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: email@example.com.
?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
was performedto 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 APL 2069
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
upon RA treatment, TopoII? had increased interaction with
RAR? in both cell lines at 24 h.
Previous reports showed that 1 ?M RA upregulated
TopoII? protein levels in the HL-60 cell line at 48 h (1). We
wanted to assess whether RA upregulated TopoII? protein
levels in the NB4 and NB4-MR2 APL cell lines. Western blot
analysis shows that TopoII? levels are upregulated at 48 h in
NB4 cells; however, the NB4-MR2 cells showed increased
TopoII? levels as early as 24 h (Fig. 3C). Real-time PCR
analysis did not demonstrate any significant elevations in
TopoII? mRNA levels after 24 or 48 h of 1 ?M RA treatment
(Fig. 3D), indicating that increased TopoII? levels result from
regulation of translation or protein stability.
Inhibition of TopoII? leads to increased acetylation on ly-
sine 9 of histone 3. To investigate whether the level of TopoII?
affects chromatin markers associated with gene activation, such
as histone acetylation, we performed ChIP assays on the pro-
moter region of RAR?. We decreased TopoII? expression by
using the TopoII inhibitor, ICRF-193, which inhibits the cat-
alytic activity of TopoII?, as well as induces degradation
through a SUMO-dependent pathway (25). A Western blot
analysis confirmed degradation of the TopoII? protein by
ICRF-193 in both cell lines at as early as 1 h (Fig. 4A).
By ChIP assay using real-time PCR analysis, we first demon-
strated that inhibition with ICRF-193 alone or in combination
region in both NB4 and NB4-MR2 cells (Fig. 4B). Conversely,
upon 24-h RA treatment, we saw an increase in TopoII? occu-
pancy levels at the RARE of the RAR? gene promoter region in
both cell lines (Fig. 4B). We performed real-time PCR with prim-
ers flanking upstream (?1 kb) of the RARE region to demon-
strate that TopoII? binds specifically at the RARE region of the
at the ?1 kb region (Fig. 4B).
We next assessed the effects of TopoII? inhibition on the
acetylation of lysine 9 on histone 3, which is linked with an
open chromatin configuration, such as that found at transcrip-
tionally active promoters. Our ChIP analysis showed an in-
crease 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 acety-
FIG. 3. TopoII? interacts with the 5? RARE region of the RAR?
gene. (A) Diagram of the RAR? promoter and real-time PCR ChIP
analysis showing increased interaction of TopoII? at the promoter
region of RAR? in NB4-MR2 compared to NB4. Treatment with 24-h
RA treatment leads to increased recruitment of TopoII? at the RARE
of RAR? in both cell lines. Cross-linked chromatin preparations were
immunoprecipitated with an antibody against TopoII?. Immunopre-
cipitated and input materials were analyzed by PCR using primers
corresponding to the RARE region of the RAR? promoter. PCR
primers flanking the region at ?1 kb upstream of the RARE region
were used to demonstrate that TopoII? binds specifically the RAR?
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 TopoII? and RAR? upon 24-h RA
treatment in NB4 and NB4-MR2 cells. Nuclear extracts were incu-
bated with RAR? antibody. Interacting proteins were separated by
SDS-PAGE and subjected to Western blotting using an antibody
against TopoII?. 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 TopoII? levels after RA treatment. NB4 and
NB4-MR2 cells left untreated or treated with 1 ?M RA for 24 and
48 h. Total nuclear protein was separated by SDS-PAGE and subjected
to Western blotting using TopoII? antibody. ?-Actin antibody was
used as a loading control. Results are representative of three experi-
ments. (D) Real-time PCR analysis of TopoII? mRNA levels by using
the GAPDH gene as a reference gene. mRNA expression for TopoII?
was analyzed in response to 24- and 48-h treatments with 1 ?M 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.
VOL. 28, 2008TopoII? NEGATIVELY MODULATES RAR? FUNCTION IN APL 2071
at McGill Univ on March 23, 2009
lated lysine 9 when RA was combined with the TopoII? inhib-
itor ICRF-193 compared to the level with RA alone (Fig. 4C).
Of note, treatment with a lower concentration of RA (0.1 ?M
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 ?M) in order to
visualize increased acetylation. This observation reflects the
FIG. 4. Inhibition of TopoII? 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 TopoII?, total nuclear proteins were subjected to Western
blotting using TopoII? antibody. (B and C) NB4 and NB4-MR2 cells were left untreated or treated with 1 ?M RA, 150 nM ICRF-193, or a
combination of both for 24 h. Cross-linked chromatin preparations were immunoprecipitated with an antibody against TopoII? (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 RAR? promoter. PCR primers flanking the region at ?1 kb upstream of the RARE region were used to demonstrate that TopoII?
binds specifically to the RAR? gene RARE region (B) and the increased H3K9 acetylation upon RA treatment and inhibition of TopoII? occurs
specifically at the RAR? 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.
