the addition of two volumes of ethanol. The DNA precipitate was collected by centrifugation and the pellet air dried prior to resuspension in 15 µl TE buffer [10mM Tris-HCl, 1mM EDTA (pH 8.0)]; 5 µl of loading buffer was added, and the samples were heated to 70°C for 2 minutes prior to running on a 0.8% agarose gel in TAE buffer [40 mM Tris, 2 mM EDTA and 0.11% (v/v) glacial acetic acid (pH 8.3)] in the presence of ethidium bromide. Gels were visualized on a Bio-Rad EZ gel doc and bands were quantified by ImageLab v.5.2.1 (Bio-Rad).

Cell Culture.

NB4 and K562 cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin (Thermo Fisher Scientific, Paisley, UK). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂. Experiments were conducted on exponentially growing cells (2–5 × 10⁵ cells/ml). NB4 (ACC-207) and K562 ( CCL-243) cell lines (Lozzio and Lozzio, 1975; Lanotte et al., 1991) were originally sourced from the German Collection of Microorganisms and Cell Cultures GmbH and American Type Culture Collection, respectively. Cells were routinely tested for mycoplasma infection.

Undifferentiated induced pluripotent stem cells (iPSCs) were maintained in feeder-free culture with GFR Matrigel (Corning) and mTeSR1 (StemCell Technologies, Cambridge, UK) with 1% penicillin/streptomycin (Pen/Strep); these cells were tested monthly for mycoplasma and were originally sourced from Lonza (Basel, Switzerland). Differentiation to induced pluripotent stem cell–derived cardiomyocytes was performed using an established protocol followed by metabolic purification (Lian et al., 2013; Tohyama et al., 2013). In brief, iPSCs were cultured as described earlier to form a confluent monolayer; at this point (day 1 of the protocol), the medium was changed to a base differentiation medium consisting of RPMI 1640 supplemented with B27 minus insulin and 1% Pen/Strep (Life Technologies) and 9 µM CHIR99021 trihydrochloride (Tocris, Abingdon UK). On day 2, the medium was changed to base medium. On day 4, the medium was changed to a mixture of fresh basal medium and the existing medium from the culture in a 1:1 ratio; this was supplemented with 5 µM IWP2 (Tocris). The medium was changed to base medium on day 6, and on day 8 the medium was changed to maintenance medium consisting of RPMI 1640 supplemented with 2% B27 and 1% Pen/Strep. On day 10, the medium was changed to metabolic purification medium consisting of RPMI 1640 without glucose, supplemented with 2% B27 and 1% Pen/Strep. On day 15, the medium was replaced with maintenance medium. Thereafter, the maintenance medium was changed every 2 to 3 days. Cardiomyocytes generated using this protocol were characterized by fluorescence-activated cell sorter analysis of SIRPA gene expression (Dubois et al., 2011) followed by quantitative polymerase chain reaction quantification of sarcomeric marker genes (ACTN2, TNNT2, TNNI3, MYH6, and MYH7) and detection of their respective proteins by immunohistochemistry (data not shown). Cardiomyocyte function was assessed by observation of spontaneous contraction and recording spontaneous calcium transients.

TARDIS Assay.

