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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 7 (April 1), 1999:
pp. 2369-2379
Constitutive Activation of the JAK2/STAT5 Signal Transduction Pathway
Correlates With Growth Factor Independence of Megakaryocytic
Leukemic Cell Lines
By
Richard Y. Liu,
Chun Fan,
Roy Garcia,
Richard Jove, and
Kenneth S. Zuckerman
From the Division of Medical Oncology and Hematology, the Department
of Internal Medicine and the Department of Biochemistry and Molecular
Biology, University of South Florida College of Medicine, and Molecular
Oncology and Hematologic Malignancy Programs, H. Lee Moffitt Cancer
Center and Research Institute, Tampa, FL.
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ABSTRACT |
The factor-independent Dami/HEL and Meg-01 and factor-dependent Mo7e
leukemic cell lines were used as models to investigate JAK/STAT signal
transduction pathways in leukemic cell proliferation. Although Dami/HEL
and Meg-01 cell proliferation in vitro was independent of and
unresponsive to exogenous cytokines including granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), IL-6, thrombopoietin (TPO), and tumor necrosis factor-
(TNF-), the growth of Mo7e cells was dependent on hematopoietic
growth factors. When these cell lines were cultured in medium without
cytokines, a constitutively activated STAT-like DNA-binding factor was
detected in nuclear extracts from both Dami/HEL and Meg-01 cells.
However, the STAT-like factor was not detectable in untreated Mo7e
cells, but was activated transiently in Mo7e cells in response to
cytokine treatments. The constitutively activated and cytokine-induced STAT-like DNA-binding factor in these three cell lines was identified as STAT5 by oligonucleotide competition gel mobility assays and by
specific anti-STAT antibody gel supershift assays. Constitutive activation of JAK2 also was detected in the factor-independent cell
lines, but not in Mo7e cells without cytokine exposure. Meg-01 cells
express a p185 BCR/ABL oncogene, which may be responsible for the
constitutive activation of STAT5. Dami/HEL cells do not express the
BCR/ABL oncogene, but increased constitutive phosphorylation of Raf-1
oncoprotein was detected. In cytokine bioassays using growth
factor-dependent Mo7e and TF-1 cells as targets, conditioned media from
Dami/HEL and Meg-01 cells did not show stimulatory effects on cell
proliferation. Our results indicate that the constitutive activation of
JAK2/STAT5 correlates with the factor-independent growth of Dami/HEL
and Meg-01 cells. The constitutive activation of JAK2/STAT5 in Dami/HEL
cells is triggered by a mechanism other than autocrine cytokines or the
BCR/ABL oncoprotein.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
SURVIVAL, PROLIFERATION, and
differentiation of normal hematopoietic cells are controlled by a
number of growth factors that interact with members of the
hematopoietic growth factor receptor family or with one of several
receptor tyrosine kinases. In contrast, leukemic cells or leukemic cell
lines frequently grow in a factor-independent manner. The mechanisms
that result in the conversion of factor-dependent cells to
factor-independent cells are poorly understood. Some investigators have
reported that mutations of growth factor receptor(s) or signal
transduction proteins cause constitutive activation of signal
transduction pathways and induce growth factor-independent cell
proliferation.1-4 Autocrine growth factor-stimulated growth
of human leukemic cells or cell lines due to aberrant cytokine
production by the malignant cells has been detected
infrequently.5-7
The signal transduction pathways triggered by growth factor ligands
binding to cell surface receptors are involved in the reversible
phosphorylation of receptors, cytosolic nonreceptor kinases, and other
cellular proteins. Among the many known signal transduction pathways,
the JAK (Janus kinase)/STAT (signal transducer and activator of
transcription) cascade and the MAPK (mitogen-activated protein kinase)
cascade are activated after exposure of megakaryocytic cells to
cytokines.8-12 Although the significance of
cytokine-induced activation of these signaling pathways remains under
active investigation, several reports have shown that phosphorylation
and activation of JAK and STAT proteins appears to play a critical role
in thrombopoietin-induced signal transduction.8,13,14 JAK
proteins are members of a family of cytoplasmic kinases, which is known
to have four members: TYK2, JAK1, JAK2, and JAK3. JAK2 is activated in
normal megakaryocyte precursors and megakaryocytic cell lines by
several specific hematopoietic cytokines and has been reported to be
required for megakaryocytic cell proliferation.14,15 The
STATs, of which at least seven distinct family members and several
alternative RNA splicing-derived variants have been described so far,
are cytosolic transcription factors, which are tyrosine-phosphorylated
after ligand activation.16 They dimerize and translocate
into the nucleus, where they bind to specific DNA sequences and
activate gene transcription. Originally, STAT5, which was described as
a transcription factor activated by prolactin and required for the
induction of transcription of milk caseins, now has been identified as
two closely related proteins, STAT5a and STAT5b.17 Recent
studies indicate that they are widely expressed in many
tissues.18 STAT5 is known to be activated by the JAKs and
Bmx tyrosine kinases,19,20 and the activated STAT5 dimers
recognize and bind to a specific palindromic DNA sequence,
TTCNNNGAA.16 This oligonucleotide sequence is found not
only in the  -casein promoter, but also in a number of other genes,
such as those encoding interferon regulatory factor-1 (IRF-1), interferon consensus sequence binding protein (ICSBP), rat serine protease inhibitor, human interstitial cell adhesion molecule, high
affinity immunoglobulin G receptor, Oncostatin M, and cytokine-induced SH2 protein (CIS).21-24 All of
these findings suggest that STAT5 is important in a broad variety of
cellular functions.
In this study, we have investigated the activation of the JAK2/STAT5
signal transduction pathway in growth factor-dependent and
factor-independent megakaryocytic cell lines. We found that the
JAK2/STAT5 signaling pathway is activated constitutively in factor-independent Dami/HEL and Meg-01 cells, but is transiently cytokine-induced in factor-dependent Mo7e cells. The constitutive activation of the JAK2/STAT5 signaling pathway appears to correlate with the factor-independence of Dami/HEL and Meg-01 cell lines. Although BCR/ABL oncogene transcripts are detected in Meg-01 cells, Dami/HEL cells do not express the BCR/ABL oncogene. Constitutive phosphorylation of Raf-1 occurs in Dami/HEL, but not Meg-01 cells. Thus, constitutive activation of JAK/STAT5 and proliferation may be
triggered by different mechanisms in Dami/HEL and Meg-01 cells.
