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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 2 (July 15), 1998:
pp. 374-382
Mutations in the E-Domain of RAR Portion of the PML/RAR
Chimeric Gene May Confer Clinical Resistance to All-trans
Retinoic Acid in Acute Promyelocytic Leukemia
By
Masue Imaizumi,
Hoshiro Suzuki,
Miyako Yoshinari,
Atsushi Sato,
Toshiaki Saito,
Akira Sugawara,
Shigeru Tsuchiya,
Yoshiro Hatae,
Takeo Fujimoto,
Akira Kakizuka,
Tasuke Konno, and
Kazuie Iinuma
From the Departments of Pediatrics and Second Internal Medicine,
Tohoku University School of Medicine, Sendai, Japan; the Department of
Pediatric Oncology, Institute of Development, Aging and Cancer, Tohoku
University, Sendai, Japan; the Department of Pediatrics, National
Sapporo Hospital, Sapporo, Japan; the Department of Pediatrics, Aichi
Medical University, Aichi, Japan; and The 4th Department, Osaka
Bioscience Institute, Osaka, Japan.
 |
ABSTRACT |
The binding of all-trans retinoic acid (ATRA) to the
ligand-binding region in the E-domain of retinoic acid receptor-
(RAR) modifies the transcriptional activity of RAR protein. ATRA
probably induces differentiation of acute promyelocytic leukemia (APL) cells by binding to the E-domain of the RAR portion
(RAR/E-domain) of PML/RAR chimeric protein. Therefore, molecular
alteration in the RAR/E-domain of the chimeric gene is one mechanism
by which patients with APL may acquire resistance to ATRA therapy. In
this study using reverse transcription-polymerase chain reaction and
single-strand conformation polymorphism, DNA segments amplified from
the RAR/E-domain in fresh APL cells of 23 APL patients (8 males and
15 females from 4 to 76 years of age) were screened for mutations. Of
those patients, 3 patients (1 with de novo and 2 with relapse) had
clinical resistance to ATRA therapy. We found mutations in the
RAR/E-domain of PML/RAR chimeric gene exclusively in the 2 patients who exhibited ATRA-resistance at relapse, whereas the
mutations were not detected at their initial onset. Interestingly, these patients received a prolonged or intermittent administration of
ATRA before relapse with ATRA-resistance. The mutations lead to the
change of amino acid in the ligand-binding region of RAR/E-domain, Arg272Gln, or Met297Leu according to the amino acid sequence of RAR,
respectively. Further study demonstrated that the in vitro ligand-dependent transcriptional activity of the mutant PML/RAR protein was significantly decreased as compared with that of wild-type PML/RAR. These findings suggest that mutations in the
RAR/E-domain of the PML/RAR chimeric gene may confer clinical
resistance to ATRA therapy in patients with APL.
| |
INTRODUCTION |
ACUTE PROMYELOCYTIC leukemia (APL) is a
unique subtype of leukemia characterized by a distinct chromosomal
t(15;17) translocation with breakpoints within the retinoic acid
receptor- (RAR) gene on chromosome 17 and the PML gene, a
putative transcription factor, on chromosome 15.1-3 The
translocation generates a PML/RAR chimeric gene and, thereby, a
PML/RAR fusion protein.4,5 Because the chimeric gene is
present invariably and specifically in APL, the formation of PML/RAR
is thought to be involved in the primary pathogenesis.3
Recently, it was shown that the PML/RAR fusion protein inhibits
differentiation or promotes the survival of myeloid precursor cells in
vitro, suggesting a role of the fusion protein in the pathogenesis of
APL.6,7
In the genomic structure of the PML/RAR chimeric gene, the
breakpoints in the RAR gene localize exclusively within the second intron of the gene, and thereby the chimeric transcripts consistently retain not only the C-domain of the RAR for DNA-binding, but also
the E-domain of the RAR that is required for ligand-binding and
receptor-dimerization.8,9 Because of this structural property, the function of the PML/RAR fusion protein may be
dependent on the binding of a specific ligand, all-trans
retinoic acid (ATRA), to the E-domain of the RAR portion
(RAR/E-domain), as is the nuclear RAR.9-12
Furthermore, it appears that this ligand-dependent transcription
mechanism may underlie the cytodifferentiation of APL cells induced by
pharmacological concentrations of ATRA.11
APL cells are induced by ATRA to differentiate in vitro and in vivo to
mature myeloid cells.13-17 ATRA therapy induces complete remission in a high percentage of patients without severe complications associated with marrow hypoplasia or hemostatic
impairment.17,18 However, most patients who relapse after
ATRA therapy exhibit an apparent acquired
ATRA-resistance.19-22 This acquired ATRA-resistance may be
associated with several mechanisms such as insufficient plasma levels
of ATRA concentration primarily caused by an increased oxidative
catabolism of ATRA by cytochrome P-450 enzyme
activity,23-26 increases in cellular retinoic acid binding
protein (CRABP),27 or multidrug-resistance (MDR) gene
product.28 More importantly, APL cells of patients with in
vivo ATRA-resistance also exhibit frequently an ATRA-resistance or a
decreased ATRA-sensitivity in vitro.20,27 These results
indicate that ATRA-resistance could be due to an acquired cellular
mechanism by which APL subclones survive and proliferate in vivo under
the condition with pharmacological concentrations of ATRA.
