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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 2969-2976
Wilms' Tumor Gene (WT1) Competes With Differentiation-Inducing
Signal in Hematopoietic Progenitor Cells
By
Kazushi Inoue,
Hiroya Tamaki,
Hiroyasu Ogawa,
Yoshihiro Oka,
Toshihiro Soma,
Toyoshi Tatekawa,
Yusuke Oji,
Akihiro Tsuboi,
Eui
Ho Kim,
Manabu Kawakami,
Tetsu Akiyama,
Tadamitsu Kishimoto, and
Haruo Sugiyama
From the Departments of Medicine III and of Clinical Laboratory
Science, Osaka University Medical School, Osaka, Japan; and The
Research Institute for Microbial Diseases, Osaka University, Osaka,
Japan.
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ABSTRACT |
The WT1 gene is a tumor-suppressor gene that was isolated as a gene
responsible for Wilms' tumor, a childhood kidney neoplasm. We have
previously reported that the WT1 gene is strongly expressed in leukemia
cells with an increase in its expression levels at relapse and an
inverse correlation between its expression levels and prognosis, thus
making it a novel tumor marker for leukemic blast cells. Furthermore,
WT1 antisense oligomers have been found to inhibit the growth of
leukemic cells. These results strongly suggested the involvement of the
WT1 gene in human leukemogenesis. The present study was performed to
prove our hypothesis that the WT1 gene plays a key role in
leukemogenesis and performs an oncogenic function in hematopoietic
progenitor cells, rather than a tumor-suppressor gene function. 32D
cl3, an interleukin-3-dependent myeloid progenitor cell line,
differentiates into mature neutrophils in response to granulocyte
colony-stimulating factor (G-CSF). However, when transfected wild-type
WT1 gene was constitutively expressed in 32D cl3, the cells stopped
differentiating and continued to proliferate in response to G-CSF. As
for signal transduction mediated by G-CSF receptor (G-CSFR), Stat3
was constitutively activated in wild-type WT1-infected 32D cl3 in
response to G-CSF, whereas, in WT1-uninfected 32D cl3, activation of
Stat3 was only transient. However, most interesting was the fact
that G-CSF stimulation resulted in constitutive activation of Stat3
only in wild-type WT1-infected 32D cl3, but not in WT1-uninfected 32D
cl3. Thus, WT1 expression constitutively activated both Stat3 and
Stat3. A transient activation of Stat1 was detected in both
wild-type WT1-infected and uninfected 32D cl3 after G-CSF stimulation,
but no difference in its activation was found. No activation of MAP
kinase was detected in both wild-type WT1-infected and uninfected 32D
cl3 after G-CSF stimulation. These results demonstrated that WT1
expression competed with the differentiation-inducing signal mediated
by G-CSFR and constitutively activated Stat3, resulting in the blocking
of differentiation and subsequent proliferation. Therefore, the data
presented here support our hypothesis that the WT1 gene plays an
essential role in leukemogenesis and performs an oncogenic function in
hematopoietic progenitor cells and represent the first demonstration of
an important role of the WT1 gene in signal transduction in
hematopoietic progenitor cells.
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INTRODUCTION |
WILMS' TUMOR GENE (WT1) was identified
as a tumor-suppressor gene responsible for Wilms tumor, a childhood
kidney neoplasm.1,2 The WT1 gene encodes a zinc finger
transcription factor and represses transcription of growth factor
(PDGF-A chain,3 CSF-1,4 and IGF-II5) and growth factor receptor (IGF-IR6)
genes, and the other genes (RAR-,7 c-myc,8
and bcl-28). We have previously reported high expression of
wild-type WT1 in fresh leukemia cells regardless of the disease
type, 9-11 an inverse correlation between WT1 expression
levels and prognosis,9 increased expression of WT1 at
relapse in acute leukemia,12 and growth inhibition of
leukemic cells by WT1 antisense oligomers.13 These results
suggested that WT1 plays an important role in leukemogenesis and may
have an oncogenic function rather than a tumor-suppressor gene function
in hematopoietic progenitor cells.
