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. Author manuscript; available in PMC: 2011 Dec 1.
Published in final edited form as: Eur J Cancer. 2010 Oct;46(15):2771–2780. doi: 10.1016/j.ejca.2010.05.010

Chromatin Remodeling at the Topoisomerase II-beta Promoter is Associated with Enhanced Sensitivity to Etoposide in Human Neuroblastoma Cell Lines

Chandra M Das 1,○, Peter E Zage 1,2,○, Pete Taylor 1, Dolly Aguilera 1,3, Johannes EA Wolff 1, Dean Lee 1, Vidya Gopalakrishnan 1,2,*
PMCID: PMC3025305  NIHMSID: NIHMS212200  PMID: 20886683

Abstract

Etoposide, an inhibitor of topoisomerase II, promotes DNA damage and apoptosis of cancer cells and is a component of standard therapy for neuroblastoma. Resistance to etoposide has been observed in neural tumor cells expressing lower levels of topoisomerase II. In the present study, we have examined the contribution of epigenetic modulation of gene expression in the potentiation of etoposide-mediated cytotoxicity in neuroblastoma cells. Specifically, we studied the effects of histone deacetylase inhibition with valproic acid on topoisomerase II gene expression and apoptosis in response to etoposide. Using human neuroblastoma cell lines SK-N-AS and SK-N-SH, we show that although the combination of valproic acid and etoposide promoted a reduction in cell growth compared to either alone in both SK-N-SH and SK-N-AS, the effect was substantially enhanced in in SK-N-SH cells compared to SK-N-SH cells. An increase in histone H3 acetylation and p21 expression was observed in both cell lines, however, upregulation of topoisomerase II-beta gene expression and an increase in PARP cleavage was observed in SK-N-AS cells only. Furthermore, chromatin immunoprecipitation assays revealed an increase in acetylation of histone H3 at the cognate topoisomerase II-beta gene after treatment with valproic acid in SK-N-AS cells. These results suggest a potential epigenetic mechanism of regulation of the topoisomerase II-beta gene and a possible role for its increased expression in the sensitivity of SK-N-AS neuroblastoma cells to etoposide.

Keywords: neuroblastoma, valproic acid, etoposide, topoisomerase, histone deacetylase inhibitor

Introduction

Neuroblastoma is the most common extracranial solid tumor of childhood, with an incidence of approximately 8 cases per million children. Children with disseminated, high-risk neuroblastoma have very poor long-term outcomes, even with intensive multimodal treatment strategies [1]. The poor outcome in response to current treatment strategies clearly indicates a need for improved treatment strategies and new therapeutic combinations in order to improve long-term survival in children with neuroblastoma.

Etoposide is an inhibitor of topoisomerase II and a component of standard chemotherapeutic regimens used to treat neuroblastoma. Inhibition of topoisomerase II activity disrupts cell growth and DNA repair mechanisms, leading to DNA damage and cell death. Etoposide has anti-tumor effects both as a single agent and as part of multi-drug regimens, but with side effects ranging from myelosuppression to increased risk of secondary myeloid leukemias observed at high doses [2,3]. Resistance to etoposide has been observed in neural tumor cells expressing lower levels of topoisomerase II [4], and increased levels of topoisomerase II have been associated with responses to etoposide in medulloblastoma tumor cells [5]. The mechanisms by which topoisomerase II gene expression is regulated in these cell types, however, are not fully understood.

A number of genes involved in cell cycle control (p21/WAF1, gelsolin, p27Kip1, and p16Ink4a) and apoptosis (BAD, TRAIL, DR5, and FasL) have been shown to be regulated by epigenetic modifications in many types of cancers [6,7]. These epigenetic modifications include DNA methylation, histone acetylation and histone methylation [reviewed in 8]. Epigenetic modification of gene expression has been described in neuroblastoma tumor cells and primary tumor samples [9-13] and gene methylation in neuroblastoma tumors has been shown to be associated with poorer overall outcomes [9,13]. Histone deacetylase inhibitors (HDACIs) promote chromatin remodeling through decreased histone deacetylation, leading to changes in gene expression patterns, and inhibition of HDAC has been shown to be effective against neuroblastoma cells in preclinical models [14-16].

