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Molecular and Cellular Biology, March 2008, p. 2066-2077, Vol. 28, No. 6
0270-7306/08/$08.00+0     doi:10.1128/MCB.01576-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Topoisomerase IIβ Negatively Modulates Retinoic Acid Receptor {alpha} Function: a Novel Mechanism of Retinoic Acid Resistance{triangledown}

Suzan McNamara, Hongling Wang, Nessrine Hanna, and Wilson H. Miller Jr.*

Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital Segal Cancer Center, and McGill University Department of Oncology, Montreal, Quebec, Canada

Received 27 August 2007/ Returned for modification 4 October 2007/ Accepted 8 January 2008


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ABSTRACT
 
Interactions between retinoic acid (RA) receptor {alpha} (RAR{alpha}) and coregulators play a key role in coordinating gene transcription and myeloid differentiation. In patients with acute promyelocytic leukemia (APL), the RAR{alpha} gene is fused with the promyelocytic leukemia (PML) gene via the t(15;17) translocation, resulting in the expression of a PML/RAR{alpha} fusion protein. Here, we report that topoisomerase II beta (TopoIIβ) associates with and negatively modulates RAR{alpha} transcriptional activity and that increased levels of and association with TopoIIβ cause resistance to RA in APL cell lines. Knockdown of TopoIIβ was able to overcome resistance by permitting RA-induced differentiation and increased RA gene expression. Overexpression of TopoIIβ in clones from an RA-sensitive cell line conferred resistance by a reduction in RA-induced expression of target genes and differentiation. Chromatin immunoprecipitation assays indicated that TopoIIβ is bound to an RA response element and that inhibition of TopoIIβ causes hyperacetylation of histone 3 at lysine 9 and activation of transcription. Our results identify a novel mechanism of resistance in APL and provide further insight to the role of TopoIIβ in gene regulation and differentiation.


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INTRODUCTION
 
Nuclear receptors are a superfamily of ligand-activated transcription factors which modulate the expression of specific genes. The retinoid nuclear receptors (retinoic acid [RA] receptor {alpha} [RAR{alpha}], RARβ, RAR{gamma}, retinoid X receptor {alpha} [RXR{alpha}], RXRβ, and RXR{gamma}) function as ligand-inducible transcription factors in the form of RAR/RXR heterodimers and bind to RA response elements (RAREs) on target genes (33, 41, 52). When not bound to a ligand, RAR{alpha} interacts with a corepressor complex which includes NCoR/SMRT-TBLR1-histone deacetylase 3 (HDAC3) (5, 6, 23, 34, 49, 54). This corepressor complex hypoacetylates histones, creating a more condensed state of chromatin that is less accessible to transcriptional machinery. Binding of all-trans RA to RAR{alpha} induces a conformation change which triggers the release of the corepressor complex and exposes a binding site for coactivators that possess histone acetylace activity to promote transcriptional activation (3, 24, 46). Coactivators, including SRC-1/NCoA-1, GRIP-1/TIF-2/NCoA2, p/CIP/AIB-1/ACTR, and CBP-p300, contain a signature LXXLL motif which is necessary and sufficient to permit the interaction between receptors and coactivators (21, 44, 50). Interestingly, several corepressors possess an LXXLL motif and function to attenuate transcription through ligand-bound nuclear receptors. These corepressors include NRIP1/RIP140 (4), LCoR (15), and PRAME (13), which was recently identified as a ligand-dependent repressor of RA signaling.

Differentiation induced by RA in patients with acute promyelocytic leukemia (APL) has provided one of the first examples of a successful therapy that targets the molecular cause of an aggressive malignancy. APL is associated with a specific chromosomal translocation, t(15;17), which fuses the RAR{alpha} gene with the promyelocytic leukemia (PML) gene (10, 29, 38, 45). In patients with APL, the PML/RAR{alpha} fusion protein has a dominant negative effect on RAR{alpha} function by preventing the release of corepressors at physiological concentrations of RA. This results in transcriptional repression of target genes and a block in granulocytic differentiation (18, 32, 43). Pharmacological concentrations of RA relieve the differentiation block by allowing dissociation of corepressors and recruitment of coactivators needed to activate transcription (17, 20, 35, 47). Treatment with RA in APL patients has led to clinical remissions in a high percentage of patients (14). However, RA treatment alone does not induce a durable remission; APL cells will ultimately develop resistance to RA both in patients and in vitro (9, 11, 12).

RA-sensitive and -resistant APL cell lines have proven useful to study retinoid receptor function, as well as to investigate new therapies to overcome RA resistance. Our lab has previously isolated RA-resistant subclones from the parental RA-sensitive cell line NB4 (47, 48). These resistant cell lines have a partial loss of RA-induced gene expression and are highly resistant to the differentiation and growth-inhibitory effects of RA. Mutational analysis detected mutations in the ligand binding domain (LBD) of PML/RAR{alpha} in one of our RA-resistant subclones (48). However, cells from a significant number of APL patients and cell lines continue to express wild-type PML/RAR{alpha} and RAR{alpha} protein yet are resistant to RA-induced differentiation (11, 16, 47). In two such RA-resistant cell lines, there is an apparent increased molecular weight of RA-bound PML/RAR{alpha} complexes, as shown by high-performance liquid chromatography (47). We hypothesized that the altered pattern of wild-type PML/RAR{alpha} complexes in these RA-resistant cells might reflect abnormal binding of coregulators.

We sought to identify mechanisms of RA resistance by characterizing the altered PML/RAR{alpha} complexes in our RA-resistant cell lines. In this study, we show a novel association between topoisomerase II beta (TopoIIβ) and retinoid receptors. Notably, we identify that TopoIIβ is overexpressed in an RA-resistant cell line. By investigating the effects of TopoIIβ down-regulation and overexpression, we show that TopoIIβ can inhibit granulocytic differentiation through negatively modulating RAR{alpha} transcriptional activity. Thus, our work reveals a new role for TopoIIβ in the regulation of RAR{alpha} transcription and uncovers a mechanism of RA resistance in APL cell lines.