Acute myelocytic leukemia (AML) is an acute myeloproliferative disease
characterized by maturation arrest within the myeloid lineage. The
majority of AML cells, like their normal counterparts, proliferate
dependently on growth factors,14 but are refractory to
differentiation induction.15 Most responsive AML cells, for example, proliferate without differentiation in response to granulocyte colony-stimulating factor (G-CSF),16 suggesting the
alteration in the G-CSF signaling pathway in AML cells.
32D cl3, an interleukin-3 (IL-3)-dependent myeloid progenitor cell
line, differentiates into mature neutrophils in response to G-CSF like
their normal myeloid cell counterparts.17 Thus, transfection of the WT1 gene into 32D cl3 provides us with an experimental system suitable for the elucidation of WT1 gene function in hematopoietic progenitor cells.
The present study was performed to prove our hypothesis that the WT1
gene plays a key role in leukemogenesis and performs an oncogenic
function in hematopoietic progenitor cells. We describe here that 32D
cl3 transfected with the WT1 gene proliferates without differentiation
in response to G-CSF and that this proliferative response is associated
with activation of both Stat3 and Stat3.
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MATERIALS AND METHODS |
Construction of retroviral vectors.
Murine retroviral vectors capable of expressing full-sized, nonspliced
form [ie, 17 amino acids (+), KTS (+)] of human WT1 with or without
the deletion of the third zinc finger domain18,19 was
constructed with pM5Gneo retroviral vector, which was kindly provided
by Dr Carol Stocking (Heinrich-Pette-Institut, Hamburg, Germany). This
vector was derived from murine proliferative sarcoma virus and
contained viral long terminal repeats (LTRs) and a neomycin resistance
(NeoR) gene under the control of herpes virus thymidine
kinase promoter. A full-sized, nonspliced type human WT1 cDNA
(EcoRI-EcoRI fragment) with or without the deletion of
the third zinc finger domain region18 was placed
immediately downstream of the viral LTRs by digestion of the viral
vector with EcoRI. The structure of retroviral vector is shown
in Fig 1.
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| Fig 1.
Structure of recombinant retroviral vectors. Human
full-sized, nonspliced type WT1 cDNA with or without the deletion of
the third domain of zinc finger region was inserted into a pM5GNeo retroviral vector. The solid bar within WT1 cDNA represents the deleted
region of mutant WT1. K, Kpn I; E, EcoRI; H,
HindIII.
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Cells and viruses.
The murine IL-3-dependent 32D cl3 cell line17 was
maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 50 U/mL recombinant murine IL-3 (rmIL-3). Recombinant retroviruses encoding WT1 were prepared by transfection of
pM5Gneo/WT1 DNA into  2 cells by the calcium phosphate method and
selected for G418 resistance. The virus stock was prepared from
supernatants of drug-resistant cell line. Viral titers were determined
by infection of NIH3T3 cells and scored for G-418 resistant colonies.
The expression of the human WT1 cDNA was confirmed by the detection of
both human WT1 transcript and protein. Polymerase chain
reaction-single-stranded conformation polymorphism
(PCR-SSCP) analyses confirmed that the WT1 mRNA expressed
by the retroviral vector contained neither point mutation nor deletion.
32D cl3 cells were infected with the recombinant retroviruses by
coculturing the cells with virus-producing 2 cells (virus titer,
~105 cfu/mL) irradiated by x-rays for 48 hours and then
grown in RPMI 1640 medium containing 10% FBS, 50 U/mL rmIL-3, and 500 µg/mL G418. G418-resistant clones were selected in 96-well microtiter plates. For induction of differentiation, exponentially growing cells
were washed twice with serum-free RPMI 1640 medium and resuspended in
RPMI 1640 medium containing 10% FBS and 20 ng/mL recombinant human
G-CSF (rhG-CSF).
