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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2452-2458
Restoration of Retinoid Sensitivity by MDR1 Ribozymes in Retinoic
Acid-Resistant Myeloid Leukemic Cells
By
Hiromichi Matsushita,
Masahiro Kizaki,
Hiroyuki Kobayashi,
Hironori Ueno,
Akihiro Muto,
Nobuyuki Takayama,
Norihiro Awaya,
Kentaro Kinjo,
Yutaka Hattori, and
Yasuo Ikeda
From the Division of Hematology, Keio University School of Medicine,
Tokyo; and the Department of Laboratory Medicine, National Defense
Medical College, Saitama, Japan.
 |
ABSTRACT |
Complete remission is achieved in a high proportion of patients with
acute promyelocytic leukemia (APL) after all-trans retinoic acid (RA) treatment, but most patients relapse and develop RA-resistant APL. We have previously reported that both RA-resistant HL-60 (HL-60R)
and APL cells express P-glycoprotein and MDR1 transcripts; and these
cells differentiate to mature granulocytes after culture with RA and
P-glycoprotein antagonist. Ribozymes have been shown to be able to
intercept a target RNA by catalytic activity. To address the role of
MDR1 in overcoming RA-resistance in APL cells, we investigated the
biologic effects of ribozymes against the MDR1 transcript in HL-60R
cells. These ribozymes efficiently cleaved MDR1 mRNA at a specific site
in vitro. The 196 MDR1 ribozyme was cloned into an expression vector,
and stably transfected (HL-60R/196Rz) cells were obtained. Expression
of MDR1 transcripts was decreased in HL-60R/196Rz cells compared with
parental HL-60R and empty vector-transfected (HL-60R/neo) cells.
Interestingly, RA inhibited cellular proliferation and induced
differentiation of HL-60R/196Rz cells in a dose-dependent manner,
suggesting reversal of drug resistance in HL-60R cells by the MDR1
ribozyme. These data are direct evidence that P-glycoprotein/MDR1 is
responsible in part for acquired resistance to RA in myeloid leukemic
cells. The MDR1 ribozyme may be a useful tool for investigating the
biology of retinoid resistance and may have therapeutic potential for
patients with RA-resistant APL.
| |
INTRODUCTION |
RETINOIC ACID (RA) has a critical role in
proliferation and differentiation of a wide variety of cell types,
especially hematopoietic cells.1,2 All-trans RA and
its stereoisomer, 9-cis RA, induce differentiation and inhibit
proliferation of HL-60 cells and fresh leukemic cells from patients
with acute promyelocytic leukemia (APL) in vitro.3-5 Recent
clinical studies have shown that a high proportion of patients with APL
achieve complete remission after treatment with all-trans
RA.6-9 However, most patients who receive continuous
treatment with RA relapse and develop RA-resistant disease.10 Although the mechanisms for development of
RA-resistance in APL are unclear, several hypotheses are possible. We
have previously reported that RA-resistant HL-60 (HL-60R) and
RA-resistant APL cells express P-glycoprotein and MDR1 transcripts
unlike wild-type HL-60 and fresh APL cells.11 RA-resistant
cells differentiate to mature granulocytes after culture with
all-trans RA combined with verapamil, a P-glycoprotein
antagonist, but not with either agent alone.11 Resistance
to multiple chemotherapeutic agents is related to the expression of
P-glycoprotein, a transmembrane drug efflux pump that is encoded by the
MDR1 gene.12,13 Therefore, we speculated that
P-glycoprotein may be important in the development of RA-resistance in
myeloid leukemic cells.
Studies have shown that ribozymes are able to target RNA by catalytic
activity in a specific manner.14-16 The hammerhead ribozyme can be engineered to cleave any triplet of NUX (N = any nucleotide, X = A, C, or U), by changing the flanking sequence.16 We have designed two hammerhead ribozymes targeted against the MDR1 transcript, 179 MDR1 ribozyme and 196 MDR1 ribozyme, which cause recovery of drug
sensitivity in multidrug-resistant tumor cells.17,18 Ribozyme-mediated inactivation of MDR1 mRNA may overcome RA-resistance in myeloid leukemic cells. Therefore, we investigated the biologic effects of ribozymes against MDR1 transcript in RA-resistant HL-60 cells.
| |
MATERIALS AND METHODS |
Cells and chemicals.
