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Experimental Therapeutics, Molecular Targets, and Chemical Biology |
Department of Interdisciplinary Oncology, Experimental Therapeutics Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida
Requests for reprints: Pamela N. Munster, Department of Interdisciplinary Oncology, Experimental Therapeutics Program, H. Lee Moffitt Cancer Center, MRC-4 East 12902 Magnolia Drive, Tampa, FL 33612. Phone: 813-745-8948; Fax: 813-745-1984; E-mail: Munstepn{at}moffitt.usf.edu.
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Abstract |
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Introduction |
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A class of compounds that modulate chromatin dynamics are the HDAC inhibitors. We have previously shown that acetylation of histones by the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) leads to conformational changes of DNA and chromatin decondensation. SAHA-induced chromatin decondensation was associated with potentiation of DNA damage induced by topo II inhibitors in a sequence-specific manner (11). Similar studies have been reported by others (12). Whereas these studies are important for the clinical development of HDAC inhibitors, they only provided limited insight into a potential mechanism by which the SAHA-induced histone hyperacetylation leads to chromatin decondensation.
Here, we expanded these studies to evaluate the effects of the antiseizure drug valproic acid (VPA, 2-propylpentanoic acid) on histone acetylation and propose a mechanism by which VPA and other HDAC inhibitors may induce chromatin remodeling and promote access of DNA-damaging agents to their target sites. Furthermore, the effects of VPA on the efficacy of DNA-damaging agents were evaluated in a breast cancer xenograft model.
Our data indicate that VPA induced chromatin decondensation in breast cancer cells by repression of SMC 1 to 5 and SMC-associated proteins, DNMT-1, and HP1. VPA-induced chromatin decondensation was associated with an increased sensitivity of DNA to nucleases and increased the association of DNA with intercalating agents. Furthermore, VPA potentiated DNA damage and apoptosis induced by cytotoxic agents that require access to the DNA for activity. VPA led to histone hyperacetylation in vivo and in vitro. However, unlike the VPA-induced histone hyperacetylation, which was maximal by 1 hour, the regulation of genes involved in chromatin decondensation required prolonged exposure to VPA, suggesting that the modulation of chromatin maintenance proteins are an important additional step in the regulation of chromatin structure. Modulation of heterochromatin maintenance proteins was reversible upon drug withdrawal and was obligatory for potentiation of DNA-damaging agents. Studies in a breast cancer xenograft model indicated that these effects occur at clinically relevant concentrations.
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Materials and Methods |
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Cell lines
All cell lines were purchased from American Type Culture Collection (Manassas, VA) and maintained in DMEM with 10% heat-inactivated fetal bovine serum, 2 mmol/L glutamine, and 50 units/mL penicillin and 50 µg/mL streptomycin. For experimental procedures, cells were incubated in a humidified atmosphere with 5% CO2 at 37°C.
Histone acetylation
Cells were exposed to experimental conditions and acetylated histones were visualized using antiacetylated histone H3 and H4 antibodies by Western blot analysis as described previously (13).
Microarray
Alterations in gene expression induced by VPA were evaluated by microarray using Affymetrix U74Av2 Genechips (900343; Affymetrix Inc., 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 increases or decreases in mRNA abundance in a direct comparison between two samples (statistical algorithms description document). Two samples of each condition were evaluated and each experiment was repeated at least twice. This analysis is based on the behavior of 16 different oligonucleotide probes designed to detect the same gene. Probe sets that yielded a change in P value of <0.04 were identified as changed (increased or decreased) and those that yielded a P value between 0.04 and 0.06 were identified as marginally changed.
Electron microscopy
Cells were fixed with 4% paraformaldehyde for 1 hour at 37°C. Samples were rinsed with PBS, dehydrated in increasing ethanol concentrations (50%, 70%, 80%, 90%, and 100%), and embedded in water-permeable LR White plastic. Cells were sectioned with a diamond knife at 90 nm thickness and stained for 2 minutes in 1% uranyl acetate and for 5 minutes with lead citrate, and finally examined at 60 kV on a Philips 100M electron microscope at 14,000- and 32,000-fold magnification.