Reverse transcriptase-PCR (RT-PCR).
Extraction of total RNA and cDNA synthesis was performed as
described previously.9 PCR for WT1 was performed
as described previously.9 For detection of murine
myeloperoxidase (MPO), lactoferrin (LF), and -actin, specific
primers were synthesized. The primer sequences are as follows:
murine MPO: sense, 5 -ACAACATTGACATC-TGGATGG-3, antisense,
5-GGTCTCCTTCCAGGAAGTCA-3; murine LF: sense,
5-GACAAGGTGGAAGTCCTTCAG-3, antisense,
5-ACAACATTGACATCTGGATGG-3; murine
-actin: sense, 5-GTGGGCCGCCCTAGGCACCAG-3,
antisense, 5-CTCTTTGATGTCACGCACGATTTC-3. PCR were performed under the following conditions. For detection of MPO
mRNA, denaturation at 94°C for 1 minute, primer annealing at
61°C for 1 minute, chain elongation at 72°C for 1.5 minutes, and 19 cycles of PCR. For detection of LF mRNA, denaturation at 94°C for 1 minute, primer annealing at 64°C for 1 minute, chain elongation at 72°C for 1.5 minutes, and 23 cycles of PCR. For detection of -actin mRNA, denaturation at 94°C for 1 minute, primer annealing at 60°C for 1 minute, chain elongation at 72°C for 1.5 minutes, and 20 cycles of PCR.
Fluorescence-activated cell sorting (FACS) analysis.
Fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody (MoAb)
RB6-8C5 (anti-Gr-1) and FITC-conjugated MoAb M1/70.15 (anti-CD11b,
Mac-1) were purchased from Pharmingen (San Diego, CA) and
Caltag Laboratories (San Francisco, CA), respectively. Fluorescence intensity measurement was performed with a FACScan (Becton
Dickinson, Mountain View, CA).
Western blot analysis.
Western blot analysis was performed as described previously.
13 In brief, approximately 20 µg of cell lysates was
transferred onto Immobilon polyvinylidene difluoride
(PVDF) (Millipore, Bedford, MA), probed with anti-WT1
polyclonal antibodies,13 anti-Stat3 antibodies
(Transduction Laboratories, Lexington, KY), anti-phosphorylated Stat3
antibodies (New England Biolabs, Beverly, MA), anti-Stat1 antibodies
(Santa Cruz Biotechnology, Santa Cruz, CA), or a mixture of anti-ERK1
and anti-ERK2 antibodies (Santa Cruz Biotechnology) and then with
alkaline phosphatase- or peroxidase-conjugated anti-Ig antibodies.
After washing, the filters were immersed in the buffer containing
nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP; for WT1 protein) or enhanced chemiluminescence (ECL; for STAT
and ERK proteins) for 10 to 60 minutes.
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RESULTS |
Introduction of the WT1 gene into 32D cells by retroviral infection.
32D cl3 (here designated 32D) cells were infected with recombinant
retrovirus containing only a neomycin resistance gene, with a neomycin
resistance gene plus a human full-sized, nonspliced type WT1 cDNA
(wild-type WT1), or with a neomycin resistance gene plus a human
full-sized, nonspliced type WT1 cDNA whose third zinc finger domain
region was deleted (mutant WT1; Fig 1). G418-resistant clones were then
isolated in medium containing IL-3 and G418 (V4, V5, and V6 were isolated as control clones
expressing only the neomycin resistance gene; W2,
W3, W4, W10, and W12
were isolated as wild-type WT1-infected clones; and Z5,
Z6, and Z7 were isolated as mutant WT1-infected
clones).
