|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 6 (March 15), 2000:
pp. 2118-2125
NEOPLASIA
STAT5 activation contributes to growth and viability in
Bcr/Abl-transformed cells
Christian Sillaber,
Franck Gesbert,
David A. Frank,
Martin Sattler, and
James D. Griffin
From the Department of Adult Oncology, Dana Farber Cancer Institute,
Harvard Medical School, Boston, MA.
 |
Abstract |
The transcription factor STAT5 is constitutively tyrosine
phosphorylated and activated after transformation of hematopoietic cells by p210Bcr/Abl. A truncated form of STAT5B ( STAT5; aa, 1-683)
that lacks tyrosine 699 and the transcriptional activation domain was
introduced into Ba/F3p210 cells under the control of a
tetracycline-inducible promoter. Treatment of these cells with doxycycline, a tetracycline analogue, induced expression of STAT5 and inhibited STAT5-dependent transcription. STAT5 coprecipitated with STAT5 and decreased Bcr/Abl-dependent tyrosine phosphorylation of
endogenous STAT5. Induction of STAT5 inhibited growth of Ba/F3p210 cells (26%-52% of control levels at 4 days) but did not cause cell-cycle arrest. STAT5 reduced viability of Ba/F3p210 cells and
increased sensitivity of the cells to the cytotoxic drugs hydroxyurea
and cytarabine. These results indicate that high-level expression of
STAT5, as achieved here by using a tetracycline-inducible promoter,
inhibits STAT5 activity, reduces the growth rate of Ba/F3p210 cells by
inhibiting viability, and results in increased sensitivity to
chemotherapeutic drugs. It is therefore likely that STAT5 activation
plays a role in the transformation of hematopoietic cell lines by
p210Bcr/Abl.
(Blood. 2000;95:2118-2125)
© 2000 by The American Society of Hematology.
| |
Introduction |
Chronic myelogenous leukemia (CML) is a chronic
myeloproliferative disorder caused by a translocation of chromosomes 9 and 22 [t(9;22)] that fuses the BCR gene from chromosome 22 to the ABL gene from chromosome 9.1 Transcription
of this hybrid gene generates a 210-kd fusion protein (p210Bcr/Abl),
which has enhanced tyrosine kinase activity.2 Bcr/Abl
transforms primary murine hematopoietic cells and converts interleukin
3 (IL-3)-dependent cell lines to growth-factor independence. Several
studies have identified signaling pathways activated by Bcr/Abl,
including activation of p21Ras3,4 PI3-kinase,5
c-jun, and c-myc.6 Also, we and others
showed that STAT5 is phosphorylated in Bcr/Abl-transformed cells,7,8 although the importance of STAT5 tyrosine
phosphorylation has remained unclear.
Two closely related forms of STAT5 exist, STAT5A and STAT5B, which are
encoded by 2 different genes.9 Both are latent
transcription factors, which are coordinately tyrosine phosphorylated
in normal hematopoietic cells in response to several cytokines,
including erythropoietin, IL-3, and granulocyte-macrophage colony
stimulating factor (GM-CSF).9 Also, several oncogenes have
been shown to be associated with constitutive phosphorylation of
STAT5,10,11 and STAT5 has been reported to be
phosphorylated in a variety of malignant disorders other than CML.
Tyrosine phosphorylation of STATs is believed to induce
dimerization,12 translocation to the nucleus, and binding
of STAT5 dimers to specific DNA motifs. Only a small number of genes
have so far been shown to be regulated by STAT5, including
 -casein,13 CIS,14
OSM,15 and
BclX.16 The role of STAT5 in
hematopoiesis of adult mice is not clear, since mice with a targeted
disruption of the STAT5A and STAT5B genes have largely normal blood
counts.17 However, these mice had reduced myeloid
progenitor counts, suggesting that STAT5 might be important for
maintenance of the stem-cell compartment.