The TARDIS assay was used to quantify the level of stabilized TOP2-DNA covalent complexes in situ, conducted as previously described (Willmore et al., 1998; Cowell et al., 2011). In brief, cells were treated for 1 hour with the desired TOP2 poison. Cells were pelleted (1000g, 5 minutes) and washed in ice-cold PBS. After recentrifugation, cells were mixed in an equal volume of molten 2% low melting point agarose (Lonza) in PBS and spread evenly onto agarose-coated slides. Agarose-embedded cells were lysed [1% (w/v) SDS, 20 mM sodium phosphate, 10 mM EDTA, pH 6.5], and noncovalently bound DNA proteins were removed using 1 M NaCl. Stabilized TOP2 covalent complexes were detected by immunofluorescence using anti-TOP2 rabbit polyclonal antibodies followed by Alexa Fluor 488–coupled anti-rabbit secondary antibodies (Thermo Fisher Scientific). Slides were counterstained with Hoechst 33258 (Thermo Fischer Scientific) to visualize DNA. Images were captured for Hoechst 33258 and Alexa Fluor 488 using an epifluorescence microscope (IX-81, 10× objective; Olympus) fitted with an Orca-AG camera (Hamamatsu) and suitable narrowband filter sets. Image capture and automated slide scoring was performed using Volocity 6.3 software (PerkinElmer). In brief, acquired images were each processed to identify nuclei by thresholding the Hoechst 33258 signal. After filtering to separate or remove touching/overlapping objects (nuclei), the immunofluorescent signal emanating from each nucleus was determined to produce a table of integrated fluorescence per nucleus. Parameters to select individual nuclei were set up at the beginning of the analysis for one slide, and these parameters were then used for all other slides in the experiment. Data are represented using GraphPad Prism 8.0 or 8.1 (GraphPad Software) and R (The R Foundation, San Diego, CA).

Immunofluorescence Analysis of γH2AX.

After drug treatment, cells were washed and pelleted in ice-cold PBS and spotted on poly-l-lysine–coated slides. Cells were fixed in 4% paraformaldehyde in PBS for 10 minutes and permeabilized using KCM+T buffer [120 mM KCl, 20 mM NaCl, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.1% Triton X-100] for 15 minutes. After blocking in (KCM+T, 2% bovine serum albumin, 10% dry milk powder), cells were probed with anti-γH2AX in blocking buffer followed by Alexa Fluor 594 anti-mouse secondary antibodies. Slides were counterstained with DAPI (4',6 diamidine 2 phenylindole). Images were captured for DAPI and Alexa Fluor 594 and quantification was performed using Volocity 6.3 (PerkinElmer) as described for the TARDIS assay.

Anthracyclines Targeting TOP2 Are Inefficient at Inducing Stable TOP2-DNA Complexes.

Similar to etoposide and mitoxantrone, anthracyclines, including doxorubicin (dox) and epirubicin (epi), are TOP2 poisons on the basis of their ability to increase the steady-state concentration of the covalent TOP2-DNA complexes by impeding religation of DNA (Pommier et al., 2010; Vos et al., 2011). Each of these drugs stimulates cleavage of DNA substrates by TOP2 in in vitro assays (Bodley et al., 1989; Capranico et al., 1993; Binaschi et al., 1998). However, unlike etoposide and mitoxantrone, the anthracyclines have not been reported to induce the formation of large numbers of stable TOP2-DNA complexes in cells that can be detected using the TARDIS assay or the in vivo complex of enzyme assay (Montaudon et al., 1997; Willmore et al., 2002; Swift et al., 2006; Nitiss et al., 2012). For example, while the anthracycline idarubicin induced a small dose-dependent increase in TOP2A complexes up to 1 µM and a marginal increase in TOP2B complexes over control cells, at 20 µM fewer TOP2-DNA complexes were seen (Willmore et al., 2002). Doxorubicin-induced TOP2-DNA complexes were not detected in this previous TARDIS study (Willmore et al., 2002).

In this study, we were able to detect doxorubicin- and epirubicin-induced TOP2A complexes in human NB4 cells using the TARDIS assay (Fig. 2A). This discrepancy may be due to the improved sensitivity of the assay in the current study resulting from the use of brighter, more stable fluorochromes and more sensitive camera technology. However, median fluorescence levels per nucleus did not exceed 20% of the level recorded for the positive control treatment (100 µM etoposide), and no significant dox or epi signal was observed for TOP2B (Fig. 2A). For both dox and epi, only a small increase in median fluorescent signal was observed for TOP2A between 1.0 and 10 µM. Subsequent experiments were carried out at 10 µM for both drugs. In line with the reduced level of stabilized TOP2 complexes compared with 100 µM etoposide, dox and epi both resulted in lower γH2AX induction than etoposide (Fig. 2B). This is similar to a previous finding (Huelsenbeck et al., 2012) in rat glioblastoma cells, where doxorubicin-induced H2AX phosphorylation was maximal at 2 µM and was reduced compared with the extent of phosphorylation induced by 100 µM etoposide. Since TARDIS analysis provides fluorescence intensity data from individual nuclei, the resulting data can also be presented as the percentage of cells with a signal above a given threshold (Huelsenbeck et al., 2012). We chose 25% of the value of the median signal obtained for 100 µM etoposide as a convenient threshold. For TOP2A (4566), approximately 5% and 10% of 10 µM dox- and epi-treated cells exceeded this cutoff (Fig. 2C).

