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
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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