Valproic acid (VPA) is a commonly used anti-convulsant and has been shown to function as an HDACI [17,18]. VPA has been used with success in the treatment of children with neural tumors [19,20] and has also been shown to have efficacy against neuroblastoma tumor cells in preclinical models [21,22]. In the current study, we analyzed the ability of VPA to potentiate the cytotoxic effects of etoposide on neuroblastoma cells. We show that the sensitization of neuroblastoma cells to the effects of etoposide is associated with chromatin remodeling at the topoisomerase-IIβ gene and an elevation in nuclear levels of topoisomerase II-beta protein.

Materials and methods

Cell lines and reagents

The characteristics of the neuroblastoma cell lines SK-N-AS and SK-N-SH used in this study have been previously described [23,24]. Neuroblastoma cell lines were grown at 37°C in 5% CO2 in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (USB, Minneapolis, MN), L-glutamine, and penicillin, streptomycin, and amphotericin (Sigma, St. Louis, MO). VPA (Sigma Aldrich, St Louis, MO, USA) was prepared as a 1.2 M stock solution in DMSO. Etoposide solution (20 mg/ml) in 30% ethanol (Ben Venue Labs, Bedford OH, USA) was diluted with culture medium prior to use.

Drug treatment

For dose-response experiments, cell lines were seeded at a density of 3,000 cells per well in 96 well tissue culture plates and incubated for 24 hours at 37°C at 5% CO2. Increasing concentrations of either VPA or etoposide or both were added to cells. Dilutions of VPA were made with growth media to obtain final concentrations ranging from 0.75 to 12 mM. Serial dilutions of the stock solution of etoposide were made to obtain final drug concentrations in the range of 0.01 μM to 100 μM. Cells were incubated in the presence of drug for 48, 72, or 96 hours. In control experiments equal volumes of solvent (DMSO or ethanol) were added. Metabolic activity was measured using MTT assays as previously described [25] and absorbance was measured at 570 nm. For co-incubation experiments, 1.5mM VPA and etoposide ranging from 0.01 μM to 100 μM were added to cells and incubations carried out for 48, 72, or 96 hours. Cell viability was measured by MTT assays. Each experiment was repeated four times and experimental variation for each measurement was found to be <10%.

Statistical analysis

IC50 values from the MTT assays were calculated using best-fit trendlines for each cell line and incubation time. Synergy calculations were performed using the Product Method of Webb as described in the text.. The effect of drug synergy across a range of concentrations was assessed using Two-way Repeated Measures ANOVA. Statistical analysis was performed using Prism 5 for Mac OS X, version 5.0c (GraphPad Software, La Jolla, CA).

Cell cycle analysis

Neuroblastoma cells were seeded at a density of 100,000 cells per well of a six well plate and incubated for 24 hours prior to addition of drugs. Cells were either untreated or treated with VPA alone (1.5 mM) or etoposide alone (0.1 μM). or co-incubated with 1.5 mM VPA and 0.1 μM etoposide for 72 hours. Cells were trypsinized and harvested by centrifugation. Pellets were washed with 1 ml of phosphate buffered saline (PBS), resuspended in 0.5 ml of 1 mg/ml sodium citrate, 0.1% Triton X-100, and 0.05 mg/ml propidium iodide and stored overnight at 4°C. Stained cells were analyzed using a Becton Dickinson FacsScan (Franklin Lakes, NJ, USA) following manufacturer’s instructions. The fraction of G0/G1, S, G2/M and sub-G1 cells in the populations was determined using CellQuest 3.2 software (Becton Dickinson Flowcytometry System).

Immunoblotting

Neuroblastoma cells were treated with VPA or etoposide for various time periods and lysed with Laemmli buffer [25]. Equal amounts of protein were subjected to SDS polyacrylamide gel electrophoresis, followed by western blotting using antibodies against topoisomerase II (Cell Signaling Technologies, Danvers, MA), topoisomerase II-beta (Abcam, Cambridge, MA), total histone H3 and acetyl-histone H3 and H4 (Millipore, Waltham, MA), cleaved poly-ADP ribose polymerase (PARP) (BD Pharmingen, San Jose, CA), p21 (Cell Signaling), and actin (Cell Signaling). Following incubation with HRP-conjugated secondary antibodies, the immunocomplexes were visualized using enhanced chemiluminescence assays (Pierce, Holmdel, NJ, USA).