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Fig. 2.

Dose response for formation of TOP2-DNA complexes and histone H2AX phosphorylation induced by doxorubicin and epirubicin. (A) NB4 cells were treated with the indicated concentrations of anthracycline or etoposide (Etop). TOP2A- and TOP2B-DNA complexes were quantified as in Fig. 1. The number of cells analyzed for each treatment is indicated in italics at the top of each graph. (B) Cells were treated as in (A) and were analyzed by immunofluorescence for γH2AX. The results are displayed as the means of the medians of replicate experiments ±S.D, normalized to 100 μM etoposide. The number of replicates for each condition is indicated above each graph. (C) Proportion of cells treated with 10 μM dox or epi that exhibit TOP2A TARDIS fluorescence above a threshold of 25% of the median value obtained with 100 μM etoposide. Error bars indicate S.D. values.

The data obtained for Figs. 1 and ​and22 involved treating cells for 60 minutes with TOP2 poison before carrying out the TARDIS assay. To determine whether the relatively low level of stable TOP2-DNA complexes observed with epi was due to slow complex formation compared with etoposide, a TOP2A TARDIS time-course experiment was performed. Median fluorescence levels induced by epi did not increase with drug incubation times from 30 to 120 minutes (Fig. 3A). The TARDIS assay data shown in Figs. 1, ​,2,2, and ​and3A3A used antibodies raised to the divergent C-terminal regions of the TOP2A and TOP2B proteins, which allows the two isoforms to be analyzed independently (4566 and 4555, respectively; see Supplemental Fig. 1). We were concerned that the low signal observed with the anthracyclines compared with etoposide could be due to epitope masking by post-translational modification or rapid proteolytic removal of the C-terminal domain following anthracycline drug treatment. To test this possibility, we repeated the TARDIS assay with antibody 4882, which was raised to the conserved N-terminal 140 kDa of calf thymus TOP2 and detects both isoforms of human TOP2 (see Supplemental Fig. 1). Using this antibody, 10 and 100 µM etoposide produced a robust signal in the TARDIS assay. However, no significant increase in median fluorescence was observed for 10 µM dox or epi compared with the no-drug control (Fig. 3, B and C), whereas an intermediate level of signal was observed for 20 µM mitoxantrone. Considering the results obtained with antibodies 4566 (TOP2A C-terminal domain), 4555 (TOP2B C-terminal domain), and 4882 (TOP2A and TOP2B N-terminal 140 kDa), the low anthracycline-induced TOP2A signal and lack of TOP2B signal in the TARDIS assay are not likely to be due to loss or masking of the C-terminal domain, indicating inefficient trapping of stable TOP2 complexes by dox and epi.

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Fig. 3.

Low anthracycline-induced TOP2-DNA complex TARDIS signal is not due to short drug incubation time or specific loss or masking of TOP2 C-terminal domain epitopes. (A) Cells were treated with 10 μM epi for the times indicated or with etoposide (Etop; 100 μM) for 60 minutes, and TOP2A complexes were quantified using antibody 4556 as in Fig. 1. (B and C) Cells were treated with the indicated TOP2 poisons for 60 minutes, and TOP2 complexes were analyzed using anti-TOP2 antibody 4882 (raised to N-terminal 140 kDa of calf thymus TOP2). Data are shown as scatterplots for one replica experiment (B) and as the means of the median values obtained from three replica experiments ±S.D. for (C). Significance values refer to comparison with untreated (control) cells by one-way ANOVA with Dunnett’s correction for multiple comparisons. Error bars indicated S.D. values. Mtx, mitoxantrone.