To confirm the production of WT1 protein from the infected WT1 gene,
cell lysates were subjected to Western blot analysis using anti-WT1
antibodies. As shown in Fig 2A, wild-type
WT1-infected clones produced various amounts of WT1 protein of 54 kD,
whereas no WT1 protein was detected in 32D clones that were infected
with the empty recombinant retrovirus containing only a neomycin
resistance gene. Mutant WT1-infected 32D clones produced a short WT1
protein corresponding to the deletion of the third zinc finger domain. Because PCR primers used here amplified both human and mouse WT1 and
the size of PCR products was the same, we could not discriminate between human and mouse WT1. However, because digestion of the PCR
products with a restriction enzyme HaeIII occurred in human WT1, but not in mouse WT1, we could discriminate between the two PCR
products. The results showed that the WT1-infected clones produced only
human, but not mouse WT1 mRNA (Fig 2B). On the other hand, in both the
parental 32D cells and the empty vector-infected clones
(V4, V5, and V6) used as controls,
neither human nor mouse WT1 mRNA was detected.
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| Fig 2.
WT1 expression in WT1-infected 32D clones. (A) Western
blot analysis of WT1 protein. V4, V5, and
V6 , empty vector-infected-32D clones; W2 , W3 , W4 , W10, and W12
, wild-type WT1-infected 32D clones; Z5 and Z7,
mutant WT1-infected 32D clones. (B) RT-PCR analysis of WT1 transcript.
RT-PCR was performed as described previously.9 WT1 PCR
products were digested with a restriction enzyme HaeIII and
electrophoresed on a 1.5% agarose gel. Lane 1, K562 (a human leukemia
cell line); lane 2, digestion of 1; lane 3, mouse kidney cells; lane 4, digestion of 3; lane 5, a WT1-infected 32D clone (W2); lane
6, digestion of 5; lane 7, an empty vector-infected 32D clone
(V4).
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Proliferative response of wild-type WT1-infected 32D cells to G-CSF.
In the presence of IL-3, no significant difference in cell growth was
found between controls and WT1-infected 32D clones
(Fig 3A). It is well-known that deprivation
of IL-3 from the medium induces apoptosis in 32D cells. In the current
study, both control and WT1-infected 32D cells underwent apoptosis and
died within 7 days after deprivation of IL-3 from the medium (Fig 3B).
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| Fig 3.
Growth kinetics of WT1-infected 32D clones. Cells were
cultured in 10% FBS-containing RPMI 1640 medium in the presence of IL-3 (50 U/mL; A), in complete deprivation of cytokines (B), or in the
presence of G-CSF (rhG-CSF at 20 ng/mL; C), and viable cells were
counted at the indicated time.
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To determine the effects of WT1 expression on granulocytic
differentiation pathways induced by G-CSF, control and WT1-infected 32D
clones were removed from the IL-3-containing medium and cultured in
G-CSF-containing medium. Control and mutant WT1-infected 32D clones
completely differentiated into phenotypically matured neutrophils during 10 days of culture, whereas wild-type WT1-infected 32D clones
stopped differentiating and began to proliferate (Figs 3C and
4). An important finding was that the
growth rate of the wild-type WT1-infected 32D clones in
G-CSF-containing medium increased in parallel with the expression
levels of the WT1 protein. In this context, a decrease in the
expression levels of the WT1 protein in the wild-type WT1-infected 32D
clones was accompanied by the appearance of phenotypically more
differentiated cells during the culture in G-CSF-containing medium
(Fig 4). Differentiated cells were frequently found in the
W4 clone, which expressed low levels of WT1 protein.
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| Fig 4.
Morphology of wild-type WT1-infected 32D clones cultured
in IL-3- or G-CSF-containing medium. Cells maintained in rmIL-3 (50 U/mL)-containing medium (top) were washed for deprivation of IL-3, seeded in G-CSF (20 ng/mL)-containing medium, and cultured for 10 days
(bottom). Cytospin cells were stained with May-Grünwald-Giemsa stain solution.
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Inhibition of G-CSF-induced differentiation of 32D cells by
constitutive expression of the WT1 gene.