As a model for an acquired resistance to ATRA, recent studies using
ATRA-resistant myeloid leukemia cell lines showed the presence of point
mutations in the E-domain of the RAR gene in HL-60 subclones that
acquired ATRA-resistance in vitro.29-31 More recently, a
NB4 subclone with ATRA-resistance had missense mutation also in the
RAR/E-domain of PML/RAR chimeric gene, which abolished the
ATRA-binding activity of the chimeric protein.32 Therefore, molecular alteration in the RAR/E-domain of the chimeric protein may
impair ATRA binding, leading to an acquired ATRA-resistance of APL
cells. On the basis of these findings, alteration of the RAR/E-domain of the chimeric gene has been anticipated, but not yet
demonstrated, as a possible mechanism for ATRA-resistance in patients
with APL.
In this study, we investigated molecular alteration of the
RAR/E-domain in 23 patients with APL, including 3 with
ATRA-resistance. We identified missense mutations in the
RAR/E-domain of the PML/RAR chimeric gene at relapse in 2 patients who had ATRA-resistance after a prolonged or
intermittent ATRA therapy. Our study may be useful to the
understanding of the mechanism(s) by which patients with APL
become ATRA-resistance during or after ATRA therapy.
| |
PATIENTS |
Clinical, hematological, and cytogenetic features of 23 patients with
APL (8 males and 15 females with an average age of 27.4 years; range, 4 to 76 years) were shown in Table 1. Of
those, 19 patients were with de novo APL and 4 were with bone marrow relapse. All patients except 2 received ATRA therapy for remission induction. Patients with ATRA-resistance were defined as those whose
APL cells failed to show maturation in vivo during remission induction
with ATRA therapy or those who relapsed during ATRA therapy. Using this
criteria, there were 3 patients who exhibited ATRA-resistance: 1 with de novo APL and 2 with relapse who received either a
prolonged ATRA therapy or a repeated alternative treatment with ATRA
administration and chemotherapy before relapse. Two major isoforms
of the PML/RAR chimeric gene, a long (or B) type with fusion
between PML exon 6 and RAR exon 3 and a short (or A) type with
fusion between PML exon 3 and RAR exon 3 were determined as
previously described.4,33
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MATERIALS AND METHODS |
Cells.
After having obtained informed consent, we obtained bone marrow or
peripheral blood samples from the patients with APL before treatment.
Mononuclear cells were separated by a density-centrifugation with
Ficoll-Paque (density, 1.077; Pharmacia, Uppsala, Sweden) and washed
with phosphate-buffered saline (GIBCO BRL, Gaithersberg, MD).
RNA extraction and cDNA synthesis.
Total RNA was isolated from mononuclear cells using an acid guanidinium
thiocyanate-phenol-chloroform extraction method.34 cDNA was
then synthesized by incubating 10 µg of total RNA with 20 U of
AMV-reverse transcripitase (Life Sciences, St Petersburg, FL) in 20 µL of reaction mixtures containing 50 mmol/L NaCl, 20 mmol/L Tris-Cl
(pH 7.4), 8 mmol/L MgCl2, 50 ng/µL of oligo-dT (Pharmacia), 2 mmol/L deoxynucleotide triphosphates, 5 mmol/L dithiothreitol, and 2 U/mL of RNAsin (Takara Syuzo, Osaka, Japan) at
42°C for 1 hour.