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MATERIALS AND METHODS
 
Materials. RPMI 1640 and fetal bovine serum were purchased from Invitrogen (Burlington, ON, Canada). All-trans RA, nitroblue tetrazolium (NBT) dye and puromycin were obtained from Sigma (Oakville, ON, Canada). ICRF-193 was obtained from Biomol (Plymouth Meeting, PA). G418 was obtained from Invitrogen (Burlington, ON, Canada). TopoIIβ antibody (catalogue no. 611493) was obtained from BD Biosciences (San Diego, CA). RAR{alpha} (catalogue no. SC-551) and PML protein (catalogue no. SC-9862) antibodies were supplied by Santa Cruz Biotechnology (Santa Cruz, CA). Acetylated H3-K9 antibody (catalogue no. 06-942) was obtained from Upstate Biotechnology (Lake Placid, NY).

Cell culture. Cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum. Cells were treated with 10–6 M all-trans RA and 150 nM ICRF-193 unless otherwise specified. Cell growth was quantified by using a standard hemocytometer technique with a trypan blue exclusion assay.

In vitro GST pull-down assay. Glutathione S-transferase (GST)-PML/RAR{alpha}, GST-RAR{alpha}, GST-RAR{alpha}(LBD), and GST-PML fusion proteins (10 to 20 µg) were preincubated at 4°C for 1 h in binding buffer (40 mM HEPES [pH 7.8], 150 mM KCl, 0.05% NP-40, 10% glycerol, 0.1 mM ZnCl2, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) containing 1 mg/ml bovine serum albumin. The fusion proteins were then incubated with 1.2 mg nuclear extracts and 25 µl of glutathione Sepharose-4B beads (Amersham Pharmacia) for 4 h at 4°C. Beads were then washed three times with 1 ml of binding buffer containing 0.1% NP-40. For the isolation of the protein complex, the bound proteins were eluted with elution buffer and boiled in sodium dodecyl sulfate (SDS) sample buffer. Eluted proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and visualized by Coomassie blue staining or transferred to a nitrocellulose membrane for Western blotting analysis.

Mass spectrometry analysis. Selected bands from fractionated proteins on SDS-PAGE gels were sent for digestion and mass spectrometric analyses to the McGill University and Genome Quebec Innovation Center, Montreal, Quebec, Canada. Mass spectrometric analyses of digested proteins were performed on a liquid chromatography-quadrupole time-of-flight tandem mass spectrometer (Micromass) that provides peptide masses and sequence tag information.

Western blot analysis. Nuclear extracts were diluted 1:1 with 2x SDS sample buffer. Proteins were then fractionated by electrophoresis on 10% SDS polyacrylamide gels and were transferred on nitrocellulose membranes (Bio-Rad Laboratories, Mississauga, ON, Canada). Membranes were probed with anti-TopoIIβ antibody at a dilution of 1:5,000 to 1:10,000 in 5% milk in phosphate-buffered saline and detected by using the ECL system (Amersham Pharmacia).

Coimmunoprecipitation assays. Nuclear extracts (750 µg to 1,500 µg) from untreated or treated cells were incubated with radioimmunoprecipitation assay buffer and precleared with 20 µl protein G beads for 1 h at 4°C. The nuclear extracts were incubated with 2.5 µg to 5 µg of either the anti-RAR{alpha} or anti-PML antibody overnight at 4°C. Protein G beads (30 µl) were then added for 4 h at 4°C and washed three times with 1 ml radioimmunoprecipitation assay buffer. The bound proteins were eluted with 2x SDS buffer, boiled, fractionated by electrophoresis on 10% SDS-PAGE gels, and transferred to a nitrocellulose membrane (Bio-Rad) for Western blotting.

Differentiation assays. Cells to be used in NBT reduction assays and for fluorescence-activated cell sorter analysis of differentiation markers were seeded at 3 x 104 cells/ml well in six-well plates. NBT assays were performed as previously described (40). Immunofluorescence staining of the cell surface myeloid-specific antigens CD11c and CD14 (PharMingen, Mississauga, Ontario, Canada) by flow-assisted cell cytometry was performed according to the antibody manufacturer's specifications (PharMingen) with the FACSCalibur flow cytometer (BD BioSciences, Mississauga, Ontario, Canada). Background staining was controlled using an isotype control phycoerythrin-conjugated mouse IgG1 (PharMingen). In each sample, viable cells were gated, and expression of CD11c and CD14 surface markers of 5 x 103 cells was evaluated.

Transient transfections. NB4, NB4-MR2, and U937 cells (1 x 107 cells/transfection) were transfected by electroporation with 5 µg of the reporter plasmid βRARE-tk-CAT or with the pTB114 plasmid which contains full-length TopoIIβ isoform fused to GFP in the pEGFP-C3 vector as previously described (39). After electroporation, cells were replenished in media and grown for 48 h in the absence or presence of treatments. Cos-1 cells were transfected by using FuGENE (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer's guidelines with 1 µg of βRARE-tk-CAT, 1 µg RAR{alpha} vector, 0.5 to 1.0 µg of TopoIIβ vector, and 0.5 µg of shRNAmir constructs against TopoIIβ (clone no. V2HS_94084 and V2HS_94089; Open Biosystems, Huntsville, AL). The chloramphenicol acetyltransferase (CAT) activity was measured by means of a modified protocol of the organic diffusion method. The CAT counts were normalized with protein concentration to obtain the relative CAT activity.