Wild-type and RA-resistant HL-60 cells (HL-60R: a generous gift from Dr
R.E. Gallagher, Montefiore Medical Center, Bronx, NY), were maintained
in RPMI 1640 medium (GIBCO-BRL, Gaithersburg, MD) with 10% fetal
bovine serum (Cytosystems, New South Wales, Australia), 100 U/mL
penicillin, and 100 µg/mL streptomycin in a humidified atomosphere
with 5% CO2. All-trans RA and 9-cis RA were purchased from Sigma Chemical Co (St Louis, MO) and Wako Pure
Chemical Industries Ltd (Tokyo, Japan), respectively. Both were
dissolved in 100% ethanol to a stock concentration of 1 mmol/L, stored
at  20°C, and protected from light. All-trans RA was
added to the maintenance culture medium of HL-60R cells.19
The morphology of differentiated-cells was evaluated from cytospin
slide preparations stained with Giemsa.
Synthesis of MDR1 ribozymes.
Production of the 179 and 196 MDR1 ribozymes and its RNA substrate are
described elsewhere.17,18 Briefly, the 196 MDR1 ribozyme
(Fig 1) was produced from two synthetic
oligonucleotides, 5 -TCT TTC AGT TTC GTC CTC ACG GAC TCA TCA GAA
TGG CAA CCC CTA TAG TGA GTC GTA TTA CAT G-3, and 5-CAT
GTA ATA CGA CTC ACT ATA GGG-3. The oligonucleotides were mixed
and heated at 80°C for 2 minutes and slowly cooled to room
temperature to form a hemiduplex. They were incubated at 37°C for 4 hours with 5 U/µL T7 RNA polymerase (New England Biolabs, Beverly,
MA), 1.2 U/µL RNase inhibitor (Promega, Madison, WI), and 2 mmol/L
each adenosine triphosphate (ATP), guanosine triphosphate (GTP),
cytidine triphosphate (CTP), and uridine triphosphate
(UTP). After incubation with 0.1 U/µL RQ1 RNase-free
DNase (Promega) for 15 minutes, ribozyme was extracted, and the pellet
was dissolved with diethyl pyrocarbonate (DEPC)-treated water.
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| Fig 1.
Hammerhead ribozyme targeted against MDR1 RNA. 196 MDR1
ribozyme (196Rz) cleaved MDR1 transcript at codon 196, as indicated by
the arrow. Codon 196 is one of the substrate cleavage sites.
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Ribonuclease protection assay.
To address the cleavage activity of MDR1 ribozymes, MDR1 mRNA was
analyzed by ribonuclease protection assay RPAII (Ambion, Austin, TX)
using total cellular RNA extracted from MOLT-3/trimetrexate (TMQ) 800 cells.20 After cDNA was synthesized
from this RNA with reverse transcription (RT), exons 6 (5-TTC
ATG CTA TAA TGC GAC AGG AGA TA-3) through 8 (5-TTC TTT
ATC AGT AAA TGA AGA TAG TA-3) of the MDR1 gene was amplified by
polymerase chain reaction (PCR). The [ -32P]CTP-labeled
MDR1 RNA probe used in this assay was synthesized from the opposite
orientation of the PCR product of MDR1 in pT7Blue T-vector by T3 RNA
polymerase.
196 MDR1 ribozyme transfection in HL-60R cells.
The cDNA of ribozyme with Sal I and HindIII restriction
sites on both ends was cloned into the expression vector pH APr-1-neo (kindly provided by Dr L. Kedes, University of Southern California, Los
Angeles, CA) (Fig 2).21 HL-60R
cells were transfected with plasmid containing 196 MDR1 ribozyme or an
empty vector by electroporation using an Electroporator II (Invitrogen,
San Diego, CA) at 350 V, 960 µF. A pulse was delivered to a 0.5-mL
suspension containing 5 × 106 cells in RPMI 1640 without fetal bovine serum and 20 µg of plasmid DNA. After an
additional 10 minutes incubation at room temperature, cells were
diluted with prewarmed RPMI 1640 with 10% fetal bovine serum. Three
days after transfection, cells were selected by culture with G418 (1 mg/mL; GIBCO-BRL). Selective medium was replaced every 5 days, and
stably transfected polyclonal cell populations were isolated after 4 weeks of selection with G418.