The mRNA expression levels of MPO20 and LF21
genes, which are the respective differentiation markers for
promyelocytes and metamyelocytes to segmented cells, were examined on
the control and wild-type WT1-infected 32D clones cultured in
G-CSF-containing medium (Fig 5). In the
control clone V4, MPO mRNA expression increased, reached a
peak 5 days after the treatment with G-CS F, and then
decreased to the initial level 10 days after G-CSF treatment. In
striking contrast, MPO mRNA expression increased in wild-type
WT1-infected 32D clones and peaked at 5 days after G-CSF treatment, but
then stayed at that level. In the control clone V4, LF mRNA
expression increased during the 10 days of culture in G-CSF-containing
medium, whereas in two wild-type WT1-infected 32D clones,
W2 and W10, that expressed relatively high
levels of WT1 protein, LF mRNA expression did not significantly change
during the 10 days of culture in G-CSF-containing medium. In
W4 that expressed low levels of WT1 protein, LF mRNA
expression increased 10 days after G-CSF treatment, suggesting the
differentiation of a part of the cells. In addition, the effects of WT1
expression on cell surface differentiation antigens were tested
(Fig 6). In control clones (V4,
V5, and V6) treated with G-CSF, expression of
both Mac-1 and Gr-1 increased along with differentiation. In contrast,
in wild-type WT1-infected clones (W2, W3,
W4, and W10), both expressions remained
unchanged during the 10-day culture period. These results showed that
constitutive expression of the WT1 gene blocked the G-CSF-induced
differentiation of the 32D cells. They also demonstrated that wild-type
WT1-infected 32D cells can differentiate to the promyelocyte or
near-promyelocyte stage, which is a little more differentiated than
that of the parental 32D cells, but are blocked from differentiating
further and are frozen at that stage, although WT1-low expressing 32D cells (W4) can differentiate in part into mature cells.
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| Fig 5.
Quantitative RT-PCR analysis of MPO and LF transcripts
after stimulation with G-CSF. Control and wild-type WT1-infected 32D clones were cultured in G-CSF-containing medium for the indicated days, and MPO and LF mRNAs were quantitated by RT-PCR under the optimized conditions.
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| Fig 6.
Inhibition of Mac-1 and Gr-1 differentiation antigen
induction by WT1 expression. Control (V4, V5,
and V6) and wild-type W T1-infected (W2,
W4, W10, and W12) 32D clones were
cultured in IL-3 (100 U/mL)-containing or G-CSF (20 ng/mL)-containing
medium for 10 days. The cells were stained with FITC-conjugated
M1/70.15 MoAb (CD 11b, Mac-1) or FITC-conjugated RB6-8C5 MoAb (Gr-1),
and then FACS-analyzed. Means ± 2 SD of positive cells in three
control clones and those in four WT1-infected clones are shown.
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Constitutive activation of Stat3 by WT1 expression.
We examined the effects of WT1 expression on two major signals, STAT
(signal transducers and activators of transcription)22,23 and MAP kinase,24 mediated by the G-CSF receptor (G-CSFR;
Fig 7). First, Western blot analysis using
antibodies directed against phosphorylated (P-) Stat3 was performed. In
the control clone V4 , only P-Stat3 was detected 10 minutes after G-CSF stimulation, after which it became undetectable.