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MATERIALS AND METHODS |
Reagents.
Recombinant human thrombopoietin (TPO), interleukin-3 (IL-3), IL-6,
granulocyte-macrophage colony-stimulating factor (GM-CSF), and
epidermal growth factor (EGF) were purchased from PeproTech (Rocky
Hill, NJ). Anti-IL-3 and anti-TPO neutralizing antibodies were
purchased from R & D Systems (Minneapolis, MN). Antiphosphotyrosine antibody-agarose and anti-JAK2 antiserum were purchased from Upstate Biotechnology (Lake Placid, NY). Phosphotyrosine Western blotting kit
(chemiluminescence) was purchased from Boehringer Mannheim Biochemicals
(Indianapolis, IN). Antigoat IgG and antimouse IgG antibodies labeled
with fluorescein isothiocyanate were purchased from Zymed (South San
Francisco, CA). Anti-STAT1, -STAT2, -STAT3, -STAT4, -STAT5 -STAT6, and
anti-Raf-1 antibodies were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). PhosphoPlus p44/p42 MAPK antibody kit containing
antitotal and anti-T202/Y204 phosphorylated p42/p44 MAPK antibody were
purchased from New England Biolabs (Beverly, MA).
[Methyl-3H]-thymidine ([3H]-TdR; specific
activity, 70 to 86 Ci/mmol) and [32P]-deoxyadenosine
triphosphate (dATP) (specific activity, >3,000 µCi/mmol) were purchased from Amersham Life Science (Arlington Heights, IL).
Cell lines.
Human megakaryocytic leukemic cell line, Dami, originally described by
Greenberg et al,25 was obtained from American Type Culture
Collection (ATCC, Rockville, MD) and was maintained in Iscove's Modified Dulbecco's Medium (IMDM; GIBCO-BRL, Grand Island, NY) containing 10% horse serum. Recently, ATCC and DSMZ (German Collection of Microorganisms and Cell Cultures) have determined that
all samples of the Dami cell line that were available for them to
analyze were genetically and karyotypically identical to the human
erythroleukemia cell line (HEL) described previously,26 which has both erythroid and megakaryocytic
characteristics.27 For clarity and for comparison to prior
reports, we are designating this cell line as Dami/HEL in this report.
Human megakaryoblastic leukemic cell line, Meg-01, originally described
by Ogura et al,28 was obtained from ATCC and was maintained
in RPMI 1640 medium (GIBCO-BRL) with 10% heat-inactivated fetal bovine
serum (FBS). Human factor-dependent megakaryoblastic leukemic cell
line, Mo7e, originally described by Avanzi et al,29 was
obtained from Genetics Institute (Boston, MA) and was maintained in
IMDM with 10% FBS, 1% L-glutamine and 5 ng/mL GM-CSF. All of the
cells were incubated at 37°C in a fully humidified atmosphere with
5% CO2 in medium alone or in the presence of various
concentrations of cytokines. In experiments to detect the effects of
known cytokines or potential cytokines in Dami/HEL or Meg-01
conditioned media, Mo7e and TF-1 cells were prepared by washing cells
three times with medium and starved for 18 hours in medium without
growth factors before cytokine or conditioned medium treatments, as
described by Dusanter-Fourt et al.30 Dami/HEL cells were
cultured in IMDM without serum, but with 1X Nutridoma-HU (Boehringer
Mannheim Biochemicals), and Meg-01 cells in RPMI 1640 with 1X
Nutridoma-HU in experiments in which cytokine production and specific
cytokine effects were being studied.
Effects of cytokines on cell growth.
To determine the proliferative response of cells to cytokines, DNA
synthesis was measured by [3H]-thymidine (TdR)
incorporation. The assays were performed in triplicate, using 4 × 105 cells for Mo7e and Meg-01 or 2 × 105
cells for Dami/HEL cells. Mo7e cells in IMDM with 10% FBS and Dami/HEL
and Meg-01 cells in medium with 1X Nutridoma-HU were cultured for 72 hours without or with cytokines. Subsequently, the cells were labeled
with 2 µCi/mL of [3H]-TdR for an additional 4 hours.
Incorporation of [3H]-TdR into newly synthesized DNA
(counts per minute; cpm) was determined by liquid scintillation
counting, according to a previously described protocol.31
For the analysis of cell ploidy, cytokine-starved Mo7e cells (1 × 106 cells/mL) were cultured in IMDM with 10% FBS plus
growth factors. Dami/HEL and Meg-01 cells (1 × 106/mL) were cultured in medium with 1X Nutridoma-HU plus
growth factors, at 37°C for 1 to 10 days. The treated cells were
harvested on days 3, 5, 7, and 10. The DNA content of the cells was
quantitated with a FACScan flow cytometer (Becton Dickinson,
Rutherford, NJ), following a published protocol.32 Isolated
human lymphocytes were used as a diploid control.
Immunoprecipitation and Western blotting.
Unstimulated or cytokine-stimulated cells were lysed in modified
radioimmunoprotection assay (RIPA) buffer with 1% NP-40
and extracts were immunoprecipitated as described.30 The
detergent-soluble proteins were incubated with antiphosphotyrosine
agarose for 3 hours at 4°C with shaking. The immunoprecipitates
were washed four times with modified RIPA buffer, lysed in sodium
dodecyl sulfate (SDS) buffer and separated by 7.5% nonreducing
SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were then
transferred to nitrocellulose membrane and probed sequentially with
rabbit anti-JAK2 antibody and antirabbit IgG antibody labeled with
peroxidase. Phosphorylated JAK2 was visualized with enhanced
chemiluminescence techniques according to the manufacturer's
recommended procedure. For the detection of phosphorylation of Raf-1, a
band shift assay was used in Western blot using an anti-Raf-1 antibody
(Santa Cruz Biotechnology) following a previously reported
procedure.33 For detecting the phosphorylation of cellular
proteins, cells treated with or without cytokines were lysed in SDS
buffer, and 40 to 80 µg of cellular proteins was loaded in each
sample lane, separated by 10.5% SDS-PAGE. For detecting total and
phosphorylated MAPK, the SDS-PAGE-separated proteins were transferred
to polyvinylide difluoride (PVDF) membranes and probed
with antitotal and antiphosphorylated p42/p44 MAPK, with
antiphosphorylated STAT3 antibody (New England Biolabs), or with
antiphosphotyrosine antibody (Boehringer Mannheim).
