Topoisomerase IIbeta negatively modulates retinoic acid receptor alpha function: a novel mechanism of retinoic acid resistance.
ABSTRACT Interactions between retinoic acid (RA) receptor alpha (RARalpha) and coregulators play a key role in coordinating gene transcription and myeloid differentiation. In patients with acute promyelocytic leukemia (APL), the RARalpha gene is fused with the promyelocytic leukemia (PML) gene via the t(15;17) translocation, resulting in the expression of a PML/RARalpha fusion protein. Here, we report that topoisomerase II beta (TopoIIbeta) associates with and negatively modulates RARalpha transcriptional activity and that increased levels of and association with TopoIIbeta cause resistance to RA in APL cell lines. Knockdown of TopoIIbeta was able to overcome resistance by permitting RA-induced differentiation and increased RA gene expression. Overexpression of TopoIIbeta 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 TopoIIbeta is bound to an RA response element and that inhibition of TopoIIbeta 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 TopoIIbeta in gene regulation and differentiation.
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ABSTRACT: Retinoic acid resistance results in refractory disease, and recovery in acute promyelocytic leukemia remains a challenge in clinical practice, with no ideal chemotherapeutic drug currently available. Here we report on the effect of an active compound of Sophora flavescens called matrine (0.1 mmol/L) combined with all-trans retinoic acid (1 µmol/L) in alleviating retinoic acid resistance in acute promyelocytic leukemia-derived NB4-LR1 cells by differentiation induction, as can be seen by an induced morphology change, increased CD11b expression, and nitro blue tetrazolium reduction activity, and a decreased expression of the promyelocytic leukemia-retinoic acid receptor α fusion gene and protein product. We further explored the probable mechanism of how matrine promotes the recovery of differentiation ability in NB4-LR1 cells when exposed to all-trans retinoic acid. We observed that the combination of all-trans retinoic acid and matrine can increase the level of cyclic adenosine monophosphate and protein kinase A activity, reduce telomerase activity, and downregulate the protein expression of topoisomerase II beta in NB4-LR1 cells. The results of this study suggest the possible clinical utility of matrine in the treatment of retinoic acid-resistant acute promyelocytic leukemia.Planta Medica 03/2014; · 2.35 Impact Factor
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ABSTRACT: Plant growth is continuous and modular, a combination that allows morphogenesis by cell division and elongation and serves to facilitate adaptation to changing environments. The pleiotropic phenotypes of the harlequin (hlq) mutant, isolated on the basis of ectopic expression of the abscisic acid (ABA)- and auxin-inducible proDc3:GUS reporter gene, were previously characterized. Mutants are skotomorphogenic, have deformed and collapsed epidermal cells which accumulate callose and starch, cell walls abundant in pectins and cell wall proteins, and abnormal and reduced root hairs and leaf trichomes. hlq and two additional alleles that vary in their phenotypic severity of starch accumulation in the light and dark have been isolated, and it is shown that they are alleles of bin3/hyp6/rhl3/Topoisomerase6B. Mutants and inhibitors affecting the cell wall phenocopy several of the traits displayed in hlq. A microarray analysis was performed, and coordinated expression of physically adjacent pairs/sets of genes was observed in hlq, suggesting a direct effect on chromatin. Histones, WRKY and IAA/AUX transcription factors, aquaporins, and components of ubiquitin-E3-ligase-mediated proteolysis, and ABA or biotic stress response markers as well as proteins involved in cellular processes affecting carbon partitioning into secondary metabolites were also identified. A comparative analysis was performed of the hlq transcriptome with other previously published TopoVI mutant transcriptomes, namely bin3, bin5, and caa39 mutants, and limited concordance between data sets was found, suggesting indirect or genotype-specific effects. The results shed light on the molecular mechanisms underlying the det/cop/fus-like pleiotropic phenotypes of hlq and support a broader role for TopoVI regulation of chromatin remodelling to mediate development in response to environmental and hormonal signals.Journal of Experimental Botany 05/2014; · 5.79 Impact Factor
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ABSTRACT: Rearrangements involving the RUNX1 gene account for approximately 15% of balanced translocations in therapy-related acute myeloid leukemia (t-AML) patients and are one of the most common genetic abnormalities observed in t-AML. Drugs targeting the topoisomerase II (TOP2) enzyme are implicated in t-AML; however, the mechanism is not well understood and to date a single RUNX1-RUNX1T1 t-AML breakpoint junction sequence has been published. Here we report an additional five breakpoint junction sequences from t-AML patients with the RUNX1- RUNX1T1 translocation. Using a leukemia cell line model, we show that TOP2 beta (TOP2B) is required for induction of RUNX1 chromosomal breaks by the TOP2 poison etoposide and that, while TOP2 alpha (TOP2A) and TOP2B proteins are both present on RUNX1 and RUNX1T1 chromatin, only the TOP2B enrichment reached significance following etoposide exposure at a region on RUNX1 where translocations occur. Furthermore, we demonstrate that TOP2B influences the separation between RUNX1 and two translocation partners (RUNX1T1 and EVI) in the nucleus of lymphoid cells. Specifically, we identified a TOP2B-dependent increase in the number of nuclei displaying juxtaposed RUNX1 and RUNX1T1 loci following etoposide treatment. © 2013 Wiley Periodicals, Inc.Genes Chromosomes and Cancer 02/2014; 53(2):117-28. · 3.84 Impact Factor
MOLECULAR AND CELLULAR BIOLOGY, Mar. 2008, p. 2066–2077
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 28, No. 6
Topoisomerase II? Negatively Modulates Retinoic Acid Receptor ?
