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Clinical Cancer Research Vol. 11, 8467-8475, December 1, 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Preclinical

Synergistic Interaction between Histone Deacetylase and Topoisomerase II Inhibitors Is Mediated through Topoisomerase IIß

Douglas C. Marchion, Elona Bicaku, Joel G. Turner, Adil I. Daud, Daniel M. Sullivan and Pamela N. Munster

Authors' Affiliation: Experimental Therapeutics Program, Department of Interdisciplinary Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida

Requests for reprints: Pamela N. Munster, H. Lee Moffitt Cancer Center, 12902 Magnolia Drive, MRC 4E, Tampa, FL 33612. Phone: 813-745-8948; Fax: 813-745-1984; E-mail: Munstepn{at}moffitt.usf.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Background: DNA topoisomerase II inhibitors and poisons are among the most efficacious drugs for the treatment of cancer. Sensitivity of cancer cells to the cytotoxic effects of topoisomerase II targeting agents is thought to depend on the expression of the topoisomerase II{alpha} isoform, and drug resistance is often associated with loss or mutation of topoisomerase II{alpha}. Histone deacetylase inhibitors (HDACi) are a novel class of compounds that potentiate the antitumor effects of topoisomerase II–targeting agents.

Methods: The interaction between HDACi and topoisomerase II–targeting agents in cancer cells was evaluated as a function of topoisomerase II{alpha} and topoisomerase IIß expression. Topoisomerase II isoforms were selectively depleted using small interfering RNA and antisense. Drug-induced formation of cleavable complexes involving topoisomerase II{alpha} and topoisomerase IIß was evaluated by trapped-in-agarose DNA immunostaining and band depletion assays in the presence and absence of HDACi.

Results: Preexposure to HDACi increased the cytotoxicity of topoisomerase II poisons. This was associated with a down-regulation of topoisomerase II{alpha} expression but had no effects on topoisomerase IIß. In the setting of HDACi-induced chromatin decondensation and topoisomerase II{alpha} depletion, topoisomerase II poison cytotoxicity was mediated through topoisomerase IIß cleavable complex formation. The HDACi-induced sensitization was also observed in cells with target-specific resistance to topoisomerase II poisons.

Conclusions: The recruitment of topoisomerase IIß as a target may overcome primary or emergent drug resistance to topoisomerase II–targeting agents and hence may broaden the applicability of this important class of anticancer agents.

Type II topoisomerases are essential enzymes that regulate the topological state of DNA to facilitate several biological processes. Topoisomerase II exists in two isoforms, topoisomerase II{alpha} and topoisomerase IIß. Although these enzymes have high sequence homology (1) and a similar mechanism of action (2), they are regulated independently and have been associated with different cellular functions. Topoisomerase II{alpha} is necessary for many biological processes involving double-stranded DNA, including replication, mitosis, and chromatin condensation (3, 4). Topoisomerase II{alpha} is mainly located in the nucleus and peaks in expression during G2-M (5, 6). In contrast, topoisomerase IIß expression remains stable throughout the cell cycle (7) and may have an important role in transcription (8). Although it was reported that topoisomerase IIß may not be essential in mitosis, topoisomerase II{alpha} knockdown experiments have shown that topoisomerase IIß may only partially substitute topoisomerase II{alpha} during chromatin decondensation and cell segregation (9).

Due to their essential roles, topoisomerase II enzymes have been successfully targeted for the treatment of cancer. To maintain genetic integrity during the cleavage and religation of DNA, both topoisomerase isoenzymes form transient bonds with the cleaved DNA called the cleavable complex (10). The stabilization and persistence of these cleavable complexes by topoisomerase II poisons have been associated with DNA damage and cell death (11, 12). Multiple in vitro and in vivo studies have suggested that sensitivity of tumor cells to topoisomerase II poisons depends on access to the targets, as well as target location and expression levels. For clinical use, topoisomerase II{alpha} has been considered the more relevant target (13–17).

A class of drugs that may enhance access to DNA and thereby increase the antitumor activity of topoisomerase II–targeting agents are the histone deacetylase inhibitors (HDACi; refs. 14, 18, 19). We previously reported a sequence-specific and time-dependent potentiation of topoisomerase II–targeting agents by the HDACi, suberoylanilide hydroxamic acid (SAHA), and valproic acid (14, 20). Our data indicated that prolonged treatment of cancer cells with an HDACi resulted in the depletion of proteins involved in the maintenance of heterochromatin and led to subsequent chromatin decondensation. Chromatin decondensation increased the access of topoisomerase II–targeting agents to the DNA substrate, which potentiated cell death induced by topoisomerase II–targeting agents. Topoisomerase II drug cytotoxicity is largely dependent upon topoisomerase II{alpha} expression. Here, we investigated the relevance of topoisomerase II{alpha} and topoisomerase IIß as targets of topoisomerase II poisons in the presence of HDACi. We show that in the setting of HDACi pretreatment, the formation of cleavable complexes involving topoisomerase IIß rather than topoisomerase II{alpha} leads to topoisomerase II poison–induced cell death. This may broaden the clinical applicability of topoisomerase II poisons and overcome drug resistance due to acquired depletion and mutations of topoisomerase II{alpha}.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and antibodies. Trichostatin A, sodium butyrate (NaB), valproic acid, mitoxantrone, and teniposide (VM-26) were purchased from Sigma Chemical Co. (St. Louis, MO). SAHA was provided by Aton Pharma (Merck, Whitehouse Station, NJ). Epirubicin was purchased from Pfizer, Inc. (New York, NY). XK469 was a kind gift from Dr. Robert Snapka. All other reagents were of analytic grade and purchased from standard suppliers. Antibodies used were as follows: Ki-S1 (monoclonal, Chemicon, Temecula, CA) and 454 (polyclonal, developed by Dr. Dan Sullivan) for topoisomerase II{alpha}, anti-topoisomerase IIß (monoclonal, BD Biosciences, San Jose, CA) and JAB (polyclonal, developed by Dr. Dan Sullivan), and acetylated histone H3 antibody (Upstate Biotechnology, Chicago, IL).