Preparation of nuclear extracts and gel mobility shift assays.
Preparation of nuclear extracts and electrophoretic gel mobility shift
assay (EMSA) was performed according to methods described previously.34 Briefly, equal amounts of nuclear proteins (5 to 10 µg) for each sample were incubated for 30 minutes at 30°C with 10,000 dpm of [32P]-labeled double-strand DNA
fragment (IRF-1 GAS; 5'-GCCTGATTTCCCCGAAATGACGGCA) containing the
interferon- activation site (GAS) that binds to interferon
regulatory factor (IRF-1).35 IRF-1 GAS contains an identical sequence to that of Bovine mammary gland factor element (MGFe), TTCCCCGAA. For competition assays, unlabeled c-Fos intragenic regulatory element (FIRE; 5'-AGCGCCTCCCCGGCCGGGGAG),
interferon-stimulated response element (ISG15 ISRE;
5'-GATCGGGAAAGGGAAACCGAAACTGAAGCC), and sis-inducible element
(SIE, 5'-AGCTTCATTTCCCGTAAATCCCTAAGC) were used as potential
DNA-protein binding competitors. The competition was performed by
adding 50X molar excess of each unlabeled DNA fragment along with the
[32P]-labeled IRF-1 GAS oligonucleotide probe. Unlabeled
MGFe was also used as a specific competitor for STAT5 binding to IRF-1 GAS. For gel mobility supershift assays, nuclear extracts were coincubated with the indicated specific anti-STAT antibodies and the
[32P]-labeled oligonucleotide probes. The DNA-protein
complexes and unbound probe were separated electrophoretically on 5%
native polyacrylamide gels in 0.5X TBE buffer (44.5 mmol/L Tris, pH
8.0, 1 mmol/L EDTA, and 44.5 mmol/L boric acid) for 3 hours at constant 140 V. The gels were fixed and dried, and the DNA-protein complexes were visualized by autoradiography at  70°C with Kodak X-OMAT film (Eastman Kodak, Rochester, NY) and a DuPont Cranex lightning-plus intensifying screen (DuPont, Wilmington, DE).
Reverse transcription-polymerase chain reaction (RT-PCR).
Cellular mRNA was prepared with RNAzol (Cinna/Biotecx Lab, Houston, TX)
following the protocol suggested by the manufacturer. A commercially
available primer set (TransPrimers, Calbiochem, CA) was used to assess
the presence of BCR/ABL transcripts. RT-PCR for normal c-ABL also was
performed to check RNA quality. A RNA PCR Core kit (GeneAmp; Perkin
Elmer, Branchburg, NJ) was used to perform RT-PCR following the
manufacturer's protocol. A 1.8% agarose gel was used for
electrophoretic analysis of the PCR products. RT-PCR was performed at
least twice to confirm the findings.
Bioassays of stimulatory cytokines.
Factor-independent Dami/HEL and Meg-0l cells were examined for the
capacity to produce autocrine cytokines. Mo7e and TF-1 cell lines, the
survival of which depend on the presence of any of several growth
factors, including GM-CSF, IL-3, IL-6, IL-9, TPO, steel factor/c-kit
ligand, tumor necrosis factor- (TNF-), and EPO etc, were used as
bioassay systems to detect possible stimulatory factors that might be
produced by Dami/HEL or Meg-01 cells. Conditioned medium was prepared
by collecting supernatants from 3-day cultures of Dami/HEL or Meg-01
cells grown in serum-free conditions. The conditioned medium was
incubated with freshly prepared Mo7e or TF-1 cells for 72 hours and
cell proliferation was determined by [3H]-TdR
incorporation into DNA, following the procedure described in the prior section.
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RESULTS |
Dami/HEL and Meg-01 cells grow in a factor-independent manner.
To determine effects of cytokines on DNA synthesis of the proliferating
cells, various concentrations of cytokines were incubated with
Dami/HEL, Meg-01, and Mo7e cells for 3 days, and the cells were labeled
subsequently with [3H]-TdR for the final 4 hours of
culture. When 0 to 200 ng/mL of TPO was incubated with the
cytokine-independent cell lines, Dami/HEL and Meg-01, no significant
stimulation of their basal [3H]-TdR incorporation was
observed (Fig 1, Dami/HEL and Meg-01). The
cells also were tested for their response to other cytokines, including
GM-CSF, IL-3, IL-6 and TNF-, and neither Dami/HEL nor Meg-01 cell
proliferation was affected by any of these cytokines (data not shown).
Addition of anti-IL-3 or anti-TPO neutralizing antibodies did not
inhibit [3H]-TdR incorporation or cell proliferation
(data not shown). However, when factor-dependent Mo7e cells were
incubated with various concentrations of TPO, incorporation of
[3H]-TdR was stimulated significantly, in a
dose-dependent manner (Fig 1, Mo7e). At the concentration of 50 ng/mL,
TPO regularly stimulated the incorporation of
[3H]-thymidine 4.5-fold over that of control Mo7e cells
at 3 days (Fig 1, Mo7e). IL-3, GM-CSF, IL-6, and TNF- also
stimulated Mo7e cell proliferation (data not shown).
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| Fig 1.
Effects of TPO on DNA synthesis in human leukemic cell
lines. Dami/HEL, Meg-01, and Mo7e cells were incubated with the
indicated concentrations of TPO for 72 hours and labeled with 2 µCi/mL [3H]-TdR for 4 hours. The cpm of
[3H]-TdR incorporation into newly synthesized DNA was
determined by counting the radioactivity from triplicate samples and
expressed as the mean cpm. This figure is representative of the data
from three separate experiments.
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To investigate effects of cytokines on the maturation of these
megakaryocytic leukemic cell lines, the cells were fixed and stained
with propidium iodide, and cell ploidy was analyzed by a FACscan flow
cytometer. In the untreated Dami/HEL, Meg-01, or Mo7e cells, 95% of
cells were diploid. When the cells were incubated with 100 to 400 ng/mL
of TPO for 1 to 10 days, no significant increase of cell ploidy or
morphologic signs of differentiation were observed in any of these cell
lines (data not shown).
STAT-like DNA-binding factors are activated constitutively in
Dami/HEL and Meg-01 cells.