Micrococcal nuclease digestion
MCF-7 cells (1 x 106) were Dounce homogenized in RSB buffer [10 mmol/L Tris-HCl (pH 7.4), 10 mmol/L NaCl, 3 mmol/L MgCl2, 0.5% NP40] containing 10 µg/mL aprotinin and leupeptin, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L Na3VO4, and 1 mmol/L DTT, and incubated on ice for 15 minutes. Samples were washed twice with RSB buffer and digested with 15 units of micrococcal nuclease in digestion buffer [15 mmol/L Tris-HCl (pH 7.5), 60 mmol/L KCl, 15 mmol/L NaCl, 1 mmol/L CaCl2, 3 mmol/L MgCl2, 20% glycerol, 15 mmol/L ß-mercaptoethanol] for 5 minutes. Digestion was stopped by adding 1 volume of stop solution [50 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 50 mmol/L EDTA, 0.3% SDS]. DNA was extracted using 1 volume of phenol/chloroform/isoamyl alcohol (25:24:1) followed by 1 volume chloroform/isoamyl alcohol (24:1) and precipitated with 100% isopropyl alcohol. The DNA was washed once with 70% ethanol, resuspended in Tris-HCl (pH 8.0)/EDTA buffer, and separated using 1.2% agarose gels.
DNase hypersensitivity
Cells (2 x 106) were fixed with 1.5% paraformaldehyde for 15 minutes on ice, washed with cold PBS, and incubated for 16 hours in 70% ethanol at –20°C. Cells were then washed once with PBS and once with ISNT buffer [25 mmol/L MgCl2, 50 mmol/L Tris-HCl (pH 8.0), 10 mmol/L ß-mercaptoethanol, 10 µg/mL bovine serum albumin (BSA); ref. 14]. DNA nicks were generated by incubation with 5 units of DNase I in ISNT buffer for 25 minutes. Cells were washed with PBS and incubated in polymerase buffer (16 µmol/L dATP, 16 µmol/L dCTP, 16 µmol/L dGTP, 16 µmol/L dUTP-FITC, 7 units/mL DNA polymerase I) for 90 minutes at 37°C and then analyzed by fluorescence-activated cell sorting (FACS) for the incorporation of dUTP-FITC.
DNA intercalation with acridine orange
MCF-7 cells (2 x 105) were treated with 2 mmol/L VPA for 48 hours and the nuclei purified as described by Nusse et al. (15). Nuclei were exposed to increasing concentrations of acridine orange (0-10 µmol/L) for 10 minutes and fluorescence intensity was measured with the appropriate filter set (excitation 485 nm, emission 535 nm). Similarly, DNA intercalation of epirubicin was measured after cells were exposed for 48 hours to increasing concentrations of VPA followed by a 4-hour epirubicin exposure (1 µmol/L). Fluorescence intensity of epirubicin was assessed as previously described (excitation 485 nm, emission 535 nm; ref. 16).
Comet assay
The alkaline comet assay was done and used according to manufacturer's recommendations (Trevigen, Gaithersburg, MD). Slides were then incubated for 1 hour in alkaline electrophoresis solution (pH > 13). Comet tails were generated by a 40-minute electrophoresis at 20 V, 4°C. Slides were stained for 20 minutes with SYBR green and the comet moment was quantified using the LAI Comet Analysis System.
Evaluation of apoptosis
Apoptosis was scored by the presence of nuclear chromatin condensation and DNA fragmentation and evaluated with fluorescence microscopy using bis-benzimide staining. Cells were 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; Molecular Probes, Eugene, OR). Two hundred cells were counted per experiment in five different fields and scored for apoptosis (nuclei / all nuclei x100). Each experiment was repeated at least thrice; error bars depict SE.