2072MCNAMARA ET AL.MOL. CELL. BIOL.
at McGill Univ on March 23, 2009
altered RA-induced gene expression in the NB4-MR2 cell line
and demonstrates how down-regulation of TopoII? at the pro-
moter region of RAR? can restore RA-induced gene expres-
sion in this cell line.
To verify that treatment with RA and inhibition of TopoII?
increase acetylation at lysine 9 specifically at the RARE region
of the RAR? 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 sig-
nificant increase at the ?1 kb region (Fig. 4C).
These data are consistent with our RAR? transcriptional
studies and support our finding that downregulation of
TopoII? at the RARE region of the RAR? gene leads to an
increase in RAR? transcriptional regulation.
TopoII? is a negative regulator of RA target genes. We next
investigated the effects of TopoII? 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 RAR?, 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 TopoII? can enhance activation of several RA target genes
in APL cell lines.
Down-regulation of TopoII? restores RA sensitivity to per-
mit 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 TopoII? 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
TopoII? expression on myeloid differentiation. NBT analyses
shown in Fig. 6A indicated a more rapid induction of differ-
entiation with ICRF-193 and RA in NB4 cells and a restora-
tion of RA-induced differentiation after 5 days of combined
treatment in NB4-MR2 cells.
In order to further verify the link between TopoII? expres-
sion and RA-induced granulocytic differentiation, we stably
FIG. 5. TopoII? negatively regulates RA target genes. Real-time PCR analysis of RAR?, 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 RAR?, RIGI, ICAM1, and
HOXA1 in NB4 were analyzed in response to 24-h treatment with 0.1 ?M RA, 150 nM ICRF-193, or a combination of both. (B) For NB4-MR2
cells, RAR? mRNA expression was analyzed in response to 24-h treatment with 1 ?M 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 ?M 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).
VOL. 28, 2008TopoII? NEGATIVELY MODULATES RAR? FUNCTION IN APL2073
at McGill Univ on March 23, 2009
FIG. 6. Down-regulation of TopoII? 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.
2074MCNAMARA ET AL.MOL. CELL. BIOL.
at McGill Univ on March 23, 2009
transfected the NB4 and NB4-MR2 cells with shRNAs against
TopoII?. A Western blot analysis confirmed downregulation
of TopoII? protein in the NB4 and NB4-MR2 cells stably
expressing shRNA (Fig. 6B). Inhibition of TopoII? 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 TopoII? was also
examined. Figure 6D shows that 5-day RA treatment in the
TopoII?-knockdown cell lines had increased CD11c expres-
sion compared to that of the nonsilencing control cell lines.
The increased differentiation observed in the TopoII? shRNA-
expressing stable cell lines was comparable to that observed
with inhibition of TopoII? by ICRF-193. These results confirm
that down-regulation of TopoII? restores RA sensitivity in the
resistant NB4-MR2 cells, suggesting that the high levels of
TopoII? mediate RA resistance to granulocytic differentiation
in this cell line.
In this report, we demonstrate a novel role for TopoII? in
RAR? transcriptional function. Our studies show that
TopoII? interacts with RAR? and negatively modulates
RAR? activity and that increased levels of TopoII? lead to a
block in RA-induced granulocytic differentiation. Evidence
supporting this model consists of the following observations. (i)
TopoII? associates with RAR? and the RAR? portion of
PML/RAR? in vitro and in vivo. (ii) We observed increased
levels of TopoII? and interaction in the RA-resistant cell line
NB4-MR2. (iii) Expression of TopoII? caused inhibition of a
?RARE-tk-CAT reporter gene in several cell lines. (iv) Down-
regulation of TopoII?, by shRNA or the inhibitor ICRF-193,
caused an increase in RA-induced gene expression and gran-
ulocytic differentiation and restored RA sensitivity in NB4-
MR2 cells. (v) Overexpression of TopoII? in clones of the
RA-sensitive cell line NB4 conferred RA resistance with a
significant reduction in RA-induced differentiation and induc-
tion of the RA target gene, RAR?. (vi) ChIP experiments
indicated that TopoII? is associated with the promoter of
RAR?, and inhibition of TopoII? leads to increased histone 3
lysine 9 acetylation at this promoter region.
TopoII? is an isomerase that is able to modulate the topo-
logical 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 TopoII? is required for the signal-depen-
dent activation of gene transcription by nuclear receptors (27).