The fact that the Dami/HEL and Meg-01 cell lines grow in a
factor-independent manner led us to investigate whether an aberrant signal transduction pathway might be involved. Because JAK and STAT
proteins have been reported to have a role in cytokine-induced proliferation of megakaryocytic and leukemic cells,8 we
analyzed STAT DNA-binding factor activation by EMSA with a
[32P]-labeled oligonucleotide containing the IRF-1 GAS
consensus STAT binding site, as the probe. DNA-binding protein(s) were
detected in the nuclear extracts from Dami/HEL and Meg-01 cells in the absence of cytokine exposure (Fig 2).
Addition of as much as 400 ng/mL of TPO or 40 ng/mL IL-3 did not have
any further effect on the constitutive DNA-binding protein activity in
these growth factor-independent cell lines (Fig 2). Exposure of these
cells to GM-CSF, IL-6, erythropoietin (EPO), or TNF-
also did not result in significant enhancing or inhibitory effects on
the constitutive DNA-binding factor activity (data not shown). In
contrast, no STAT-like DNA-binding factor was detectable in untreated
control Mo7e cells; however, addition of 400 ng/mL TPO (Fig 2) or 40 ng/mL of GM-CSF or IL-3 (Fig 3)
significantly induced a STAT-like transcriptional factor binding to
IRF-1 GAS.
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| Fig 2.
Constitutive activation of a STAT-like DNA-binding factor
in Dami/HEL and Meg-01 cells. Nuclear extracts from each cell line
cultured in the absence of cytokine (CTL) or in the presence of as much
as 400 ng/mL TPO or 40 ng/mL IL-3, were incubated with
[32P]-labeled, double-stranded IRF-1 GAS oligonucleotide.
The DNA-binding complex and unbound probes were separated
electrophoretically on 5% nondenaturing polyacrylamide gels. The
autoradiograph shows the STAT-like DNA-binding factor (DBF) and the
nonspecific bands (NS).
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| Fig 3.
Effects of cytokines on activation of a STAT-like
DNA-binding factor in Mo7e cells. Nuclear extracts from Mo7e cells
grown in the absence of cytokine (CTL), or in the presence of 40 ng/mL
EPO, 400 ng/mL TPO, 40 ng/mL GM-CSF or 40 ng/mL IL-3, were incubated
with [32P]-labeled IRF-1 GAS oligonucleotide. The
STAT-like DNA-binding complex and unbound probe were separated on 5%
native polyacrylamide gel. The autoradiograph shows the DBF, NS, and
free probes (P).
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The constitutive and cytokine-activated STAT-like factor binds
specifically to DNA containing the IRF-1 GAS sequence.
The question remained whether the constitutively activated STAT-like
DNA-binding factor in Dami/HEL or Meg-01 cells is the same one as that
activated by cytokines in Mo7e cells. We performed experiments to
identify the specific STAT protein activated in these leukemic cell
lines. First, we used a set of oligonucleotides to attempt to inhibit
competitively the binding of the STAT-like factor to
[32P]-labeled IRF-1 GAS. When Dami/HEL nuclear extracts
were coincubated with the labeled probe plus a 50-fold molar excess of
unlabeled FIRE, ISRE, or SIE oligonucleotides, which do not contain
TTCNNNGAA sequence, no competitive inhibition of the formation of
DNA-protein complexes was observed (Fig 4,
Dami). However, a 50-fold molar excess of the unlabeled MGFe, which
contains the same TTCCCCGAA STAT-binding sequence as IRF-1 GAS,
completely abolished the formation of the labeled DNA-protein
complexes. Similar results were found with Meg-01 cells (data not
shown).
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| Fig 4.
Specificity of binding of STAT-like DNA-binding factors
in Dami/HEL and Mo7e cells to DNA containing the IRF-1 GAS sequence.
(A) Dami/HEL cell nuclear extracts were incubated with
[32P]-labeled IRF-I GAS probe (lane 1) or with
[32P]-labeled probe plus a 50-fold excess of unlabeled
oligonucleotide FIRE, ISRE, SIE, or MGFe, respectively (lanes 2 to 5).
The DNA-binding complexes were separated in a 5% nondenaturing
polyacrylamide gel. (B) Mo7e cell nuclear extracts from untreated
control cells (lane 1) or cells treated with 400 ng/mL TPO (lane 2)
were incubated with [32P]-labeled IRF-1 GAS probe or with
[32P]-labeled probe plus a 50-fold excess of unlabeled
oligonucleotide FIRE, ISRE, SIE, or MGFe, respectively (lanes 3 to 6).
The DNA-binding complexes were separated in a 5% nondenaturing
polyacrylamide gel. The autoradiograph shows the STAT-like DBF
and NS.
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We then investigated whether the cytokine-induced DNA-binding factor in
Mo7e cells has similar features as that in Dami/HEL cells. When the
nuclear extracts from TPO-treated Mo7e cells were incubated with
[32P]-labeled IRF-1 GAS probe or the
[32P]-labeled probe plus a 50-fold molar excess of
unlabeled FIRE, ISRE, SIE, or MGFe oligonucleotides, the
cytokine-induced DNA-binding factor in Mo7e cells had the same features
as the constitutively activated STAT-like factor in Dami/HEL cells.
Both of the factors were able to bind to the IRF-1 GAS probe, and their
binding activity could be abolished completely by unlabeled MGFe, but
not by oligonucleotides that do not contain the TTCNNNGAA sequence.
The GAS-binding factor activated constitutively in Dami/HEL and
Meg-01 cells and induced transiently by cytokine in Mo7e cells is
STAT5.
Because different STAT transcription factors may share the same
binding site to some of the DNA probes, the data from the gel mobility
shift assays with IRF-1 GAS as the probe may not confirm specifically
what STAT protein is activated, although the pattern is most consistent
with activated STAT5. To identify the specific DNA-binding factor(s),
gel mobility supershift assays with antibodies to specific STAT
proteins were used to examine the type of STAT(s) involved in the
DNA-protein complexes. When anti-STAT5 antiserum was used, supershift
of the DNA-protein complexes was observed with nuclear extracts from
Dami/HEL and Meg-01 cells cultured in medium without cytokines or from
TPO-treated Mo7e cells (Fig 5), but not in
untreated Mo7e cells (data not shown). When anti-STAT3 antiserum was
used, no supershift of DNA-protein complexes occurred in TPO-treated
Mo7e or unstimulated Dami/HEL or Meg-01 cells (Fig 5). However,
anti-STAT1, anti-STAT3, and anti-STAT5 antibodies induced supershift of
the DNA-protein complex in nuclear extracts from EGF-treated SK-BR-3
cells (Fig 5), in which STAT1, STAT3, and STAT5 have been shown
previously to be activated.36 Reactions of the DNA-protein
complexes with anti-STAT1, anti-STAT2, anti-STAT4 and anti-STAT6
antibodies also were investigated, and no supershifted DNA-protein
complexes were observed (data not shown). These results indicate that
the predominant or exclusive constitutively activated (in Dami/HEL and
Meg-01 cells) or TPO- or GM-CSF-induced (in Mo7e cells) STAT
DNA-binding factor in these megakaryocytic leukemic cell lines is
STAT5.