Chromatin immunoprecipitation (ChIP) assays

SK-N-AS and SK-N-SH cells were treated with VPA (1.5mM) for 72 hours and analyzed for changes in the acetylation of chromatin-associated histone H3 and H4 using antibodies specific to acetylated histone H3 and H4 (Millipore) or control IgG as previously described [26]. Immunoprecipitated DNA was purified using the QiaQuick PCR purification kit (Qiagen, Valencia, CA) following manufacturer’s instructions and analyzed by SYBR GREEN quantitative RT-PCR analyses using primers specific for the transcription start site and 0.5 kb upstream of the start site in the human topoisomerase II-alpha and -beta gene promoters. Significance in DNA pull-down between VPA treated and untreated samples was calculated using Statistica 6.0 software (Statsoft, Tulsa, OK)

Results

Effects of etoposide on human neuroblastoma cells

The effects of etoposide on SK-N-SH and SK-N-AS human neuroblastoma tumor cells were determined by treating the cells with different concentrations of etoposide for various time periods. IC50 values for SK-N-SH cells treated with etoposide ranged from 0.3 to 1μM at 96, 72 and 48 hours (Figure 1A, Table 1). SK-N-AS cells were much less sensitive to etoposide, with IC50 values between 0.6 and 80μM at 96, 72, and 48 hours (Figure 1B, Table 1).

Figure 1.

Figure 1

Figure 1

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Sensitivity of neuroblastoma tumor cells to etoposide. (A) SK-N-SH and (B) SK-N-AS neuroblastoma tumor cells were exposed to etoposide at varying concentrations for 48, 72, and 96 hours each. Cytotoxicity was determined by MTT assays. Cell numbers are represented as percentages normalized to untreated control cells. IC50 values are indicated by vertical lines. (C) SK-N-SH (left) and SK-N-AS (right) neuroblastoma tumor cells were treated with 0.1 μM etoposide (Etop) for 72 hours or left untreated (UT), and cell lysates were collected and analyzed by western blotting for cleaved PARP, p21, topoisomerase II-alpha, and actin.

TABLE 1.

IC50 Values For Neuroblastoma Cells Treated with Etoposide

Cell Line Time (H) IC50 (μM)
SK-N-SH 48 1
72 0.5
96 0.3
SK-N-AS 48 80
72 1.8
96 0.6
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To further investigate the mechanism underlying the response of neuroblastoma cells to etoposide, they were treated with 1μM etoposide for 72 hours and whole cell extracts were analyzed by western blotting for changes in the levels of the 85 kD cleaved form of poly-ADP ribose polymerase (PARP), p21 and topoisomerase II-alpha. As expected, etoposide treatment resulted in an elevation in the 85 kD cleaved PARP band (Figure 1C), suggesting that neuroblastoma cells undergo apoptosis in response to etoposide. However, while etoposide treatment resulted in an increase in p21 levels in SK-N-SH cells, a similar change in p21 expression in SK-N-AS cells was not observed. Topoisomerase II-alpha levels remained essentially unchanged in both cell lines, whereas topoisomerase II-beta was not detected in either cell line (Figure 1C and data not shown).

Effects of valproic acid and etoposide on human neuroblastoma cells

Since HDACIs are known to modulate gene expression and alter tumor cell response to chemotherapy, we evaluated the ability of VPA to alter the response of SK-N-SH and SK-N-AS neuroblastoma cells to etoposide. SK-N-SH and SK-N-AS cells were treated with various concentrations of valproic acid for 48, 72, and 96 hour time periods and the effects on cell growth were assessed by MTT assays. IC50 values for SK-N-SH cells treated with valproic acid were ranged between 1 and 2.5 mM at 96, 72 and 48 hours (Figure 2A, Table 2). SK-N-AS cells were less sensitive to valproic acid, with IC50 values between 1.2 to 6.0 mM at 96, 72, and 48 hours (Figure 2B, Table 2).

