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NEOPLASIA
From the Lady Davis Institute for Medical Research, Sir
Mortimer B. Davis Jewish General Hospital, and the McGill University
Department of Oncology and Medicine, Montreal, Quebec, Canada;
Montefiore Medical Center and Albert Einstein Cancer Center, Bronx, NY;
and Dipartimento di Istologia ed Embriologia Medica, Università
di Roma "La Sapienza," Rome, Italy.
Acute promyelocytic leukemia (APL) is characterized by a specific
translocation, t(15;17), that fuses the promyelocytic leukemia (PML) gene with the RA receptor RAR Acute promyelocytic leukemia (APL) accounts for
approximately 10% of all cases of acute myeloid leukemia. APL is a
unique subtype of leukemia characterized by a distinct chromosomal
translocation, t(15;17), with breakpoints within the retinoic acid
receptor alpha (RAR The fusion protein consistently retains both the DNA-binding C-domain
of the RAR RA therapy induces complete remission in a high percentage of
patients with APL.15 Unfortunately, the duration of
response is short, and further therapy with this agent is less
effective, suggesting the development of drug
resistance.16-19 Explanations for this resistance include
progressive reduction of RA plasma concentration, which may be
explained by an increased level of cellular RA-binding protein
(CRABP),20 an increased oxidative catabolism of RA by
cytochrome P450 enzyme activity, or multidrug-resistance (MDR) gene
product.21,22 But additional genetic mechanisms of
retinoid resistance, such as mutations in nuclear retinoid receptors,
have previously been found in HL-60 myeloid leukemic cells.23-25
In vitro studies on APL cells are provided by a cell line, NB4, derived
from an APL patient.26 Our laboratory, and others, have
developed RA-resistant NB4 subclones to study cellular or molecular
mechanisms that mediate retinoid response or
resistance.27-29 We have reported RA-resistant subclones
that are highly resistant to natural and synthetic retinoids that do
not bind CRABP and are not metabolized by P450
enzymes.29,30 We identified a point mutation in the
ligand-binding domain (LBD) of the RAR Recently, mutations in the RAR At the same time, Ding et al38 found
PML/RAR The goal of this study was to characterize the phenotype of these
clinically observed mutations and determine how these mutations, found
in RA-resistant APL patients, could impair the ligand-induced and
transcriptional functions of the RAR Cell culture
Plasmid constructs
Assay for ligand-binding activity The Cos-1 cells were transiently transfected by electroporation with the expression vectors containing either wild-type or mutant PML/RAR . Nuclear extracts were prepared from 1 to
5 × 108 cells and incubated for 18 hours at 4°C with
10 nmol/L [3H]-RA (50.7 Ci/mmol; DuPont-NEN, Boston) or
with [3H]-RA in the presence of 200-fold excess of
unlabeled RA, as previously described.11 The extracts were
subsequently fractionated at 4°C by HPLC by means of a
superose 6 HR 10/30 size exclusion column (Pharmacia, Uppsala,
Sweden). The flow rate was 0.4 mL/min; fractions of 0.4 mL
were collected; and radioactivity was determined by means of a liquid
scintillation counter. The HPLC system was calibrated by means of a
series of molecular weight (MW) markers, consisting of the following:
blue dextran, MW 2 000 000; thyroglobulin, MW 669 000;  -amylase,
MW 200 000; bovine serum albumin, MW 66 000; and ovalbumin,
MW 45 000.
Limited proteolytic digestion of translated fusion proteins Wild-type and mutant PML/RAR fusion proteins were synthesized
in vitro by means of a coupled transcription and translation reticulocyte lysate system for 90 minutes at 30°C as suggested by the
manufacturer (Promega). The reaction was performed in the presence of
[35S]-methionine (NEN; Streetsville, Ontario, Canada) to
produce radioactive fusion proteins. We incubated 5 µL of in
vitro synthesized wild-type or mutated PML/RAR proteins with 1 µmol/L of all-trans RA (Sigma, St Louis, MO) for up to 30 minutes at room temperature in the dark. After treatment with 0.5 µL
of different concentrations of trypsin (Sigma-Aldrich Canada Ltd;
Oakville, Ontario, Canada) for 10 minutes at room temperature, 20 µL
of denaturing loading dye was added. An equal volume of water
was added for the undigested controls or untreated samples. The samples
were denatured and directly analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (10% wt/vol). The gels
were then dried and exposed for autoradiographic analysis.