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| Fig 5.
Identification of the constitutively activated
DNA-binding factor in Dami/HEL and Meg-01 cells and the
cytokine-induced DNA-binding factor in Mo7e cells by gel
electrophoretic mobility supershift assays. Nuclear extracts prepared
from Dami/HEL cells without cytokine exposure were incubated with
[32P]-labeled IRF-1 GAS plus anti-STAT3 or anti-STAT5
antibodies (lanes 1 and 2). Nuclear extracts from Meg-01 cells without
cytokine exposure were incubated with the [32P]-labeled
probe plus anti-STAT3 or anti-STAT5 antibodies (lanes 3 and 4). Nuclear
extracts from Mo7e cells without cytokine exposure (lane 5) or treated
with 400 ng/mL TPO (lanes 6 to 8), were incubated with
[32P]-labeled IRF-1 GAS probe (lanes 5 and 6), or with
[32P]-labeled probe plus anti-STAT3 or anti-STAT5
antibodies (lanes 7 and 8). Nuclear extracts, from SK-BR-3 cells
incubated without cytokine exposure (lane 9) or with 20 ng/mL EGF
(lanes 10 to 14), were incubated with [32P]-labeled IRF-1
GAS probe alone (lanes 9, 10, and 13), or with
[32P]-labeled probe plus anti-STAT1, anti-STAT3, or
anti-STAT5 antibodies (lanes 11, 12, and 14). The EGF-treated SK-BR-3
cells, which were reported by our group to have activated STAT1, STAT3,
and STAT5,36 were used to show the antibody-induced
supershift of STAT1, STAT3, and STAT5. The autoradiograph shows the
STAT-like DBF, NS, and free IRF-1 GAS probe (P). The arrowheads
indicate the supershifted complexes. These results are representative
of three separate experiments.
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JAK2 is activated constitutively in Dami/HEL and Meg-01 cells.
To examine protein phosphorylation patterns and to identify the
mechanism for constitutive STAT5 activation, Dami/HEL and Meg-01 cells
were incubated in medium without serum, and Mo7e cells were deprived of
growth factors in medium with 10% FBS for 18 hours, and cells were
then exposed to TPO or GM-CSF for 10 minutes. Two growth factor-induced
tyrosine-phosphorylated protein bands of approximately 130 kD and 116 kD were observed in Mo7e cells (Fig 6,
lanes 2 and 3, Mo7e). When Dami/HEL cells were examined, there was no
detectable difference in the protein phosphorylation patterns in
control Dami/HEL cells or cells treated with cytokines (Fig 6, lanes 1 through 3, Dami). Interestingly, the pattern of tyrosine
phosphorylation in untreated Dami/HEL cells was very similar to that of
the cytokine-treated Mo7e cells (compare Fig 6, lane 1, Dami and lanes
2 and 3, Mo7e). The results show that phosphorylated protein bands,
which are undetectable or barely detectable in control Mo7e cells, but
are induced by TPO or GM-CSF, are phosphorylated constitutively in
untreated control Meg-01 (data not shown) and Dami/HEL cells. Because
it has been reported that JAK2 is activated by TPO or other cytokines
in megakaryocytic cells,14,15 we examined the tyrosine
phosphorylation of JAK2 in Dami/HEL, Meg-01, and Mo7e cells. Lysates
from cells treated with or without cytokines were immunoprecipitated
with antiphosphotyrosine agarose. The results show that JAK2 is
phosphorylated constitutively in control Dami/HEL
(Fig 7, lane 1, Dami) and Meg-01 cells
(data not shown). However, JAK2 was unphosphorylated in control Mo7e cells (Fig 7, lane 1, Mo7e) and phosphorylated in cytokine-treated Mo7e
cells (Fig 7, lanes 2 and 3, Mo7e). No further effect on tyrosine-phosphorylation of JAK2 was observed when Dami/HEL cells were
exposed to TPO or IL-3 (Fig 7, lanes 2 and 3, Dami). These results show
that JAK2 is phosphorylated constitutively in the two growth
factor-independent megakaryocytic leukemic cell lines and that its
phosphorylation is induced by TPO and IL-3 in factor-dependent Mo7e
cells.
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| Fig 6.
Tyrosine phosphorylation of cellular proteins in Dami/HEL
and Mo7e cells. Cell lysates from Dami/HEL cells (A) and Mo7e cells (B)
were cultured in medium without cytokines (CTL), or with 400 ng/mL TPO
or 40 ng/mL GM-CSF (GM). The cellular proteins were lysed in SDS
buffer, separated on SDS-PAGE, transferred onto PVDF membrane, and
probed with antiphosphotyrosine antibodies. The arrowheads indicate
cytokine-induced tyrosine-phosphorylated protein bands in Mo7e cells,
which are constitutively expressed in unstimulated control Dami/HEL
cells.
|
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| Fig 7.
Phosphorylation of JAK2 in Dami/HEL and Mo7e cells. (A)
Cellular proteins (500 µg/mL) from untreated Dami/HEL or Mo7e cells
(CTL), or from cells treated with 400 ng/mL TPO or 40 ng/mL IL-3, were
precipitated with antiphosphotyrosine agarose. The immunoprecipitates
were subjected to SDS-PAGE, transferred to PVDF membrane, and probed
with antihuman JAK2 antibody to detect tyrosine-phosphorylated JAK2.
(B) Cellular proteins (20 µg) from untreated Dami/HEL or Mo7e cells
(CTL), or cells treated with 400 ng/mL TPO or 40 ng/mL IL-3, were
subjected directly to 7.5% SDS-PAGE and the blotted PVDF membrane was
probed with antihuman JAK2 antiserum to detect total JAK2 protein as a
control. Molecular mass markers are indicated in
kilodaltons.
|
|
Proto-oncogene Raf-1 is constitutively phosphorylated in Dami/HEL
cells.