Oligonucleotide primers.
For screening the presence of mutation, two segments in the
RAR/E-domain, named E2E4 and EF1 in Fig
1, were amplified using two pairs of
synthetic oligonucleotide primers, E2-E4a and EF1-EF1a, respectively
(Sawaday Technology, Tokyo, Japan). Segments for single-strand
conformation polymorphism (SSCP) analysis were designed to be no longer
than 300 bp in length. The segments, E2E4 or EF1, correspond to the region at the middle part of E-domain or to the
region spanning both of the carboxyl-terminal of E-domain and the
F-domain of RAR gene, respectively. Because both of sense and
antisense primers of these oligonucleotides correspond to the regions
within the E and F-domains of RAR gene, it was expected that
polymerase chain reaction (PCR) products would be a mixture of DNA
fragments amplified from both of the RAR gene and PML/RAR chimeric gene. To distinguish which gene, either RAR or PML/RAR, the DNA segments were derived from, we designed the other two pairs of
oligonucleotide primers with sense primers corresponding either to exon
1 of the RAR gene (RAR-1A) or to exon 3 of the PML gene (PML-C) (Fig
1). Using a common antisense oligonucleotide primer (E2a) at the
E-domain of the RAR gene, DNA segments could be differentially
amplified from the RAR or PML/RAR genes.
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| Fig 1.
Primer designs for RT-PCR/SSCP analysis (EF1 and EF1a
primers for EF1 segment and E2 and E4a primers for E2E4 segment) or for
differential amplification of the E-domain of RAR (RAR-1A and E2a)
or of PML/RAR gene (PML-C and E2a).
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PCR.
PCR reactions were performed as described, with
modification.35 Reaction mixtures containing 0.2 µL of
cDNA, 200 mmol/L deoxynucleotides, 1.0 mmol/L oligonucleotide primers,
and 0.6 U of Taq polymerase (Perkin-Elmer Cetus, Norwalk, CT) in 20 µL of PCR buffer (10 mmol/L Tris-Cl, 50 mmol/L KCl, 1.5 mmol/L
MgCl2, 5% dimethyl sulfoxide) were applied to Gene Amp PCR
system 2400 (Perkin-Elmer Cetus) in 30 cycles. The conditions were
denaturation for 30 seconds at 94°C, annealing for 30 seconds at
61°C for the paired primers of RAR-1A/E2a or at 57°C for other
pairs of primers, and extension for 30 seconds at 72°C, respectively.
The sequences of oligonucleotide primers were as follows: E2 (sense),
5 -GCATCATTAAGACTGTGGAG-3; E4a (antisense), 5-GCGAAGGCAAAGACCAGG-3;
EF1 (sense), 5-AGATTACTGACCTGCGAAGC-3; EF1a (antisense),
5-GTAGAAAGGCAGAGAAAAGC-3; RAR-1A (sense), 5-ATGGCCAGCAACAGCAGCTCCTGCCCGAC-3; PML-C (sense),
5-CCGATGGCTTCGACGAGCTT-3; E2a (antisense),
5-CAGCCCCGTCTCCGCATCAT-3.
SSCP.
For screening the presence of mutation, we used a nonradioisotopic
modification of SSCP as described.36,37 Aliquots (2 µL)
of PCR products were subsequently diluted with an equal volume of 95%
formamide (Sigma, St Louis, MO) denatured at 90°C for 5 minutes and
then electrophoresed in nondenaturing 8% polyacrylamide gel containing
10% glycerol (40 cm × 40 cm × 0.4 mm; Wako Pure Chemical,
Osaka, Japan). Electrophoresis was performed at room temperature with
the power constant at 20 W for 7 hours using 1× TBE. After
electrophoresis, DNA bands were visualized by silver staining (Bio-Rad
Laboratories, Hercules, CA).
DNA sequencing.
Reverse transcription-PCR (RT-PCR) products amplified from
the patients with positive SSCP finding were subcloned into ddT-tailed pBluescript (Strategene, La Jolla, CA), and more than 5 subclones were
selected randomly and sequenced using a T7 sequencing kit (U.S.