Cell lines. SKBr-3, MCF-7, KM12C, A375, BT-474, and MDA-MB-361 cells were purchased from the American Type Culture Collection (Manassas, VA). Doxorubicin-resistant MCF-7 cells (MCF:Dox) were a kind gift from Dr. Fred Hausheer. Cell lines were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mmol/L glutamine, and 50 units/mL penicillin, and 50 µg/mL streptomycin (Life Technologies Bethesda Research Laboratories, Carlsbad, CA). Cells were incubated in a humidified atmosphere with 5% CO2 at 37°C.

Cytotoxicity assays. Topoisomerase II poison cytotoxicity was evaluated in the presence of HDACi by apoptotic and clonogenic assays.

Apoptosis was scored by the presence of nuclear chromatin condensation and DNA fragmentation and evaluated with fluorescence microscopy using bis-benzimide staining. Briefly, cells were treated with an HDACi for 48 hours, after which the media was removed and replaced with media containing the indicated concentrations of a topoisomerase II poison. After 4 hours, the topoisomerase II poison was removed, and the cells were cultured for an additional 48 hours. Cells were harvested using a Cell Scraper (Fisher, Hampton, NH), fixed in 4% paraformaldehyde for 10 minutes at room temperature, and washed with PBS. Cell nuclei were stained with 0.5 µg/mL of bis-benzimide trihydrochloride (Hoechst #33258, Molecular Probes, Eugene, OR). Two hundred cells were counted for each experiment and evaluated for apoptotic scores (apoptotic nuclei/all nuclei x 100). Each experiment was repeated thrice, and the SE was calculated.

For clonogenic assays, cells were plated on six-well dishes at a density of 150 per well and allowed to adhere for 24 hours. For drug combination studies, cells were incubated with medium containing 0.5 mmol/L valproic acid. At this concentration of valproic acid, valproic acid had no discernable effects on growth or apoptosis. After a 48-hour treatment with valproic acid, the medium was removed and replaced with medium containing 0, 1, 10, 25, 50, or 100 nmol/L epirubicin for 4 hours. Epirubicin was then removed, and colonies were allowed to grow for 14 to 21 days to a maximal size of 2 to 3 mm in diameter, stained with 2% crystal violet in methanol, and counted. Colonies were included in the assessment if measuring at least 0.2 mm. For single-drug samples and untreated controls, saline was used in lieu of valproic acid or epirubicin or both. All experiments were done in duplicates and repeated at least thrice. The concentration of epirubicin required for IC50 was determined using the CalcuSyn software as described previously (14). Synergistic effects versus additive effects were also determined by the CalcuSyn program. The fractional inhibition of valproic acid was depicted as the number of observed cells (treatment group) divided by the number of expected cells (untreated control) with a range of 0 to 1.

Microarray. Expression levels of topoisomerase II{alpha} mRNA were evaluated by microarray analysis using Affymetrix Genechips (Affymetrix, Santa Clara, CA) by standard protocols (Moffitt, Molecular Biology Core). Hybridization to Affymetrix chips was analyzed using Affymetrix Microarray Suite 5.0 software. Signal intensity was scaled to an average intensity of 500 before comparison analysis. The MAS 5.0 software uses a statistical algorithm to assess changes in mRNA abundance in a direct comparison between two samples (Statistical algorithms description document. http://www.affymetrix.com/support/technical/whitepapers.affx). This analysis is based on the behavior of 16 different oligonucleotide probes designed to detect the same gene. Using the programmed default values, probe sets that yielded a change P < 0.04 were identified as changed (increased or decreased), and those that yielded a P between 0.04 and 0.06 were identified as marginally changed.