Reverse transcriptase (RT)-PCR assay for MDR1
transcript.
Expression of the MDR1 gene was investigated by the RT-PCR method. The
RT reaction was performed using 1 µg of total cellular RNA, 100 pmol
of random hexamer (Boehringer-Mannheim, Indianapolis, IN), 10 U RNase
inhibitor (Promega), 200 U moloney murine leukemia virus-RT
(GIBCO-BRL), and deoxynucleotides (final concentration, 0.5 mmol/L
each; Pharmacia, Tokyo, Japan). After cDNA synthesis, 30 cycles of PCR
were sequentially performed using MDR1 and -actin as follows: for
MDR1, sense, 5-ATG TTG AGC CGG GCA GTG TGC-3 and
antisense, 5-CTG AAG AGC TGT CTG GGC TGT-3,12
and for -actin, sense, 5-ATG GAT GAT GAT ATC GCC GCG-3
and antisense, 5-AAA GAA CAC GGC TAA GTG
TGC-3.22 PCR was performed using denaturing steps at
95°C for 1 minute, annealing steps at 55°C for 1 minute, and
extending steps at 72°C for 2 minutes. At the end of 30 cycles,
further extension for 7 minutes at 72°C was included. All reactions
were performed in a total volume of 50 µL containing 1 µL of cDNA
(of 10 µL total RT reaction), 1 mmol/L of each primer, 200 mmol/L of
each deoxynucleotide, 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 2.5 mmol/L MgCl2, and 0.5 U Taq DNA polymerase (Perkin
Elmer-Cetus, Norwalk, CT). PCR products were electrophoresed on a 2%
NuSieve/1% Seakem agarose (FMC, Rockland, MI) gel and visualized by
staining with ethidium bromide. The specificity of PCR amplification
was also examined by Southern blotting of the amplified cDNA with
hybridization to nonradioactive probes (ECL 3-oligolabeling and
detection system; Amersham Japan, Tokyo, Japan).
Assays for cellular proliferation.
Cellular proliferation was measured by cell viability and a
nonradioactive cell proliferation assay system (MTT assay)
according to the manufacturer's specifications (Boehringer-Mannheim).
Flow cytometric analysis.
For analysis of cellular differentiation, expression of cell surface
antigens was measured by direct immunofluorescence staining technique.
Cells were incubated for 30 minutes with human AB serum (Sigma) to
block Fc receptors and then were stained with phycoerythrin (PE)-conjugated mouse antihuman CD11b antibodies
(Becton-Dickinson, Mountain View, CA). For P-glycoprotein detection,
cells were stained with monoclonal antibody MRK16 (Kyowa Medex Co Ltd,
Tokyo, Japan), followed by staining with goat antimouse antibody
conjugated to fluorescein isothiocyanate (FITC). Control studies were
performed with nonbinding control mouse IgG isotype antibody
(Becton-Dickinson). Cells were analyzed by a Cytoron Absolute Flow
Cytometer (Ortho Diagnostic Systems, Raritan, NJ).
| |
RESULTS |
Cleavage of MDR1 mRNA by ribozymes.
We have designed two hammerhead ribozymes to cleave the GUC sequence at
codons 179 and 196 of MDR1 mRNA.17,18 We already reported
that the ribozymes efficiently cleaved target RNA substrates in a
cell-free system.18 Furthermore, we analyzed the cleavage activity of ribozymes in mRNA extracted from MOLT-3/TMQ 800 cells using
ribonuclease protection assay (Fig 3). The
196 MDR1 ribozyme cleaved MDR1 mRNA into two fragments of 124 bases of
5 fragment (5F) and 133 bases of 3 fragment
(3F) by using 32P-labeled MDR1 RNA probe (Fig 3).
The 196 MDR1 ribozyme was more active than the 179 MDR1 ribozyme (Fig
3); thus, we used the 196 MDR1 ribozyme in this study.
