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Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 2634-2642
SPECIAL FOCUS
Reduced Retinoic Acid-Sensitivities of Nuclear Receptor Corepressor
Binding to PML- and PLZF-RAR Underlie Molecular Pathogenesis and
Treatment of Acute Promyelocytic Leukemia
By
Fabien Guidez,
Sarah Ivins,
Jun Zhu,
Mats Söderström,
Samuel Waxman, and
Arthur Zelent
From the Leukaemia Research Fund Centre at the Institute of Cancer
Research, Chester Beatty Laboratories, London, UK; the Department of
Cell Biology, Faculty of Health Sciences, University of
Linköping, Linköping, Sweden; and the Department of
Medicine, Mount Sinai School of Medicine, New York, NY.
 |
ABSTRACT |
Typical acute promyelocytic leukemia (APL) is associated with
expression of the PML-RAR fusion protein and responsiveness to
treatment with all-trans retinoic acid (ATRA). A rare, but recurrent, APL has been described that does not respond to ATRA treatment and is associated with a variant chromosomal translocation and expression of the PLZF-RAR fusion protein. Both PML- and PLZF-RAR possess identical RAR sequences and inhibit ATRA-induced gene transcription as well as cell differentiation. We now show that
the above-mentioned oncogenic fusion proteins interact with the nuclear
receptor corepressor N-CoR and, in comparison with the wild-type RAR
protein, their interactions display reduced sensitivities to ATRA.
Although pharmacologic concentration of ATRA could still induce
dissociation of N-CoR from PML-RAR , it had a very little effect on
its association with the PLZF-RAR fusion protein. This
ATRA-insensitive interaction between N-CoR and PLZF-RAR was mediated
by the N-terminal PLZF moiety of the chimera. It appears that
N-CoR/histone deacetylase corepressor complex interacts directly in an
ATRA-insensitive manner with the BTB/POZ-domain of the wild-type PLZF
protein and is required, at least in part, for its function as a
transcriptional repressor. As the above-noted results predict, histone
deacetylase inhibitors antagonize oncogenic activities of the
PML-RAR fusion protein and partially relieve transcriptional
repression by PLZF as well as inhibitory effect of PLZF-RAR on ATRA
response. Taken together, our results demonstrate involvement of
nuclear receptor corepressor/histone deacetylase complex in the
molecular pathogenesis of APL and provide an explanation for
differential sensitivities of PML- and PLZF-RAR -associated leukemias to ATRA.
 |
INTRODUCTION |
ACUTE PROMYELOCYTIC leukemia
(APL) is associated with the t(15;17)(q22;q21) reciprocal chromosomal
translocation that causes the fusion of the retinoic acid receptor (RAR ) locus with a gene of unknown function called PML (for
promyelocytic leukemia) and expression of PML-RAR chimeric proteins
in all leukemic cells.1-4 The wild-type PML protein
localizes onto nuclear bodies (NBs; also called PML oncogenic domains
or PODs).5-7 Expression of the PML-RAR chimeric protein
causes delocalization of PML and other components of NBs to a
microspeckled nuclear structure and differentiation of APL cells with
all-trans-retinoic acid (ATRA) results in restoration of their
normal localization in NBs.5-8 At present, it is not clear
what relationship, if any, this phenomenon has to the pathogenesis and
treatment of APL.
Expression of the PML-RAR protein in transgenic mice results in the
development of APL, which, as the human disease, can be induced into
remission by ATRA treatment.9 These results and studies
with APL cells in vitro, or cells exogenously expressing PML-RAR ,
suggest that this oncoprotein is also a primary target of ATRA
action.10,11 Therefore, in addition to being the first human cancer to be successfully treated with differentiation therapy, APL is also the only example of a successful application of a therapy
targeting the activities of a specific oncoprotein.
To date, three other APL-associated translocations of the RAR gene
have been characterized at the molecular level. The
t(5;17)(q35;q21),12 t(11;17)(q23;q21),13,14
and t(11;17) (q13;q21)15 fuse RAR to nucleophosmin
(NPM), promyelocytic leukemia zinc finger (PLZF), and nuclear mitotic
apparatus (NuMA) genes, respectively. So far, the t(5;17)(q35;q21) and
t(11;17)(q13;q21) have only been reported in index cases and, as APL
with t(15;17), appeared to respond to treatment with
ATRA.12,15 In contrast, APL with t(11;17)(q23;q21) has been
reported on recurrent basis, albeit at very low frequency, and has been
found to be consistently unresponsive to ATRA therapy.16,17 The molecular basis for the lack of response of this APL variant to
ATRA is not understood.