Clonogenic assays
Cells were treated with 0.5 mmol/L VPA for 48 hours followed by a 4-hour epirubicin exposure. Colonies were evaluated after 14 to 21 days in culture without retreatment, stained with 2% crystal violet in methanol, and counted if measuring at least 0.2 mm. All experiments were repeated at least thrice.
Isobologram analysis
Fractional inhibition of growth was determined using colony-forming assays and isobologram analysis with the CalcuSync software program as described by Chou et al. (17) to determine synergistic, additive, or antagonistic effects from drug combinations. IC50s were calculated as the concentrations required for 50% inhibition of growth.
Animal studies
Four- to six-week old nu/nu athymic female mice were obtained from the National Cancer Institute-Frederick Cancer Center (Frederick, MD) and maintained in ventilated caging. Experiments were done under an Institutional Animal Care and Use Committee–approved protocol (R2462) and institutional guidelines for animal welfare. Estrogen pellets (60-day slow release, 0.72 µg, Innovative Research, Sarasota, FL) were placed under the dorsal skin 7 days before tumor inoculation. MCF-7 cells (5 x 106) mixed 1:1 with Matrigel were injected s.c. on the right and left flanks. Once tumors reached 5 mm in the largest diameter, mice were treated in the following cohorts: saline only, VPA only, epirubicin only, and VPA followed by epirubicin. Mice receiving VPA were injected with 500 mg/kg/d VPA in 0.1 mL saline i.p. twice daily for 5 doses. Epirubicin (3 mg/kg in 0.1 mL saline) was injected once after the last dose of VPA. The respective control groups received corresponding saline injections.
Tumor growth. Tumor growth (four to eight mice per cohort) was monitored three to four times per week by measuring two perpendicular tumor diameters with a caliper. Tumor volumes were calculated as V = 
/ 6 x length x width2. Animals were sacrificed when the largest tumor diameter reached 13 mm. Statistical analysis was done using paired Student's t test and multivariate analysis by ANOVA for comparisons within and among groups.
Histone acetylation. Tumor cells isolated from mice (three mice per experimental group) injected with saline or 500 mg/kg/d VPA for 48 hours were evaluated for histone H3 acetylation by immunofluorescence. Briefly, tumors were dissected with care taken to remove the extraneous connective tissue. Tumors were minced in 1 mL PBS containing 200 units of type III collagenase and incubated for at 37°C for 1 hour. Single cells were acquired by passage through a 35 µm nylon mess. Cells were washed in PBS, adhered to glass slides using Cytospin Funnels, and fixed with 95% ethanol, 5% acetic acid for 1 minute. Slides were then incubated with antiacetylated H3 antibody in 2% BSA for 1 hour, washed, and then incubated with fluorescent-labeled secondary antibody for 1 hour and nuclei were counterstained with 0.5 µg/mL bis-benzimide (Hoechst). Images were acquired by confocal microscopy acquired as TIFF files, and H3 staining was analyzed for square pixel surface area. Statistical analysis was done by Student's t test.
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Results |
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epirubicin, 32.3 (±3.1), P < 0.05; these results suggest that epirubicin-induced comet moments were significantly increased in the presence of VPA. To determine if the potentiation of anthracycline-induced DNA damage by VPA translated into cell death, epirubicin- and aclarubicin-induced apoptosis were assessed in the presence of VPA. MCF-7 cells were treated with VPA for 48, 24, 12, 4, or 0 hours before being exposed to an anthracycline (Fig. 5A). Potentiation of epirubicin- and aclarubicin-induced apoptosis was only observed after prolonged VPA preexposure (48 hours) and was abrogated with shorter preexposure or when concomitantly treated. VPA-induced potentiation of epirubicin was also evaluated by clonogenic assays. As depicted by the fractional inhibition, cells treated with 0.5 mmol/L VPA before epirubicin showed a decrease in survival when compared with cells treated with epirubicin alone (Fig. 5B). Isobologram analysis indicated that the combination was synergistic across all dose levels tested (Fig. 5C).