To examine the phosphorylation of Raf-1, Western blot with a specific
anti-Raf-1 antibody was used to examine the band shift of Raf-1. The
band shift of Raf-1 in Western blot has been identified to be induced
by the phosphorylation of Raf-1.33,37 When cell lysates
were prepared from cells treated with or without GM-CSF, and equal
amounts of cellular proteins were subjected to SDS-PAGE, band shift of
Raf-1 was detected by Western blot in Mo7e cells in response to GM-CSF
treatment (Fig 8). In the cell lysates
prepared from Dami/HEL and U937 cells without cytokine exposure,
phosphorylation (band shift) of Raf-1 was detected (Fig 8, Dami and
U937, CTL). Treating Dami/HEL cells with GM-CSF had no further effects
on the phosphorylation of Raf-1 (Fig 8, Dami, GM). In Meg-01 cells without GM-CSF exposure, no significant band shift was observed (Fig 8,
Meg-01, CTL). GM-CSF treatment also failed to induce any significant
phosphorylation of Raf-1 in Meg-01 cells (Fig 8, Meg-01, GM). U937
cells, which have been shown previously to have increased phosphorylation of Raf-1 in cells without cytokine
exposure,37 were used as the control to locate the
phosphorylated Raf-1.
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| Fig 8.
Phosphorylation of proto-oncogene Raf-1 and activation of
MEK in Mo7e, Meg-01, and Dami/HEL cells. (A) Phosphorylation of Raf-1
was examined by band shift on Western blot. Equal amounts of cellular
proteins (40 µg) from Mo7e, Dami/HEL, Meg-01, and U937 cells
incubated with no cytokine (lanes marked as C) or 40 ng/mL GM-CSF
(lanes, GM) were subjected directly to 10.5% SDS-PAGE. The separated
proteins were transferred to a PVDF membrane and probed with
anti-Raf-1 antibody. The arrow indicates the location of
unphosphorylated Raf-1 and the arrowhead indicates the band shift of
phosphorylated Raf-1. (B) Activation of MEK was detected by the
kinase-induced phosphorylation of a kinase-defective
p42mapk (K52R) labeled with [32P]-ATP. Equal
amounts of cellular proteins (30 µg) from Mo7e, Meg-01, and Dami/HEL
cells incubated with no cytokine (lanes marked as C) or 40 ng/mL GM-CSF
(lanes, GM) were used to detect MEK activity. Phosphorylation of p42 is
used as the readout to detect MEK activity.
|
|
No increased activity of MAP kinase kinase (MEK) is detected in
Meg-01 or Dami/HEL cells.
It has been reported that TPO and other cytokines stimulate both the
MAPK and JAK/STAT signaling pathways in megakaryocytic cells. Because
we observed the constitutive activation of the JAK2/STAT5 signaling
pathway in Dami/HEL and Meg-01 cells and the increased Raf-1
phosphorylation in Dami/HEL cells, it was of interest to see whether
the MEK also was activated constitutively in these cells. Our data show
that all of the cells had a low level of MEK functional activity and no
difference of the MEK activity could be distinguished among Mo7e,
Meg-01, and Dami cells without any cytokine exposure (Fig 8B, lanes 1, 3, and 5). However, a significant increased level of MEK activity was
detected transiently in all three cell lines after treatment with
GM-CSF (lanes 2, 4, and 6), IL-3, TPO, or IL-6 (data not shown).
Furthermore, when phosphorylation of p44/p42 MAPK was investigated in
control and cytokine-treated cells to confirm the cytokine-induced MEK
activation, the data showed that there was minimal phosphorylated MAPK
detectable in the absence of any cytokine, and strong cytokine-induced
phosphorylation of MAPK was observed in all three cell lines (data not shown).
No BCR/ABL oncogene product is detected in Mo7e or Dami/HEL cells.
There are several reports showing that the BCR/ABL chimeric oncoprotein
activates STATs and usually JAKs and induces cytokine-independent growth in cells transfected with the BCR/ABL oncogene.38-40
Our data show no BCR/ABL transcripts detectable in factor-dependent Mo7e cells with or without cytokine exposure
(Fig 9A, lanes 3 and 4). Although
constitutive activation of STAT5 and phosphorylation of JAK2 are
observed in Dami/HEL cells without cytokine exposure, no BCR/ABL
transcript was detected in this cell line, either without (Fig 9, lane
5) or with (Fig 9, lane 6) TPO treatment. BCR/ABL transcripts were
observed in Meg-01 and K562 cell lines (Fig 9, lanes 1, 2, 7, and 8),
both of which are cell lines that were derived from patients with
chronic myelogenous leukemia (CML). In Meg-01 cells, a smaller BCR/ABL
PCR product was detected (consistent with p185 BCR/ABL), compared with
that of K562 (consistent with p210 BCR/ABL) (compare Fig 9, lanes 7, 8 and 1, 2).
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| Fig 9.
RT-PCR analysis of BCR/ABL and C-ABL transcripts in
Dami/HEL, Meg-01, and K562 leukemic cell lines. (A) C-ABL expression
was analyzed by RT-PCR using 1 µg total RNA from K562 (lane 1), Mo7e
(lane 3), Dami/HEL (lane 4), and Meg-01 (lane 5) cell lines. RT-PCR was
performed without cellular RNA as a negative control (lane 2). (B)
BCR/ABL expression was analyzed by RT-PCR using 0.01 µg or 1 µg
total RNA from K562 cells as positive controls (lanes 1 and 2), or
using 1 µg of total RNA from Mo7e cells treated without (lane 3) or
with 100 ng/mL TPO (lane 4), from Dami/HEL cells treated without (lane
5) or with 100 ng/mL TPO (lane 6), and from Meg-01 cells treated
without (lane 7) or with 100 ng/mL TPO (lane 8). The sizes of PCR
products are indicated in base pairs.
|
|
No detectable stimulatory factors are secreted by Dami/HEL or Meg-01
cells.
To investigate further the mechanism for the constitutive activation of
JAK2 and STAT5 in Dami/HEL and Meg-01 cells, we performed experiments
to examine whether the cytokine-independent Dami/HEL and Meg-01 cell
lines acquired the capacity to produce cytokines that may result in
autocrine stimulation of cell proliferation and JAK2/STAT5 activation.