Biochemical, Cleveland, OH) by a DNA sequencer (Pharmacia, Piscataway,
NJ) by the dideoxy chain termination method.38
Allele-specific oligonucleotide (ASO) hybridization.
ASO hybridization was used to confirm the presence of mutation and to
distinguish in which gene, either RAR or PML/RAR, the point
mutations were present. DNA fragments were amplified differentially
from either the RAR or PML/RAR gene, transferred onto nylon
membrane after separation by gel electrophoresis with 2% agarose, and
then hybridized with fluorescein-dUTP labeled 20 bp length of
oligonucleotide probes identical to either wild-type or mutant sequence
by using ECL kit and the manufacturer's instruction (Amersham
International, Buckinghamshire, UK). After washing, chemiluminescence
reaction was performed on the membrane, and then autoradiography was
performed at room temperature for 30 minutes. To reduce nonspecific
binding of oligonucleotide probes, an excess amount of nonlabeled
wild-type or mutant oligonucleotide probes was added to the
hybridization mixtures with fluorescein-dUTP labeled either mutant or
wild-type oligonucleotide probes, respectively.
Transient transfection experiments for transcriptional activity.
cDNA clones of mutant PML/RAR with either G815A or A889T mutation
were derived from pCMX expression vector harboring a short form of the
wild-type PML/RAR cDNA using a site-directed mutagenesis kit
(Stratagene, Cambridge, UK). These cDNA clones were then used in
transient transfection experiments for evaluating in vitro transcriptional activity. African green monkey kidney, CV-1 cells, were
grown in monolayer cultures at 37°C under 5% CO2 in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
fetal calf serum, 100 U/mL penicillin, and 100 µg/mL streptomycin.
When CV-1 cells became 80% confluent, they were grown in DMEM with 1%
calf serum treated with resin and charcoal (stripped
medium).39 After 4 hours of incubation, cells were
transfected with expression plasmid (2 µg), R-140 luc reporter
plasmid containing a  RARE sequence40 (1.2 µg), and
CMV--galactosidase control plasmid (pCMV; Clontech, Palo Alto,
CA; 0.8 µg) per 3.5-cm plate using the calcium phosphate
precipitation method.41 Cells then were grown for 36 hours
in the absence or presence of ATRA. Luciferase and -galactosidase
activities of the cell extracts were assayed to correct for
transfection efficiency.42 The corrected luciferase activities of samples were expressed as fold increases of luciferase activity to that of the samples in the absence of ligand (1-fold basal).
| |
RESULTS |
SSCP analysis for EF1 and E2E4 segments.
At first, we screened for mutation in the EF1 segment because this
segment contains the region where mutations were detected in HL-60 or
NB4 subclones with an acquired resistance to ATRA. SSCP analysis for
EF1 segment showed single-strand bands with normal electrophoretic
mobility in all patients (data not shown). These results indicated that
mutation in the EF1 region of either the RAR or the PML/RAR
chimeric gene was unlikely. By contrast, SSCP analysis for the E2E4
segment showed the bands with normal and altered mobility in 2 patients
(Fig 2). Interestingly, these 2 patients
were those who exhibited resistance to ATRA therapy at relapse. Because
this SSCP finding was obtained at relapse in both patients, we examined
whether their blood materials obtained at their initial onset would
have the same SSCP findings. As shown in Fig
3, SSCP findings at the initial onset did
not show altered bands in both patients and were identical to that of
HL-60 without mutation. This indicated that the appearance of altered
bands in SSCP analysis was not a primary, but an acquired event during their clinical course.
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| Fig 2.
A representative result of SSCP analysis of E2E4
segments. In addition to bands with normal mobility, bands with altered
mobility were detected in lanes 3 (case no. 1) and 4 (case no. 2),
which were marked with arrow heads.
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| Fig 3.
SSCP analysis of E2E4 segments amplified from the
patients (cases no. 1 and 2) at the initial onset. Lanes 1 and 4, HL-60 without mutation; lanes 2 and 3, cases no. 1 and 2 at the initial onset, respectively.
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DNA sequence of missense mutations.