Doxorubicin, Epirubicin, and Mitoxantrone Can Inhibit Stable TOP2-DNA Complexes in Competition Assays.

Various lines of evidence, including in vitro DNA cleavage data, H2AX phosphorylation, and TARDIS data (Bodley et al., 1989; Willmore et al., 2002; Smart et al., 2008; Huelsenbeck et al., 2012), support the idea that intercalating anthracycline TOP2 poisons and mitoxantrone inhibit TOP2 activity at higher concentrations. Competition between poisoning and inhibition may then explain the relatively inefficient generation of stable TOP2-DNA complexes by anthracyclines. To further study this, etoposide-induced TOP2 complexes were quantified using the TARDIS assay in NB4 cells that had been preincubated with 10 µM dox, 10 µM epi, 20 µM mitoxantrone, or the well characterized bisdioxopiperazine TOP2 catalytic inhibitor ICRF-193 (100 µM) (Patel et al., 2000). As noted previously, the anthracyclines on their own induced a low TOP2A signal compared with that obtained with etoposide, whereas 20 µM mitoxantrone alone induced a small but significant TOP2A signal. ICRF-193 did not induce TOP2A or TOP2B complexes significantly above basal levels. However, preincubation with each of these drugs significantly inhibited both TOP2A and TOP2B covalent complex formation by etoposide (Fig. 4, A and B). Indeed, the level of inhibition achieved by the anthracyclines and mitoxantrone was similar to that observed for ICRF-193. Since NB4 cells contain a high level of myeloperoxidase activity, which increases TOP2 complex formation via oxidative activation of etoposide, we repeated the experiments shown in Fig. 4, A and B using the myeloperoxidase nonexpressing cell line K562 (Fig. 4, C and D). Very similar results were observed in K562 cells; the anthracyclines efficiently inhibited etoposide-induced TOP2A and TOP2B complex formation at 10 and 100 µM etoposide. Mitoxantrone also significantly inhibited etoposide-induced TOP2B complex formation and TOP2A complexes at 100 µM etoposide.

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Fig. 4.

Anthracyclines and mitoxantrone inhibit etoposide-induced TOP2A- and TOP2B-DNA complex stabilization. NB4 cells (A and B) or K562 cells (C and D) were preincubated for 1 hour with doxorubicin (Dox), epirubicin (Epi), mitoxantrone (Mtx), or ICRF-193 at the concentration shown in the top-left corner of each graph. Cells were then treated with 10 or 100 μM etoposide (Etop; or vehicle control) for a further hour. TOP2A (A and C) and TOP2B (B and D) complexes were quantified as in Fig. 1. The first bar in each group (gray) corresponds to treatment with etoposide alone. Data in each graph are the means of the medians from replica experiments ±S.D. The number of replicates for each condition is shown at the top of each graph. Significance tests were performed by two-way ANOVA using Dunnett’s correction for multiple comparisons (comparisons were made to etoposide treatment alone).

Since TOP2 complexes are processed in cells to DSBs that elicit γH2AX formation, we also determined the ability of dox, epi, and mitoxantrone to inhibit etoposide-induced H2AX phosphorylation. As observed in Fig. 2B, the anthracyclines alone at 10 µM led to H2AX phosphorylation (Fig. 5); however, no signal above background was observed for 100 µM ICRF-193. Levels of histone H2AX phosphorylation induced by 10 μM etoposide were not significantly increased or decreased by pretreatment with dox or epi compared with etoposide alone. Whereas dox and epi alone produce an H2AX signal, no additive effect on DSB formation was observed when the anthracyclines were coincubated with 10 μM etoposide. In addition, H2AX phosphorylation induced by 10 μM etoposide was not reduced by anthracycline treatment. However, preincubation with epi, but not dox, did significantly reduce the level of H2AX phosphorylation by approximately 25% following treatment with 100 µM etoposide. In contrast, etoposide-induced H2AX phosphorylation was strongly suppressed by mitoxantrone at both concentrations of etoposide. Indeed, this suppression was greater than that observed for ICRF-193 with 100 µM etoposide, although the efficacy of inhibition by ICRF-193 may be limited by its solubility in aqueous solution.