STAT5 has several important structural domains, including a DNA-binding
sequence, an SH2-domain, and a transactivation domain. Phosphorylation
of tyrosine 694 of STAT5A and tyrosine 699 of STAT5B creates a binding
site for the SH2 domain of another STAT5 molecule. Deletions of various
domains within the STAT5 molecule have been found to modify STAT5
function. In particular, expression of a STAT5B mutant truncated at
position 683 was found to exert a dominant negative effect on
endogenous STAT5.18
In the current study, a STAT5 complementary DNA encoding STAT5B
truncated at aa 683 was introduced into Ba/F3p210 cells under the
control of an inducible promotor. Induction of the truncated STAT5 gene
( STAT5) was found to inhibit STAT5 activity and STAT5-induced gene
expression. Using this system, we analyzed the role of STAT5 in
transformation by p210Bcr/Abl.
| |
Materials and methods |
Cell lines and cell culture
Ba/F3 is an IL-3-dependent murine hematopoietic precursor cell
line. Ba/F3 cells were maintained in RPMI 1640 medium (Mediatech Cellgro, Herndon, VA) supplemented with 10% (vol/vol) fetal calf serum
and 10% (vol/vol) WEHI-3B-conditioned medium as a source of IL-3.
Ba/F3 cells containing the reverse Tet transactivator pUHD172-1
(Ton.BaF.119) and Ton B.210.1 cells,19 in which p210Bcr/Abl can be induced by the addition of doxycycline, were grown
in the same medium. Transformed Ba/F3p210 cells are growth-factor independent and were maintained without WEHI-3B supernatant unless otherwise stated. All cell lines were grown in a humidified incubator at 37°C (5% carbon dioxide).
Introduction of a truncated STAT5 mutant into Ba/F3p210 cells
A truncated form of the murine STAT5B (STAT5; aa, 1-683, in
plasmid pBABE) was obtained from A. L. Mui18 (DNAX Research Institute, Palo Alto, CA). The construct was cloned into pTRE plasmid
(Clontech, Palo Alto, CA) by using EcoRI and XbaI
sites. The pTRE plasmid containing the truncated STAT5 construct was then cotransfected with a p210Bcr/Abl plasmid (pGD21020)
into Ton.BaF.1 cells containing the reverse Tet
transactivator.19 Cell lines were then selected for
growth-factor independence in the absence of doxycycline.
Factor-independent sublines were analyzed for doxycycline-inducible
STAT5 expression by Western blotting, as well as for expression of
p210Bcr/Abl.
STAT5 reporter gene assay
STAT5-dependent transcription was measured by using a reporter gene
construct (GAS-luciferase) containing 4 tandem -casein-like GAS
elements from the -globin locus control region cloned into the pGL2
luciferase vector (from A. D'Andrea, Boston, MA).21 This
construct (20 µg) was transiently transfected into Ba/F3p210 cells.
Twelve hours later, transfected cells were divided into 2 equal
cultures and then maintained in either the presence or absence of
doxycycline (1 µg/mL) for the next 24 hours. After being washed twice
with phosphate-buffered saline (PBS) at 4°C, cells were resuspended
in lysis buffer E397A (Promega, Madison, WI). The lysates (20 µL)
were then incubated with 300 µL of luciferase assay buffer (25 mmol/L
of glycylglycine [pH 7.8], 15 mmol/L of potassium phosphate [pH
7.8], 15 mmol/L of magnesium sulfate, 4 mmol/L of ethylene glycol
tetraacetic acid, 2 mmol/L of adenosine triphosphate, and 1 mmol/L of
dithiothreitol) and 100 µL of D-luciferin (0.3 mg/mL;
Pharmingen, San Diego, CA). Luciferase activity was assessed with an
automated liminometer (Lumat LB 9507; EG&G Berthold, Gaithersburg, MD).
The plasmid CMV-GAL (20 µg; Invitrogen, San Diego, CA) was used as
a reporter for transfection efficiency, and -GAL activity was
measured with a -GAL assay kit (Invitrogen, Carlsbad, CA). When
STAT5 reporter gene activity in 2 different cell lines was compared,
luciferase activity was reported as a ratio of luciferase activity to
GAL activity.