PCR products derived from the patients with positive SSCP findings were
subcloned into ddT pBluescript, and DNA sequence was determined in more
than five subclones randomly selected on an LB agarose plate. As shown
in Fig 4, two of five subclones examined in
1 patient (case no. 1) showed a missense mutation of G815A according to
the nucleotide sequence of RAR cDNA, which generated a new
Pst I site. However, the other three subclones had no mutation, suggesting a possibility that these subclones were amplified either from the normal RAR or wild-type PML/RAR gene. Similarly, the presence of missense mutation, A889T, was shown in the other patient (case no. 2) with a positive SSCP finding by DNA sequencing
(data not shown).
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| Fig 4.
DNA sequence of a subcloned E2E4 segment derived from
case no. 1 who acquired ATRA resistance at relapse. The presence of G815A missense mutation was detected as compared with that of normal
RAR/E-domain sequence. The position number of nucleotides was noted
according to the sequence of normal RAR cDNA.
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Mutations present exclusively in PML/RAR chimeric gene.
For determining which gene, either RAR or PML/RAR chimeric gene,
had the mutations, DNA segments were amplified differentially either
from the RAR or the PML/RAR gene and then examined by digesting
with Pst I restriction enzyme or by hybridizing with wild-type
or mutant oligonucleotide probes. In case no. 1 with G815A mutation, an
additional Pst I site was detected in DNA segment amplified
from the PML/RAR gene, but not in that derived from the RAR gene
(Fig 5). These results indicated that G815A
mutation was present exclusively in the RAR/E-domain of PML/RAR
chimeric gene. Similarly, in case no. 2 with A889T mutation, the
oligonucleotide probe containing A889T mutation was exclusively
hybridized to DNA segments amplified from the PML/RAR chimeric gene,
but not to that derived from RAR gene (Fig
6). However, in contrast to case no. 1, case no. 2 had the PML/RAR chimeric gene that could hybridize to
both wild-type and mutant probes, suggesting that two distinct
subclones of APL cells with or without A889T mutation in the
RAR/E-domain of PML/RAR gene were present simultaneously in this
patient at relapse.
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| Fig 5.
Detection of G815A mutation by Pst I digestion in
case no. 1. DNA segments amplified differentially from RAR in lanes
1 through 4 or from PML/RAR chimeric gene in lanes 5 through 8. Lanes 1 and 3, HL-60; lanes 5 and 7, an ATRA-sensitive control patient harboring a short PML/RAR isoform; lanes 2, 4, 6, and 8, case no. 1. PCR products in lanes 2, 4, 6, and 8 were digested with Pst I
before electrophoresis. The Pst I digestion pattern of
RAR-derived segments in case no. 1 (lanes 3 and 4) was similar to
that of HL-60 (lanes 1 and 2). By contrast, the 873-bp segment derived from PML/RAR in case no. 1 was digested with Pst I into
three fragments due to two Pst I sites including one additional
site (lanes 7 and 8), which was not detected in a control patient
without mutation (lanes 5 and 6).
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| Fig 6.
Detection of A889T by ASO hybridization in case no. 2. Using a control sample (ATRA-sensitive APL cells with a short
PML/RAR isoform) (lanes 1 and 3) and case no. 2 (lanes 2 and 4), DNA
segments amplified differentially from RAR (lanes 1 and 2) or
PML/RAR chimeric gene (lanes 3 and 4). (A) Ethidium bromide staining
of agarose gel electrophoresis. (B) The membrane blotted from (A) was
hybridized with oligonucleotide probe specific for the wild-type sequence. (C) The membrane same as (B) was hybridized with
oligonucleotide probe for A889T. The sequence of wild-type probe was
5-CGGACCCAGATGCACAACGC-3, the center adenine of which was replaced
with thymidine in the mutant probe. The mutant probe positively
hybridized only to the DNA segment that was amplified from PML/RAR
chimeric gene of case no. 2 (lane 4).
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Ligand-dependent transcriptional activity.
As shown in Fig 7,
ligand-dependent transcriptional activity of the wild-type PML/RAR
chimeric protein was increased in a manner dependent on ATRA
concentrations. By contrast, the mutant PML/RAR chimeric proteins
harboring either G815A or A889T mutation showed no increase of
luciferase activity in the presence of ATRA as compared with that of
wild-type PML/RAR with significant difference.
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| Fig 7.