Western blot analysis. Samples were prepared using SDS lysis buffer [2% SDS, 10% glycerol, 0.06 mol/L Tris (pH 6.8)] and evaluated for protein concentration using the bicinchoninic acid method (Pierce, Rockford, IL). Proteins (50 µg) were separated on 8% SDS-PAGE gels and transferred to nitrocellulose membranes. Membranes were blocked in tris-buffered saline containing 0.05% Tween 20 (TBST), 5% nonfat milk and incubated with primary antibody in TBST, 5% nonfat milk, overnight at 4°C. Membranes were washed thrice for 10 minutes with TBST and incubated with the appropriate secondary antibody in TBST, 5% nonfat milk for 90 minutes at room temperature. Antibody binding was visualized by chemiluminescence on autoradiography film. Relative expression of proteins was determined by densitometry analysis of at least two scanned autoradiography films from independent experiments using Photoshop software.

Immunofluorescence. MCF-7 cells were treated with 2 mmol/L valproic acid for 48 hours. Cells were harvested by trypsinization, washed in PBS, and adhered to glass slide using Cytospin Funnels (Shandon, Pittsburgh, PA) at a density of 1 x 105/mL. Slides were blocked with 2% bovine serum albumin (BSA) in PBS for 1 hour and probed with antibodies specific for topoisomerase II{alpha}, KiS1 (monoclonal, 1:50), and acetylated histone H3 (polyclonal, 1:100) in 1% BSA/PBS for 1 hour at room temperature in a humidified chamber. Slides were washed in PBS and incubated with anti-rabbit Alexa Fluor 488 and anti-mouse Alexa Fluor 546 (Molecular Probes) diluted 1:100 in 1% BSA/PBS containing goat serum for 1 hour at room temperature in a humidified chamber. Slides were washed with PBS and coverslipped using Prolong Gold mounting media (Molecular Probes). Images were acquired by confocal microscopy. The square pixel surface area for the respective protein expression was analyzed in at least 50 cells per treatment using Photoshop software. For each measured nucleus, the background staining was measured in the immediate vicinity and subtracted from the average square pixel surface area. Evaluation of secondary antibody staining only served as internal staining control. Levels for the fluorescence detection were set accordingly. Experiments were repeated at least twice.

Antisense/small interfering RNA. Topoisomerase II{alpha} and topoisomerase IIß protein expression was blocked with antisense oligonucleotides and small interfering RNA (siRNA) duplexes. Antisense: The DNA sequences for the oligonucleotide probes were as follows: 5'-CTGCAATGGTGACACTTCCAT-3' for topoisomerase II{alpha} and 5'-TTTGTAGTGGACAGAAACACAGTA-3' for topoisomerase IIß as described by Towatari et al. (21). Oligonucleotide (1 µg) was suspended in 100 µL Optimem (Life Technologies Bethesda Research Laboratories) containing 2 µL Superfect transfection reagent (Qiagen, Valencia, CA). The oligonucleotide suspension was raised to a total volume of 700 µL by the addition of DMEM containing 10% fetal bovine serum and incubated on cell monolayers (1 x 105 cells) for 24 hours. The oligonucleotide mixture was replaced the following day, and the cells were incubated for an additional 4 hours before experimental procedures. Controls included topoisomerase II{alpha} and topoisomerase IIß sense oligonucleotides as well as incubation of cells with Superfect in the absence of oligonucleotide. siRNA: RNA duplexes for topoisomerase II{alpha} (sense, GGUAUUCCUGUUGUUGAAC) and topoisomerase IIß (sense, GGUUACCUUUGUGCCAGGU) were purchased from Ambion (Austin, TX). Cells were suspended in 0.1 mL siPort electroporation buffer (3 x 106/mL, Ambion), mixed with 1 µg siRNA, and pulsed with 300 V for 0.5 milliseconds. Pulsed cells were incubated at 37°C for 15 minutes before experimentation. The Silencer Negative Control #2 siRNA (Ambion), a nonsense siRNA duplex, was used as a control.

Trapped-in-agarose DNA immunostaining assay. The trapped-in-agarose DNA immunostaining assay (TARDIS) was done as described by Willmore et al. (22) with modifications. Cells (2 x 104 per slide) were imbedded in agarose and lysed [2.5 mol/L NaCl, 100 mmol/L EDTA (pH 10), 10 mmol/L Tris, 1% sarkosyl, 1% Triton X-100] in the presence of protease inhibitors (2 µg/mL pepstatin, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 0.2 mmol/L Na3VO4, and 1 mmol/L phenylmethylsulfonyl fluoride) for 30 minutes. Imbedded cells were washed for 20 minutes with 1 mol/L NaCl in the presence of protease inhibitors and incubated in 2% BSA in PBS for 1 hour. Immunostaining consisted of anti-topoisomerase II{alpha} (454, polyclonal, 1:200) and anti-topoisomerase IIß (monoclonal, 1:100) in 0.5% BSA for 1 hour at room temperature in a humidified chamber. Slides were washed twice for 10 minutes in 0.5% BSA and incubated with the appropriate secondary antibodies (anti-rabbit 546 and anti-mouse 488; Molecular Probes) at a concentration of 1:200 in 0.5% BSA containing goat serum (1:100) for 1 hour at room temperature. Slide were washed with 0.5% BSA for 10 minutes, dried, and counterstained with 0.5 µg/mL of bisbenzimide trihydrochloride (Hoechst #33258, Molecular Probes). Images were acquired by confocal microscopy and the square pixel surface area of the respective protein was analyzed for a minimum of 50 cells per treatment group. Statistical analysis was done by ANOVA. Experiments were repeated at least twice.