Preparation of cell lysates and Western blotting
Cells were washed twice in Dulbecco's PBS at 4°C and
resuspended in lysis buffer (1 mL/108 cells) consisting of
50 mmol/L of Tris (pH 8.0), 150 mmol/L of sodium chloride (NaCl), 1%
(vol/vol) NP-40, 0.5% (wt/vol) deoxycholic acid, 0.1% (wt/vol) sodium
dodecyl sulfate (SDS), 100 mmol/L of sodium fluoride, 1 mmol/L of
phenylmethylsulfonyl fluoride, 20 µg/mL of aprotinin, 40 µg/mL of
leupeptin, and 1 mmol/L of sodium orthovanadate. Lysates were then
incubated on ice for 30 minutes (resuspended vigorously every 5 minutes), centrifuged for 15 minutes (12 000g) to remove
insoluble particles, and either subjected to immunoprecipitation or
analyzed directly by immunoblotting. For immunoprecipitation, lysates
from 2 × 107 cells were incubated with an
anti-STAT5 antibody (Ab) (C-17; Santa Cruz Biotechnology, Santa Cruz,
CA) and protein G Sepharose beads (Pharmacia, Uppsala, Sweden) in lysis
buffer for 3 hours at 4°C and washed 3 times with 1% (vol/vol)
NP-40 at 4°C. Lysates and immunoprecipitates were then separated
under reducing conditions by 7.5% SDS-polyacrylamide gel
electrophoresis and electrophoretically transferred to a nitrocellulose
membrane (Protran; Schleicher & Schuell, Keene, NH) in buffer
containing 25 mmol/L of Tris, 192 mmol/L of glycine, and 20% (vol/vol)
methanol at 4°C. The membrane was blocked for 1 hour in 5%
(wt/vol) notfat dry milk powder in Tris-buffered saline (10 mmol/L of
Tris-hydrochloric acid [pH 8.0] and 150 mmol/L of NaCl). The membrane
was incubated sequentially with the primary and the secondary
horseradish peroxidase (HRP)-coupled Ab (1:5000 dilution in
Tris-buffered saline; Amersham, Piscataway, NJ) for 1 hour. HRP
activity was detected by using HRP substrates (Renaissance; NEN,
Boston, MA) and X-Omat film (Kodak, Rochester, NY).
The following Abs were used: the anti-STAT5 Ab, Ab 89 (Transduction
Laboratories, Lexington, KY), and C-17 (Santa Cruz Biotechnology); anti-myc Ab A-14 (Santa Cruz Biotechnology);
antiphosphotyrosine Ab 4G10 (Brian Druker, Oregon Health Sciences
University, Portland, OR); anti-CIS Ab (Santa Cruz Biotechnology);
anti-BclX Ab 2H12 (Transduction Laboratories); and
anti-Bcl2 Ab C-21 (Santa Cruz Biotechnology). The anti-Abl monoclonal
Ab 3F12 was obtained from Ravi Salgia, Dana Farber Cancer Institute,
Boston, MA.
Cell-cycle analysis
For each sample, 0.5 × 106 cells were washed in
Dulbecco's PBS at 4°C and resuspended in 500 µL of staining
solution containing 50 µg/mL of propidium iodide (PI), 0.1%
(vol/vol) NP-40, and 0.1% (wt/vol) sodium citrate. Cells were
incubated at 4°C in the dark for 15 minutes and then analyzed by
flow cytometry (Epics XL flow cytometer; Coulter Corp, Miami, FL).
Combined annexin V/PI staining procedure
Viability was assessed by using an annexin V staining kit (FLUOS;
Boehringer Mannheim, Indianapolis, IN) according to the manufacturer's
recommendations. Binding of fluorescein-conjugated annexin V and PI was
measured by fluorescence-activated cell separation. In some
experiments, cells were exposed to cytarabine for 36 hours (Bedford
Laboratories, Bedford, OH) or hydroxyurea (Sigma, St Louis, MO) for 5 hours before annexin V/PI staining.