Stable transfectants. For stable short hairpin RNA (shRNA) transfectants, NB4 and NB4-MR2 cells (1 x 107 cells/transfection) were transfected by electroporation with 5 to 10 µg of shRNAmir constructs against TopoIIβ (clone no. V2HS_94084 and V2HS_94089; Open Biosystems, Huntsville, AL). For the TopoIIβ-overexpressing cells, NB4 cells (1 x 107 cells/transfection) were transfected by electroporation with 5 µg of pTB114 plasmid. After electroporation, cells were replenished in media and grown for 48 h. The shRNA-stable transfectant cells were placed under selection with 2 µg/ml puromycin for 2 months. The stably pTB114-transfected TopoIIβ-overexpressing cells, designated pTB-1 and pTB-2 clones, were placed under selection with 800 µg/ml of G418 for 1 month.

mRNA analysis. Total mRNA was isolated by using the TRIzol method (Invitrogen). Reverse transcription was performed on 5 µg total RNA, after heating at 65°C for 5 min, with random hexamer primers. The reaction was carried out at 42°C for 50 min in the presence of SuperScript II reverse transcriptase (Invitrogen). cDNA was amplified for RARβ and RIGI by real-time PCR analysis (ABI Prism7500; Applied Biosystems) using hybridization probes. cDNA was amplified for ICAM1 and HOXA1, by real-time PCR analysis (ABI Prism7500; Applied Biosystems) using primer sets as follows: for ICAM1, 5' TGG CCC TCC ATA GAC ATG TGT 3' (sense) and 5' TGG CAT CCG TCA GGA AGT G 3' (antisense); and for HOXA1, 5' ACC CCG CCA GGA AAC G 3' (sense) and 5' GGC GAA GAG CTG GAC TTC TCT 3' (antisense).

ChIP. Chromatin immunoprecipitation (ChIP) for analysis of TopoIIβ and histone 3 acetylation was carried out as follows. Nuclei were prepared from 2 x 106 cells. Formaldehyde was added to a final concentration of 1%. Sonicated chromatin was precleared with 60 µl of protein A-agarose for 1 h at 4°C in immunoprecipitation buffer (16.7 mM NaCl, 16.7 mM Tris [pH 8.1], 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100, protease inhibitors). Chromatin was immunoprecipitated overnight with 5 µg of antibody; the next day, 60 µl of protein A-agarose beads was added for 4 h at 4°C. The protein A-agarose was washed five times, and bound material was eluted with elution buffer (0.1 M NaHCO3, 1% SDS). Unbound chromatin in the sample without antibody was used as the input. DNA from both unbound and eluted chromatins was purified with the Qiaquick PCR purification kit (Qiagen). For the real-time PCR, the DNA product was measured by Sybr green fluorescence (Sybr green master mix; Applied Biosystems). Primers for real-time PCR methods of the RARβ promoter were 5' TCC TGG GAG TTG GTG ATG TCA G 3' (sense) and 5' AAA CCC TGC TCG GAT CGC TC 3' (antisense). Primers for real-time PCR methods of the –1 kb upstream region of the RARβ gene RARE region were 5' AGT GGC CAC CAA CAC TCT GTG 3' (sense) and 5' GCA GTG TCT CAG CCT CCT GT 3' (antisense).


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RESULTS
 
Identification of proteins interacting with PML/RAR{alpha}. The RA-resistant subclone NB4-MR2 was previously isolated from the RA-sensitive human APL cell line NB4, and it expresses levels of wild-type RAR{alpha} and PML/RAR{alpha} mRNA and protein that do not differ from those of RA-sensitive NB4 clones (47). Our previous study using high-performance liquid chromatography assays and radiolabeled RA suggested that the NB4-MR2 cell line had higher-molecular-weight PML/RAR{alpha} complexes than the NB4 cell line (47). In order to assess differences in nuclear proteins that interact with PML/RAR{alpha}, we incubated nuclear extracts from untreated NB4 and NB4-MR2 cells with GST-tagged PML/RAR{alpha} and GST alone. The nuclear extracts were resolved by one-dimensional SDS-PAGE and stained with Coomassie blue. We observed three protein bands that had increased interaction with the GST-PML/RAR{alpha} in the NB4-MR2 lane compared to that in the NB4 lane or the GST control lane. To identify the protein bands, we sent the SDS-PAGE gel for mass spectrometric analysis. The three gel bands were subjected to in-gel digestion by trypsin and analyzed by liquid chromatography-quadrupole time-of-flight mass spectrometry, which provides tandem mass spectrometry data for the subsequent identification of peptides from complex mixtures by using Mascot (Matrix Science). Of these peptides, eight proteins were identified and are listed in Table 1.


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  		TABLE 1. Results of the mass spectrometry analysis identifying nuclear proteins interacting with GST-PML/RAR{alpha}

TopoIIβ associates with RAR{alpha}. We elected to verify the interaction between TopoIIβ and PML/RAR{alpha}. We confirmed the mass spectrometry results by performing a coimmunoprecipitation for untreated NB4 and NB4-MR2 cell lines, using a RAR{alpha} antibody, which pulls down both wild-type RAR{alpha} and PML/RAR{alpha}. Figure 1A shows TopoIIβ interacting with RA receptors in both cell lines. An increased interaction of TopoIIβ with RA receptors in the NB4-MR2 cell line compared to that in the NB4 cell line was observed (Fig. 1A). Western blotting was performed to assess whether the increased interaction between TopoIIβ and RA receptors was due to increased total nuclear TopoIIβ protein levels in NB4-MR2. Indeed, Fig. 1A shows increased TopoIIβ in NB4-MR2 cells compared to the amount of TopoIIβ in NB4 cells. Real-time PCR analysis did not demonstrate any significant differences in TopoIIβ mRNA levels between the NB4 and NB4-MR2 cell lines (Fig. 1A), indicating that increased TopoIIβ protein levels in NB4-MR2 may result from regulation of translation or protein stability.