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| Fig 3.
Cleavage reaction of MDR1 mRNA with ribozymes.
Ribonuclease protection assay was performed to study for the cleavage
of MDR1 mRNA by the ribozyme expressed in MOLT-3/TMQ 800 cells. Lane 1, probe only; lane 2, probe hybridized with yeast tRNA, digested with
ribonucleases; lane 3, protected fragment of MDR1 mRNA; lane 4, 1 µg
of mRNA extracted from MOLT-3/TMQ 800 cells plus 179 MDR1 ribozyme;
lane 5, 1 µg of mRNA extracted from MOLT-3/TMQ 800 cells plus 196 MDR1 ribozyme.
|
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Expression of P-glycoprotein and MDR1 mRNA in ribozyme-transfected
HL-60R cells.
The 196 MDR1 ribozyme was cloned into the mammalian expression vector
(pHAPr-1-neo) (Fig 2) and transfected into HL-60R cells by
electroporation. After G418 selection, stably transfected polyclonal cells were obtained (HL-60R/neo and HL-60R/196Rz cells). Morphologic changes did not occur in HL-60R/neo and HL-60R/196Rz cells and a
similar phenotype was observed in all transfected cells; and these
cells were resistant to 1 mg/mL G418, and their growth rate was similar
to parent HL-60R cells (data not shown). Expression of P-glycoprotein
was low in parental HL-60R, HL-60R/neo and HL-60R/196Rz cells (data not
shown). Therefore, to test the ability of the 196 MDR1 ribozyme to
cleave MDR1 mRNA in HL-60R/196Rz cells, we investigated the expression
of MDR1 transcripts in both parental and ribozyme-transfected HL-60R
cells by the RT-PCR technique. The expression of MDR1 transcripts
decreased in HL-60R/196Rz cells compared with parental HL-60R and
HL-60R/neo cells, and this was confirmed by subsequent Southern
blotting of the RT-PCR products using a probe specific for the MDR1
gene (Fig 4). -actin was amplified
simultaneously and showed comparable amounts of RNA in the cells. The
disabled ribozyme is considered to be an appropriate control to
determine the possible effects of functional ribozymes. However, we
have already shown that a disabled 196 MDR1 ribozyme was neither
capable of specific cleavage in vitro nor of decreasing the MDR1
transcripts in MOLT-3/TMQ 800 cells.18 These data therefore suggest that the 196 MDR1 ribozyme specifically inhibits the expression of the MDR1 transcripts in HL-60R cells.
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| Fig 4.
Expression of MDR1 transcript in parental HL-60R,
HL-60R/neo, and HL-60R/196Rz cells by RT-PCR analysis and subsequent
Southern blotting. 196 MDR1 ribozyme was cloned into the expression
vector, pHAPr-1-neo and transfected into HL-60R cells by
electroporation (HL-60R/196Rz). Control clone was derived by
transfection of empty pHAPr-1-neo alone (HL-60R/neo). A total of 1 µg of total RNA was reverse transcribed to cDNA and amplified by PCR
with primers specific for MDR1 and -actin as an internal control;
the amplification products were electrophoresed, transferred, and
hybridized with probes specific for MDR1 (upper bands) and -actin
(lower bands) genes. Lane 1, HL-60R cells; lane 2, HL-60R/neo; lane 3, HL-60R/196Rz cells.
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Effects of ribozyme on cellular proliferation of HL-60R cells.
Cell growth was assessed by absorbance in the MTT cellular
proliferation assay system. We have already reported that neither all-trans RA nor its stereoisomer, 9-cis RA, inhibited
proliferation or induced differentiation of HL-60R cells into either
mature granulocytes or monocytes.11,23 Parental HL-60R,
HL-60R/neo, and HL-60R/196Rz cells were cultured for 4 days in the
presence of various concentrations of retinoids (10-10 to
10-6 mol/L) (Fig 5).
All-trans RA did not affect the number of viable cells or the
absorbance of MTT by parental HL-60R and HL-60R/neo cells (Fig 5 and
data not shown). However, all-trans RA and 9-cis RA
inhibited cellular proliferation in HL-60R/196Rz cells in a dose-dependent manner (10-10 to 10-6 mol/L),
suggesting reversal of drug resistance in HL-60R cells by 196 MDR1
ribozyme. 9-cis RA showed slightly more inhibition of the
growth of HL-60R/196Rz cells.