Transient transfection experiments for transcriptional activity Cos-1 cells were grown in RPMI-1640 with 10% FCS and were seeded at 120 000 cells per well in 6-well plates 1 day before transfection. Cells were rinsed with serum-free OPTI-MEM (Life Technologies) and transfected by the lipofectamine method (Life Technologies) with 0.7 µg of wild-type or mutant fusion protein plasmids, 1 µg of the reporter plasmids DR5-tk-CAT41 or TREpal-tk-CAT,41 and 0.3 µg of pCMV-Galactosidase
(Gal) as an internal control for transfection efficiency. Cells were
transfected for 5 hours, replenished with 2 mL of RPMI-1640 with 10%
FCS, and grown for 24 hours in the absence or presence of different
concentrations of RA. The chloramphenicol acetyltransferase (CAT)
activity was measured by means of a modified protocol of the organic
diffusion method.42 Briefly, 50 µL of cell extract was
incubated at 37°C for 1 to 5 hours with 200 µL of 1.25 mmol/L cold
chloramphenicol (ICN; Costa Mesa, CA) dissolved in 100 mmol/L Tris-Cl,
pH 7.8, and 0.25 µCi [3H]-labeled acetyl coenzyme A
(NEN). The reaction was extracted with Ready Organic Scintillation
Cocktail (Beckman; Mississauga, Ontario, Canada), and 750 µL of the
organic phase was counted on a scintillation counter. The CAT counts
were normalized with Gal activity43 to obtain the
relative CAT activity.
Electrophoretic mobility shift assays Electrophoretic mobility shift assays were performed with the use of a direct-repeat 5 (DR5) RARE. The nucleotide sequence of the [32P]-labeled oligonucleotide duplex used was as follows: 5'-agcttcAGGTCAccaggAGGTCAgagagct-3'. [35S]-labeled wild-type and mutant PML/RAR fusion
proteins were synthesized by means of a coupled transcription and
translation reticulocyte lysate system, according to the
manufacturer's recommended protocol (Promega). Equal counts of in
vitro translated wild-type and mutant fusion proteins, purified
glutathione S-transferase (GST) fusion proteins (1 µg), and
[32P]-labeled DR5 (100 000 cpm) were incubated with or
without RA, as indicated with the following: 0.3 µg poly (dI:dC) for
30 minutes at room temperature in a 24-µL reaction containing 100 mmol/L KCl; 6% glycerol; 10 mmol/L Tris, pH 8.0; 0.05% NP-40; and 1 mmol/L dithiothreitol. Where specified, bacterially expressed and
purified GST fusions containing the receptor interacting domains of
SMRT (GST-SMRT-IDII; amino acid 1073-1168; 1 µg/lane) or
ACTR (GST-ACTR-RID; amino acid 621-821; 1 µg/lane) were
added.13,44 The protein-DNA complexes were
resolved on a 4.5% native polyacrylamide gel electrophoresis in 0.5X
TBE and visualized by autoradiography.
PML/RAR mutations associated with RA resistance in
cell lines (numbers 1-5) and in APL patients (numbers 6-11). In this
study, the phenotypes of the first PML/RAR natural
mutations identified in RA-resistant APL patients were characterized.
Transcriptional properties of the mutations, indicated by number 6, 8, 9, 10, and 11 in Figure 1A, were examined to discover the extent of
functional impairment. The locations of the mutations were described
with reference to normal protein of RAR1,32 because
their position in the fusion molecule depends on the isoforms of
PML/RAR messenger RNA. Approximate locations of the
evaluated mutations in the 3-dimensional structure of the RAR LBD are
shown in Figure 1B.