Band depletion. The band depletion assay was done as described by Xiao et al. (8). Briefly, 5 x 105 cells were lysed in alkaline lysis solution (200 mmol/L NaOH, 2 mmol/L EDTA), and the lysate was neutralized [neutralization buffer: 1 mol/L HCl, 600 mmol/L Tris (pH 8.0)]. The neutralized lysate was then mixed with 3x SDS sample buffer [150 mmol/L Tris-HCl (pH 6.8), 6 mmol/L EDTA, 45% sucrose, 9% SDS, 10% ß-mercaptoethanol], and the lysates were separated on 8% SDS-PAGE gels.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Interactions of topoisomerase II poisons and histone deacetylase inhibitors. We previously reported an increased sensitivity of tumor cells to topoisomerase II–targeting agents after pretreatment with an HDACi in vitro and in vivo (14, 20). Treatment with HDACi led to dose- and time-dependent histone acetylation and subsequent modulation of genes and proteins essential for the maintenance of heterochromatin. The ensuing chromatin decondensation was associated with an increased binding of topoisomerase II poisons to the DNA substrate and in the presence of topoisomerase II, with increased DNA damage and cell death.

The sensitivity of cancer cells to topoisomerase II–targeting agents has been linked to expression of the topoisomerase II{alpha} isoform, whereas studies on the role of topoisomerase IIß have been more limited. Our experimental data indicate that preexposure of tumor cells to HDACi potentiates the apoptosis induced by the topoisomerase II poisons epirubicin and mitoxantrone (Fig. 1A). Potentiation was not limited to a certain class of HDACi and was observed with selective (SAHA and trichostatin A) as well as with nonselective (valproic acid) HDACi. Similarly, potentiation occurred with other topoisomerase II–targeting agents, such as doxorubicin, etoposide, and VM-26 (see below; data not shown; and refs. 14, 20).



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  		Fig. 1. HDACi potentiate topoisomerase II inhibitor cytotoxicity. MCF_7 breast cancer cells were treated with 2 mmol/L valproic acid (VPA) or 0.5 µmol/L SAHA for 48 hours followed by exposure to 0.5 µmol/L epirubicin or mitoxantrone for 4 hours. Cells were harvested 24 hours later and evaluated for nuclear condensation and fragmentation. Columns, mean from at least three replicate experiments; bars, SE.

 
Densitometry analysis of Western blots (n = 3) evaluating cancer cells derived from different organs, including breast, colon, and melanoma, showed that the examined cell lines with decreased or mutated topoisomerase II{alpha}, required higher concentration of epirubicin for IC50 (Table 1) as assessed by colony-forming assays. As reported by other investigators, these data suggest that not organ specificity but rather expression of the topoisomerase II isoforms was predictive for response (13).


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  		Table 1. Percentage expression of topoisomerase II{alpha} and topoisomerase IIß relative to MCF-7 cells and the IC50 dose (95% confidence intervals) of epirubicin by clonogenic analysis with and without a 48-hour pretreatment with the indicated valproic acid dose in the breast cancer cell lines SKBr-3, MCF-7, BT-474, MDA-361, and MCF:Dox; the colon cancer cell line KM12C; and 0.25 mmol/L valproic acid in the melanoma cell line A375

 
A 48-hour preexposure of these cells to the HDACi valproic acid resulted in a decrease in the IC50 of epirubicin. The concentration of valproic acid used for these experiments affected <10% of cells, resulting in a fractional inhibition of cell growth by valproic acid alone of Fi < 0.1 compared with untreated cells (Table 1). Isobologram analysis using the CalcuSyn software suggested the interaction between valproic acid and epirubicin was synergistic in SKBr3, MCF-7, A375, BT-474, KM12C, and MCF:Dox cells (data not shown). A decrease in IC50 was not observed in MDA-361 cells with a near depletion of both topoisomerase II isoforms topoisomerase II{alpha} and topoisomerase IIß. In contrast, in the epirubicin-resistant cell line MCF:Dox, the IC50 of epirubicin was lowered by valproic acid despite a mutation and down-regulation of the topoisomerase II{alpha} protein. Although a potential role of multidrug resistance genes in the potentiation of epirubicin by valproic acid cannot be ruled out in the scope of these experiments, limited microarray experiments do not suggest a change in the expression of MDR and MDR-related proteins (data not shown).