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Fig. 5.

Anthracyclines and mitoxantrone (Mitox) inhibit etoposide (Etop)-induced H2AX phosphorylation. NB4 cells were pretreated with anthracyclines, mitoxantrone, or ICRF-193 before treatment with etoposide, as in Fig 4. Histone H2AX phosphorylation was assessed by quantitative immunofluorescence. Data in each graph are the means of the medians from replica experiments ±S.D. Significance tests were performed as for Fig. 4.

Doxorubicin Failed to Induce Detectable TOP2B-DNA Complexes in Cardiomyocytes Derived from iPSCs but Did Suppress Etoposide-Induced Complex Formation.

Although anthracyclines are widely used and effective anticancer drugs, their side effect profile includes potentially serious cardiac damage (van Dalen et al., 2005). Using iPSC-derived cardiomyocytes, we examined the expression of TOP2B and TOP2A by immunofluorescence. As anticipated for fully differentiated cells, only about 5% of the cardiomyocytes expressed significant levels of TOP2A, but all cells expressed abundant TOP2B (Fig. 6A). Whereas 100 µM etoposide efficiently induced TOP2B-DNA complexes in the cardiomyocyte cells, doxorubicin did not induce TOP2B complexes significantly above background levels (Fig. 6B). Moreover, preincubation with 10 µM doxorubicin suppressed etoposide-induced TOP2B complexes in cardiomyocyte cells, confirming that doxorubicin attenuates TOP2 activity in cardiomyocytes. Thus, the inhibition observed by doxorubicin treatment is not limited to a single cell type.

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Fig. 6.

TOP2 expression and induction of stable TOP2B-DNA complexes in human cardiomyocytes. (A) Induced human iPSC cardiomyocytes were stained with anti-sarcomeric actin (green) and TOP2A or TOP2B antibodies (red) and counterstained with DAPI (blue). All cells stained positive for TOP2B. Most cells were negative for TOP2A; the proportion of cells weakly (+) or strongly (++) positive for TOP2A are indicated in the table below (A). (B) Cardiomyocytes were pretreated with doxorubicin as indicated and then incubated with 100 μM etoposide (Etop). TOP2B-DNA stabilized complexes were then quantified using the TARDIS assay as in Fig. 1. Significance values refer to comparison with control (untreated) cells (Kruskal-Wallace test with Dunn’s correction for multiple comparisons).

In Vitro Inhibition of TOP2 Decatenation Activity by Mitoxantrone, Doxorubicin, Epirubicin, or ICRF-193.

In vitro, TOP2 decatenation activity can be analyzed using highly catenated kDNA. As shown in Fig. 7, high-molecular-weight kDNA remains in the well of an agarose gel (first lane of each panel), and in the absence of drugs, TOP2A and TOP2B catalyze the decatenation of kDNA, resulting in the migration of decatenated circles into the gel (second lane of each panel). However, addition of mitoxantrone, dox, or epi resulted in a decline in the TOP2-mediated decatenation activity with increasing concentrations of drugs (Fig. 7). The effect of the catalytic inhibitor ICRF-193 is shown for comparison.

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Fig. 7.

Mitoxantrone, doxorubicin, and epirubicin inhibit TOP2A- and TOP2B-mediated in vitro decatenation activity. kDNA decatenation assays using recombinant TOP2A (left panels) or recombinant TOP2B (right panels) were performed in the presence of mitoxantrone (Mtx) (A), doxorubicin (B), or epirubicin (C). The gel positions of catenated and decatenated kDNA are indicated. The last lane in each gel (M) contains a marker consisting of catenated and decatenated kDNA.