| |
Results |
Generation of stable Ba/F3p210 cells inducibly expressing
STAT5
The Ba/F3 cell line is a nonleukemic hematopoietic cell line that
requires murine IL-3 for growth and viability. Transfection of Ba/F3
cells with p210Bcr/Abl makes these cells leukemic and growth-factor
independent.22 To investigate the contribution of STAT5
activation to Bcr/Abl transformation, a series of cell lines was
created in which a truncated, dominant negative, mutant form of
STAT518 (STAT5, a C-terminal deletion that retains aa
1-683 of STAT5B) was expressed under the control of a
doxycycline-inducible promotor. Ba/F3 cells stably expressing the
reverse Tet transactivator pUHD172-1 (Ton.BF.119) were
cotransfected with a STAT5 construct (pTRE-STAT5) and p210Bcr/Abl
(pGD210) and selected for growth-factor independence. These cells were
designated Ba/F3p210STAT5 cells. As a control, TonBaF.1 cells were
transfected with pGD210 and the empty pTRE plasmid (designated
Ba/F3p210Ctrl cells). Eight subclones of Ba/F3p210STAT5 cells were
obtained by single-cell cloning. On induction with doxycycline
(1µg/mL), the truncated form of STAT5 (STAT5 [78 kd]) was
induced in 3 of 8 clones tested. Inducible STAT5 expression in these
3 subclones (C7.14, C7.28, and C7.30) is shown in Figure
1 (lower panel). Doxycycline did not induce
any form of STAT5 protein in Ba/F3p210Ctrl cells. In the absence of
doxycycline, STAT5 expression in Ba/F3p210STAT5 cell clones was
minimal but detectable, indicating a small degree of leakiness in this
system. Each clone expressed endogenous (full-length) STAT5A and
STAT5B (Figure 1, lower panel), p210Bcr/Abl, and c-abl (upper
panel). Growth-factor independence was due specifically to
Bcr/Abl, since each of these clones died rapidly in the presence of
the Bcr/Abl kinase inhibitor STI57123 (1µmol/L). The
expression levels of c-abl and Bcr/Abl in the 3 Ba/F3p210STAT5 cell clones and in Ba/F3p210Ctrl cells were
approximately equivalent.
View larger version (31K):
[in this window]
[in a new window]
| Fig 1.
Expression of STAT5 and p210Bcr/Abl in Ba/F3-p210
cells.
Ba/F3 cells transfected with p210Bcr/Abl and either the
empty pTRE plasmid (Ba/F3p210Ctrl) or pTRE STAT5 (Ba/F3p210STAT5)
were maintained in either the presence or absence of doxycycline (1 µg/mL) for 18 hours and then subjected to Western blot analysis.
Although all cell lines were found to express both forms of STAT5
(STAT5A and STAT5B), incubation with doxycycline induced the truncated
form of STAT5 (estimated molecular weight, 78 kd) in 3 clones of
Ba/F3p210STAT5 (lower panel). No induction of STAT5 protein was
found in Ba/F3p210Ctrl cells. Probing with an Abl antibody
revealed expression of c-abl (145 kd) and of p210Bcr/Abl (210 kd) (upper panel).
|
|
Interference with tyrosine phosphorylation of endogenous STAT5 by
expression of STAT5
Expression of truncated forms of STAT5 was previously shown to
inhibit STAT5 activity.13,18 STAT5 was also shown to
inhibit tyrosine phosphorylation of STAT5 on stimulation with IL-3
receptor.19 Therefore, in Ba/F3p210STAT5 cells, tyrosine
phosphorylation of STAT5 was measured before and after induction of
STAT5 with doxycycline for 24 hours. Immunoprecipitation of STAT5 by
using a STAT5 Ab directed against the C-terminal of STAT5 precipitated full-length STAT5A and STAT5B (Figure 2,
middle panel). When probed with antiphosphotyrosine Ab 4G10, STAT5 was
found to be phosphorylated in unstimulated Ba/F3p210STAT5 cells as
well as in Ba/F3p210Ctrl cells (Figure 2, upper panel). Tyrosine
phosphorylation of STAT5 was reduced in Ba/F3p210STAT5 cells
when STAT5 was induced by incubation with doxycycline for 24 hours. In Ba/F3p210Ctrl cells, addition of doxycycline had no effect on
tyrosine phosphorylation of STAT5.
View larger version (31K):
[in this window]
[in a new window]
| Fig 2.
Reduction of phosphorylation of endogenous STAT5 by
STAT5 expression.