Function: a Novel Mechanism of Retinoic Acid Resistance?
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
Interactions between retinoic acid (RA) receptor ? (RAR?) and coregulators play a key role in coordinating
gene transcription and myeloid differentiation. In patients with acute promyelocytic leukemia (APL), the RAR?
gene is fused with the promyelocytic leukemia (PML) gene via the t(15;17) translocation, resulting in the
expression of a PML/RAR? fusion protein. Here, we report that topoisomerase II beta (TopoII?) associates
with and negatively modulates RAR? transcriptional activity and that increased levels of and association with
TopoII? cause resistance to RA in APL cell lines. Knockdown of TopoII? was able to overcome resistance by
permitting RA-induced differentiation and increased RA gene expression. Overexpression of TopoII? 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 TopoII? is bound to an RA response
element and that inhibition of TopoII? 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 TopoII? in gene regulation and differentiation.
Nuclear receptors are a superfamily of ligand-activated tran-
scription factors which modulate the expression of specific
genes. The retinoid nuclear receptors (retinoic acid [RA] re-
ceptor ? [RAR?], RAR?, RAR?, retinoid X receptor ?
[RXR?], RXR?, and RXR?) 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? 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 tran-
scriptional machinery. Binding of all-trans RA to RAR? in-
duces a conformation change which triggers the release of the
corepressor complex and exposes a binding site for coactiva-
tors that possess histone acetylace activity to promote tran-
scriptional 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 neces-
sary and sufficient to permit the interaction between receptors
and coactivators (21, 44, 50). Interestingly, several corepres-
sors possess an LXXLL motif and function to attenuate tran-
scription through ligand-bound nuclear receptors. These core-
pressors include NRIP1/RIP140 (4), LCoR (15), and PRAME
(13), which was recently identified as a ligand-dependent re-
pressor of RA signaling.
Differentiation induced by RA in patients with acute pro-
myelocytic leukemia (APL) has provided one of the first ex-
amples 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?
gene with the promyelocytic leukemia (PML) gene (10, 29, 38,
45). In patients with APL, the PML/RAR? fusion protein has
a dominant negative effect on RAR? 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). Phar-
macological 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 remis-
sions 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 mu-
tations in the ligand binding domain (LBD) of PML/RAR?
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? and RAR? pro-
tein 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? com-
plexes, as shown by high-performance liquid chromatogra-
phy (47). We hypothesized that the altered pattern of wild-
type PML/RAR? complexes in these RA-resistant cells
might reflect abnormal binding of coregulators.
* Corresponding author. Mailing address: Lady Davis Institute for
Medical Research, Sir Mortimer B. Davis Jewish General Hospital,
Segal Cancer Center, 3755 Chemin de la Co ˆte–Ste-Catherine, Mon-
treal, Quebec, Canada H3T 1E2. Phone: (514) 340-8260. Fax: (514)
340-7576. E-mail: firstname.lastname@example.org.
?Published ahead of print on 22 January 2008.
at McGill Univ on March 23, 2009
We sought to identify mechanisms of RA resistance by char-
acterizing the altered PML/RAR? complexes in our RA-resis-
tant cell lines. In this study, we show a novel association be-
tween topoisomerase II beta (TopoII?) and retinoid receptors.
Notably, we identify that TopoII? is overexpressed in an RA-
resistant cell line. By investigating the effects of TopoII? down-
regulation and overexpression, we show that TopoII? can
inhibit granulocytic differentiation through negatively modu-
lating RAR? transcriptional activity. Thus, our work reveals a
new role for TopoII? in the regulation of RAR? transcription
and uncovers a mechanism of RA resistance in APL cell lines.