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| Fig 5.
Effects of retinoids on proliferation of parental HL-60R,
HL-60R/neo, and HL-60R/196Rz cells. Cells (2 × 103) were
incubated in 96-well plates with various concentrations (10 10 to 106 mol/L) of all-trans
RA or 9-cis RA for 4 days, and MTT incorporation was then
measured. The absorbance at 570 nm (optical density [OD] 570) was recorded using an enzyme-linked immunosorbent assay plate reader. Results are expressed as percent of control absorbance of three
experiments, and standard deviation (SD) was within 10% of the mean.
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Effects of ribozyme on differentiation of HL-60R cells.
Induction of differentiation of parental HL-60R, HL-60R/neo, and
HL-60R/196Rz cells into mature granulocytes by retinoids was assessed
by morphology (Fig 6) and expression of
CD11b antigen (Fig 7). Morphologically,
exposure of wild-type HL-60 cells to either 10-7 mol/L
all-trans RA or 9-cis RA for 4 days resulted in
differentiation towards mature granulocytes (Fig 6) and increased
expression of CD11b (Fig 7). However, retinoid treatment did not change
the morphology or expression of CD11b antigen in parental HL-60R and HL-60R/neo cells (Figs 6 and 7). In contrast, expression of CD11b antigen in HL-60R/196Rz cells was increased by 10-7 mol/L
all-trans RA or 9-cis RA (Fig 7). Incubation with
either retinoid resulted in differentiation of HL-60R/196Rz cells as evidenced by nuclear maturation and lobulation (Fig 6), suggesting that
retinoids could partially differentiate HL-60R/196Rz cells, but not
parental HL-60R and HL-60R/neo cells, toward granulocytes. 9-cis RA was slightly more potent than all-trans RA in
inducing differentiation of HL-60R/196Rz cells.
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| Fig 6.
Morphologic changes induced in wild-type HL-60, parental
HL-60R, HL-60R/neo, and HL-60R/196Rz cells by either all-trans
RA or 9-cis RA. Cells were treated with 107
mol/L of either retinoid for 4 days, and cytospin slides were then
prepared and stained with Giemsa. Original magnification × 1,000.
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| Fig 7.
Expression of CD11b antigen in wild-type HL-60, parental
HL-60R, HL-60R/neo, and HL-60R/196Rz cells by fluorescence-activated cell sorting (FACS) analysis. Cells were cultured with
10-7 mol/L of either all-trans RA or 9-cis
RA for 4 days. Cells were incubated for 30 minutes with human AB serum
to block Fc receptors, and then stained with PE-conjugated mouse
antihuman CD11b antibody. Control studies were performed with control
mouse IgG isotype antibody.
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DISCUSSION |
All-trans RA is now widely used in the treatment of patients
with APL as a differentiation-inducing therapy.6-9 Although a high proportion of patients with APL achieve complete remission with
RA, a minority of patients are primarily resistant to retinoids, and
relapsed patients frequently develop RA-resistant
disease.10 Relapse and acquired resistance to treatment
with all-trans RA have been hypothesized to result from
alteration of drug metabolism and increased expression of proteins
related to retinoid metabolism.24,25 HL-60 and APL cells
have different genetic changes. HL-60 cells do not have an APL-specific
15;17 translocation resulting in a PML/RAR chimeric gene, however,
both cells can be induced to differentiate into mature granulocytes by
all-trans RA. Therefore, HL-60 cells can provide a model to
help study the mechanisms of induction of differentiation in response
to retinoids. We have previously reported that RA-resistant HL-60
(HL-60R) and APL cells, but not RA-sensitive cells, express MDR1 gene
transcripts and P-glycoprotein.11 Moreover, the combination
of retinoids and a P-glycoprotein antagonist, verapamil, but neither of
them alone, induced morphologic and functional differentiation of
RA-resistant leukemic cells.11 P-glycoprotein is an
energy-dependent drug efflux pump that decreases intracellular
accumulation of various lipophilic compounds12; we
speculated that retinoid, a fat soluble vitamin, may be a substrate for
P-glycoprotein. Recent studies have shown significantly lower
P-glycoprotein expression in APL than in other types of AML.26,27 Therefore, a metabolic pathway for retinoids may exist that depends on P-glycoprotein expression; P-glycoprotein induction may be responsible in part for retinoid resistance in myeloid
leukemic cells.