Three days after G-CSF stimulation, P-Stat3 became distinctly
detectable again, peaked at 5 days after G-CSF stimulation, and once
more became undetectable. In the wild-type WT1-infected clone
W2, P-Stat3 was also detected 10 minutes after G-CSF
stimulation, then became undetectable, and 3 days after G-CSF
stimulation became detectable again. However, in contrast
to in V4, P-Stat3 was detected continuously from days 3 through 7 after G-CSF stimulation. The most striking contrast was seen
in P-Stat3,25 which became detectable in W2
3 days after G-CSF stimulation and persisted from day 3 through 7 after G-CSF stimulation. Next, the amounts of Stat3 and Stat3 were examined with Western blot analysis using anti-Stat3 antibodies directed against the sequence common to Stat3 and Stat3. In V4, the amounts of Stat3 and Stat3 did not
significantly change at any time, and those of Stat3 were
significantly greater than those of Stat3. In W2, on the
other hand, the quantity of Stat3 was significantly greater than
that of Stat3 at 0, 10, and 60 minutes after G-CSF stimulation. One
day after stimulation, the amounts of Stat3 had become equivalent to
those of Stat3, but then quantity of Stat3 exceeded that of
Stat3 from days 3 through 7 after G-CSF stimulation, so that the
ratio of Stat3 to Stat3 was reversed. As for Stat1, a transient
activation was detected in both V4 and W2, but
neither quantitative nor qualitative differences were found between
V4 and W2 at any time after G-CSF stimulation (data not shown). Examination of the second signal, MAP kinase showed
no significant difference in the amounts of MAP kinase between
V4 and W2 after G-CSF stimulation, and we did
not detect any active forms of MAP kinase in either V4 or
W2 (Fig 7). Altogether, these data demonstrated that WT1
expression constitutively activated both Stat3 and Stat3 and that
this activation might block differentiation and induce proliferation.
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| Fig 7.
Constitutive activation of Stat3 in wild-type
WT1-infected 32D cells. IL-3-deprived 32D cells were stimulated with
IL-3 (100 U/mL) or G-CSF (20 ng/mL). Cell lysates were prepared at the
indicated time and subjected to Western blot analysis, using antibodies directed against phosphorylated Stat3 protein (top panel) or Stat3 protein (middle panel), or using a mixture of antibodies directed against ERK1 or ERK2 (bottom panel). Arrows indicate activated ERK2.
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DISCUSSION |
Mutational studies of G-CSFR have shown that the cytoplasmic domain of
G-CSFR consists of two distinct functional domains. The
membrane-proximal region, which contains two subdomains designated boxes 1 and 2, is responsible for the transduction of the proliferation signal and the C-terminal domain, called box 3, transduces the signal
for cell differentiation.26-28 32D cells differentiate into neutrophils in G-CSF-containing medium. This indicates that the differentiation-inducing signal mediated by G-CSFR is transduced, whereas the proliferation signal transduction is inhibited. Our present
study demonstrated that 32D cells stopped differentiating into
neutrophils and continued to proliferate in G-CSF-containing medium in
the presence of constitutive WT1 expression. This indicated that
constitutive WT1 expression competed with the differentiation-inducing signal mediated by G-CSFR and blocked transduction of the
differentiation-inducing signal and, instead, transduced only the
proliferation signal mediated by G-CSFR. Thus, the results clarified
that WT1 involves in the determination of whether
differentiation-inducing signal mediated by G-CSFR can be transduced.
Fresh leukemic cells from patients with AML usually show proliferation,
rather than differentiation, in response to G-CSF.14-16
This proliferative response of AML cells to G-CSF can now be explained
by the high levels of WT1 expression in AML cells,9-11
which allow for transduction of only the proliferation signal, but not
of the differentiation-inducing signal mediated by G-CSFR.