Topoisomerase II{alpha} expression is down-regulated by histone deacetylase inhibitor. It is thought that sensitivity of cancer cells to topoisomerase II–targeting agents is predominantly mediated through topoisomerase II{alpha} (13–17). However, we observed an HDACi-induced sensitization in cells with depleted topoisomerase II{alpha} but not in cells depleted of both topoisomerase II isoforms. The effects of HDACi on the expression of topoisomerase II{alpha} and topoisomerase IIß were evaluated across sensitive and resistant cell lines. As reported previously, we found that HDACi-induced sensitization of cells to topoisomerase II poisons was maximal after a 24- to 48-hour preexposure to the HDACi (14). Time course evaluating the protein expression of topoisomerase II{alpha}, topoisomerase IIß, and topoisomerase I showed that after a 48-hour exposure to 2 µmol/L SAHA, rather than an up-regulation, a depletion of topoisomerase II{alpha} protein was observed (Fig. 2A). The depletion of topoisomerase II{alpha} expression was not limited to SAHA but was observed with the short-chain fatty acids valproic acid and NaB as well as the hydroxamic acid trichostatin A (Fig. 2B). The decrease in nuclear protein expression corresponded to a time-dependent reduction in the expression of topoisomerase II{alpha} mRNA (Fig. 2C) and not a change in cellular location (Fig. 2D). Furthermore, HDACi had no significant effects on the expression of topoisomerase I and topoisomerase IIß (Fig. 2A and B). Expression levels of topoisomerase II{alpha} vary with alterations in cell cycle distribution with peak expression in G2-M. To rule out a causal link between HDACi-induced cell cycle block and the observed reduction in topoisomerase II{alpha} expression, topoisomerase II{alpha} levels were studied over a wide range of HDACi concentrations. Topoisomerase II{alpha} was depleted at concentrations of HDACi that did not perturb cell cycle progression (Fig. 2B; data not shown), suggesting that HDACi-induced cell cycle arrest and HDACi-induced topoisomerase II{alpha} depletion are independent events (data not shown). Furthermore, a SAHA-induced depletion of topoisomerase II{alpha} was observed at concentration of SAHA (>5 µmol/L) that were shown to result in a G2-M arrest, the cell cycle phase where topoisomerase II{alpha} is increased (23).



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  		Fig. 2. HDACi exposure depletes topoisomerase II{alpha} (topo II{alpha}) but not topoisomerase IIß. A, Western blot depicting changes in the expression of topoisomerase II{alpha} in log-phase MCF-7 cells after exposure to 2 µmol/L SAHA for the indicated times. B, expression of topoisomerase II{alpha} and topoisomerase IIß in log-phase MCF-7 cells treated with 2 mmol/L valproic acid (VPA), 0.5 µmol/L SAHA, 0.05 µmol/L trichostatin A (TSA), and 2 mmol/L NaB for 48 hours. C, microarray analysis showing the relative topoisomerase II{alpha} mRNA expression after 0, 4, 24, and 48 hours of incubation with 2 mmol/L valproic acid. D, expression and location of topoisomerase II{alpha} and acetylation of histone H3 in MCF-7 cells after exposure to 2 mmol/L valproic acid for 48 hours using confocal microscopy at a x63 magnification.

 
Relevance of topoisomerase IIß as a target. Potentiation of topoisomerase II targeting agents by HDACi in the setting of topoisomerase II{alpha} depletion argued for the involvement of the alternative topoisomerase II isoform topoisomerase IIß. Synergy between an HDACi and a topoisomerase II–targeting agent was preserved when using the topoisomerase IIß–specific poison, XK469, signifying a potential role of topoisomerase IIß as a target for drug therapy. A potentiation of XK469 by preexposure to valproic acid was found in several examined breast cancer cell lines, including SKBr-3, MCF-7, and the topoisomerase II{alpha}–depleted MCF:Dox cells (Fig. 3A). To further define the involvement of topoisomerase IIß in the interaction between HDACi and topoisomerase II poisons, both topoisomerase II isoforms were alternatively depleted by siRNA or antisense and exposed to HDACi before administration of a topoisomerase II poison. We have shown above that treatment with HDACi resulted in a down-regulation of topoisomerase II{alpha} (Fig. 2). Treatment of MCF-7 cells with siRNA resulted in significant depletion of topoisomerase II{alpha} and topoisomerase IIß protein levels without affecting viability (Fig. 3B; data not shown). Topoisomerase II{alpha} was further depleted with the addition of valproic acid (Fig. 3B). Depletion of topoisomerase II{alpha} by siRNA did not effect the potentiation of epirubicin, mitoxantrone, or VM-26 by valproic acid (Fig. 3C). In contrast, the synergistic activity between HDACi and these topoisomerase II–targeting drugs was abrogated by the depletion of topoisomerase IIß (Fig. 3C). These findings were mirrored when antisense was used to deplete topoisomerase II{alpha} or topoisomerase IIß or when SAHA was used in lieu of valproic acid (data not shown).