Ligand-dependent transcriptional activity of wild-type or
mutant PML/RAR chimeric protein on RARE. Luciferase activity of samples was normalized to -galactosidase activity and then
calculated as fold increases of luciferase activity to that of samples
in the absence of ATRA. Each point represents the mean of triplicated results, and bars denote SD. *Significant difference as compared with
the luciferase activities of wild-type PML/RAR in the presence of
the corresponding concentrations of ATRA.
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Summary of the patients with missense mutations.
Similarly and interestingly, as shown in Table
2, both patients with missense mutations
expressed a short PML/RAR isoform and had received a prolonged ATRA
therapy before relapse with ATRA-resistance. Case no. 1, a 10-year-old
boy, could not continue to receive chemotherapy because of liver
abscess after chemotherapy for remission induction, and, thereafter, he
had been undergoing ATRA therapy alone for 7 months until he relapsed
with ATRA-resistance. Case no. 2, a 10-year-old boy, was successfully
induced into complete remission by differentiation-induction therapy
with ATRA and was then treated with an alternative regimen consisting
of an intensive chemotherapy and an intermittent administration of ATRA
(45 mg/m2 of ATRA for 2 weeks in every 2 months) for 11 months before relapse with ATRA-resistance. These 2 patients, cases no.
1 and 2, showed missense mutation, G815A or A889T, in the
RAR/E-domain of the chimeric gene, which lead to amino acid
replacement, Arg272Gln or Met297Leu, according to the amino acid
sequence of RAR protein, respectively.
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Table 2.
Profile of the Patients With an Acquired ATRA-Resistance
Who Had Missense Mutations in the RAR/E-Domain of the PML/RAR
Chimeric Gene
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DISCUSSION |
In this study, we demonstrated the presence of point mutations in the
E-domain of the RAR portion of PML/RAR chimeric gene in APL with
ATRA-resistance, the characteristics of which were the following.
First, the mutations were detected exclusively in patients who
exhibited an acquired ATRA-resistance, but not in patients who
responded to ATRA therapy or exhibited poor response to the initial
ATRA therapy. Second, the mutations were detected at the relapse with
ATRA-resistance, but not at the initial onset. Third, the mutations
were present in the E-domain of the RAR portion of PML/RAR
chimeric gene, but not in the normal RAR gene. These findings
clearly indicated that the appearance of APL subclone with these
mutations was not a primary, but an acquired event, and is closely
related to the relapse of APL patients exhibiting an acquired
resistance to ATRA therapy.
A continuous intake of pharmacological doses of ATRA may induce
alteration(s) in the systemic response of ATRA metabolism, which may
result in the lowering plasma ATRA levels by a rapid clearance of ATRA
from the plasma.23,43 With a prolonged 7-month period of
ATRA administration in case no. 1, it was likely that the patient's
plasma concentrations of ATRA at relapse might be lower than that
required for in vitro cytodifferentiation of APL cells. However, the
treatment protocol applied for case no. 2 has been demonstrated to
prevent a progressive reduction of the plasma concentration of ATRA by
the combination of an intermittent administration of ATRA with
multidrug-combined chemotherapy alternatively.43 More
importantly, APL cells of most patients with a clinical resistance to
ATRA are either resistant or less sensitive to cytodifferentiation induced by ATRA in vitro.19,27 Thus, although we did not
measure the plasma levels of ATRA in our patients, the intracellular
alteration(s) of APL cells should be thought more significant as the
mechanism(s) that causes an acquired ATRA-resistance in patients with
APL.20
In APL cells, the intracellular ATRA can be oxidized to inactive
metabolites by cytochrome P-450 isoform(s), and this process may be
facilitated by CRABPs, which can be induced with an intake of
pharmacological doses of ATRA.20,24 Alternatively, ATRA can
be inactivated by the oxidative interaction with lipid
hydroperoxides.20,26 Delva et al27 have
reported an increased expression of CRABP-II at the time of relapse of
APL as compared with the levels before ATRA therapy, suggesting an
involvement of CRABP-mediated metabolism in the mechanism(s) of an
acquired resistance to ATRA. By contrast, Kizaki et al28
reported more recently that the levels of cytochrome P-450 activities
were not significantly different between wild-type and ATRA-resistant
HL-60 cells and, more interestingly, that multidrug-resistance-1 gene
was expressed in ATRA-resistant APL cells, as compared with ATRA-sensitive cells. Although these findings obviously indicate that
an altered intracellular metabolism(s) could underlie an acquired
ATRA-resistance by APL cells, it remains to be answered whether
alteration(s) in intracellular metabolism of ATRA would be
quantitatively sufficient to prevent pharmacological doses of ATRA from
reaching into the nucleus as an unbound form.20
Another possible mechanism is an acquired molecular alteration of the
nuclear retinoid receptors of APL cells, which may impair an
interaction of ligand to retinoid receptors. This possibility has been
suggested by previous studies using ATRA-resistant APL cell lines that
were developed by growing cells in the continuous presence of ATRA in
vitro. HL-60 subclones with an acquired ATRA-resistance showed either
missense or nonsense point mutations in the middle region or in the
carboxyl-terminal of the RAR/E-domain,
respectively.29-31 A truncated RAR protein caused by the
latter nonsense mutation lacks 52 amino acids of the carboxyl end of
the RAR protein, leading to a loss of transcription
activity.29 More recently, Shao et al32 have
reported an ATRA-resistant subclone of NB4 with a missense point
mutation in the carboxyl-terminal of the RAR/E-domain in the
PML/RAR chimeric gene, but not of the normal RAR gene.