Figure 2.

Figure 2

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Sensitivity of neuroblastoma tumor cells to valproic acid. (A) SK-N-SH and (B)SK-N-AS neuroblastoma cells were exposed to valproic acid at varying concentrations for 48, 72, and 96 hours each. Cytotoxicity was determined by MTT assays. Cell numbers are represented as percentages normalized to untreated control cells. IC50 values are indicated by vertical lines.

TABLE 2.

IC50 Values For Neuroblastoma Cells Treated with VPA

Cell Line Time (H) IC50 (mM)
SK-N-SH 48 2.5
72 1.5
96 1.0
SK-N-AS 48 6.0
72 2.0
96 1.25
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We next asked if treatment with VPA altered the response of SK-N-SH and SK-N-AS tumor cells to etoposide. Cells were treated with 1 mM VPA and etoposide ranging in concentration from 0.0014 μM to 0.14 mM for 48, 72, and 96 hours. IC50 values for these cell lines in the presence of etoposide alone or etoposide and VPA together are shown in Tables 3A and 3C. We observed that VPA treatment caused a 8-fold increase and 10-fold decrease in IC50 values for SK-N-SH and SK-N-AS cells respectively at 48 hours, The change in IC50 value upon co-treatment with VPA at 72 hours was less than 2-fold for both cells, while VPA co-treatment for 96 hours promoted a 3-fold and 10-fold decrease in IC50 values for SK-N-SH and SK-N-AS cells respectively. These results suggest that while VPA antagonized the effects of etoposide on SK-N-SH cells at early time points, it sensitized both cells to etoposide at longer time-periods of incubation, although the extent of sensitization was more significant for SK-N-AS cells (10-fold) at 96 hours.

TABLE 3A.

SK-N-SH Cells: IC50 Values for Etoposide Treatment in the Presence and Absence of VPA

Time (H) −VPA (μM) +VPA (μM)
48 1.0 8.0
72 0.12 0.075
96 0.12 0.045
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TABLE 3C.

SK-N-AS Cells: IC50 Values for Etoposide Treatment in the Presence and Absence of VPA

Time (H) −VPA (μM) +VPA (μM)
48 13 1.25
72 1.0 0.6
96 0.7 0.07
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To determine if the observed effects were synergistic, we applied the fractional product method of Webb as described previously (25). This method uses the formula: γ12=>< γ1 γ2, where γ1,2 is the fraction of cell surviving treatment with both drugs, γ1 and γ2 are the fraction of cells surviving treatment with VPA and etoposide alone respectively and γ1γ2 is the predicted survival in the presence of both drugs. Cell killing is considered synergistic if the predicted survival (γ1γ2) is greater than the observed survival γ12, additive when γ1γ2=γ12, and antagonistic if γ1γ2 is less than γ12. As seen in Table 3B, the predicted survival (γ1γ2) for SK-N-SH cells, are 0.45, 0.31 and 0.24 for the combination of drugs at 48, 72 and 96 hours which is less than the observed survival (γ1,2) of 0.67, 0.38 and 0.27 at these time points. These numbers suggest clear antagonism at 48 hours and an additive effect at 72 and 96 hours for SK-N-SH cells. In contrast, the predicted survival (γ1γ2) of 0.6, 0.48 and 0.54 at 48, 72 and 96 hours of treatment with both drugs for SK-N-AS cells was equal to or greater than the observed survival (0.59, 0.41 and 0.27) at these time points. These values indicate an additive effect at 48 hours and modest to strong synergy at 72 and 96 hours respectively. Together, these data suggest that VPA caused a more significant decrease in survival of SK-N-AS cells compared to SK-N-SH cells.

TABLE 3B.