The PLZF gene encodes a protein with N-terminal BTB/POZ-domain and nine
C-terminal Krüppel-like Zinc(Zn)-fingers.13 The PLZF-RAR fusion protein consists of the N-terminus of the PLZF protein, including two of its nine Zn-fingers linked to the DNA (region
C) and ligand binding (region E) domains of the RAR protein (see
Fig 1A for schematic representation).
Although PLZF, PML, NPM, and NuMA are not structurally related, it is
worth noting that RAR fusion proteins possess identical RAR
sequences, which include the DNA, corepressor, coactivator, and ATRA
binding regions (see below). Interestingly, localization of the PML
protein and integrity of NBs remain undisturbed in APL cells expressing
either PLZF-RAR 18 or NuMA-RAR 15 proteins.
Nevertheless, both PML-RAR and PLZF-RAR localize to the same
microspeckled structures and the wild-type PLZF protein colocalizes
completely with the PML-RAR oncoprotein in APL cells.18

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| Fig 1.
Differential ATRA sensitivities of N-CoR association with
RAR and its PML and PLZF fusions. (A) Schematic representation of
PML, PLZF, RAR 1, PML-RAR , PLZF-RAR , and N-CoR. RI, RII, and
RIII, N-CoR repression domains. RIDII and RIDI, N-CoR interaction domains with RAR . CoR-box, corepressor interaction domain in the
RAR . Solid boxes represent the Sin3 interaction domains of N-CoR.
Numbers correspond to the amino acids flanking various functional
domains, indicated with different patterns, within a given protein.
Fusion points between RAR and PLZF or PML are indicated by
arrowheads. (B) Binding of RAR , PML-RAR , and PLZF-RAR to
GST-N-CoR corepressor (amino acids 1679-2453). Radiolabeled receptors,
synthesized in vitro, were incubated with immobilized GST-N-CoR over a
range of ATRA concentrations, as indicated. Bound proteins were
analyzed by SDS-PAGE and autoradiography.
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RARs are ligand regulated transcription factors that affect many
physiologic processes,19-21 including
hemopoiesis.22 They exert their effect on gene expression
by binding as heterodimers with the retinoid X receptors (RXRs) to the
retinoic acid response elements (RAREs) located in promoter/enhancer
regions of specific genes and either activating or repressing basal
transcription.23-26 In the absence of ATRA, RARs remain
associated with the nuclear receptor corepressors, N-CoR (negative
coregulator)27 or SMRT (silencing mediator for retinoid and
thyroid-hormone receptors),28 and repress basal
transcription. Both N-CoR29,30 and SMRT31 have
been shown to associate with the mammalian homologues (Sin3A and Sin3B)
of yeast global transcriptional repressor SIN332-34 and
histone deacetylase35 (HDAC1 or 2) and to repress
transcription through histone deacetylation, rendering the nearby
chromatin inaccessible to transcriptional activators and/or
basal transcription factors. Sin3A, Sin3B, and histone deacetylases
have also been implicated in transcriptional repression by
Mad/Max30,36,37 or Max/Mxi29 heterodimers.
Both PML-RAR and PLZF-RAR can bind to an RARE as homodimers or,
in combination with RXR, as multimeric complexes.38-40 Both fusion proteins inhibit the activity of the wild-type RAR in a
dominant negative manner.1,2,8,39-41 Previous studies have shown that the dominant negative activities of PML-RAR and
PLZF-RAR on ATRA-inducible transcription are inherent properties of
the RAR chimeric proteins and are not observed upon expression of N-terminal truncation of RAR and/or N-terminal PLZF or PML
sequences.1,2,41 Because the PML- and PLZF-RAR chimeric
proteins bind ATRA with near wild-type affinities,40 we set
out to test whether their impaired abilities to be activated by ATRA
could be due to inefficient association with RAR coactivators
and/or too strong interaction with the corepressors. We have
now shown that, in contrast to RAR and PML-RAR , the association
of PLZF-RAR with N-CoR is insensitive to ATRA. This is due to a
ligand-insensitive association of N-CoR corepressor with the N-terminus
of the wild-type PLZF protein. Furthermore, the ATRA sensitivity of
N-CoR association with the PML-RAR is lower than with the wild-type
RAR , ie, sensitive to pharmacologic but not physiologic
concentrations of ATRA. These results support the above-noted
hypothesis and suggest that abnormal interactions between RAR fusion
proteins and nuclear receptor corepressors play a major role in the
pathogenesis of APL and its response to ATRA treatment.
 |
MATERIALS AND METHODS |
Expression plasmids and glutathione-S-transferase (GST) fusion
proteins.