Interestingly, this mutation is located close to the nonsense mutation
of the HL-60 subclone with truncated RAR and impairs the
ATRA-binding activity of the mutated PML/RAR fusion protein.32
The mutations detected in our study were missense point mutations of
G815A or A889T according to the sequence of RAR cDNA, respectively,
which lead to amino acid replacement of Arg272Gln or Met297Leu using
the amino acid sequence of RAR protein, respectively. These
mutations, which are close to each other (74 bp apart) in the middle
region of the E-domain, make a marked contrast to the location of
mutations detected in ATRA-resistant HL-60 or NB4 subclones, the
carboxyl-terminal of the E-domain. Conversely, the mutations of our
patients are localized close to the other missense mutation detected in
another subclone of ATRA-resistant HL-60.31 Therefore, the
collection of these mutations of our patients and ATRA-resistant APL
cell lines may imply the presence of two particular regions, either the
middle or the carboxyl-terminal of the E-domain, where mutations may
cluster in ATRA-resistant APL cells (Fig
8). Because the amino acid sequence of the
RAR/E-domain is evolutionary conserved,44 any missense
mutations may alter the essential function of the E-domain, which could
be the case demonstrated in our patients exhibiting an acquired
resistance to ATRA.
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| Fig 8.
A comparison between TR- gene with mutations in RTH
and RAR or PML/RAR genes on the alignment of sequence homology.
The mutations in TR- gene of RTH were clustering almost exclusively in the two regions, I and II, of the hormone binding
domain.46 The mutations in the RAR/E-domain of
ATRA-resistant APL were indicated as follows: 1, case no. 1; 2, case
no. 2; 3 and 5, ATRA-resistant HL-60 subclones29-31; and 4, a ATRA-resistant NB4 subclone.32
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The ligand-dependent transcriptional activity of the PML/RAR
chimeric protein is shown in vitro to be increased in a manner similar
to that of normal RAR, the E-domain of which functions for
ligand-binding and receptor-dimerization.45 With transient transfection analysis in vitro, we demonstrated that the mutant PML/RAR lacked the activity of ligand-dependent transcription in
vitro, suggesting that the change of the amino acid, Arg272Gln or
Met297Leu, of RAR portion in the chimeric protein may impair these
functions of PML/RAR.
An analogy between APL with an acquired ATRA-resistance and an
inherited disorder with resistance to thyroid hormone (RTH) may give us
important clues for understanding the function of the mutations shown
by this study. RTH is an inherited disorder characterized with an
impaired TH binding to thyroid hormone receptor- (TR-), a member
of steroid/thyroid hormone receptor family with a homology to RAR
gene.46 RTH is primarily caused by inherited mutations of
TR- gene, which cluster almost exclusively in the two regions of the
ligand-binding domain of TR- (Fig 8).46,47 These
mutations biochemically inactivate the ligand-binding of the receptor,
while preserving other functions, such as receptor-dimerization or
DNA-binding, that are indispensable to the dominant negative action of
the mutant receptor.46 Interestingly, in the alignment of
TR- ligand-binding domain and the RAR/E-domain on sequence homology, the mutations in ATRA-resistant patients and APL cell lines
may cluster in accordance with the regions in RTH, denoted as I or II
in Fig 8, respectively. Therefore, it is suggested that these two
regions may play roles indispensable to the function of ATRA-binding,
but not receptor-dimerization, of the PML/RAR chimeric protein.