RA-binding activity of the wild-type and mutant PML/RAR alter the
function of the LBD, we examined their RA-binding activity. Binding of
[3H]-RA to nuclear extracts from Cos-1 cells transiently
transfected with the wild-type or the mutant PML/RAR expression
vectors was analyzed. The size exclusion HPLC profile of extracts from
cells expressing the wild-type form of PML/RAR is characterized by 3 main peaks as previously described.11 The 50-kd
peak represents the endogenous RARs in Cos-1 cells. The 110-kd peak
characterizes the binding of PML/RAR monomers, and the approximately
670-kd peak represents macromolecular complexes formed by the
interaction of PML/RAR with itself and/or other nuclear proteins.
The HPLC profiles of extracts from cells expressing the PML/RAR
mutations R394W and M413T are similar to the pattern observed with the
wild-type form, indicating that these mutations in the LBD of
PML/RAR do not substantially impair the binding of ligand (Figure
2). Nuclear extract from cells expressing
the PML/RAR mutations M297L and R272Q showed HPLC profiles
consistent with the elution of PML/RAR macromolecular nuclear
complexes in fractions corresponding to MWs of about 400 to 200 kd. In
addition, the mutation R272Q of PML/RAR decreased RA binding to the
fusion protein. Specific RA-binding activity was not detectable in
nuclear extracts prepared from cells transiently transfected with the
PML/RAR containing the mutation L290V (Figure 2A).
Proteolytic analysis of the wild-type and mutant
PML/RAR did not differ in digestion pattern (Figure 3). In the absence of RA, a 32-kd
fragment was more resistant to trypsin digestion. After 1 µmol/L RA
treatment, the 32-kd fragment disappeared, whereas 2 fragments of 36 to
37 kd were more resistant to protease treatment. As with human
RAR,47 human progesterone
receptor,47 and human estrogen
receptor,48 ligand binding causes a
conformational change of PML/RAR, resulting in the modification of
accessibility of trypsin cleavage sites. Analysis of the mutations
M297L and M413T showed that their trypsin digestion patterns are
identical to that of the wild-type. The digestion patterns for the
mutated R272Q and R394W PML/RAR differ from that of the wild-type by the presence of an intense 32-kd resistant fragment after RA treatment. These results indicate that RA can still bind to the mutated fusion protein LBD, but induces a different conformational change than in the
wild-type LBD. In the case of the mutation L290V, the trypsin digestion
pattern after RA treatment is completely different from that of the
wild-type: the 32-kd fragment is still present and the 36- to 37-kd
fragments are absent. The identical digestion patterns in the absence
and presence of RA indicate that the mutated fusion protein does not
bind the ligand, which is consistent with our ligand-binding analysis
(Figure 2A). The same results were obtained with the mutation found in
NB4-R4 cells, PML/RAR (m4), which we have previously shown is not
able to bind RA.31
Ligand-dependent transcriptional activity The conformational changes induced by certain mutations might also be expected to alter protein-protein interactions that are required for RARE binding and activation. Thus, we compared the transcriptional activity of PML/RAR mutants with that of the wild-type fusion
protein on 2 retinoid-responsive elements. Fusion protein plasmids were
cotransfected into Cos-1 cells with tk-CAT reporters driven by either a
DR5 RARE (Figure 4A) or a palindromic
thyroid response element TREpal (Figure 4B). Figure 4 showed that RA
slightly induced transcription of both DR5-tk-CAT and TREpal-tk-CAT
reporters cotransfected with the empty vectors (pSG5 and pCMX),
consistent with the known endogenous RAR present in Cos-1 cells. The
wild-type PML/RAR S-form and L-form acted as dominant negative
inhibitors of the control transcriptional activity for both DR5-tk-CAT
and TREpal-tk-CAT reporters (Figure 4), reducing the baseline CAT
activity as compared with the empty vectors. This dominant negative
activity of the fusion protein is maintained in all PML/RAR mutants,
as exhibited by a smaller baseline CAT activity than was seen with the
empty vectors. As shown in Figure 4, ligand-dependent transcriptional