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  		Fig. 3. Topoisomerase IIß (topo IIß) is the prime target of topoisomerase II inhibitors. A, % apoptotic nuclei in cells treated with 2 mmol/L valproic acid (VPA) for 48 hours followed by 0, 0.1, and 0.5 mmol/L of the topoisomerase IIß–specific inhibitor XK469 for 48 hours in the cell lines MCF-7 and SKBr-3 and the topoisomerase II{alpha}–depleted MCF:Dox. B, expression of topoisomerase II{alpha} and topoisomerase IIß in MCF-7 cells after exposure to siRNA and 2 mmol/L valproic acid for 48 hours. Densitometry analysis of the protein bands is listed as % expression relative to control samples. C, % apoptotic nuclei in cells treated with 2 mmol/L valproic acid followed by 0.5 µmol/L epirubicin, 0.5 µmol/L mitoxantrone, or 1 µmol/L VM-26 for 4 hours, after depletion of topoisomerase II{alpha} and topoisomerase IIß by siRNA. Columns, mean from at least three different replicate experiments; bars, SE.

 
Although the topoisomerase II{alpha} siRNA was exquisitely specific, the topoisomerase IIß siRNA showed a slight off-target effect on the expression of topoisomerase II{alpha}. This off-target effect unlikely contributed to the abrogation of synergy between valproic acid and topoisomerase II poisons because the direct depletion of topoisomerase II{alpha} by siRNA in the absence of HDACi effects and the further depletion of topoisomerase II{alpha} by the addition of valproic acid did not sensitize cells to topoisomerase II poisons.

Hence, in the presence of an HDACi, topoisomerase II poison cytotoxicity seems mediated through topoisomerase IIß.

Histone deacetylase inhibitor increases the formation of DNA/topoisomerase IIß cleavable complexes. Treatment with HDACi result in chromatin decondensation and increased access of topoisomerase II–targeting agents to the DNA substrate (14, 20). Topoisomerase II poisons are thought to confer cytotoxicity through the stabilization of topoisomerase II/DNA cleavable complexes. We evaluated whether the presence of HDACi resulted in the involvement of a specific topoisomerase II isoform in the cleavable complexes stabilized by topoisomerase II poisons.

The cleavable complexes involving topoisomerase II{alpha} or topoisomerase IIß induced by epirubicin in the presence and absence of a 48-hour pretreatment with 2 mmol/L valproic acid were evaluated by trapped-in-agarose DNA immunostaining assay.

Immunofluorescence analysis of confocal microscopy images showed that at concentrations of epirubicin with minimal effect on the stabilization of cleavable complexes involving topoisomerase II{alpha} (P > 0.05), there was a 3-fold increase in the accumulation of topoisomerase IIß–containing cleavable complexes stabilized by epirubicin in the presence of valproic acid (Fig. 4A and B). This difference reached statistical significance (*, P < 0.001). Similar results were observed when mitoxantrone was used in lieu of epirubicin (data not shown). Valproic acid by itself did not induce the formation of cleavable complexes. As described above, valproic acid treatment results in depletion of topoisomerase II{alpha}.



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  		Fig. 4. Topoisomerase IIß (topo IIß) is the effector protein. A, trapped-in-agarose DNA immunostaining analysis of cleavable complexes involving topoisomerase II{alpha} (red) and topoisomerase IIß (green) in cells treated with 0.5 µmol/L epirubicin (Epi) for 4 hours in the presence and absence of a 48-hour pretreatment with 2 mmol/L valproic acid (VPA) by confocal microscopy. B, immunofluorescence analysis of square pixel surface area per replicate sample depicting changes in topoisomerase II{alpha} and topoisomerase IIß cleavable complexes for the indicated treatment groups. Squares, mean; square brackets, 95% confidence interval. *, P ≤ 0.001. C, band depletion assay of topoisomerase II{alpha} and topoisomerase IIß in MCF-7 cells incubated with 50 µmol/L VM-26 after pretreatment with 0 or 2 mmol/L valproic acid and densitometry analysis relative to untreated cells. Topoisomerase II{alpha} and topoisomerase IIß were depleted in the presence of VM-26, suggesting the formation of cleavable complexes. Topoisomerase IIß was further depleted when cells were exposed to valproic acid before VM-26, suggesting an enhancement of cleavable complex formation. The decrease in topoisomerase II{alpha} induced by valproic acid was not due to the formation of cleavable complexes but due to down-regulation of protein expression (see Fig. 2B and Fig. 3B). Therefore, the depletion of topoisomerase II{alpha} in the VPA:VM-26 sample was a reflection of protein down-regulation by valproic acid and cleavable complex formation induced by VM-26 alone and did not signify an enhancement of VM-26-induced cleavable complex formation.

 
To further establish whether these findings were intrinsic to the anthracycline family members (intercalating topoisomerase II poisons), band depletion assays were performed using the nonintercalating topoisomerase II poison, VM-26. This drug has been reported to induce cell death with the preferential formation of topoisomerase II{alpha}–containing cleavable complexes (24). The band pattern of topoisomerase II{alpha} and topoisomerase IIß was evaluated in MCF-7 cells exposed to VM-26 and the HDACi valproic acid (Fig. 4C). Band depletion assays allow detection of topoisomerase II{alpha} and topoisomerase IIß complexed with DNA. The topoisomerase enzymes that are covalently bound to DNA (cleavable complexes) are prohibited from entering the acrylamide gel resulting in a band depletion, whereas topoisomerase II enzymes not bound to DNA readily enter the gel. Exposure of cells to VM-26 resulted in a 52% depletion of topoisomerase IIß band, whereas no effects were seen with valproic acid alone. In cells preexposed to 2 mmol/L valproic acid, VM-26 induced an 81% reduction in the topoisomerase IIß band, suggesting an increase in cleavable complex formation Fig. 4C (bottom).