Synergy Calculations For SK-N-SH Cells

Time (H) Survival (1.5
mM VPA)
(γ1)		 Survival (1.5
μM Etop)
(γ2)		 Predicted Survival
(VPA + Etop)(γ1γ2)		 Observed Survival
(VPA + Etop) (γ12)
48 0.90 0.50 0.45 0.67
72 0.62 0.51 0.31 0.38
96 0.51 0.49 0.24 0.27
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Additive: γ1γ2 = γ12

Synergy: γ1γ2 ≥ γ12

Antagonism: γ1γ2 ≤ γ12

Since maximal synergy in SK-N-AS cells was observed at 96 hours, we performed our analyses described below after 72 hours of treatment with VPA and etoposide so as to detect molecular events preceding maximal cell death. To determine whether the reduction in cell growth after treatment with VPA and etoposide resulted from induction of apoptosis, we evaluated treated cells for cleavage of PARP. As shown in Figure 4, the 85kD cleaved PARP band did not significantly increase after treatment with both agents compared to etoposide alone in SK-N-SH cells. However, a substantial enhancement in PARP cleavage was observed in SK-N-AS cells in the presence of VPA and etoposide compared to either drug alone. Western blot analyses also revealed an increase in the acetylation of histone H3 and expression of p21 in the presence of VPA, while total histone H3 and actin levels remained constant under all conditions (Figure 4A). Furthermore, cell cycle analysis demonstrated treatment of SK-N-SH and SK-N-AS cells with VPA and etoposide resulted in an increase in the percentage of cells in the sub-G1 population (16% and 25% respectively at 72 hours) (Figure 4B).

Figure 4.

Figure 4

Figure 4

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Measurement of apoptosis in the presence of VPA and etoposide. (A) SK-N-SH (left) and SK-N-AS (right) neuroblastoma tumor cells were treated with 1mM VPA, 0.1 μM etoposide (Etop) or both VPA and etoposide (Both) for 72 hours. Cell lysates were collected and studied by western blotting for cleaved PARP, p21, acetylated and total histone H3, and actin. (B) SK-N-SH (top) and SK-N-AS (bottom) cells were treated with 1 mM VPA, 0.1 μM etoposide, or both VPA and etoposide for 72 hours and analyzed for cell cycle progression by flow cytometry.

Effects of valproic acid on topoisomerase-II expression

Etoposide is an inhibitor of topoisomerase II function, and increased levels of topoisomerase II have been associated with responses to etoposide in neural tumor cells [5]. We therefore explored the effects of valproic acid on topoisomerase II expression levels in neuroblastoma tumor cells. SK-N-SH and SK-N-AS cells were treated with 1mM VPA for 72 hours and whole cell extracts were studied by western blotting for changes in the levels of topoisomerase II alpha and -beta proteins. Topisomerase II alpha was detected in both SK-N-SH and SK-N-AS cells and its levels were unaffected by VPA treatment. While topoisomerase-II beta was not detected in western blots of whole cell extracts in either cell type, nuclear preparations revealed low but detectable levels of the protein in SK-N-SH cells but not in SK-N-AS cells (Figures 5A and B). VPA treatment caused perhaps a two-fold upregulation of topoisomerase-II beta protein expression in SK-N-SH cells, whereas a substantial increase in its levels were observed in SK-N-AS cells that was detectable in both whole cell and nuclear extracts (Figures 5A and B)

Figure 5.

Figure 5

Figure 5

Figure 5

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Valproic acid remodels chromatin and increases topoisomerase-II beta expression. (A) SK-N-SH (left) and SK-N-AS (right) neuroblastoma cells were treated with 1 mM VPA for 72 hours and cell lysates were collected and analyzed for topoisomerase II-alpha and -beta and actin. (B) SK-N-SH (left) and SK-N-AS (right) cells were treated with 1 mM VPA for 48 hours, nuclear lysates prepared and analyzed for topoisomerase II-beta protein and total histone H3 (loading control). (C, D) Chromatin immunoprecipitation assay to study changes in the acetylation of histones H3 and H4 within the Top2A and Top2B promoters. SK-N-SH (left panels) and SK-N-AS (right panels) neuroblastoma cells were treated with 1mM VPA for 48 hours (+VPA) or left untreated (-VPA) and subjected to chromatin immunoprecipitation using anti-acetyl histone H3 or H4 antibodies or control non-immune sera (IgG). SYBR GREEN RT-PCR amplification of the transcription start site (TS) and 500bp upstream (−0.5) of the Top2A (C) and Top2B (D) promoters was performed using primers flanking these regions. Relative binding of acetylated histones H3 and H4 to these regions was calculated by subtracting the signal obtained with control IgG from that obtained with specific antibodies following normalization to input DNA. Significance was calculated using Statistica 6.0 software.