activity of both S-form and L-form of the wild-type PML/RAR chimeric
proteins was increased in a manner dependent on RA concentrations. The PML/RAR L-form stimulated the RA-induced transcription more
efficiently than the S-form (Figure 4). The PML/RAR chimeric
proteins harboring either M297L or R394W mutations showed a
ligand-dependent transcriptional activity on both tk-CAT reporters that
was similar to the wild-type S-form fusion protein (Figure 4). In
contrast, the mutant R272Q and L290V fusion proteins had an altered
transcriptional activity on DR5 and TREpal reporters in the presence of
RA. The mutant R272Q showed no increase in CAT activity in the presence
of 0.01 µmol/L RA on the DR5 (Figure 4A) and on the TREpal
(Figure 4B). In the presence of 0.1 µmol/L RA, both reporters
produced increased transcriptional activity that remained less than
that of the wild-type PML/RAR. The mutated form, PML/RAR L290V,
has significantly lost ligand-dependent transcriptional activity on
both reporters (Figure 4). As previously reported, PML/RAR (m4) does
not activate transcription in response to even 1 µmol/L RA, on all
reporters tested (Figure 4 and data not shown). The PML/RAR fusion
protein harboring M413T mutation showed a loss of approximately half of the CAT activities on RARE reporters, as compared with the wild-type L-form PML/RAR, in the presence of RA (Figure 4). Similar results were obtained with the use of a DR5-tk-LUC reporter (data not shown).
Ligand-dependent corepressor SMRT release by wild-type and mutant
PML/RAR bound a radio-labeled DR5 element in
the absence of SMRT-IDII. Addition of purified GST-SMRT-IDII shifted
the bound complex, as indicated by an arrow in Figure
5. Binding of GST-SMRT-IDII to each of
the mutants was similar to the wild-type PML/RAR in the absence of
RA. In contrast, ligand-induced dissociation of SMRT-IDII from certain
mutants was very different from the wild-type. As previously reported,
the wild-type PML/RAR fusion proteins completely dissociated
SMRT-IDII at RA concentrations between 10 6 and
105 mol/L (Figure 5). PML/RAR harboring the mutations
M297L or R394W required about 10-fold higher concentrations of RA to
dissociate the corepressor SMRT than did the wild-type (Figure 5A). The
fusion proteins harboring the mutations R272Q, L290V, M413T, or
PML/RAR (m4) could not be dissociated from the corepressor SMRT-IDII
even at 10 µmol/L RA concentration (Figure 5), indicating that these mutants required more than 100-fold higher concentrations of RA to
dissociate SMRT than did the wild-type.
Recruitment of the coactivator ACTR by mutants
PML/RAR could normally recruit the coactivator ACTR, the
central receptor-interacting domain of ACTR (ACTR-RID) fused to the GST
protein was expressed in bacteria. Purified GST-ACTR-RID and in vitro
translated fusion proteins were then employed in a gel-shift study of
wild-type and mutated PML/RAR to a DR5 probe (Figure
6). In the absence of RA, ACTR-RID did
not form a complex with the wild-type or mutated fusion proteins. The
wild-type PML/RAR-ACTR-RID complex was first noted at 0.01 µmol/L
and was maximal at 0.05 µmol/L RA. The M297L mutant appeared to be
similar to the wild-type in its ability to recruit ACTR-RID over the RA
concentrations tested (Figure 6A). The mutant M413T required about
10-fold greater RA concentrations, relative to the wild-type, to begin
the recruitment of ACTR-RID (Figure 6B). The protein harboring the
mutation R394W started recruiting the coactivator at 0.01 µmol/L as
did the wild-type, but its association is maximal only at 1 µmol/L
RA, as compared with 0.05 µmol/L for the wild-type. The mutant R272Q
and L290V fusion proteins were least able to associate with the
coactivator. The mutation R272Q is fully associated with ACTR-RID only
at 1 µmol/L RA, which is 20-fold greater than for the wild-type
association. PML/RAR harboring the mutation L290V showed only
minimal recruitment of ACTR at 1 µmol/L RA, which indicates that the
coactivator needs more than 100-fold RA concentrations for its
association with this mutated fusion protein. The mutation PML/RAR
(m4) showed that even with 1 µmol/L RA, ACTR-RID was not able to bind
the fusion protein (Figure 6B).