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 In-
vitrogen (Burlington, ON, Canada). TopoII? antibody (catalogue no. 611493)
was obtained from BD Biosciences (San Diego, CA). RAR? (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 (cata-
logue 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?6M 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?,
GST-RAR?, GST-RAR?(LBD), and GST-PML fusion proteins (10 to 20 ?g)
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 ?l
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 mem-
brane 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 2? SDS sample
buffer. Proteins were then fractionated by electrophoresis on 10% SDS poly-
acrylamide gels and were transferred on nitrocellulose membranes (Bio-Rad
Laboratories, Mississauga, ON, Canada). Membranes were probed with anti-
TopoII? 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 ?g to 1,500 ?g) from
untreated or treated cells were incubated with radioimmunoprecipitation assay
buffer and precleared with 20 ?l protein G beads for 1 h at 4°C. The nuclear
extracts were incubated with 2.5 ?g to 5 ?g of either the anti-RAR? or anti-PML
antibody overnight at 4°C. Protein G beads (30 ?l) 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 2? SDS buffer, boiled, fractionated by electro-
phoresis 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 ? 104cells/ml well in six-well plates. NBT assays were performed as previ-
ously 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 manu-
facturer’s specifications (PharMingen) with the FACSCalibur flow cytometer
(BD BioSciences, Mississauga, Ontario, Canada). Background staining was con-
trolled using an isotype control phycoerythrin-conjugated mouse IgG1 (Phar-
Mingen). In each sample, viable cells were gated, and expression of CD11c and
CD14 surface markers of 5 ? 103cells was evaluated.
Transient transfections. NB4, NB4-MR2, and U937 cells (1 ? 107cells/
transfection) were transfected by electroporation with 5 ?g of the reporter
plasmid ?RARE-tk-CAT or with the pTB114 plasmid which contains full-length
TopoII? 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 manufac-
turer’s guidelines with 1 ?g of ?RARE-tk-CAT, 1 ?g RAR? vector, 0.5 to 1.0 ?g
of TopoII? vector, and 0.5 ?g of shRNAmir constructs against TopoII? (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 nor-
malized with protein concentration to obtain the relative CAT activity.
Stable transfectants. For stable short hairpin RNA (shRNA) transfectants,
NB4 and NB4-MR2 cells (1 ? 107cells/transfection) were transfected by elec-
troporation with 5 to 10 ?g of shRNAmir constructs against TopoII? (clone no.
V2HS_94084 and V2HS_94089; Open Biosystems, Huntsville, AL). For the
TopoII?-overexpressing cells, NB4 cells (1 ? 107cells/transfection) were trans-
fected by electroporation with 5 ?g of pTB114 plasmid. After electroporation,
cells were replenished in media and grown for 48 h. The shRNA-stable trans-
fectant cells were placed under selection with 2 ?g/ml puromycin for 2 months.
The stably pTB114-transfected TopoII?-overexpressing cells, designated pTB-1
and pTB-2 clones, were placed under selection with 800 ?g/ml of G418 for 1
mRNA analysis. Total mRNA was isolated by using the TRIzol method (In-
vitrogen). Reverse transcription was performed on 5 ?g 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 RAR? and RIGI by real-time PCR anal-
ysis (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 TopoII? and
histone 3 acetylation was carried out as follows. Nuclei were prepared from 2 ?
106cells. Formaldehyde was added to a final concentration of 1%. Sonicated
chromatin was precleared with 60 ?l 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 ?g of antibody; the next day, 60 ?l 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 RAR? pro-
moter 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 RAR? 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).
Identification of proteins interacting with PML/RAR?. 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? and PML/RAR? mRNA and protein
that do not differ from those of RA-sensitive NB4 clones (47).
Our previous study using high-performance liquid chromatog-
raphy assays and radiolabeled RA suggested that the NB4-
MR2 cell line had higher-molecular-weight PML/RAR? com-
plexes than the NB4 cell line (47). In order to assess differences
in nuclear proteins that interact with PML/RAR?, we incu-
bated nuclear extracts from untreated NB4 and NB4-MR2
VOL. 28, 2008TopoII? NEGATIVELY MODULATES RAR? FUNCTION IN APL2067
at McGill Univ on March 23, 2009
cells with GST-tagged PML/RAR? and GST alone. The nu-
clear 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? 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 com-
plex mixtures by using Mascot (Matrix Science). Of these pep-
tides, eight proteins were identified and are listed in Table 1.