Lysates from Ba/F3p210STAT5 cells (maintained in either the presence
or absence doxycycline [1 µg/mL] for 24 hours) were incubated with
anti-STAT5 antibody C17 and protein G beads for 3 hours and then
subjected to 7.5% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and Western blotting. Probing with an
anti-STAT5 antibody raised against the SH2 and SH3 domains of STAT5
revealed equal amounts of STAT5A and STAT5B (middle panel). When probed
with antiphosphotyrosine antibody 4G10, STAT5 was found to be
constitutively phosphorylated in both Ba/F3p210Ctrl cells and
Ba/F3p210STAT5 cells (upper panel). Induction of STAT5 for 24 hours was associated with a reduction of tyrosine phosphorylation of
STAT5. A longer exposure of the blot revealed that STAT5
coprecipitated with endogenous STAT5 in Ba/F3p210STAT5 cells (lower
panel). Neither endogenous STAT5 nor STAT5 coprecipitated when
lysates were incubated with normal rabbit serum and beads.
|
|
Because STAT5 is believed to dimerize after tyrosine phosphorylation,
the possible formation of STAT5/STAT5 dimers was
investigated. Immunoprecipitation was performed with an
anti-STAT5 C-terminal Ab (this epitope is missing in STAT5). This
was followed by immunoblotting with a different STAT5 Ab that
recognizes endogenous STAT5 as well as STAT5. A small amount of
STAT5 coprecipitated with endogenous (full-length) STAT5 (Figure 2,
lower panel), suggesting that STAT5 can form heterodimers with wtSTAT5.
Inhibition by STAT5 of Bcr/Abl-dependent activation of a STAT5
reporter gene
Phosphorylation of STAT5 leads to dimerization and translocation
into the nucleus, where STAT5 dimers then activate transcription through binding to specific DNA sequences. The consensus STAT5 binding
site is TTCNNNGAA.24 Genes known to have STAT5 binding sites in their promotors include -casein, IRF1, CIS, and
BclX.24 The STAT5 binding site of the
-casein gene has previously been used for STAT5 reporter
gene constructs.13,18
In the current study, we used a reporter gene construct
(GAS-luciferase) in which a tandem repeat of 4 STAT binding motifs was
cloned upstream of the luciferase gene in the pGL2 promotor vector.
Using this reporter gene construct, we analyzed p210Bcr/Abl-transformed cells for STAT5 reporter gene activity and compared the results with
findings in nontransformed Ba/F3 cells. A -GAL reporter gene
construct was used as a transfection control. STAT5 reporter gene
activity was considerably higher in Ba/F3p210 cells (Figure 3A), (ratio of STAT5 to -GAL,
1533 ± 423) compared with untransformed Ba/F3 cells (ratio of
STAT5 to -GAL, 157 ± 30). Increased STAT5 reporter gene
activity could directly be attributed to Bcr/Abl, since incubation with
STI571 (1 µmol/L for 12 hours) inhibited STAT5 activity (ratio of
STAT5 to -GAL, 312 ± 51) in Ba/F3p210 cells but had no effect
in untransformed Ba/F3 cells (Figure 3A), (ratio of STAT5 to -GAL,
157 ± 28).
View larger version (1314K):
[in this window]
[in a new window]
| Fig 3.
Induction of STAT5 reporter gene activity with Bcr/Abl.
(A) Ba/F3p210 cells and untransformed Ba/F3 cells in RPMI plus WEHI
(10%) were transfected with the -casein luciferase reporter gene
(together with the -GAL construct). Twelve hours after transfection,
cells were split and maintained in either the presence or absence of
Bcr/Abl kinase inhibitor STI571 (1 µmol/L). Cells were then
harvested, and lysates were assayed for luciferase and -GAL
activities, expressed as a ratio of luciferase activity to
-GAL-activity. Ba/F3p210 cells expressed significantly more STAT5
reporter gene activity than did Ba/F3 cells, and the increased STAT5
activity was due directly to Bcr/Abl, since it could be abrogated by
Bcr/Abl kinase inhibitor STI571. (B) Untransformed Ba/F3 cells and
TonB.210 cells were transfected with the -casein luciferase reporter
gene (together with the -GAL construct). Twelve hours after
transfection, cells were split, maintained in either the presence or
absence of doxycycline (1 µg/mL), and assayed for luciferase and
GAL activities. Induction of Bcr/Abl in Ton B.210.1 cells
by doxycycline increased STAT5 reporter gene activity. Doxycycline
had no effect on STAT5 reporter gene activity in untransformed Ba/F3
cells.
|
|
The ability of Bcr/Abl to induce STAT5-dependent gene transcription was
confirmed by using a cell line (TonB.210) in which p210Bcr/Abl can be
induced by the addition of doxycycline.19 Doxycycline (1 µg/mL for 24 hours) significantly increased STAT5 reporter gene
activity (Figure 3B) (ratio of STAT5 to -GAL, 2089 ± 468 for
the control and 10 216 ± 1805 for doxycycline-treated cells).