The constitutive WT1 expression in 32D cells increased the amount of
Stat3 protein, resulting in the reversion of ratio of Stat3 to
Stat3 and activated both Stat3 and Stat3. It is known that
Stat3 results from a truncation of the C-terminal 55 amino acids of
Stat3 and the addition of 7 new C-terminal amino acids encoded by
3 sequence of Stat3.25 Chakraborty et
al29 investigated whether G-CSF signaling in immature
normal and leukemic human myeloid cells diverges at the level of
activation of STAT protein. In their experiments, the isoform
composition of Stat3 proteins activated by G-CSF was examined on
immature normal and leukemic human myeloid cells. In CD34+
cells and in a leukemic myeloid cell line capable of differentiating in
response to G-CSF, only Stat3 was activated, whereas in leukemic cells not capable of differentiating in response to G-CSF, both Stat3 and Stat3 were activated. In acute myeloid leukemia cell line AML-193, which proliferates, but does not differentiate in response to G-CSF, there was activation of both Stat3 and Stat3. These results suggested that the balance of the two Stat3 isoforms in
myeloid cells may influence the cellular pattern of gene activation and
consequently the ability of these cells to differentiate in response to
G-CSF, because the transcriptional activity of Stat3 is distinct
from Stat3.29,30 Therefore, wild-type WT1-infected 32D
cells are leukemia cell-like in the sense that they are
activated in both Stat3 and Stat3 and proliferate in response to
G-CSF. Taken together, it may be assumed that blocking of
differentiation and induction of proliferation in response to G-CSF in
wild-type WT1-expressing 32D cells mimics in vivo leukemogenesis, in
which a normal myeloid progenitor cell might be disturbed to
differentiate and be turned to proliferation in response to
physiological concentration of G-CSF when constitutive WT1 expression
happened in the cell by as yet undetermined causes.
The hypothesis that the WT1 gene has basically two functional aspects,
namely that of a tumor-suppressor gene and that of an oncogene, but in
leukemic cells performs an oncogenic rather than a tumor-suppressor
gene function, can be established on the basis of the following data:
(1) high expression of wild-type WT1 in fresh leukemic
cells,9-11,31-34 (2) an inverse correlation between WT1
expression levels and prognosis,9 (3) increased expression
of WT1 at relapse as compared with that at diagnosis in acute
leukemia,12 (4) growth inhibition of leukemic cells by WT1
antisense oligomers,13 and (5) our results presented here.
Because it is known that WT1 protein interacts with P5335
or par-436 protein, differences in the interaction of WT1
protein with other regulator proteins might determine whether the WT1
gene acts as a tumor-suppressor gene or performs an oncogenic function.
Transfection of v-abl,37 v-src,38
v-ras,39 v-myb,40 or c-myb41
oncogenes blocked the G-CSF-induced granulocytic differentiation of
32D cells and caused them to proliferate in response to G-CSF. The v-abl- or v-src-infected 32D cells could proliferate independently from IL-3, whereas, like the WT1 gene, the v-ras, v-myb, or c-myb oncogene could not abrogate the dependency of the infected 32D cells on
IL-3 for proliferation. The morphology of the rapidly proliferating
cells of the wild-type WT1-infected 32D clones in G-CSF-containing
medium was a little more differentiated than that of the parental 32D
cells maintained in IL-3-containing medium (Fig 4). In addition, MPO
expression levels increased after G-CSF stimulation (Fig 5). These
results suggested that wild-type WT1-infected 32D cells can
differentiate to promyelocyte or near-promyelocyte stage, which is a
little more differentiated than that of the parental 32D cells, but are
blocked from differentiating further and frozen at that stage, although
WT1 expression higher than that in the wild-type WT1-infected 32D
clones examined here might never allow differentiation. Transfection of
v-ras or c-myb induced MPO in the 32D cells in response to G-CSF,
whereas transfection of v-abl, v-src, or v-myb did not induce MPO.
Thus, the WT1 gene might have an aspect similar to the oncogenic
function of the v-ras or c-myb oncogene.
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FOOTNOTES |
Submitted August 5, 1997;
accepted November 24, 1997.
Address reprint requests to Haruo Sugiyama, MD, Department
of Clinical Laboratory Science, Osaka University Medical School, 1-7, Yamada-Oka, Suita City, Osaka 565, Japan.
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 Dr Hisamaru Hirai (Tokyo University, Tokyo, Japan)
for providing us with 32D cl3. We also thank Tsuyomi Yajima for typing
the manuscript and Machiko Mishima for her skillful technical asistance
with the PCR.
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Abstract
Introduction
Abstract