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Valproic acid–induced histone acetylation and chromatin decondensation in a breast cancer xenograft model. To determine if the effects of VPA on cancer cell lines are reproducible in an in vivo model, we evaluated histone acetylation and tumor growth in a breast cancer xenograft model. Mice bearing MCF-7 tumors were treated with 0 or 500 mg/kg/d VPA twice a day for 48 hours. Tumor cells were isolated 4 hours after the last dose of VPA and assayed for acetylated histone H3 by immunofluorescence as described in Materials and Methods. Tumor cells isolated from mice treated with VPA showed a 2.7-fold increase in the square pixel surface area of acetylated histone H3 staining when compared with controls (Fig. 6A). VPA potentiated the antitumor effects of epirubicin in this xenograft model. As shown in Fig. 6B, tumor progression was significantly inhibited (P < 0.05) in animals treated with VPA followed by epirubicin compared with mice treated with saline, epirubicin, or VPA alone. The concentration of VPA used in this study reflects plasma concentrations of 
3 mmol/L (26, 27). VPA plasma concentrations typically used for the chronic treatment of migraine headaches or seizures range from 0.3 to 1.1 mmol/L [Food and Drug Administration (FDA) package insert]. Higher concentrations are achievable for shorter exposure times without significant toxicities.1
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Discussion |
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VPA has been reported to function as an HDAC inhibitor, leading to the acetylation of histone tails (23, 30). Histone acetylation results in attenuation of the electrostatic charge interactions between histones and DNA and has been associated with chromatin decondensation (11, 12, 31). However, other studies postulate that histone acetylation alone may have minimal effects on chromatin structure (32). Epigenetic silencing is accomplished by condensation of the chromatin in the form of heterochromatin and has been associated with hypoacetylation and hypermethylation. Furthermore, the expression of proteins involved in the maintenance of heterochromatin, such as the SMC proteins, SMC-associated proteins, DNMT1, and HP1, affects the dynamics of the chromatin structure. As reported in other cell systems (18–23, 28–30), we observed a rapid, but reversible, histone hyperacetylation in the examined breast cancer cells and the MCF-7 xenograft model with clinically relevant concentrations of VPA. In the cultured cells, the maximal effects occurred by 1 hour of exposure (Fig. 1A). In addition, we found that VPA caused a decrease in the expression of mRNA encoding SMC proteins 1 to 5, several SMC-associated proteins, DNMT1, and HP1 (Fig. 2A) with subsequent depletion of the corresponding proteins (Fig. 2B). Whereas our data is most consistent with a transcriptional regulation of these genes, the possibility that VPA and SAHA may decrease the stability of mRNA transcripts or target them for proteasomal degradation cannot be entirely ruled out.
Down-regulation of these proteins resulted in profound changes in the chromatin structure as determined by electron microscopy (Fig. 3A). These chromatin structure changes correlated with increased sensitivity of DNA to nucleases, further indicating decondensation of the chromatin (Fig. 3B-D). Whereas we observed maximal histone acetylation within 1 hour at concentration of VPA as low 0.25 mmol/L (Fig. 1A and B), chromatin decondensation required higher concentrations of VPA as well as longer exposure times (at least 24-48 hours; Fig. 3). The kinetics and concentrations of VPA required to induce changes in the chromatin structure imply that the attenuation of electrostatic charges between DNA and histones induced by hyperacetylation of histones is not sufficient to explain chromatin decondensation. The results of this study indicate that the VPA-induced modulation of heterochromatin maintenance proteins is an important additional step in the control of the chromatin structure and its access to regulatory factors. Chromatin decondensation was also seen with other HDAC inhibitors, such as SAHA (11), arguing against a selective or direct effect of VPA.