Constructions of PLZF, 5 PLZF (N-terminal region of PLZF, amino
acids 1-455), PLZF POZ (PLZF deleted for the BTB/POZ domain, amino
acids 1-120), PML-RAR , PLZF-RAR cDNA expression vectors, and
GST-PLZF plasmid have been previously described.40,41
Mammalian, bacterial, and in vitro expression vectors for the
PML,8 full-length and partial N-CoR,30,42
HDAC1,35 as well as Sin3A and B36 proteins were
described by others. L4-tkluc reporter construct contains four LexA
operators with two PLZF target sites (5 -GTACAGTAC-3 ), which were derived using PCR from L8-CAT plasmid43 and
cloned into the pT109luc44 vector upstream of the minimal
HSV-thymidine kinase (tk) promoter and luciferase (luc) gene. The
RARE2-tkluc reporter vector was previously
described.41 Mammalian two-hybrid expression vectors were
derived from pGALO and pNLVP16 plasmids45 by subcloning
indicated cDNAs in frame with the coding regions for the GAL4 DNA
binding and VP16 activating domains, respectively. The
GAL(RE)5-tkluc reporter was derived from the pT109luc
plasmid by inserting five copies of the GAL4 DNA binding site upstream of the minimal HSV-tk promoter.
In vitro interaction assays.
All GST fusion proteins were prepared using standard
procedures.40 35S-methionine-labeled proteins
were synthesized in vitro using coupled transcription-translation
system, TNT (Promega, Madison, WI), following the
supplier's directions. 35S-labeled proteins were incubated
with 1 µg GST or a given GST fusion protein (see below for
conditions). In the case of 35S-labeled RAR , PML-RAR ,
and PLZF-RAR , binding reactions were performed for 2 hours at
4°C in the absence or in the presence of 1 × 10 6 mol/L, 1 × 10 7 mol/L,
or 1 × 10 8 mol/L ATRA. Assays were performed
in NETN buffer (20 mmol/L Tris, pH 8.0, 100 mmol/L NaCl, 1 mmol/L EDTA,
0.5% NP-40) at 4°C for 60 minutes with gentle rocking.
Glutathione-Sepharose beads were washed five times with H buffer (20 mmol/L HEPES, pH 7.7, 50 mmol/L KCl, 20% glycerol, 0.1% NP-40). Bound
proteins were eluted in Laemmeli loading buffer and separated on a 5%
or 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). Gels were fixed in 25% isopropanol and 10% acetic acid,
dried, and exposed to Kodak Biomax film (Eastman Kodak, Rochester,
NY). Anti-Sin3A and anti-N-CoR antibodies were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA). For
coimmunoprecipitation, in vitro translated Sin3A and N-CoR were
incubated with a given 35S-labeled protein in NETN buffer
for 1 hour at 4°C. Immunocomplexes were isolated by further
incubation with an appropriate antibody preadsorbed on protein A/G
Sepharose (Pharmacia, Uppsala, Sweden), washed 5 times in
H buffer, analyzed by SDS-PAGE, and visualized by autoradiography.
Cell culture and transient expression analysis.
Transient transfections using 293T46 or CV-147
cells, maintained in Dulbecco's modified Eagle's medium
(DMEM) with 10% fetal calf serum (FCS), were
performed using either the calcium phosphate precipitation
method41 or SuperFect transfection reagent (Qiagen, San
Clarita, CA). FCS treated with dextran-coated
charcoal was used for all transfection experiments involving subsequent
additions of ATRA. Cytomegalovirus (CMV)-driven
-galactosidase gene expression plasmid (CMV-lacZ) was used as a
control for transfection efficiency. Cells were harvested for assaying
luciferase and -galactosidase activities 26 to 48 hours after
transfection. Luciferase assays were normalized using units of
-galactosidase activity. Both assays were performed using commercial
reagents according to the supplier's recommendations (Promega). Cells
were treated with either ATRA at 1 × 10 6 mol/L
(Sigma, St Louis, MO) for 20 hours alone or ATRA plus 1 mmol/L sodium butyrate (NaB; Sigma). BSM+ DNA (Stratagene)
was used as a carrier to equalize the total amount of transfected DNA.