Addition of doxycycline had no effect on untransformed Ba/F3 cells
(Figure 3B) (ratio of STAT5 to -GAL, 2476 ± 285 for the
control and 1982 ± 276 for doxycycline-treated cells).
To determine whether induction of STAT5 in Bcr/Abl-transformed cells
would inhibit STAT5 reporter gene activity, the Ba/F3p210STAT5 clones were transfected with the -casein reporter gene
construct. Twelve hours after transfection, cells were split and then
maintained in either the presence or absence of doxycycline (1µg/mL)
for 24 hours. STAT5 expression significantly reduced luciferase
reporter gene activity in each of the 3 clones tested (Figure
4), (C7.14, 23.9% ± 9.5% of
unstimulated control; C7.28, 28.2% ± 3.5% of unstimulated control; and C7.30, 42.0% ± 5.8% of unstimulated control). No reduction of STAT5 reporter gene activity was found in the
Ba/F3p210Ctrl cells (99.5% ± 18.6% of unstimulated control).
Therefore, doxycycline-induced expression of STAT5 in
Ba/F3p210STAT5 cells inhibited STAT5 reporter gene activity.
View larger version (12K):
[in this window]
[in a new window]
| Fig 4.
Inhibition of STAT5 reporter gene activity by  STAT5
expression.
Cells from 3 clones of Ba/F3p210STAT5 cells were transfected with
the -casein luciferase construct. Twelve hours after transfection,
cells were split and maintained in either the presence or absence of
doxycycline (1 µg/mL) for 24 hours. Cells were then assayed for
luciferase activity, and results are expressed as the percentage of
activity in unstimulated controls. Induction of STAT5 in
Ba/F3p210STAT5 cells by doxycycline significantly inhibited STAT5
reporter gene activity in all clones tested. No inhibition of STAT5
activity was observed when Ba/F3p210Ctrl cells were exposed to
doxycycline.
|
|
Inhibition of CIS protein expression but not of BclX
protein expression in Ba/F3p210 cells by induction of STAT5
CIS is a protein involved in the negative regulation of JAK/STAT
signaling.25 Binding of CIS protein to the cytoplasmic domain of a cytokine receptor is associated with inhibition of STAT
phosphorylation, thereby creating a negative feedback loop that limits
cytokine-induced STAT activation. The CIS gene, like the
-casein gene, has STAT5 binding sites upstream of its
initiation site,24,26 and STAT5 activation leads to CIS
expression.26 In the current study, CIS expression was
constitutively expressed in Ba/F3p210 cells (Figure
5A), and induction of STAT5 with
doxycycline for 24 hours reduced CIS protein expression (Figure 5A).
Two forms of CIS, a 37-kd form and a 45-kd form, were detected by
immunoblotting. The 45-kd form of CIS is likely to represent a
ubiquitinated form of CIS.27 These results show that
expression of STAT5 is associated with reduced transcription of a
known STAT5-inducible gene, which is consistent with the idea that
STAT5 functions as a dominant negative mutant.
View larger version (3327K):
[in this window]
[in a new window]
| Fig 5.
Differential regulation of STAT5-induced genes,
CIS and BclX, in Bcr/Abl
transformed cells.
(A) Inhibition of CIS protein expression by STAT5 expression.