VPA-induced chromatin decondensation led to enhanced sensitivity of DNA to nucleases and increased DNA binding by intercalating agents (Fig. 4A and C), signifying an increase in the access of macromolecules to DNA. This is supported by our data showing an increase in epirubicin binding to DNA and a potentiation of epirubicin- and aclarubicin-induced apoptosis and inhibition of colony growth after preexposure to VPA, which followed the kinetics of VPA-induced chromatin decondensation (Fig. 5A and B). Synergy, as determined by isobologram analysis (Fig. 5C) between epirubicin and VPA, was only observed when cells were preexposed to VPA for 48 hours. Short preexposure time or concomitant exposure abrogated this synergy. We have shown earlier that the effects of the HDAC inhibitor SAHA on tumor cell growth and differentiation were reversible and required the continuous presence of drug for biological effects (13). This is supported by the clinical evaluation of SAHA, where benefits were only observed when the drug was administered at least 3 to 5 d/wk (ref. 33 and Cancer Therapy Evaluation Program solicitation letter). The effects of VPA on tumor cells were reversible. Within 2 hours of VPA withdrawal, histone hyperacetylation was reversed; within 48 hours, the depleted chromatin structural proteins had reverted to baseline levels (Fig. 5D). In addition, the synergy between VPA and epirubicin was abrogated when VPA was washed out for 48 hours before epirubicin exposure (Fig. 5E). Whereas the VPA effects on the structural maintenance proteins are obligatory at the time of epirubicin exposure for synergy, a 48-hour preexposure seems to be optimal as the VPA pretreatment duration. Exposure of cells beyond 96 hours maintained the synergistic effects without further benefits (Fig. 5E). The latter findings are not consistent with an outgrowth of resistant cells, as one would expect an abrogation of synergy if the resistant cells were outnumbering the sensitive cells. The reversibility of HDAC inhibitor may pose limitations on their use as anticancer agents and may emphasize a more important role in sensitizing cells to cytotoxic agents.
The effects of VPA on tumor cell lines were then evaluated in vivo using a breast cancer xenograft model. Similar to in vitro experiments, treatment of tumor-bearing mice resulted in histone acetylation of tumor cells (Fig. 6A). Furthermore, we showed that a 48-hour treatment of mice with VPA-sensitized tumors to epirubicin induced growth inhibition in vivo (Fig. 6B) at concentrations where either drug alone had very little effect on tumor growth. Both drugs were used at concentrations relevant for clinical use and a clinical trial of this sequence-specific combination is currently ongoing.
In summary, we showed that VPA-induced chromatin decondensation correlated with transcriptional repression of gene and proteins involved in the maintenance of heterochromatin and the control of DNA access. The dose and duration of VPA required to induce changes in the chromatin structure indicate that the attenuation of DNA-histone charge interaction due to VPA-induced histone acetylation is not sufficient for chromatin decondensation, but rather a downstream effect of HDAC inhibition. We propose that the VPA-induced modulation of the chromatin structural proteins rather than histone hyperacetylation results in chromatin decondensation. VPA-induced chromatin decondensation may enhance the activity of targeting agents that depend on access to the target site.
These findings have several clinical implications: Histone hyperacetylation is a rapid event and may occur at lower concentrations than those required for chromatin decondensation. Hence, histone hyperacetylation may not be a reliable predictive surrogate or even pharmacologic marker of HDAC inhibitor when used as antitumor agents. The effects of VPA on chromatin structure were reversible, but obligatory for the potentiation of epirubicin-induced DNA damage. Therefore, maximal benefits in combinations between HDAC inhibitor and DNA-damaging agents are likely schedule dependent and may necessitate higher drug concentrations than those observed for histone acetylation. Moreover, the VPA-induced modulation of the chromatin structure proteins may be an alternative surrogate when evaluating HDAC inhibitors in translational studies.
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Acknowledgments |
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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.
We thank Dr. Nikola Valkov for his assistance with the electron micrographs and Joel Turner for his assistance with the micrococcal nuclease.
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Footnotes |
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Received 7/15/04. Revised 11/15/04. Accepted 1/13/05.
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. Int J Oncol 2002;20:97–106.[Medline]
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), VPA alone (
), epirubicin alone (
), or VPA followed by epirubicin (
). Tumor growth was significantly inhibited (P > 0.05) in animals treated with VPA followed by epirubicin compared with all other treatments.