TopoII? associates with RAR?. We elected to verify the
interaction between TopoII? and PML/RAR?. We confirmed
the mass spectrometry results by performing a coimmunopre-
cipitation for untreated NB4 and NB4-MR2 cell lines, using a
RAR? antibody, which pulls down both wild-type RAR? and
PML/RAR?. Figure 1A shows TopoII? interacting with RA
receptors in both cell lines. An increased interaction of
TopoII? with RA receptors in the NB4-MR2 cell line com-
pared to that in the NB4 cell line was observed (Fig. 1A).
Western blotting was performed to assess whether the in-
creased interaction between TopoII? and RA receptors was
due to increased total nuclear TopoII? protein levels in NB4-
MR2. Indeed, Fig. 1A shows increased TopoII? in NB4-MR2
cells compared to the amount of TopoII? in NB4 cells. Real-
time PCR analysis did not demonstrate any significant differ-
ences in TopoII? mRNA levels between the NB4 and NB4-
MR2 cell lines (Fig. 1A), indicating that increased TopoII?
protein levels in NB4-MR2 may result from regulation of
translation or protein stability.
APL cell lines express RAR? and PML/RAR? protein. The
RAR? portion of the PML/RAR? fusion protein does not
contain the A domain of RAR?. To confirm that TopoII?
interacts with both wild-type RAR? and the RAR? portion of
PML/RAR?, we performed GST pull-down experiments using
purified GST, GST-tagged PML protein, a GST-tagged
RAR?, and a GST-tagged LBD portion of RAR? lacking the
A domain [GST-RAR(LBD)]. As shown in Fig. 1B, TopoII?
interacts with GST-PML/RAR?, GST-RAR?, 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? but does not express the PML/
RAR? fusion protein. The results shown in Fig. 1C confirm
that TopoII? interacts with RAR?.
TopoII? overexpression negatively regulates RAR? tran-
scriptional function and mediates RA resistance in the NB4
cell line. To test whether TopoII? can act as a transcriptional
regulator of RAR? function, we cotransfected NB4 cells with
a reporter gene that contains a retinoid response element,
?RARE-tk-CAT, along with a TopoII? expression plasmid.
Figure 2A shows a strong activation of reporter gene transcrip-
tion in the presence of RA. Interestingly, coexpression of
TopoII? resulted in attenuation of RA-induced ?RARE-tk-
CAT reporter gene activation (Fig. 2A). This suggests that
TopoII? acts as a repressor of RAR? transactivation in APL
Next, we tested whether TopoII? repressed RAR? transac-
tivation in cell lines that express only the RAR? protein. The
?RARE-tk-CAT reporter gene and the TopoII? expression
plasmid were cotransfected in the monocytic leukemia cell line
U937 and the Cos-1 cell line. Figure 2B shows that overexpres-
sion of TopoII? represses RAR? transactivation of a ?RARE-
tk-CAT reporter gene in U937 cells. Transfection of increasing
concentrations of TopoII? along with a RAR? vector in Cos-1
cells caused a concentration-dependent inhibition of ?RARE-
tk-CAT reporter gene expression (Fig. 2C). In order to show
specific TopoII?-mediated repression of the ?RARE-tk-CAT
reporter gene, we transiently knocked down TopoII? by using
shRNA in Cos-1 cells. Figure 2D shows that knockdown of
TopoII? was able to overcome the repressive effects of
TopoII? overexpression on the ?RARE-tk-CAT reporter
gene. Taken together, these data show that expression of
TopoII? results in repression of ligand-dependent transcrip-
tion mediated by RAR? in a variety of cell types.
To further characterize the effects of TopoII? overexpres-
sion in APL cell lines, we stably transfected the TopoII? ex-
pression plasmid pTB114 in the NB4 cell line. Figure 2E shows
overexpression of TopoII? by Western blot analysis of two
stably transfected NB4 clones, designated pTB-1 and pTB-2.
We first examined whether TopoII? overexpression would af-
fect mRNA expression of the RAR? gene, an RA target gene
that is up-regulated during RA-induced differentiation. Figure
2E shows that both overexpressing clones have a reduced RAR?
differentiation in APL cells. Therefore, we next examined the
effects of TopoII? overexpression on granulocytic differentiation.
To test this, we treated the NB4 control cells and the TopoII?-
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 TopoII?-overexpressing subclones
compared to that of the NB4 control cell line. These results
support evidence that increased expression of TopoII? is neces-
sary and sufficient for the inhibition of RA-induced gene expres-
sion and differentiation in an APL cell line.
TopoII? interacts with the 5? RARE region of the RAR?
gene. We next investigated whether TopoII? binds to the
RARE region of RAR? target genes. We performed ChIP
assays by using primers flanking the 5? RARE promoter
TABLE 1. Results of the mass spectrometry analysis identifying
nuclear proteins interacting with GST-PML/RAR?