Figure 1
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  		FIG. 1. TopoIIβ associates with RAR{alpha}. (A) Coimmunoprecipitation analysis using nuclear extracts incubated with RAR{alpha} antibody. Interacting proteins were separated by SDS-PAGE and subjected to Western blotting using an antibody against TopoIIβ and RAR{alpha}. Western blotting of total nuclear protein from NB4 and NB4-MR2 cell lines was performed by using an antibody against TopoIIβ and β-actin as a loading control. The input is equivalent to 5% of the nuclear extracts used for the coimmunoprecipitations. Real-time PCR analysis was performed for the TopoIIβ mRNA levels, using the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene as a reference gene. mRNA expression for TopoIIβ in NB4 and NB4-MR2 cells was analyzed. Results shown are representative of three experiments performed in triplicate. Error bars represent standard deviations. No significant differences (P > 0.05) in TopoIIβ mRNA levels between NB4 and NB4-MR2 cell lines were found. IP, immunoprecipitation; IB, immunoblot; IgG, immunoglobulin G. (B) GST pull-down assay to identify which domain of PML/RAR{alpha} interacts with TopoIIβ. Purified GST, GST-PML/RAR{alpha}, GST-RAR(LBD), GST-RAR{alpha}, and GST-PML protein were incubated with nuclear extracts from NB4 and NB4-MR2 (B) and HL-60 (C) cells. The input is equivalent to 5% of the nuclear extracts used for the GST pull-down experiment. Interacting proteins were separated by SDS-PAGE and subjected to Western blotting using antibodies against TopoIIβ and GST. Results are representative of three experiments.

APL cell lines express RAR{alpha} and PML/RAR{alpha} protein. The RAR{alpha} portion of the PML/RAR{alpha} fusion protein does not contain the A domain of RAR{alpha}. To confirm that TopoIIβ interacts with both wild-type RAR{alpha} and the RAR{alpha} portion of PML/RAR{alpha}, we performed GST pull-down experiments using purified GST, GST-tagged PML protein, a GST-tagged RAR{alpha}, and a GST-tagged LBD portion of RAR{alpha} lacking the A domain [GST-RAR(LBD)]. As shown in Fig. 1B, TopoIIβ interacts with GST-PML/RAR{alpha}, GST-RAR{alpha}, and GST-RAR(LBD) in NB4 and NB4-MR2 cell lines but does not bind to GST-PML protein. To verify this interaction, we performed the GST pull-down experiment with the HL-60 leukemic cell line, which expresses RAR{alpha} but does not express the PML/RAR{alpha} fusion protein. The results shown in Fig. 1C confirm that TopoIIβ interacts with RAR{alpha}.

TopoIIβ overexpression negatively regulates RAR{alpha} transcriptional function and mediates RA resistance in the NB4 cell line. To test whether TopoIIβ can act as a transcriptional regulator of RAR{alpha} function, we cotransfected NB4 cells with a reporter gene that contains a retinoid response element, βRARE-tk-CAT, along with a TopoIIβ expression plasmid. Figure 2A shows a strong activation of reporter gene transcription in the presence of RA. Interestingly, coexpression of TopoIIβ resulted in attenuation of RA-induced βRARE-tk-CAT reporter gene activation (Fig. 2A). This suggests that TopoIIβ acts as a repressor of RAR{alpha} transactivation in APL cell lines.


Figure 2
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  		FIG. 2. TopoIIβ represses RA-dependent transcriptional activation by RAR{alpha} and mediates RA resistance in the NB4 cell line. Transiently transfected NB4 (A) and U937 (B) cells were electroporated with empty vector, βRARE-tk-CAT, and the TopoIIβ expression vector (pTB114) alone or in combination and left untreated or treated for 48 h with 1 µM RA. (C) Cos-1 cells were transiently transfected with empty vector, βRARE-tk-CAT, a TopoIIβ expression vector, or a RAR{alpha} expression vector. (D) Cos-1 cells were transiently transfected with empty vector, βRARE-tk-CAT, a TopoIIβ expression vector, a RAR{alpha} expression vector, or shRNA directed against TopoIIβ. Transfected cells were treated for 24 h with 1 µM RA. Error bars represent standard deviations. (A to C) Asterisks indicate significant differences between RA-treated cells with and without overexpression of TopoIIβ (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (C) The two asterisks above the brace indicate a significant difference between cells treated with 0.5 µg TopoIIβ and those treated with 1.0 µg TopoIIβ (P < 0.01). (D) Asterisks indicate significant differences between RA-treated cells with and without overexpression of TopoIIβ and with and without knockdown of TopoIIβ by shRNA (P < 0.001). (E) NB4 cells were stably transfected with empty vector (Control) or the TopoIIβ expression plasmid (pTB114). To analyze levels of TopoIIβ protein in the NB4 TopoIIβ-overexpressing clones (pTB-1 and pTB-2), total nuclear proteins were separated by SDS-PAGE and subjected to Western blotting using TopoIIβ antibody. (E) Real-time PCR analysis of RARβ mRNA levels of NB4 cells stably transfected with overexpressing TopoIIβ (pTB-1 and pTB-2) in response to 24-h treatment with 1 µM RA, using the GAPDH gene as a reference gene. Error bars represent standard deviations. Asterisks indicate significant differences between RA-treated cells with and without overexpression of TopoIIβ (P < 0.001). Results shown are representative of three experiments. (F) NBT reduction assay on NB4 cells stably transfected with empty vector (Control) or TopoIIβ expression plasmid. Differentiation results of NB4 control, pTB-1, and pTB-2 cells in response to 5-day exposure to RA. Results are representative of three experiments performed in triplicate. Error bars represent standard deviations. Asterisks indicate significant differences between RA-treated NB4 stably transfected cells with empty vector and RA-treated NB4 cells stably transfected with TopoIIβ (P < 0.001).

Next, we tested whether TopoIIβ repressed RAR{alpha} transactivation in cell lines that express only the RAR{alpha} protein. The βRARE-tk-CAT reporter gene and the TopoIIβ expression plasmid were cotransfected in the monocytic leukemia cell line U937 and the Cos-1 cell line. Figure 2B shows that overexpression of TopoIIβ represses RAR{alpha} transactivation of a βRARE-tk-CAT reporter gene in U937 cells. Transfection of increasing concentrations of TopoIIβ along with a RAR{alpha} vector in Cos-1 cells caused a concentration-dependent inhibition of βRARE-tk-CAT reporter gene expression (Fig. 2C). In order to show specific TopoIIβ-mediated repression of the βRARE-tk-CAT reporter gene, we transiently knocked down TopoIIβ by using shRNA in Cos-1 cells. Figure 2D shows that knockdown of TopoIIβ was able to overcome the repressive effects of TopoIIβ overexpression on the βRARE-tk-CAT reporter gene. Taken together, these data show that expression of TopoIIβ results in repression of ligand-dependent transcription mediated by RAR{alpha} in a variety of cell types.