Lysates from Ba/F3p210STAT5 cells (after treatment with or without
doxycycline [1 µg/mL] for 24 hours) were incubated with a CIS
antibody and protein G beads. Immunoprecipitates were then subjected to
SDS-PAGE (9%). Probing with anti-CIS antibody revealed constitutive
expression of CIS protein (37 kd) in Bcr/Abl-transformed cells, and
expression of STAT5 inhibited CIS protein expression. Addition of
doxycycline had no effect on CIS protein expression in Ba/F3p210Ctrl
cells. A larger, presumably ubiquitinated form of CIS protein (~ 45 kd) was also expressed in Ba/F3p210 cells. (B) Lack of effect on
BclX protein expression of induction of STAT5. Lysates
from Ba/F3 and Ba/F3p210 cells (maintained in the presence of WEHI-CM)
were subjected to SDS-PAGE. Ba/F3p210 cells expressed more BclXL
than untransformed Ba/F3 cells (upper left-hand panel). Enhanced
BclXL expression could be attributed directly to Bcr/Abl,
since incubation with Bcr/Abl kinase inhibitor STI571 (1 µmol/L for
24 hours) reversed the enhanced BclXL expression in
Ba/F3p210 cells. However, induction of STAT5 with doxycycline (1 µg/mL for 3 days) had no effect on BclXL expression in
Ba/F3p210STAT5 cells (upper right-hand panel). Induction of STAT5
in Ba/F3p210STAT5 cells is shown as a control (lower panel).
|
|
It was previously reported that the BclX
gene, like the CIS gene, contains STAT5 binding
sites,24 and induction of a constitutive active STAT5
mutant was found to enhance BclX levels.28
Bcr/Abl also induces BclX (Figure 5B, upper left panel), suggesting the possibility that BclX induction might be due
to STAT5 activation by Bcr/Abl. However, when STAT5 was induced in
Ba/F3p210STAT5 cells (using 1 µg/mL of doxycycline for 3 days), no
effect on BclX protein expression was found (Figure 5B,
upper right panel). These results suggest that, in Bcr/Abl-transformed cells, induction of CIS depends on STAT5 activity, whereas expression of BclX does not.
Effects of STAT5 expression on the growth and cell cycle
of Ba/F3p210 cells
To determine the effects of STAT5 induction on the growth of
Ba/F3p210 cells, Ba/F3p210STAT5 cells were maintained in either the
presence or absence of doxycycline (1 µg/mL), and daily cell counts
were obtained. Cells expressing STAT5 were found to grow more slowly
than untreated cells. After 4 days of culture, Ba/F3p210STAT5 cells
induced with doxycycline (1 µg/ml) grew at rates that were 26% to
52% of the growth rates of untreated cells (Figure
6). The degree of growth inhibition was
similar in each of the 3 clones tested. On day 4, the values were as
follows: C7.14, 43.2% ± 8.3% of control; C7.28,
26.2% ± 6.8% of control; and C7.30, 51.9% ± 11.8% of
control. In contrast, doxycycline had no significant effect on growth
of Ba/F3p210Ctrl cells; on day 4, the value was 87.1% ± 17.8%
of control.
View larger version (28K):
[in this window]
[in a new window]
| Fig 6.
Effects of STAT5 expression on growth of Ba/F3p210
cells.
Ba/F3p210STAT5 cells were maintained in either the presence or
absence of doxycycline (1 µg/mL), and daily cell counts were
obtained. Cell counts from cells maintained in the presence of
doxycycline are expressed as a percentage of the cell counts obtained
from cells maintained in the absence of doxycycline. Expression of
STAT5 significantly inhibited cell growth; however, the cells were
still growing although at a slower rate. No significant effect of
doxycycline was observed in Ba/F3p210Ctrl cells.
|
|
To determine whether STAT5 induced cell-cycle arrest, cell-cycle
analysis was performed by using a standard PI staining protocol and
flow cytometry. STAT5 expression did not result in G1 cell-cycle arrest or other alterations in the distribution of cells in other phases of the cell cycle (not shown).
Reduction of viability of Ba/F3p210 cells by STAT5
expression
When Ba/F3p210STAT5 cells were maintained in the presence of
doxycycline (1 µg/mL) for 2 days, a small but significant increase in
annexin V-positive (apoptotic) cells was observed in all clones tested
(Figure 7). The addition of doxycycline to
clone C7.14 increased annexin V-positive cells from 8.4% ± 1.1%
to 20.8% ± 4.9%; from 8.7% ± 0.5% to 22.5% ± 3.3% in
clone C7.28; and from 12.0% ± 1.3% to 20.5% ± 3.2% in clone
C7.30 (Figure 7). In contrast, doxycycline (1 µg/mL) had no
significant effect on viability of Ba/F3p210Ctrl cells (control,
8.4% ± 0.8% annexin V-positive cells; and doxycycline-treated
cells, 9.0% ± 1.8% annexin V-positive cells). The reduced
viability associated with STAT5 is likely to have accounted for some
or all of the reduced growth rate of Ba/F3p210STAT5 cells.