In all cotransfection experiments, the total amount of transfected
mammalian expression vector was kept constant. All transfections were
performed at least three times. The NB-4 cells48 were
maintained in RPMI with 10% FCS. Cells were treated either with ATRA
alone (500 nmol/L) or with ATRA followed by 1 mmol/L NaB. Cell
differentiation was assayed by scoring the percentage of nitro blue
tetrazolium (NBT)-positive cells in treated cells versus untreated
controls at 2 and 3 days after treatment.
 |
RESULTS |
Compared with the wild-type RAR , interactions between N-CoR and
APL-associated PLZF- or PML-RAR fusions are less sensitive to ATRA.
Inhibition and stimulation of granulocytic differentiation by dominant
negative RAR mutants49,50 (or antagonists of
RAR 51,52) and RAR -specific agonists,52,53
respectively, suggest that granulopoiesis requires activation of RAR
transcriptional activity by physiologic concentrations of ATRA. The
PML-RAR chimera expressed in APL cells inhibits granulocytic
differentiation under physiologic (10 8 mol/L) but
not pharmacologic (10 6 mol/L) concentrations of
ATRA,54,55 suggesting lower sensitivity to ATRA. The
PLZF-RAR chimera appears to be even less sensitive, or completely
insensitive, to ATRA, because APL cells with
PLZF-RAR 16,17 or myeloid cells overexpressing
transfected PLZF-RAR 56 fail to respond to ATRA.
Nevertheless, the wild-type RAR , PLZF-RAR , and PML-RAR possess
approximately equal binding affinities for ATRA.40,57 Given
this background information, we attempted to test whether the chimeric
receptors differ in their binding affinities for nuclear receptor
corepressor N-CoR and, hence, would display lower responsiveness to
ATRA. Using an in vitro interaction assay, we found that, in the
absence of ATRA radiolabeled RAR , PML-RAR and PLZF-RAR were
specifically retained on a matrix-bound fusion of GST with N-CoR
fragment containing amino acids 1679-2453 (GST-N-CoR 1679-2453; Fig
1B). Furthermore, over ATRA concentrations ranging from 1 × 10 8 mol/L to 1 × 10 6 mol/L,
binding of RAR , PML-RAR , and PLZF-RAR to GST-N-CoR displayed
dramatically different ligand sensitivities. Both RAR and PML-RAR
lost most of their N-CoR binding at increasing concentrations of ATRA,
although PML-RAR required a higher ATRA concentration to attain a
similar degree of dissociation (Fig 1B, compare lanes 2 through 6 between the upper and middle panels). In contrast, binding of N-CoR to
PLZF-RAR remained relatively unchanged, even in the presence of
pharmacologic concentrations of ATRA (Fig 1B, bottom panel).
The above-noted in vitro data were fully corroborated by results from
in vivo experiments that used the mammalian two-hybrid assay with the
N-CoR protein fused to the GAL4 DBD and RAR , PLZF-RAR , or
PML-RAR VP16 activation domain fusions. As reflected by the luciferase activities generated from the GAL(RE)5-tkluc
reporter vector, strength of in vivo interaction between
GAL4(DBD)-N-CoR and VP16-PLZF-RAR was virtually unaffected by
10 6 mol/L concentration of ATRA
(Fig 2). In contrast, when compared with
the results obtained in the absence of ATRA, the degree of in vivo
association between GAL4(DBD)-N-CoR and VP16-PML-RAR was very low at
10 6 mol/L ATRA, and GAL4(DBD)-N-CoR/VP16-RAR
interaction was nearly completely abolished with ATRA treatment (Fig
2).

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| Fig 2.