To further characterize the effects of TopoIIβ overexpression in APL cell lines, we stably transfected the TopoIIβ expression plasmid pTB114 in the NB4 cell line. Figure 2E shows overexpression of TopoIIβ by Western blot analysis of two stably transfected NB4 clones, designated pTB-1 and pTB-2. We first examined whether TopoIIβ overexpression would affect mRNA expression of the RARβ gene, an RA target gene that is up-regulated during RA-induced differentiation. Figure 2E shows that both overexpressing clones have a reduced RARβ mRNA induction compared to that of the NB4 control. RA target genes are involved in the regulation of hematopoietic granulocytic differentiation in APL cells. Therefore, we next examined the effects of TopoIIβ overexpression on granulocytic differentiation. To test this, we treated the NB4 control cells and the TopoIIβ-overexpressing pTB-1 and pTB-1 clones with RA for 5 days and performed NBT analyses. Figure 2F shows substantial decreases in NBT reduction in both TopoIIβ-overexpressing subclones compared to that of the NB4 control cell line. These results support evidence that increased expression of TopoIIβ is necessary and sufficient for the inhibition of RA-induced gene expression and differentiation in an APL cell line.

TopoIIβ interacts with the 5' RARE region of the RARβ gene. We next investigated whether TopoIIβ binds to the RARE region of RARβ target genes. We performed ChIP assays by using primers flanking the 5' RARE promoter region of the RARβ gene. Figure 3A shows that TopoIIβ interacts with the RARE region of the RARβ gene at low levels in NB4 cells and shows increased levels of TopoIIβ in the NB4-MR2 cell line. Treatment with RA for 24 h led to an increase in TopoIIβ occupancy levels at the RARβ promoter region in both cell lines. Interestingly, we observed increased TopoIIβ occupancy levels at the RARβ promoter in NB4-MR2, in the absence and presence of RA, compared to those in NB4. This increase in TopoIIβ occupancy levels at the RARβ promoter may be a consequence of the increased levels of TopoIIβ we observed in the NB4-MR2 cell line (Fig. 1A). In order to demonstrate that TopoIIβ binds specifically at the RARE region of the RARβ gene, we performed real-time PCR with primers flanking upstream (–1 kb) of the RARE-containing region. Our PCR analysis did not reveal minimal TopoIIβ occupancy at the –1 kb region (Fig. 3A).


Figure 3
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  		FIG. 3. TopoIIβ interacts with the 5' RARE region of the RARβ gene. (A) Diagram of the RARβ promoter and real-time PCR ChIP analysis showing increased interaction of TopoIIβ at the promoter region of RARβ in NB4-MR2 compared to NB4. Treatment with 24-h RA treatment leads to increased recruitment of TopoIIβ at the RARE of RARβ in both cell lines. Cross-linked chromatin preparations were immunoprecipitated with an antibody against TopoIIβ. Immunoprecipitated and input materials were analyzed by PCR using primers corresponding to the RARE region of the RARβ promoter. PCR primers flanking the region at –1 kb upstream of the RARE region were used to demonstrate that TopoIIβ binds specifically the RARβ gene RARE region. Asterisks indicate significant differences between dimethyl sulfoxide (DMSO)- and RA-treated cells in both cell lines (P < 0.001). Error bars represent standard deviations. Results shown are representative of one of three experiments performed in triplicate. IgG, immunoglobulin G. (B) Coimmunoprecipitation analysis showing increased interaction between TopoIIβ and RAR{alpha} upon 24-h RA treatment in NB4 and NB4-MR2 cells. Nuclear extracts were incu-bated with RAR{alpha} antibody. Interacting proteins were separated by SDS-PAGE and subjected to Western blotting using an antibody against TopoIIβ. Input is equivalent to 5% of the nuclear extracts used for the coimmunoprecipitations. Results are representative of one of three experiments. IP, immunoprecipitation; IB, immunoblot. (C) Western blot analysis of TopoIIβ levels after RA treatment. NB4 and NB4-MR2 cells left untreated or treated with 1 µM RA for 24 and 48 h. Total nuclear protein was separated by SDS-PAGE and subjected to Western blotting using TopoIIβ antibody. β-Actin antibody was used as a loading control. Results are representative of three experiments. (D) Real-time PCR analysis of TopoIIβ mRNA levels by using the GAPDH gene as a reference gene. mRNA expression for TopoIIβ was analyzed in response to 24- and 48-h treatments with 1 µM RA. No significant differences (P > 0.05) between DMSO-treated cells and RA-treated cells in both cell lines and no significant differences (P > 0.05) between the untreated NB4 and NB4-MR2 cell lines were found. Results shown are representative of three experiments performed in triplicate. Error bars represent standard deviations.

A coimmunoprecipitation was performed to observe whether upregulation of TopoIIβ with RA treatment also led to increased interaction with RAR{alpha}. Figure 3B shows that upon RA treatment, TopoIIβ had increased interaction with RAR{alpha} in both cell lines at 24 h.

Previous reports showed that 1 µM RA upregulated TopoIIβ protein levels in the HL-60 cell line at 48 h (1). We wanted to assess whether RA upregulated TopoIIβ protein levels in the NB4 and NB4-MR2 APL cell lines. Western blot analysis shows that TopoIIβ levels are upregulated at 48 h in NB4 cells; however, the NB4-MR2 cells showed increased TopoIIβ levels as early as 24 h (Fig. 3C). Real-time PCR analysis did not demonstrate any significant elevations in TopoIIβ mRNA levels after 24 or 48 h of 1 µM RA treatment (Fig. 3D), indicating that increased TopoIIβ levels result from regulation of translation or protein stability.