To ask if VPA modulated topoisomerase II-beta expression through increased histone H3 and H4 acetylation and chromatin remodeling chromatin at the cognate promoter, we performed ChIP assays with untreated or VPA-treated SK-N-AS and SK-N-SH cells using anti-acetyl histone H3 and H4 antibodies or control non-immune sera. The immunoprecipitated material was analyzed by SYBR Green RT-PCR using primers specific to the transcription start site (TS) and 500 bp upstream of the TS (−0.5 kb) for each gene. The levels of DNA pulled down in each case were first normalized to input DNA. Then, levels of DNA associated with non-immune sera were subtracted from that associated with antibodies against acetylated histones H3 and H4 and plotted as relative DNA levels. VPA treatment produced no significant change in the levels of acetylated histones H3 and H4 at the TS or −0.5 kb region of the topoisomerase II-alphagene promoter and the TS of topoisomerase II-beta gene promoter in both SK-N-SH or SK-N-AS cells. (Figures 5C and D). In contrast, a significant increase in levels of acetylaed histones H3 but not H4 at the −0,5 kb region of topoisomerase II-beta gene promoter was observed in SK-N-AS (Figure 5D). Similar changes were not observed in the topoisomerase II-beta gene promoter in SK-N-SH cells (Figure 5D). These results suggest that VPA may potentiate the effect of etoposide in SK-N-AS cells through histone deacetylation at the topoisomerase II-beta gene promoter and subsequent upregulation of topoisomerase II-beta expression.

Discussion

Children with disseminated, high-risk neuroblastoma have very poor longterm outcomes, despite intensive multimodal treatment strategies [1]. The poor outcomes in response to current treatment strategies clearly indicate a need for novel therapies in order to improve long-term survival in children with neuroblastoma. A number of epigenetic changes in gene expression are associated with poor overall outcomes in children with neuroblastoma [9,13], raising the possibility that reversal of these changes through epigenetic modifying agents/drugs may be effective for neuroblastoma therapy.

Acetylation of specific lysine residues on chromatin-associated histones H3 and H4 decreases chromatin compaction, making DNA more readily available to DNA damaging agents such as etoposide and modulating the expression of genes that can induce apoptosis in tumor cells. For example, the HDACIs VPA, suberoylanilide hydroxamic acid (SAHA), and trichostatin A (TSA) reactivate the expression of aberrantly silenced genes involved in cell cycle control and apoptosis and exhibit anti-tumor activity in neuroblastoma preclinical models [13-15].

In the present study we observed that SK-N-SH cells were more sensitive to treatment with VPA and etoposide alone compared to SK-N-AS cells. Although the combination of drugs modestly enhanced cell-death in SK-N-SH cells at longer incubation periods (72 and 96 hours), it caused a decline in etoposide-mediated cell killing at shorter exposure times (48 hours). The reason for this antagonistic effect is not clear at this time. In contrast, VPA synergistically increased the cytotoxicity of etoposide in SK-N-AS cells in vitro. These findings are consistent with our prior work demonstrating enhanced etoposide-mediated cytotoxicity when combined with VPA against glioma cells in vitro [25], and with other studies demonstrating increased sensitivity to etoposide in leukemia cells treated with the HDACIs TSA or sodium butyrate [27,28]. SAHA has also been shown to potentiate DNA damage by etoposide in breast cancer lines in a synergistic manner, although this synergy was dependent upon the sequence of drug administration [29]. The HDACI, depsipeptide, was shown to sensitize neuroblastoma tumor cells to etoposide by Keshelava and colleagues, although the mechanism of sensitization was not investigated [30]. Interestingly, the synergistic increase in cell death of SK-N-AS cells was associated with a strong elevation in topoisomerase II beta expression in these cells. SK-N-SH cells expressed low basal levels of topoisomerase-II beta, which may account for its increased sensitivity to etoposide compared to SK-N-AS cells. The modest increase in topoisomerase-II beta expression upon VPA treatment may potentially explain the additive increase in cell-death in SK-N-SH cells in the presence of both agents.