The comparative analysis of the crystal structure of RAR with and
without bound ligand may be a further help to the understanding of how
these cluster regions with mutations would play an essential role in
the process of ligand-binding, despite their dispositions at a distance
apart each other. After the electrostatic attraction of ATRA to the
ligand-binding pocket of -helical structures formed by the middle
part of RAR/E-domain, ATRA is held into the cavity by the reposition
of -helical structures of the carboxyl-terminal of RAR protein,
which is caused by the conformation change associated with the ligand
proceeding into the binding cavity.48 Importantly, in this
mouse trap mechanism of RAR for ligand-binding, these two
-helical structures are consistent to the two mutation cluster regions in the E-domain of RAR, which we have found in APL cells with
an acquired ATRA-resistance. More specifically, arginine at position
272 of RAR (Arg274 of RAR), which was replaced with glutamine in
case no. 1, is important in making van der Waals contact with the
carbon molecule of the acyl chain of ATRA.48 Furthermore,
the site-directed mutagenesis at Arg272 of RAR has been shown to
impair the binding of ATRA49; therefore, Arg272Gln mutation
may inhibit the ligand-binding function of PML/RAR chimeric protein.
However, the role of Met297 of RAR/E-domain of the chimeric protein
remains to be investigated.
For understanding how APL subclones with these mutations could expand
during clinical course, it may be of note that, in case no. 2, APL
subclones with either mutant or wild-type RAR/E-domain of the
chimeric gene coexisted at relapse, while the chimeric gene of APL
cells in case no. 1 was totally mutated at relapse. Thus, a biological
pressure of ATRA therapy for selecting ATRA-resistant APL subclones may
be increased as the period of ATRA administration is prolonged, because
APL subclones with the mutations could have an advantage in surviving
or proliferating under an environment with pharmacological
concentrations of ATRA, as compared with that of ATRA-sensitive APL
clone.6 However, the mechanism(s) inducing mutations in the
RAR/E-domain of PML/RAR chimeric gene remains unknown.
Further study is needed to clarify the clinical significance of these
mutations in larger populations of patients with APL. Moreover, it
remains to be answered how these mutations could impair the functions
of the RAR/E-domain in molecular levels. The answers to these
questions would be of aid in establishing therapeutic approach to
diminish the acquired resistance to ATRA, but also for understanding
ATRA-induced cytodifferentiation of APL at the molecular level.
| |
FOOTNOTES |
Submitted January 27, 1998;
accepted April 22, 1998.
Supported by grants from the Ministry of Education, Science and
Culture, and the Ministry of Health and Public Welfare, Japan.
Address reprint requests to Masue Imaizumi, MD, Department of
Pediatrics, Tohoku University School of Medicine, 1-1 Seiryo-machi Aoba-ku, Sendai 980-8574, Japan; e-mail:
mimaizumi{at}ped.med.tohoku.ac.jp.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors are grateful to Dr T.R. Breitman for his thoughtful
suggestion and Dr S. Kure for his insightful advice. We also thank the
following doctors for kindly providing us with the blood materials: Drs
K. Endo, K. Meguro, and Y. Saito (Internal Medicine, Tohoku University
School of Medicine); Drs N. Takano and M. Endo (Department of
Pediatrics, Iwate Medical University); Drs S. Yokoyama and
S. Sato (Department of Pediatrics and Third Internal Medicine, Yamagata
University); Drs E. Tamate and T. Sugawara (Department of Internal
Medicine, Furukawa City Hospital); Dr T. Kawakami (Department of
Pediatrics, Tottori University); Dr K. Asami (Department of Pediatrics,
Niigata Cancer Center); and Dr S. Sibuya (Department of Pediatrics,
Kanazawa Medical University).
| |
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[Abstract]
[Full Text]
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R.J. LIN, H.-Y. KAO, P. ORDENTLICH, and R.M. EVANS
The Transcriptional Basis of Steroid Physiology
Cold Spring Harb Symp Quant Biol,
January 1, 1998;
63(0):
577 - 586.
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Abstract
Introduction
Abstract