Ribozymes inhibit the expression of a variety of genes and may be a
novel therapeutic approach in infectious disease and
cancer.28,29 Ribozymes appear to act in an antisense
fashion and as catalytic enzymes that cleave their
substrate.15,16 One molecule of ribozyme can cleave many
molecules of target RNA, then dissociate from the products, bind to a
new target molecule, and inactivate it. Thus, ribozymes are thought to
be more potent than antisense oligonucleotides at suppressing the
expression of target genes.30 Several studies have used
ribozymes to specifically target the bcr-abl, PML/RAR, and
AML/MTG8 fusion genes in leukemic cells.31-35 These
studies have shown that ribozymes efficiently cleaved target
RNA.36
Because the MDR1 product seems to be responsible in part for retinoid
resistance in RA-resistant myeloid leukemic cells,11 we
designed hammerhead ribozymes that can cleave the GUC sequence in MDR1
mRNA. In our study, the 196 MDR1 ribozyme produced more efficient
cleavage than the 179 MDR1 ribozyme, thus, we used the 196 MDR1
ribozyme in this study. We showed that ribozyme against MDR1 could
cleave the substrate RNA extracted from MOLT-3/TMQ 800 cells using
ribonuclease protection assay . More importantly, after transfection
into RA-resistant HL-60 cells, the ribozyme inhibited expression of
MDR1 transcripts, partially inhibited proliferation, and induced
differentiation of these cells. The data described in our study
suggests that ribozyme-mediated inactivation of the MDR1 gene will
partially overcome the block of leukemic cell differentiation in HL-60R
cells and provide direct evidence that the MDR1 gene is responsible for
acquired resistance to retinoids in myeloid leukemic cells.
The partial response to retinoids after inhibiting MDR1 transcription
indicates a complex mechanism of RA-resistance in these cells. In
addition, pharmacologic levels of retinoids were required to overcome
RA-resistance even when MDR1 gene was targeted by the ribozyme,
suggesting the other defect existed in HL-60R cells. Several mechanisms
have been proposed to explain resistance to retinoids in leukemic
cells,25 including molecular alterations of the RA receptor
(RAR) gene,37,38 and pharmacologic alterations in the
metabolism of retinoids.24,25 Based on these studies, several strategies may overcome RA resistance in APL patients. To date,
however, most approaches have not yet been successful in overcoming
retinoid resistance in vivo.25 Therefore, further studies
are needed to clarify the detailed molecular mechanisms of
retinoid-resistance in APL; the combination of retinoids and 196 MDR1
ribozyme is one possible strategy to cure patients who fail the initial
differentiation-inducing therapy with all-trans RA.
In summary, retinoid resistance is a serious and important clinical
problem of differentiation-inducing therapy for the patients with APL.
However, there is redundancy and complexity of the biologic mechanisms
of RA-resistance in vivo. Our data presented in this study have direct
evidence that the MDR1 gene is responsible in development of
RA-resistance in myeloid leukemic cells. In addition, inhibition of
MDR1 transcripts by ribozymes restored the sensitivity to RA. Taken
together, we conclude that this technology may be useful as a new
therapy in the treatment of patients with RA-resistant APL and other
cancers.
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FOOTNOTES |
Submitted February 27, 1997;
accepted November 14, 1997.
Supported by grants from the Ministry of Education, Science, and
Culture in Japan, the National Grant-in-Aid for the Establishment of
High-Tech Research Center in a Private University, and the Keio
University Special Grants.
Address reprint requests to Masahiro Kizaki, MD, Division of
Hematology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan.
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 |
We thank Dr T. Ohnuma, Mount Sinai School of Medicine, New York, NY for
providing MOLT-3/TMQ 800 cells.
| |
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Abstract
Introduction
Abstract