VM-26 induced a 51% depletion in the topoisomerase II{alpha} band. There was a 46% decrease in the topoisomerase II{alpha} band after treatment with valproic acid alone. In cells treated with valproic acid before VM-26, there was a 92% depletion of the topoisomerase II{alpha} band (Fig. 4C, top). However, as discussed above, treatment with valproic acid resulted in a down-regulation of topoisomerase II{alpha} protein expression (Fig. 2A and B and Fig. 3B). The further reduction in the topoisomerase II{alpha} band by VM-26 in the presence of valproic acid resulted from the additive effects of the HDACi-induced down-regulation of the protein and the loss of topoisomerase II{alpha} band due to cleavable complex formation induced by VM-26. This is further supported by the trapped-in-agarose DNA immunostaining assay showing no increase in the topoisomerase II{alpha}–cleavable complexes induced by valproic acid alone. These findings suggest that treatment of cell with valproic acid enhanced the formation of cleavable complexes involving topoisomerase IIß but not topoisomerase II{alpha} in the presence of VM-26.


   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Topoisomerase II is an essential enzyme for cell survival, and the increased expression of topoisomerase II in many cancer cells has made these enzymes attractive targets for cancer therapy. Topoisomerase II exists in two isoforms, topoisomerase II{alpha} and topoisomerase IIß, with partial functional redundancy (9, 25–28). In yeast, either isoform may substitute for the endogenous topoisomerase II enzyme (29). Murine transgenic studies have shown that although topoisomerase II{alpha} may compensate for a loss of topoisomerase IIß during embryogenesis (30), the loss of topoisomerase II{alpha} leads to embryonic death (31). However, topoisomerase IIß seems essential for neuromuscular development after birth (30). In adult mammalian cells, disruption of topoisomerase IIß by siRNA has no detrimental effects on cell cycle progression. In addition, topoisomerase IIß may compensate, albeit only partially, for topoisomerase II{alpha} during chromatin condensation and cell segregation (9, 32). Despite similar catalytic activities of the topoisomerase II isoenzymes, cell survival has been correlated with the expression of topoisomerase II{alpha} (12, 33). The role of topoisomerase II as drug targets for topoisomerase II poisons has been evaluated in several studies; and it has been suggested that topoisomerase II{alpha} is the relevant target of topoisomerase II poisons. Disruption or mutation of topoisomerase II{alpha} was associated with drug resistance (34–36). In particular, there are several reports suggesting topoisomerase II{alpha} to be an important target for epirubicin, the drug used predominantly for this study; however, much less is known about the role of topoisomerase IIß as a drug target for epirubicin (17). There currently are several drugs approved for clinical use that predominantly target topoisomerase II{alpha}, whereas the clinical utility of topoisomerase IIß–specific drugs is still under investigation (37).

Here, we provide evidence that HDACi render topoisomerase IIß a relevant target and effector substrate for topoisomerase II poisons. We have shown that preexposure of tumor cells to HDACi led to histone acetylation and down-regulation of proteins essential for the maintenance of heterochromatin (20). The ensuing chromatin decondensation was associated with increased binding of topoisomerase II poisons to the DNA substrate (14, 20). The kinetics of the HDACi-induced chromatin decondensation suggested that a 48-hour preexposure was optimal for synergistic activity.

We now show that exposure of tumor cells to HDACi significantly reduced the concentrations of topoisomerase II poisons required for growth inhibition, even in cells manipulated for target-specific resistance to topoisomerase II–targeting agents. Sensitization was not tissue specific but was observed in breast, colon, leukemia, melanoma, and sarcoma cell lines. We observed a time-dependent reduction in the expression of topoisomerase II{alpha} mRNA and protein in the presence of HDACi that was maximal at 48 hours. In contrast, the effects of HDACi on topoisomerase IIß were minimal. Modulation of the topoisomerase II isoforms with siRNA and antisense showed that depletion of topoisomerase IIß abrogated the HDACi-induced potentiation of topoisomerase II poisons, whereas the depletion of topoisomerase II{alpha} did not affect this synergistic interaction. Furthermore, topoisomerase II poison cytotoxicity in the presence of HDACi was associated with an accumulation of cleavable complexes involving topoisomerase IIß. The depletion of topoisomerase II{alpha} and increase in topoisomerase IIß–containing cleavable complexes provide a strong argument for a more prominent role of topoisomerase IIß in the synergistic interaction between HDACi and topoisomerase II–targeting agents.