Inhibition of TopoIIβ leads to increased acetylation on lysine 9 of histone 3. To investigate whether the level of TopoIIβ affects chromatin markers associated with gene activation, such as histone acetylation, we performed ChIP assays on the promoter region of RARβ. We decreased TopoIIβ expression by using the TopoII inhibitor, ICRF-193, which inhibits the catalytic activity of TopoIIβ, as well as induces degradation through a SUMO-dependent pathway (25). A Western blot analysis confirmed degradation of the TopoIIβ protein by ICRF-193 in both cell lines at as early as 1 h (Fig. 4A).


Figure 4
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  		FIG. 4. Inhibition of TopoIIβ leads to increased acetylation on lysine 9 of histone 3 (H3K9). (A) NB4 and NB4-MR2 cells were left untreated or treated with ICRF-193 for 1, 4, 8, 24, and 48 h. To visualize down-regulation of TopoIIβ, total nuclear proteins were subjected to Western blotting using TopoIIβ antibody. (B and C) NB4 and NB4-MR2 cells were left untreated or treated with 1 µM RA, 150 nM ICRF-193, or a combination of both for 24 h. Cross-linked chromatin preparations were immunoprecipitated with an antibody against TopoIIβ (B) or acetylated lysine 9 on histone 3 (C). Immunoprecipitated and input materials were analyzed by real-time PCR using primers corresponding to the RARE region of the RARβ promoter. PCR primers flanking the region at –1 kb upstream of the RARE region were used to demonstrate that TopoIIβ binds specifically to the RARβ gene RARE region (B) and the increased H3K9 acetylation upon RA treatment and inhibition of TopoIIβ occurs specifically at the RARβ gene RARE region (C). For panel B, asterisks indicate significant differences between dimethyl sulfoxide (DMSO)- and ICRF-193- or RA-treated cells (*, P < 0.05; **, P < 0.01; ***, P < 0.001). For panel C, asterisks indicate significant differences from RA-treated cells and RA-plus-ICRF-193-treated cells (**, P < 0.01; ***, P < 0.001). Results shown are representative of three experiments performed in triplicate. Error bars represent standard deviations.

By ChIP assay using real-time PCR analysis, we first demonstrated that inhibition with ICRF-193 alone or in combination with RA decreases TopoIIβ protein levels at the RARβ promoter region in both NB4 and NB4-MR2 cells (Fig. 4B). Conversely, upon 24-h RA treatment, we saw an increase in TopoIIβ occupancy levels at the RARE of the RARβ gene promoter region in both cell lines (Fig. 4B). We performed real-time PCR with primers flanking upstream (–1 kb) of the RARE region to demonstrate that TopoIIβ binds specifically at the RARE region of the RARβ gene. Our PCR analysis did not reveal TopoIIβ occupancy at the –1 kb region (Fig. 4B).

We next assessed the effects of TopoIIβ inhibition on the acetylation of lysine 9 on histone 3, which is linked with an open chromatin configuration, such as that found at transcriptionally active promoters. Our ChIP analysis showed an increase in lysine 9 acetylation on histone 3 after 24 h of RA treatment in the NB4 and NB4-MR2 cell lines compared to the control (Fig. 4C). Interestingly, we saw an increase in acetylated lysine 9 when RA was combined with the TopoIIβ inhibitor ICRF-193 compared to the level with RA alone (Fig. 4C). Of note, treatment with a lower concentration of RA (0.1 µM RA) was used for NB4 cells in order to observe differences in acetylation between RA treatment alone and the combination of RA with ICRF-193. Conversely, the NB4-MR2 cell line required a higher concentration of RA (1.0 µM) in order to visualize increased acetylation. This observation reflects the altered RA-induced gene expression in the NB4-MR2 cell line and demonstrates how down-regulation of TopoIIβ at the promoter region of RARβ can restore RA-induced gene expression in this cell line.

To verify that treatment with RA and inhibition of TopoIIβ increase acetylation at lysine 9 specifically at the RARE region of the RARβ gene, we performed real-time PCR on the ChIP samples with primers flanking upstream (–1 kb) of the RARE-containing region. Our PCR analysis did not observe any significant increase at the –1 kb region (Fig. 4C).

These data are consistent with our RAR{alpha} transcriptional studies and support our finding that downregulation of TopoIIβ at the RARE region of the RARβ gene leads to an increase in RAR{alpha} transcriptional regulation.

TopoIIβ is a negative regulator of RA target genes. We next investigated the effects of TopoIIβ inhibition on RA-induced gene expression in the NB4 and NB4-MR2 cell lines. We studied the effects of treatment with ICRF-193, RA, and the combination on known RA target genes, the RARβ, RIGI, HOXA1, and ICAM1 genes, all of which possess a direct repeat (DR5) RARE in the promoter region. Increased mRNA induction of all four RA target genes were observed in the NB4 (Fig. 5A) and NB4-MR2 (Fig. 5B) cell lines with the combination treatment compared to induction for cells treated with RA or ICRF-193 alone. These data show that inhibition of TopoIIβ can enhance activation of several RA target genes in APL cell lines.


Figure 5
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  		FIG. 5. TopoIIβ negatively regulates RA target genes. Real-time PCR analysis of RARβ, RIGI, HOXA1, and ICAM1 mRNA levels for the NB4 cell line (A) and the NB4-MR2 cell line (B), using the GAPDH gene as a reference gene. (A) mRNA levels for RARβ, RIGI, ICAM1, and HOXA1 in NB4 were analyzed in response to 24-h treatment with 0.1 µM RA, 150 nM ICRF-193, or a combination of both. (B) For NB4-MR2 cells, RARβ mRNA expression was analyzed in response to 24-h treatment with 1 µM RA, 150 nM ICRF-193, or a combination of both. RIGI, ICAM1, and HOXA1 mRNA levels were analyzed in response to 48-h treatment with 1 µM RA, 150 nM ICRF-193, or a combination of both. Results shown are representative of three experiments. Error bars represent standard deviations. Asterisks indicate significant differences between RA-treated cells and RA-plus-ICRF-193-treated cells (**, P < 0.01; ***, P < 0.001).