Mammalian two-hybrid analysis of interactions between
N-CoR and RAR , PML-RAR , or PLZF-RAR and their ATRA
sensitivities in vivo. Cotransfections of CV-1 cells with 125 ng of
GAL(RE)5-tkluc reporter plasmid, 35 ng of GAL4(DBD)-N-CoR
expression vector (or an empty vector), 100 ng of CMV-lacZ internal
control, and 125 ng of an expression vector for a given VP16 fusion
protein, as indicated, were performed in 24-well plates using calcium
phosphate precipitation and approximately 105 cells per
well. Where indicated, cells were treated 24 to 26 hours after
transfection with 10 6 mol/L ATRA for approximately 20 hours before harvesting. Similar to the VP16-RAR control,
cotransfection of an empty GAL4(DBD) vector (pGALO) either with
VP16-PML-RAR or VP16-PLZF-RAR did not result in activation of the
luciferase gene expression (not shown).
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Nuclear receptor corepressor N-CoR interacts with the wild-type PLZF,
but not with the PML protein.
Differential sensitivities of N-CoR interaction with RAR and the two
fusion proteins could be due to stabilization of corepressor binding by
either PML or PLZF sequences in the fusion proteins. We therefore used
in vitro coimmunoprecipitation and GST-pulldown assays to investigate
if the wild-type PML and PLZF could associate with the N-CoR protein.
Although PML did not interact with N-CoR, we detected interaction
between N-CoR and the PLZF protein (Fig 3A). We then used various PLZF and N-CoR deletion mutants to identify domains within the PLZF and N-CoR proteins that were responsible for
this interaction (Fig 3B through D). The interaction was mapped to the
region of N-CoR that overlapped with the previously mapped Sin3
interaction domain29,30 and the BTB/POZ-domain of the PLZF
protein. These results were consistent with the data, described in the
preceding section, showing ATRA insensitive interaction between
GST-N-CoR (amino acids 1679-2453) and PLZF-RAR (containing amino
acids 1-455 of the PLZF protein). Because previous studies have shown
that N-CoR corepressor associates with certain nuclear receptors as a
complex with Sin3A and HDAC, we determined whether PLZF could also
interact with those molecules. Using GST-pulldown assays, we find that
in vitro PLZF also appears to interact directly with the Sin3A and
Sin3B proteins (Fig 3E) as well as HDAC1 (Fig 3F) and, to a lesser
degree, also with HDAC2 (data not shown). Nevertheless, the interaction
of the Sin3 proteins with PLZF was considerably weaker than with N-CoR.
Interaction of PLZF with HDAC1 was also weak, albeit comparable to the
interaction of HDAC1 with the Sin3A protein as detected by
coimmunoprecipitation in vitro. All interactions appeared to be
specific for PLZF, because no signal was detected using just the GST
protein as a control. The in vitro association between PLZF and N-CoR,
Sin3, or HDAC1 protein was insensitive to ATRA treatment (data not
shown).

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| Fig 3.
Mapping of the interaction domains between PLZF and
N-CoR. (A) Coimmunoprecipitation of N-CoR and PLZF. PML and PLZF were radiolabeled and incubated with in vitro translated N-CoR. After immunoprecipitation with anti-N-CoR antibody ( N-CoR), proteins were
analyzed by SDS-PAGE and autoradiography. Twenty-five percent of input
is shown (input). (B) N-CoR mutants (as indicated by amino acids
numbers) were labeled in vitro with 35S-methionine,
incubated with GST-PLZF affinity matrix, and analyzed in pulldown
assays. (C) Partial N-CoR proteins (delineated by amino acid numbers)
were expressed in bacteria as GST fusions and used in pulldown assays
for in vitro interaction with radiolabeled PLZF. Ten percent of input
is shown (input). (D) Various PLZF mutants, as indicated, were
radiolabeled and incubated with GST-N-CoR affinity matrix (amino acids
1829-1940, containing the C-terminal Sin3 interaction domain).
PLZF POZ and 5 PLZF correspond to PLZF without the BTB/POZ
domain and the region of PLZF contained in the PLZF-RAR chimeric
protein, respectively. Ten percent input is shown (input). (E)
Radiolabeled Sin3A and B proteins were incubated with GST-N-CoR (amino
acids 1679-2453), GST-PLZF, or GST affinity matrix and analyzed in
pulldown assays. Twenty-five percent input is shown (input). (F) HDAC1
was labeled in vitro with 35S-methionine and subjected to
either the pulldown assay with GST-PLZF or coimmunoprecipitation with
in vitro translated Sin3A protein and anti-Sin3A antibody ( Sin3A).
Twenty-five percent input is shown (input).
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Repression of transcription by PLZF is mediated, at least in part,
through histone deacetylation.