Our demonstration that VPA promotes chromatin remodeling at the topoisomerase II-beta promoter is novel. Most studies to date have attributed genetic changes in the topoisomerase II-alpha gene as the reason for altered sensitivity of tumor cells to etoposide [31,32], with limited evaluation of the role of topoisomerase II-beta. Our demonstration of HDACI-mediated elevation of topoisomerase II-beta levels agrees with previously described results in other model systems [25,33]. The increase in topoisomerase II-beta may enhance the sensitivity of SK-N-AS neuroblastoma cells to etoposide-mediated cytotoxicity as a result of increased target availability. Although elevated levels of topoisomerase II-beta protein may contribute to increased repair of etoposide-induced DNA damage, this seems unlikely in our studies since the upregulation in topoisomerase II-beta levels is associated with increased apoptosis in SK-N-AS cells. The observation that chromatin remodeling occurs at a region 500 base-pairs upstream of the transcription start site of the topoisomerase II beta promoter is intriguing and raises the possibility that VPA treatment may facilitate the binding of transcriptional activators to this region. In support of this possibility, a previous study attributed maximal (80%) promoter activity to the region between 500 and 481 base-pairs upstream of the transcription start site. Importantly, this region houses binding sites for the transcription factors nuclear factor-Y (NF-Y) and SP1, proteins known to be important for the transcriptional control of Top2B gene expression (34). Further, the absence of chromatin remodeling at the transcription start site also supports this possibility and suggests that elevation in topoisomerase-II beta expression probably may not occur through enhanced RNA polymerase loading but may be the result of increased recruitment of co-activators such as NF-Y and SP1. Additional studies to determine changes in binding of these transcription factors to the Top2B promoter may yield more mechanistic information.

In summary, the combination of VPA and etoposide has anti-tumor effects in neuroblastoma tumor cells in vitro. Our observations suggest a potential epigenetic mechanism for modulation of topoisomerase II-beta gene expression and may explain the relative absence of mutations in this gene in human tumors. Our findings also raise the possibility of future studies evaluating the combination of VPA and etoposide and of HDACIs and topoisomerase-II inhibitors in general in the treatment of children with neuroblastoma.

Figure 3.

Figure 3

Figure 3

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Sensitivity of neuroblastoma tumor cells to valproic acid and etoposide. SK-N-SH (A-C) and SK-N-AS (D-F) neuroblastoma tumor cells were exposed to 1 mM valproic acid and varying concentrations of etoposide for 48 (A,D), 72 (B,E), and 96 (C,F) hours. Cytotoxicity was determined by MTT assays and cell numbers represented as percentages normalized to untreated control cells. IC50 values are indicated by vertical lines. The effect of drug synergy across a range of concentrations was assessed using Two-way Repeated Measures ANOVA. P values of 0.0003 (*), 0.0001 (**), or less than 0.0001 (***) are indicated.

TABLE 3D.

Synergy Calculations For SK-N-AS Cells

Time (H) Survival
(VPA) (γ1)		 Survival
(Etop) (γ2)		 Predicted Survival
(VPA + Etop) (γ1γ2)		 Observed Survival
(VPA + Etop) (γ12)
48 0.62 0.98 0.60 0.59
72 0.58 0.82 0.48 0.41
96 0.83 0.66 0.54 0.27
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Additive: γ1γ2 = γ12

Synergy: γ1γ2 ≥ γ12

Antagonism: γ1γ2 ≤ γ12

Acknowledgements

This work was supported by funds from the Miller Foundation Neuroblastoma Research Fund and the Donne di Domani Research Fund to PEZ and Ray Fish Foundation, American Cancer Society IRG and Brain Tumor SPORE 5P50CA127001-02 grants to VG.

Abbreviations

HDAC(I)

histone deacetylase (inhibitor)

PARP

poly-ADP ribose polymerase

VPA

Valproic acid

ChIP

chromatin immunoprecipitation

SAHA

suberoylanilide hydroxamic acid

TSA

trichostatin A

Footnotes

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