Down-regulation of TopoIIβ restores RA sensitivity to permit granulocytic differentiation. We previously reported that the NB4-MR2 cell line is unable to differentiate into mature granulocytes in the presence of RA (47). We assessed whether the negative effects of TopoIIβ overexpression on RA-induced gene transcription in the NB4-MR2 cell line (Fig. 5B) were necessary for the block in myeloid differentiation. The TopoII inhibitor ICRF-193 was used to study the effects of increased TopoIIβ expression on myeloid differentiation. NBT analyses shown in Fig. 6A indicated a more rapid induction of differentiation with ICRF-193 and RA in NB4 cells and a restoration of RA-induced differentiation after 5 days of combined treatment in NB4-MR2 cells.


Figure 6
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  		FIG. 6. Down-regulation of TopoIIβ restores RA sensitivity to permit granulocytic differentiation. PE, phycoerythrin. (A) Results of the NBT assay to analyze differentiation of NB4 and NB4-MR2 cells in response to 3- and 5-day exposure to RA, ICRF-193, or a combination of both. Results are representative of three experiments performed in triplicate. Error bars represent standard deviations. Asterisks indicate significant differences between RA-treated cells and RA-and-ICRF-193-treated cells (**, P < 0.01; ***, P < 0.001). (B) The upper panels show Western blots (WB) indicating decreased levels of TopoIIβ protein in the stably shRNA-transfected cells. Shown in the lower panels are results of the NBT reduction assay to analyze differentiation of NB4 and NB4-MR2 cells stably transfected with either nonsilencing shRNA or shRNA against TopoIIβ in response to 5-day exposure to 0.1 µM RA for NB4 clones and 1 µM RA for NB4-MR2 clones. Results are representative of three experiments performed in triplicate. Error bars represent standard deviations. Asterisks indicate significant differences between RA-treated nonsilencing shRNA-transfected cells and cells stably transfected with shRNA against TopoIIβ (**, P < 0.01; ***, P < 0.001). (C) Graphs with percentages of NB4 and NB4-MR2 cells expressing CD11c in response to 5-day exposure to RA, ICRF-193, or a combination of both. Results are representative of one of three experiments performed in triplicate. (D) Graphs with percentages of NB4 and NB4-MR2 cells stably transfected with either nonsilencing shRNA (Control) or shRNA against TopoIIβ expressing CD11c in response to 5-day exposure to 1 µM RA. Results are representative of one of three experiments performed in triplicate.

In order to further verify the link between TopoIIβ expression and RA-induced granulocytic differentiation, we stably transfected the NB4 and NB4-MR2 cells with shRNAs against TopoIIβ. A Western blot analysis confirmed downregulation of TopoIIβ protein in the NB4 and NB4-MR2 cells stably expressing shRNA (Fig. 6B). Inhibition of TopoIIβ expression by shRNA and treatment with RA resulted in a small increase in NBT reduction in the stably transfected NB4 cells. However, a significant increase of NBT reduction was seen for stably transfected NB4-MR2 cells treated with RA (Fig. 6B). CD14 and CD11c, which are markers for monocytic and granulocytic differentiation, respectively, were examined by fluorescence-activated cell sorter analysis. Although expression levels of CD14 did not increase after RA and ICRF-193 treatments at 5 days (data not shown), the granulocytic marker CD11c was synergistically elevated in response to the combination at 5 days in NB4-MR2 cells, and we observed an additive effect in NB4 cells (Fig. 6C). CD11c expression in the NB4 and NB4-MR2 cells stably expressing shRNA against TopoIIβ was also examined. Figure 6D shows that 5-day RA treatment in the TopoIIβ-knockdown cell lines had increased CD11c expression compared to that of the nonsilencing control cell lines. The increased differentiation observed in the TopoIIβ shRNA-expressing stable cell lines was comparable to that observed with inhibition of TopoIIβ by ICRF-193. These results confirm that down-regulation of TopoIIβ restores RA sensitivity in the resistant NB4-MR2 cells, suggesting that the high levels of TopoIIβ mediate RA resistance to granulocytic differentiation in this cell line.


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DISCUSSION
 
In this report, we demonstrate a novel role for TopoIIβ in RAR{alpha} transcriptional function. Our studies show that TopoIIβ interacts with RAR{alpha} and negatively modulates RAR{alpha} activity and that increased levels of TopoIIβ lead to a block in RA-induced granulocytic differentiation. Evidence supporting this model consists of the following observations. (i) TopoIIβ associates with RAR{alpha} and the RAR{alpha} portion of PML/RAR{alpha} in vitro and in vivo. (ii) We observed increased levels of TopoIIβ and interaction in the RA-resistant cell line NB4-MR2. (iii) Expression of TopoIIβ caused inhibition of a βRARE-tk-CAT reporter gene in several cell lines. (iv) Downregulation of TopoIIβ, by shRNA or the inhibitor ICRF-193, caused an increase in RA-induced gene expression and granulocytic differentiation and restored RA sensitivity in NB4-MR2 cells. (v) Overexpression of TopoIIβ in clones of the RA-sensitive cell line NB4 conferred RA resistance with a significant reduction in RA-induced differentiation and induction of the RA target gene, RARβ. (vi) ChIP experiments indicated that TopoIIβ is associated with the promoter of RARβ, and inhibition of TopoIIβ leads to increased histone 3 lysine 9 acetylation at this promoter region.