The carboxy-terminal Zn-fingers of the PLZF protein possess DNA binding
activity with specificity for 5 -GTACAGTAC-3 motif, which
is present in genomic sequences and, serendipitously, in the LexA
operator (Sitterlin et al58 and our unpublished
results). Furthermore, the N-terminus of the PLZF protein
fused to the Gal4 DBD repressed transcription from a Gal4 binding
site.59 We now show that the wild-type PLZF protein, which
is transiently expressed in 293T cells, represses transcription from
its binding site within the LexA operator located upstream of the
minimal ( 109 to +52) HSV-tk promoter
(Fig 4A). The level of
repression increases with increasing amounts of cotransfected PLZF
expression vector and is completely dependent on the presence of PLZF
BTB/POZ-domain. Cotransfecting N-CoR and Sin3A expression vectors
further reduces the transcription from the L4-tkluc reporter in the
presence but not in the absence of cotransfected PLZF protein (Fig 4B).
Treatment of transfected cells with a histone deacetylase
inhibitor, such as Trichostatin A (TSA),60 relieved the
repression by about 50% (Fig 4C), suggesting that, in addition to
histone deacetylation, the PLZF protein represses transcription through
other mechanism(s).

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| Fig 4.
The BTB/POZ domain containing the N-CoR
interaction region is required for transcriptional repression by the
PLZF protein. Cell transfections were performed in 6-well plates
(~106 cells per well per transfection) using SuperFect
reagent (Promega). (A) Increasing amounts (as indicated) of expression
vectors for the wild-type PLZF and PLZF POZ (solid and open boxes,
respectively) were cotransfected with L4-tkluc (400 ng) reporter vector
and 50 ng of CMV-lacZ plasmid as a control for transfection efficiency. Results are expressed as the percentage of luciferase activity obtained
in the presence of equivalent amounts of cotransfected empty expression
vector (pSG5). (B) Cotransfection of PLZF (60 ng), N-CoR (300 ng), and
Sin3A (300 ng). Expression plasmids were transfected in the indicated
amounts and the percentage of L4-tkluc (400 ng) activity was evaluated
as described in (B). (C) Addition of TSA (200 ng/mL), a histone
deacetylase inhibitor, relieved repression of the L4-tkluc reporter
plasmid expression by the PLZF protein. In this experiment, 300 ng of
PLZF expression vector or empty vector (pSG5) control, together with
400 ng of L4-tkluc and 50 ng CMV-lacZ, were cotransfected.
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Inhibitory effects of PML- and PLZF-RAR on the activities of the
wild-type RARs correlates with the ATRA sensitivity of N-CoR binding by
these chimeric proteins and can be alleviated by histone deacetylase
inhibitors.
In a number of cell systems, the PML-RAR and PLZF-RAR chimeric
proteins were found to antagonize the function of endogenous retinoic
acid receptors and/or perform as much less potent activators when compared with the wild-type RAR .1,2,8,39-41 When
assayed side by side, PLZF-RAR was a consistently poorer activator
of transcription from the RARE2-tkluc reporter than
PML-RAR and/or a stronger inhibitor of transcription by the
wild-type RARs (Fig 5A). Involvement of
histone deacetylation in the mechanism of PML- and PLZF-RAR action
is supported by data showing that a known histone deacetylase
inhibitor, NaB,61-63 can relieve the repression of ATRA
response by these chimeric proteins (Fig 5A). Data obtained using NaB
were fully corroborated when another histone deacetylase inhibitor,
TSA, was used (not shown). Furthermore, histone deacetylase inhibitors,
NaB (Fig 5B) and TSA (data not shown), synergized with ATRA in
differentiation of t(15;17)-positive APL cells. It is worth noting
that, in the presence of ATRA, other agents, such as
hexamethylene-bisacetamide, have been shown to have a rapidly
synergistic effect on differentiation of NB-4 cells,64 raising possibilities that such compounds may also possess histone deacetylase-inhibiting activities. Combination of ATRA and NaB also
synergized to induce differentiation of the PML-RAR -negative human
myeloid leukemia HL-60 cell line,65 most likely reflecting stimulatory effects of histone deacetylase inhibitors on the activation of the wild-type RAR by retinoids.

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| Fig 5.