Their studies on the pS2 promoter show that a TopoII?–
poly(ADP-ribose) polymerase 1 (PARP-1) complex is re-
cruited 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 TopoII? inhibition with
ICRF-193 at early time points of RA induction of RAR? in
NB4 and NB4-MR2 cells. Our results are in accordance with
their findings and show a decrease in RA induction of RAR?
mRNA by 1 h, when TopoII? is inhibited, in both cell lines
(data not shown). This suggests that TopoII? has an activating
role upon early RA signaling, and conversely, at later time
points, TopoII? mediates repression. Interestingly, one report
shows TopoII? and PARP-1 serving as components of a core-
pressor complex, prior to activation of the MASH1 gene (28).
They further demonstrate that PARP-1 is activated upon in-
duction of the signaling pathway, which results in dissociation
of poly(ADP-ribosyl)ated components of the corepressor com-
plex, such as TopoII?. Hence, these data suggest that TopoII?
can be a component of an activation or repression complex
which is dependent on the time of signaling and inducible
Our studies found TopoII? protein up-regulated by RA in
APL cells. Previous reports showed that up-regulation of
TopoII? by RA corresponds with an increased hyperphospho-
rylated state and slower degradation of TopoII? protein during
RA-induced differentiation (1, 8). This suggests that RA in-
duces the phosphorylation of TopoII?, which in turn may lead
to changes in TopoII? stability, function, or interactions with
other proteins. We observed increased TopoII? occupancy
levels at the RAR? 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
TopoII? levels caused by RA may potentially act as an “off
switch” for RAR? function. In this respect, it is of interest that
RA upregulates RIP140 protein, which gives a negative feed-
back signal toward ligand-activated nuclear receptors in an
HDAC-dependent fashion (30). This leads us to speculate that
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 TopoII? 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
TopoII? in response to 5-day exposure to 0.1 ?M RA for NB4 clones and 1 ?M 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 TopoII? (**, 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 TopoII? expressing CD11c in response to 5-day exposure to 1 ?M RA. Results are
representative of one of three experiments performed in triplicate.
VOL. 28, 2008TopoII? NEGATIVELY MODULATES RAR? FUNCTION IN APL 2075
at McGill Univ on March 23, 2009
increased TopoII? protein and phosphorylation levels may
direct increased interactions with multiprotein complexes that
repress transcription, such as HDACs. This mechanism of re-
pression has been a subject of speculation regarding TopoII?,
in part due to the interaction of TopoII? with HDAC1 and
HDAC2, subunits of the chromatin remodeling complex NurD
(26, 51). Indeed, our ChIP studies showing increased acetyla-
tion on histone 3 at the RAR? promoter upon TopoII? inhi-
bition suggest this potential mechanism (Fig. 4C). In addition,
we hypothesize that the TopoII? interaction with RAR? and
its repressive function on RA target genes are mediated by
participation of TopoII? 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, splic-
ing, and/or DNA damage repair. Our ongoing studies are at-
tempting to define this complex in order to better characterize
the precise mechanisms by which TopoII? exerts gene repres-
In acute myeloid leukemia (AML) cells, inhibition of
TopoII? was shown to be associated with an increase in RA-
induced apoptosis, growth arrest, and maturation to granulo-
cytes (7), suggesting that TopoII? has a negative role in RA-
induced differentiation. We found TopoII? overexpressed in
the APL RA-resistant cell line NB4-MR2 and down-regulation
of TopoII? restored RA sensitivity, suggesting that the high
levels of TopoII? mediate RA resistance in this cell line.
TopoII? 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 TopoII? protein can mediate
resistance to RA in APL cell lines. This finding is of signifi-
cance, since TopoII is a key target for anthracyclines, which are
administered in the treatment of several malignancies, includ-
ing 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 TopoII? by RA
in APL cell lines should potentiate the sensitivity to anthracy-
clines. In addition, if APL cells begin to develop RA resistance
by increased expression of TopoII? protein, their susceptibility
to anthracyclines may be enhanced. Indeed, when RA is coad-
ministered 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 tar-
geting TopoII proteins in the treatment of cancer.
In conclusion, the present study has revealed that TopoII?
associates with retinoid nuclear receptors, and when overex-
pressed, represses RA-induced gene expression and differen-
tiation. In addition, we show that TopoII? overexpression pro-
vides a novel mechanism by which APL cells can develop
resistance to RA.
We thank Yin-Yuan Mo and William T. Beck (University of Illinois
at Chicago) for providing the pTB114 TopoII?-expressing vector. We
are grateful to Jessica Nichol and Koren K. Mann for excellent dis-
cussions 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 Sante ´ du Que ´bec. S. McNamara was supported by
a student grant from the Fonds de la Recherche en Sante ´ du Que ´bec
and the Montreal Center for Experimental Therapeutics in Cancer.
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