Replication factor C4
Ribosomal protein PO 60S
aScores are calculated as ?10 ? log(P), where P is the probability that the
observed match is a random event. Individual ion scores of ?35 indicate identity
or extensive homology (P ? 0.05).
2068MCNAMARA ET AL.MOL. CELL. BIOL.
at McGill Univ on March 23, 2009
region of the RAR? gene. Figure 3A shows that TopoII?
interacts with the RARE region of the RAR? gene at low
levels in NB4 cells and shows increased levels of TopoII? in
the NB4-MR2 cell line. Treatment with RA for 24 h led to
an increase in TopoII? occupancy levels at the RAR? pro-
moter region in both cell lines. Interestingly, we observed
increased TopoII? occupancy levels at the RAR? promoter
in NB4-MR2, in the absence and presence of RA, compared
to those in NB4. This increase in TopoII? occupancy levels
at the RAR? promoter may be a consequence of the in-
creased levels of TopoII? we observed in the NB4-MR2 cell
line (Fig. 1A). In order to demonstrate that TopoII? binds
specifically at the RARE region of the RAR? gene, we
performed real-time PCR with primers flanking upstream
(?1 kb) of the RARE-containing region. Our PCR analysis
did not reveal minimal TopoII? occupancy at the ?1 kb
region (Fig. 3A).
whether upregulation of TopoII? with RA treatment also led
to increased interaction with RAR?. Figure 3B shows that
wasperformed to observe
FIG. 1. TopoII? associates with RAR?. (A) Coimmunoprecipitation analysis using nuclear extracts incubated with RAR? antibody. Interacting
proteins were separated by SDS-PAGE and subjected to Western blotting using an antibody against TopoII? and RAR?. Western blotting of total
nuclear protein from NB4 and NB4-MR2 cell lines was performed by using an antibody against TopoII? and ?-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 TopoII? mRNA
levels, using the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene as a reference gene. mRNA expression for TopoII? 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 TopoII? mRNA levels between NB4 and NB4-MR2 cell lines were found. IP, immunopre-
cipitation; IB, immunoblot; IgG, immunoglobulin G. (B) GST pull-down assay to identify which domain of PML/RAR? interacts with TopoII?.
Purified GST, GST-PML/RAR?, GST-RAR(LBD), GST-RAR?, 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 TopoII? and GST. Results are representative
of three experiments.
VOL. 28, 2008TopoII? NEGATIVELY MODULATES RAR? FUNCTION IN APL2069
at McGill Univ on March 23, 2009
FIG. 2. TopoII? represses RA-dependent transcriptional activation by RAR? and mediates RA resistance in the NB4 cell line. Transiently
transfected NB4 (A) and U937 (B) cells were electroporated with empty vector, ?RARE-tk-CAT, and the TopoII? expression vector (pTB114)
alone or in combination and left untreated or treated for 48 h with 1 ?M RA. (C) Cos-1 cells were transiently transfected with empty vector,
?RARE-tk-CAT, a TopoII? expression vector, or a RAR? expression vector. (D) Cos-1 cells were transiently transfected with empty vector,
?RARE-tk-CAT, a TopoII? expression vector, a RAR? expression vector, or shRNA directed against TopoII?. Transfected cells were treated for
24 h with 1 ?M RA. Error bars represent standard deviations. (A to C) Asterisks indicate significant differences between RA-treated cells with and
without overexpression of TopoII? (*, 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 ?g TopoII? and those treated with 1.0 ?g TopoII? (P ? 0.01). (D) Asterisks indicate significant differences between
RA-treated cells with and without overexpression of TopoII? and with and without knockdown of TopoII? by shRNA (P ? 0.001). (E) NB4 cells were
stably transfected with empty vector (Control) or the TopoII? expression plasmid (pTB114). To analyze levels of TopoII? protein in the NB4
TopoII?-overexpressing clones (pTB-1 and pTB-2), total nuclear proteins were separated by SDS-PAGE and subjected to Western blotting using
TopoII? antibody. (E) Real-time PCR analysis of RAR? mRNA levels of NB4 cells stably transfected with overexpressing TopoII? (pTB-1 and pTB-2)
in response to 24-h treatment with 1 ?M 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 TopoII? (P ? 0.001). Results shown are representative of three
experiments. (F) NBT reduction assay on NB4 cells stably transfected with empty vector (Control) or TopoII? 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.
RA-treated NB4 cells stably transfected with TopoII? (P ? 0.001).
at McGill Univ on March 23, 2009