Histone deacetylase inhibitor NaB relieves repressing
activities of RAR chimeric proteins and synergizes with ATRA in
differentiation of APL cells. (A) Transcriptional activities of the
wild-type and mutant RARs assayed in 293T cells. Cotransfections were
performed with RARE2-tkluc reporter plasmid (200 ng), 50 ng
of CMV-lacZ control, and a pSG5 expression vectors for the
indicated proteins (50 ng each). ATRA (10 6 mol/L) was
added alone or in combination with histone deacetylase inhibitor NaB (1 mmol/L). NaB synergizes with ATRA in alleviating the transcriptional
inhibition of RARE2-tkluc reporter by PML-RAR and
PLZF-RAR . ATRA alone, or together with NaB, had a considerably lower
effect on transcriptional activation by PML-RAR , but not by the
wild-type RAR (not shown), when an equal amount of the wild-type
PLZF expression vector was cotransfected. All cell transfections were
performed in 24-well plates (~105 cells per well per
transfection) using calcium phosphate precipitation. (B) NaB
potentiated differentiating effects of ATRA on NB-4 cells. The Y-axis
indicates the percentage of NBT-positive cells assayed 2 and 3 days
after treatment with 500 nmol/L ATRA, no treatment, or treatment with
500 nmol/L ATRA followed by 1 mmol/L of NaB.
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 |
DISCUSSION |
It now appears that the strong inhibitory effect of the PLZF-RAR
chimera is based on the ability of its N-terminal BTB/POZ-domain to
associate independently, in an ATRA-insensitive manner, with the
N-CoR corepressor and possibly also with other components of the
nuclear receptor corepressor complex, such as Sin3 and HDAC. Therefore,
the PLZF-RAR protein can engage with N-CoR by means of the receptor
interaction domain (CoR-box, see Fig 1A) and the PLZF BTB/POZ
interaction domain, with the latter being completely insensitive to
ATRA. Because PML does not appear to interact with N-CoR, decreased
sensitivity of PML-RAR /N-CoR association to ATRA may be due to
steric effects that the PML moiety exerts on the N-CoR binding RAR
region (CoR-box) of the chimeric protein or some other factors that may
interact with both PML and N-CoR and stabilize the complex. In this
respect, noteworthy is our previous study reporting interaction and
colocalization between PML and PLZF as well as PLZF and PML-RAR
proteins (but not PML and PLZF-RAR ) in APL cells,18
suggesting that PLZF may play a wider role in APL than previously
supposed from the study of few patients with the t(11;17)(q23;q21)
chromosomal translocation. The hypothesis that PLZF may be such a
factor is supported by observation that overexpression of the PLZF
protein strongly inhibits responsiveness of the PML-RAR to ATRA as
well as ATRA and NaB (Fig 5A). The strong inhibitory effect of PLZF,
even in the presence of histone deacetylase inhibitor, is consistent
with a model in which transcriptional repression by PLZF is only
partially mediated through histone deacetylation. Nevertheless,
interaction between PML-RAR and N-CoR remains sensitive to treatment
with pharmacologic doses of ATRA providing a mechanism for therapeutic
response of APL with t(15;17) to this drug.
Very recently, it has also been shown that PLZF,66 as well
as BTB/POZ-domain Zn-finger protein LAZ-3/BCL-6,67 which
plays a role in the pathogenesis of diffuse large-cell
lymphoma,68,69 interact with another nuclear receptor
corepressor, SMRT. In vitro interactions of SMRT with RAR ,
PML-RAR , and PLZF-RAR also displayed differential sensitivities
to ATRA.66 LAZ-3/BCL-6 can also interact with the N-CoR
protein (data not shown), suggesting that both nuclear receptors and
BTB/POZ-domain Zn-finger proteins repress transcription through the
same general mechanism. These results also provide evidence for a
relationship between signalling through distinct families of
transcriptional regulators, such as nuclear receptors, BTB/POZ domain
Zn-finger proteins, and E-box binding Mad/Max proteins. Because
distinct regulators often possess common coregulators, by sequestration
of a shared coregulator, activation or repression by a given factor may
have the opposite effect on activity of another factor(s). For example,
activation of a nuclear hormone receptor by ligand binding causes
association of a coactivator, such as CBP (CREB-binding
protein), which is also required by a number of other activators,
including CREB, AP-1, and STAT-1 .70,71 Depending on the
relative affinities of these proteins for CBP and their concentrations,
activation of a nuclear receptor by its ligand could inhibit CREB,
AP-1, and STAT-1 activities and, hence, signalling through cAMP,
Ras, and interferon (INF ) pathways, respectively. Because
Sin3/N-CoR/HDAC are also required for transcriptional repression by
Mad/Max complex,30,36,37 which is associated with cellular
differentiation, the fusion receptors may in part contribute to
leukemogenesis by sequestration of these factors and inhibition of
Mad/Max activity in favor of growth promoting Myc/Max function.