TopoIIβ is an isomerase that is able to modulate the topological state of DNA by generating a transient break in a double-stranded DNA (dsDNA) to transport another dsDNA in an ATP-dependent manner (2). A recent report shows that a dsDNA break by TopoIIβ is required for the signal-dependent activation of gene transcription by nuclear receptors (27). Their studies on the pS2 promoter show that a TopoIIβ-poly(ADP-ribose) polymerase 1 (PARP-1) complex is recruited within 10 min of ligand binding to promote exchange of histone H1 for HMGB to stimulate transcription. This study prompted us to study the effects of TopoIIβ inhibition with ICRF-193 at early time points of RA induction of RARβ in NB4 and NB4-MR2 cells. Our results are in accordance with their findings and show a decrease in RA induction of RARβ mRNA by 1 h, when TopoIIβ is inhibited, in both cell lines (data not shown). This suggests that TopoIIβ has an activating role upon early RA signaling, and conversely, at later time points, TopoIIβ mediates repression. Interestingly, one report shows TopoIIβ and PARP-1 serving as components of a corepressor complex, prior to activation of the MASH1 gene (28). They further demonstrate that PARP-1 is activated upon induction of the signaling pathway, which results in dissociation of poly(ADP-ribosyl)ated components of the corepressor complex, such as TopoIIβ. Hence, these data suggest that TopoIIβ can be a component of an activation or repression complex which is dependent on the time of signaling and inducible locus.

Our studies found TopoIIβ protein up-regulated by RA in APL cells. Previous reports showed that up-regulation of TopoIIβ by RA corresponds with an increased hyperphosphorylated state and slower degradation of TopoIIβ protein during RA-induced differentiation (1, 8). This suggests that RA induces the phosphorylation of TopoIIβ, which in turn may lead to changes in TopoIIβ stability, function, or interactions with other proteins. We observed increased TopoIIβ occupancy levels at the RARβ promoter in the RA-resistant cell line NB4-MR2 in the absence and presence of RA compared to the levels in NB4 cells (Fig. 3A). This suggests that the increase in TopoIIβ levels caused by RA may potentially act as an "off switch" for RAR{alpha} function. In this respect, it is of interest that RA upregulates RIP140 protein, which gives a negative feedback signal toward ligand-activated nuclear receptors in an HDAC-dependent fashion (30). This leads us to speculate that increased TopoIIβ protein and phosphorylation levels may direct increased interactions with multiprotein complexes that repress transcription, such as HDACs. This mechanism of repression has been a subject of speculation regarding TopoIIβ, in part due to the interaction of TopoIIβ with HDAC1 and HDAC2, subunits of the chromatin remodeling complex NurD (26, 51). Indeed, our ChIP studies showing increased acetylation on histone 3 at the RARβ promoter upon TopoIIβ inhibition suggest this potential mechanism (Fig. 4C). In addition, we hypothesize that the TopoIIβ interaction with RAR{alpha} and its repressive function on RA target genes are mediated by participation of TopoIIβ in a distinct complex, normally present at a later time point after transcriptional activation. The proteins identified by mass spectrometry shown in Table 1 may be part of this complex, and interestingly, several of these were previously known to play roles in DNA replication, splicing, and/or DNA damage repair. Our ongoing studies are attempting to define this complex in order to better characterize the precise mechanisms by which TopoIIβ exerts gene repression.

In acute myeloid leukemia (AML) cells, inhibition of TopoIIβ was shown to be associated with an increase in RA-induced apoptosis, growth arrest, and maturation to granulocytes (7), suggesting that TopoIIβ has a negative role in RA-induced differentiation. We found TopoIIβ overexpressed in the APL RA-resistant cell line NB4-MR2 and down-regulation of TopoIIβ restored RA sensitivity, suggesting that the high levels of TopoIIβ mediate RA resistance in this cell line. TopoIIβ has also been found to be overexpressed in AML (53) and lymphoma (19) patients and has been identified as a fusion partner with NUP98 in AML patients (42). We are the first to report that increased levels of TopoIIβ protein can mediate resistance to RA in APL cell lines. This finding is of significance, since TopoII is a key target for anthracyclines, which are administered in the treatment of several malignancies, including leukemias, lymphomas, and breast, uterine, ovarian, and lung cancers. Interestingly, increased TopoII expression was found to enhance the cytotoxic activity of anthracyclines in leukemic, melanoma, and breast cancer tumor cell lines and patients (22, 31, 36, 37). The up-regulation of TopoIIβ by RA in APL cell lines should potentiate the sensitivity to anthracyclines. In addition, if APL cells begin to develop RA resistance by increased expression of TopoIIβ protein, their susceptibility to anthracyclines may be enhanced. Indeed, when RA is coadministered with anthracycline-based therapy for patients with APL, patients have increased remission and survival rates. These interactions may explain why APL patients respond favorably to the combination treatment. Taken together, these findings underline the importance of understanding and targeting TopoII proteins in the treatment of cancer.

In conclusion, the present study has revealed that TopoIIβ associates with retinoid nuclear receptors, and when overexpressed, represses RA-induced gene expression and differentiation. In addition, we show that TopoIIβ overexpression provides a novel mechanism by which APL cells can develop resistance to RA.


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ACKNOWLEDGMENTS
 
We thank Yin-Yuan Mo and William T. Beck (University of Illinois at Chicago) for providing the pTB114 TopoIIβ-expressing vector. We are grateful to Jessica Nichol and Koren K. Mann for excellent discussions and advice. We thank Michael Witcher and Anna Laurenzana for critical reading of the manuscript.

This work was supported by a grant from the Canadian Institutes of Health Research. W. H. Miller, Jr., is a Chercheur National of Fonds de la Recherche en Santé du Québec. S. McNamara was supported by a student grant from the Fonds de la Recherche en Santé du Québec and the Montreal Center for Experimental Therapeutics in Cancer.


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FOOTNOTES
 
* Corresponding author. Mailing address: Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, Segal Cancer Center, 3755 Chemin de la Côte-Ste-Catherine, Montreal, Quebec, Canada H3T 1E2. Phone: (514) 340-8260. Fax: (514) 340-7576. E-mail: wmiller{at}ldi.jgh.mcgill.ca Back

{triangledown} Published ahead of print on 22 January 2008. Back


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Molecular and Cellular Biology, March 2008, p. 2066-2077, Vol. 28, No. 6
0270-7306/08/$08.00+0     doi:10.1128/MCB.01576-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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