The Mo7e cell line, the survival of which is dependent on the presence
of at least one of several growth factors, including GM-CSF, IL-3,
IL-6, IL-9, steel factor/C-kit ligand, TNF-, and TPO, was used as a
bioassay system to detect the presence of possible stimulatory
factor(s) released into the conditioned medium of Dami/HEL or Meg-01
cells. The results show that the conditioned medium from either
Dami/HEL or Meg-01 cells did not stimulate [3H]-TdR
incorporation in freshly prepared Mo7e cells, but addition of 2 ng/mL
GM-CSF significantly increased the [3H]-TdR incorporation
(Fig 10). TF-1 cells, which grow in a
growth factor-dependent manner and have EPO receptors and are
EPO-responsive, also were used as a bioassay to examine the conditioned
media from Dami and Meg-01 cells, and no significant effects of the conditional medium from Dami/HEL or Meg-01 cells on
[3H]-TdR incorporation were observed (data not shown).
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| Fig 10.
Bioassays of possible stimulatory factors in Dami/HEL
and Meg-01 conditioned medium. Mo7e cells, pretreated for 18 hours in
medium without cytokines, were incubated for 72 hours with control or
conditioned medium from cultures of Dami/HEL cells (A) or Meg-01 cells
(B), using either (1) culture medium not exposed to Dami/HEL or Meg-01
cells (CTL), (2) conditioned medium collected from the 3-day culture of
Dami/HEL or Meg-01 cells (CM), or (3) conditioned medium plus 2 ng/mL
GM-CSF (CM + GM-CSF). The cells then were labeled with
[3H]-TdR for an additional 4 hours. CPM of
[3H]-TdR incorporation was determined from triplicate
samples and expressed as the mean. These results are representative of
three separate experiments.
|
|
| |
DISCUSSION |
Our studies show that the JAK2/STAT5 signal transduction pathway is
activated constitutively in the growth factor-independent leukemic cell
lines, Dami/HEL and Meg-01. In contrast, the activation of this
signaling pathway is strictly cytokine-induced transiently in the
growth factor-dependent leukemic cell line, Mo7e. Others also have
reported transient cytokine-dependent activation of the JAK2/STAT
signaling pathway in several growth factor-dependent cell
lines.8-12,15 Although our present study is focused on two growth factor-independent megakaryocytic leukemic cell lines (Dami/HEL cells also have erythroid characteristics), the aberrant signaling pathway could also exist in other factor-independent leukemic cell
lines. Based on the published observation that STAT-related transcription factors are activated constitutively in primary cells
from acute myeloid and lymphoid and chronic myeloid leukemia patients41-44 and given the close relationship between cell
proliferation and the activation of JAK2/STAT5, constitutive activation
of the JAK2/STAT5 pathway may be one of the important mechanisms of
leukemogenesis and of maintaining the leukemic phenotype.
Mechanisms for the growth factor-independent growth of leukemic cells
are not well understood. The BCR/ABL oncoprotein is implicated in the
pathogenesis of CML, as well as some acute leukemias, especially a
subset of acute lymphoblastic leukemias. BCR/ABL exhibits protein
kinase activity, which activates RAS, JAKs, STATs, and other signaling
pathways.38-40,45 When cytokine-dependent cells are
transfected with the BCR/ABL oncogene, constitutive activation of STAT5
and loss of a cytokine requirement for continued cell growth have been
reported.39 In cells transformed with the BCR/ABL oncogene,
the BCR/ABL oncoprotein may activate STAT5 directly, bypassing the need
for activation of JAK2.46 Others, however, reported that
the activation of STAT5 is still via the JAK
kinase.40,41,45,47 We observed that the Meg-01 cell line, which is derived from a patient with CML, contains BCR/ABL oncogene transcripts, which is a likely explanation for the constitutive activation of STAT5 in this cell line. However, no BCR/ABL transcripts were detected in either Dami/HEL or Mo7e cells, indicating that the
constitutive activation of JAK2/STAT5 in Dami/HEL cells is triggered by
a mechanism other than the BCR/ABL oncogene.
The proto-oncogene, Raf-1, has been reported to have a role in the
factor-independent growth of human myeloid leukemia cell lines.37 In our study, we did observe increased
phosphorylation of Raf-1 in Dami/HEL cells and U937 cells without
cytokine exposure, and cytokine treatment had no significant further
effects on Raf-1 phosphorylation. Although the constitutive
phosphorylation of Raf-1 may be responsible for the factor-independence
of Dami/HEL and U937 cells, the relationship of the constitutive
activation of Raf-1 and STAT5 is not clear at the present time. On the
other hand, no significant increased phosphorylation of Raf-1 or MAPK, and no increased MEK activity were observed in Meg-01 cells without cytokine exposure. This result was unexpected, as Meg-01 cells express
BCR/ABL gene transcripts, which are reported to have the capacity to
induce the activation of the Ras-Raf-MAPK signal transduction pathway.38,40 Whether the lack of constitutive
phosphorylation of Raf-1 and MAPK is related to the p185 BCR/ABL
expression in Meg-01 cells would be interesting to investigate.
STAT signaling pathways, particularly STAT3 and STAT5, are frequently
activated during oncogenesis.34,36,40,48 The initial report
of STAT activation by a specific oncoprotein showed that one STAT
family member, STAT3, is constitutively activated in rodent fibroblast
cell lines stably transformed by the oncogenic Src tyrosine
kinase.48 Although STAT3 seems to be the predominant family
member activated by Src, STAT5 was reported to be activated by various
oncoproteins in human leukemias.38-47 Recently, the fusion
of the human TEL gene on chromosome 12p13 to the JAK2 gene on
chromosome 9p24 has been reported in human leukemias.43,44 The TEL gene was initially described as a "promiscuous" gene with a role in both myeloid and lymphoid leukemias. Three variants have been
identified that fuse the TEL pointed (PNT) domain to the JAK2
JH1-kinase domain, part of, and all of the JH2 pseudokinase domain.
More interestingly, all of the human TEL/JAK2 fusion variants are
oncoproteins in vitro that strongly activate STAT5 and transform the
IL-3-dependent murine hematopoietic cell line Ba/F3 to
IL-3-independent growth.43 It will be important in future
studies to determine whether an oncogenic mutation involving JAK2 is
responsible for the constitutive activation of JAK2/STAT5 signaling
pathway in at least Dami/HEL cells.