Deregulation of histone acetylation has for some time been thought to
be associated with oncogenesis, and histone deacetylase inhibitors were
reported to possess anti-tumor activities.72,73 Association
of histone acetyltransferase, CBP, with two different leukemogenic
translocations, t(11;16)(q23;p13)74 and
t(8;16)(p11;p13)75 involving the MLL and MOZ genes,
respectively, has also been reported. In summary, our data directly
implicate for the first time the nuclear receptor corepressor/histone
deacetylase complex in carcinogenesis and provide a plausible mechanism
(Fig 6) for the molecular pathogenesis of
APL and its response to treatment with ATRA. Central to this model is
the observation that nuclear receptor corepressor binding to PML- and
PLZF-RAR displays abnormal sensitivity to ATRA, with corepressor/PLZF-RAR association being the least sensitive or not
sensitive at all. Because histone deacetylase inhibitors considerably potentiate effects of ATRA on both the activities of the RAR fusion
proteins and APL cell differentiation, they may serve as useful
adjuncts to ATRA in treatment of APL.

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| Fig 6.
Model for the role of nuclear receptor corepressor
N-CoR/Sin3A/HDAC1 complex and RAR fusion proteins in the
pathogenesis and treatment of APL. (A) In the absence of ATRA, RAR ,
PML-RAR , and PLZF-RAR associate with N-CoR/Sin3A/HDAC1
corepressor complex. The associated corepressor/RAR complex acts on
chromatin structure by deacetylation of histone tails inducing its
reorganization into a repressed state that is inaccessible to basal
transcription factors. Binding of ATRA (orange triangle) induces
conformational change in the RAR , causing dissociation of the
corepressor complex and association of a coactivator, such as SRC-1,
with intrinsic histone acetyltransferase activity.76
Acetylation (Ac) of histone tails disrupts tightly packed and repressed
chromatin structure, allowing access of the basal transcription factors
and transcriptional activation. Physiologic concentrations of ATRA
(10 8) are sufficient to induce this process. (B) In the
case of the PML-RAR protein, pharmacologic doses of ATRA
(10 6 mol/L) are required to achieve efficient
dissociation of the N-CoR corepressor complex from the chimeric protein
and transcriptional activation. (C) Because of additional,
ligand-insensitive interactions between the PLZF moiety of the
PLZF-RAR fusion protein and N-CoR (and possibly also Sin3A and
HDAC1), the corepressor/PLZF-RAR complex remains associated even in
the presence of pharmacologic concentrations of ATRA and, in the
absence of chromatin remodelling by histone acetylation, transcription
remains inhibited.
|
|
 |
FOOTNOTES |
Submitted December 3, 1997;
accepted December 29, 1997.
Supported by The Leukaemia Research Fund of Great Britain, National
Institutes of Health (Grant No. CA59936-01), and the Medical Research
Council. F.G. and J.Z. were in part supported by the TMR Programme
Marie Curie Research Training Grant from the European Commission and a fellowship from the Samuel Waxman Cancer Research Foundation, respectively.
Address reprint requests to Arthur Zelent, PhD, Leukaemia Research Fund
Centre at the Institute of Cancer Research, Chester Beatty
Laboratories, 237 Fulham Rd, London SW3 6JB, UK.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
 |
ACKNOWLEDGMENT |
The authors thank Q.-H. Huang, Z. Chen, L. Wiedemann, T. Enver, J. Licht, and M. Greaves for helpful comments and discussions. We are
grateful to P. Chambon, S. Hollenberg, C.D. Laherty, R.N. Eisenman,
C.A. Hassig, S.L. Schreiber, C.K. Glass, C.V. Dang, E.R. Fearon, T.H.
Rabbitts, M. Lanotte, and M. Yoshida for their generous gifts of
molecular clones, expression vectors, antibodies, cells, and drugs that
were used in this study.
 |
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