Combinations of HDACi and topoisomerase II–targeting agents have been reported by several investigators. Although Johnson et al. observed a decrease in sensitivity of leukemia cells to etoposide after preexposure to an HDACi (38), Tsai et al. observed a sensitization of leukemia cells to etoposide (18). This discrepancy is most like explained by differences in HDACi preexposure and drug concentration. Where Johnson et al. preexposed HL-60 cells to 100 nmol/L trichostatin A for 0.5 hours, Tsai et al. used 400 nmol/L trichostatin A for 4 hours. Since then, other groups have reported increased sensitivity of several tumor cell lines to topoisomerase II targeting agents after preexposure to an HDACi (14, 19, 20, 39, 40). Synergy was dependent on sequence and timing of drug administration and associated with HDACi-induced chromatin decondensation (14, 19).

Although topoisomerase II{alpha} has been proposed as the effector molecule for cytotoxicity for topoisomerase II poison, the specific roles of topoisomerase II{alpha} and topoisomerase IIß in the interaction between HDACi and topoisomerase II poisons have not been defined.

Fraser et al. reported an initial activation of the topoisomerase II{alpha} gene promoter with a transient up-regulation of topoisomerase II{alpha} protein expression in leukemia cells treated with the HDACi NaB for 18 to 24 hours. This promoter activation was reversed with longer HDACi exposure, resulting in topoisomerase II{alpha} promoter activity repression (41). In contrast, Kim et al. found no change in the expression or activity of topoisomerase II{alpha} in glioblastoma cells exposed to the HDACi SAHA (19); however, the exposure times may have differed. We observed an HDACi-induced depletion of topoisomerase II{alpha} mRNA and protein. Maximal effects only occurred after a prolonged exposure to the HDACi (48 hours). Topoisomerase II{alpha} depletion was not limited to a specific HDACi class but was observed with both short-chain fatty acid and hydroxamic acid HDACi. In part, the differences between the findings presented in this report and those presented by others may be explained by differences in the duration of HDACi exposure. More importantly, these findings may also suggest a limited role of topoisomerase II{alpha} in the observed synergy between an HDACi and a topoisomerase II poison. This is further supported by our findings showing that near-complete depletion of topoisomerase II{alpha} by siRNA or antisense in the presence of an HDACi did not inhibit the potentiation of topoisomerase II poisons by HDACi. The HDACi-induced depletion of topoisomerase II{alpha} mRNA and protein expression was maximal at 48 hours, which correlated to maximal HDACi-induced chromatin decondensation and the optimal timing of synergy between HDACi and topoisomerase II–targeting agents. Although topoisomerase II{alpha} was reported to be involved in chromatin condensation (42), a mechanistic link between the topoisomerase II{alpha} depletion and chromatin decondensation cannot be established by the presented data.

More evidence suggesting topoisomerase IIß as a relevant target in the potentiation of topoisomerase II–targeting agents by HDACi emerges from studies using the topoisomerase IIß–specific poison XK469. XK469 was shown to induce cleavable complexes in several cell lines in vitro; however, meaningful effects on tumor burden in vivo were not achievable at tolerable doses (43). We found that XK469 by itself only caused minimal apoptosis in breast cancer cells; however, when administered in a sequence-specific combination with HDACi, we observed a potentiation of XK469 induced apoptosis by HDACi. These experiments collectively imply that topoisomerase IIß is a relevant target in the interaction between HDACi and topoisomerase II–targeting agents. This is further supported by the findings that depletion of topoisomerase IIß abrogated the synergistic interaction, whereas depletion of topoisomerase II{alpha} did not affect synergy.

Although topoisomerase IIß may be a target of selected topoisomerase II poisons (44–46), most reports suggest that the cytotoxicity for the clinically relevant topoisomerase II–targeting agents is determined by topoisomerase II{alpha} levels (47–50). The data presented here show that HDACi potentiates the cytotoxicity of topoisomerase II{alpha} and topoisomerase IIß–specific topoisomerase II poisons by recruiting topoisomerase IIß as a target. The results of this study may have several clinical implications. HDACi may lower the concentration of topoisomerase II targeting agents required for activity, thereby limiting adverse effects. Emerging drug resistance due to a loss or mutation of topoisomerase II{alpha} may be circumvented by engaging topoisomerase IIß as an alternative target. A potentiation of cytotoxicity was not observed in fibroblast exposed to an HDACi before exposure to a topoisomerase II poison (data not shown). Furthermore, preliminary results from an ongoing phase I trial studying a combination of valproic acid and epirubicin did not suggest a potentiation of epirubicin-induced toxicity (Munster et al, ASCO 2005 #A3084). The differential effects of this combination on somatic versus tumor cells may limit tissue-specific toxicities, such as anthracycline-associated cardiotoxicity. In addition, HDACi may increase the clinical utility of ß-specific drugs.


   Acknowledgments
 
We thank Dr. Fred Hausheer (Bionumerick Pharmaceuticals Inc., San Antonio, TX) for the MCF:Dox cell line and Dr. Robert Snapka (Department of Radiology, Ohio State University, Columbus, OH) for XK469.


   Footnotes
 
Grant support: Susan G. Koman Breast Cancer Foundation and Don Shula Career Development Award.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 5/13/05; revised 8/15/05; accepted 8/31/05.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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