APL has the unique characteristic of responding in vitro and in
vivo to differentiation therapy with RA. The chimeric protein PML/RAR However, several groups have identified specific genetic lesions
associated with RA resistance, both in cell lines and in patients.
Several missense mutations in the LBD of the RAR Recently, 2 independent groups identified the presence of the first
missense mutations in the LBD of the RAR The L290V mutation is located in the In contrast, other mutations observed in cells from resistant patients
had quite varying effects on RAR Our results with the mutation R272Q of PML/RAR An additional case, the substitution of the methionine at position 413 by threonine (M413T), suggests that loss of ligand-inducible coactivator binding does not always accompany loss of corepressor release. This methionine, located in H12 of the LBD is believed to
contribute directly to stabilization of the RA binding pocket through
the formation of hydrogen bonds or van der Waals contacts with the
bound RA molecule.45 Although this substitution is not
conservative, our analysis of ligand binding, as well as our limited
proteolytic-digestion assay, indicated that this mutation has only a
minor effect on the binding of RA for PML/RAR We also characterized 2 patient mutations in PML/RAR Finally, we analyzed the substitution of the methionine at position 297 for a leucine (M297L), within H6, which presented almost the same
phenotype as that of the wild-type chimeric protein. These results
suggest that the methionine at 297 does not play a major role in the
ligand binding and in the interaction with transcriptional
coregulators. The nature of the conservative substitution may explain
the very minor effect of this mutation on the tested functional
properties of the fusion protein PML/RAR The relationship of different mutations to the severity of the clinical
phenotype of RA resistance in APL patients is difficult to establish,
in part because it was not possible to define differences in the degree
of clinical resistance among patients. A given mutation of the
PML/RAR The comparison of RA-resistant APL with the RTH syndrome may help us
understand the functional roles of these mutations. More than 70 different natural TR Since the discovery of these mutations, new mutations have been found
in the above hot spots of the LBD of the fusion protein PML/RAR It is now clear that the acquisition of mutations in PML/RAR
We thank Wei Ding for the generation of the pSG5-PML/RAR
Submitted May 3, 2000; accepted July 11, 2000.
Supported by grants from the Medical Research Council of Canada, Public Health Service Grant No. CA56771, and the Associazione Italiana per la Ricerca sul Cancro. S.C. is supported by fellowships from Fonds de la Recherche en Santé du Québec, Cancer Research Society, and Israel Cancer Research Fund. A.B. is supported by the Fondazione Italiana per la Ricerca sul Cancro. W.H.M. is a scientist of the Medical Research Council of Canada.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Wilson H. Miller Jr, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, 3755, Chemin de la Côte Ste-Catherine, Montreal, Quebec, Canada H3T 1E2; e-mail: wmiller{at}ldi.jgh.mcgill.ca.
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ligand-binding domain arising in retinoic
acid-resistant patients with acute promyelocytic leukemia
Top
Abstract
Introduction
.
) or with (+)
1 µmol/L RA, and subsequently treated with increasing trypsin
concentrations (0 to 25 µg/mL). Digestion products were analyzed by
denaturing electrophoresis. The arrows indicate the intact PML/RAR
; RA 0.01 µmol/L, 
; RA
0.1 µmol/L, 
; RA 1 µmol/L, 
.