In addition to oncoproteins, there are accumulated data indicating that
aberrant expression or mutational activation of cytokine receptors or
their downstream signal transduction pathways may have an important
role in abnormally, constitutively activated signal transduction
pathways and in factor-independent cell growth. Transfecting growth
factor-dependent cells with the TPO-receptor gene containing a point
mutation in the dimer interface homology domain of the TPO receptor
(also known as c-mpl) induces constitutive receptor activation and
factor-independent cell proliferation.1,4 By engineering
expression of the EPO receptor with a single amino acid change,
Watowich et al2 observed that mutants resulting in
disulfide-linked receptor homodimerization cause constitutive activation of the EPO receptor and induction of factor-independent cell
proliferation. Kitayama et al3 reported a point mutation of
the c-Kit receptor tyrosine kinase that confers factor-independent growth and tumorigenicity of factor-dependent hematopoietic cells. Theoretically, mutation of any cytokine receptors that signal through
JAK2 and STAT5 or downstream signal transduction pathways could cause
the constitutive activation of JAK2/STAT5 in Meg-01 and Dami/HEL cells
(Dami/HEL cells also have constitutive phosphorylation of Raf-1).
Further study is needed to examine the possibility of whether aberrant
receptors or other signaling molecules are involved in the constitutive
activation of JAK2/STAT5, as well as the growth factor-independent
survival and proliferation of Dami/HEL cells.
We provide substantial evidence to show that the constitutive
activation of the JAK2/STAT5 signaling pathway is not likely to be via
autocrine stimulatory cytokines. This conclusion is supported by the
following observations. (1) We and others showed that Dami/HEL and
Meg-01 cells grow very well, even in serum-free medium, in the absence
of exogenous cytokines; and addition of cytokines such as TPO, GM-CSF,
IL-3, IL-6, and TNF- does not result in any stimulatory effect on
cell growth. (2) The addition of anti-IL-3, anti-GM-CSF, and anti-TPO
neutralizing antibodies does not have any effect on proliferation of
Dami/HEL or Meg-01 cells. (3) Using freshly prepared Mo7e or TF-1
cells, which are often used as bioassay systems to detect stimulatory
cytokines, neither Dami/HEL nor Meg-01 cell-conditioned medium support
Mo7e or TF-1 survival or proliferation. (4) If growth
factor-independent cells produce secreted or unsecreted cytokines that
cause autocrine stimulation of the cells, one should see not only the
constitutively activated JAK2/STAT5, but also increased MEK activity
and MAPK phosphorylation, as we and others have shown that IL-3,
GM-CSF, TPO, and EPO all activate both JAK2/STAT5 and MAPK signaling
pathways in erythroleukemic (TF-1 and UT-7) and megakaryocytic (Mo7e)
cell lines.11,12 (5) Other cytokines, such as G-CSF and
those which signal through gp130 (IL-6, IL-11, etc), induce JAK and
STAT family members other than JAK2 and STAT5. (6) We and
others8,10 showed that even persistent cytokine-treatment
only induces the transient activation of JAK2/STAT5 (Fig 3). Thus, an
autocrine stimulatory factor effect seems not to be a reasonable
explanation for the constitutive activation of JAK2/STAT5 in Dami/HEL
or Meg-01 cells.
We found constitutive activation of a STAT-like DNA-binding factor in
the growth factor-independent Dami/HEL and Meg-01 cells and
cytokine-induced activation of a STAT-like DNA-binding factor in Mo7e
cells, which raised the question of whether STAT-like proteins in
factor-independent cells are the same as those induced in
factor-dependent cells. Because other STATs also have the capacity to
bind to the GAS promoter, albeit to a lesser degree, it was necessary
to identify the specific STAT(s) that is/are activated constitutively
in Dami/HEL and Meg-01 cells. Our results show clearly that the
constitutively activated STAT-like DNA-binding factor in Dami/HEL and
Meg-01 cells and the cytokine-induced STAT-like protein in Mo7e cells
is STAT5. This conclusion is based on the following findings: (1) both
the cytokine-dependent and the cytokine-independent DNA-binding factors
have the capacity to bind to the IRF-1 GAS oligonucleotide probe; (2)
the formation of DNA-protein complexes is abolished by competing
oligonucleotides containing the STAT5-binding sequence (MGFe), but not
by ISRE, SIE, and FIRE, which do not have a consensus STAT5 binding
site; (3) anti-STAT5 antibody supershifts both the constitutively
activated (from Dami/HEL and Meg-01 cells) and cytokine-induced (from
Mo7e cells) DNA-protein complexes; and (4) antibodies to STAT1, STAT2,
STAT3, STAT4, and STAT6 do not supershift the DNA-protein complexes in
nuclear extracts from either unstimulated Dami/HEL and Meg-01 cells or
TPO-stimulated Mo7e cells. Our observation that STAT5 or a STAT5-like
factor, but not STAT3, is activated shortly after exposure of Mo7e
cells to cytokines is consistent with prior reports8,10 in
which only the STAT5 activation, but no STAT3 activation, was reported in hematopoietic cell lines exposed to these cytokines.
Our data suggest that the activation of JAK2/STAT5 is triggered by
different mechanisms in Dami/HEL, Meg-01, and Mo7e cells. The
constitutive activation of JAK2/STAT5 correlates well with the
factor-independence of Dami/HEL and Meg-01 cell lines. Future detailed
investigation will be important to determine the underlying reason(s)
for the constitutive activation of the JAK2/STAT5 pathway in Dami/HEL
and Meg-01 cell lines, the relationship of the constitutively activated
Raf-1 and JAK2/STAT5 in Dami/HEL cells and the relationship between
p185BCR/ABL and JAK2/STAT5 activation in Meg-01 cells. A better
understanding of these processes will provide insight into signal
transduction events leading to uncontrolled cell survival and
proliferation in leukemias and ultimately may lead to new diagnostic
and therapeutic strategies.
| |
FOOTNOTES |
Submitted March 23, 1998; accepted November 23, 1998.
Supported by Grants No. CA56072, CA55652, and P30 CA76292 from the
National Institutes of Health/National Cancer Institute, Bethesda, MD.
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.
Address reprint requests to Richard Y. Liu, PhD, H. Lee Moffitt Cancer
Center, MRC 3007, 12902 Magnolia Dr, Tampa, FL 33612; e-mail:
liur{at}moffitt.usf.edu.
| |
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