View larger version (30K):
[in this window]
[in a new window]
| Fig 7.
Effects of STAT5 on viability of Ba/F3p210 cells.
Cells from 3 clones of Ba/F3p210STAT5 cells were maintained in
either the presence or absence of doxycycline (1 µg/mL) for 2 days,
and viability was determined by using a combined annexin V/PI staining
protocol.29,30 Compared with cells not subjected to
induction (black bars), STAT5 expression induced by doxycycline
(stippled bars) increased the percentage of annexin V-positive cells
(apoptotic cells) in all clones tested. Doxycycline had no effect on
the viability of Ba/F3p210Ctrl cells.
|
|
Because many cytotoxic drugs inhibit growth of cancer cells by inducing
apoptosis, we wondered whether the presence of STAT5 would alter the
sensitivity of Bcr/Abl-transformed cells to chemotherapeutic drugs used
to treat CML. Ba/F3p210STAT5 cells were incubated with cytarabine in
either the presence or absence of doxycycline (1 µg/mL). Thirty-six
hours later, cell viability was analyzed by using annexin V/PI
staining. Incubation of uninduced Ba/F3p210STAT5 cells with
cytarabine resulted in a dose-dependent increase in apoptotic cells, as
expected (Figure 8A). Induction of STAT5 by doxycycline enhanced the sensitivity of these cells to cytarabine and produced a moderate but reproducible shift in the
dose-response curve. Doxycycline had no effect on the sensitivity of
Ba/F3p210Ctrl cells to cytarabine. However, Ba/F3p210Ctrl cells were
less sensitive to cytarabine than were uninduced Ba/F3p210STAT5
cells. This may represent clonal variation, or it could reflect
low-level expression of STAT5 due to leakiness of the inducible
vector. Similar results were obtained when Ba/F3p210STAT5 cells were incubated with various concentrations of hydroxyurea for 5 hours. The
percentage of apoptotic cells was higher after STAT5 induction than
in cells not subjected to induction (Figure 8B). Viability in response
to hydroxyurea did not change when Ba/F3p210Ctrl cells were incubated
with doxycycline. These results show that STAT5 activation contributes
to viability in Ba/F3p210 and that withdrawal of STAT5 activity
synergizes with the cytotoxic effects of cytarabine or
hydroxyurea.
View larger version (1518K):
[in this window]
[in a new window]
| Fig 8.
Effects of STAT5 expression and coincubation with
cytarabine and hydroxyurea on viability of Ba/F3p210 cells.
(A) The effects of STAT5 expression on viability were tested after
cells were incubated with various concentrations of cytarabine
(1000-31.6 nmol/L) for 36 hours. When coincubated with doxycycline (1 µg/mL) ( ), Ba/F3p210STAT5 cells were more susceptible to
killing induced by cytarabine than were cells maintained in the absence
of doxycycline ( ). No significant effect on viability was observed
when Ba/F3p210Ctrl cells were coincubated with doxycycline and
cytarabine. (B) Ba/F3p210STAT5 cells were coincubated in either the
presence () or absence () of doxycycline (1 µg/mL) with various
doses of hydroxyurea for 5 hours. After 36 hours, they were analyzed
for viability. Coincubation with doxycycline enhanced killing induced
by various doses of hydroxyurea. No effect of doxycycline on viability
was observed when Ba/F3p210Ctrl cells were coincubated with
hydroxyurea.
|
|
| |
Discussion |
CML is a malignant disorder of hematopoietic stem cells caused by
the Bcr/Abl oncogene. The disease is characterized by an abnormal
expansion of hematopoietic progenitor cells and accumulation of mature
myeloid cells in the blood, spleen, and bone marrow. Bcr/Abl has been
shown to constitutively activate a number of different signaling
pathways that under normal circumstances are tightly regulated by such
cytokines as IL-3 or GM-CSF. The result is enhanced proliferation,
prolonged viability, altered adhesion, and increased mobility.
It was previously shown that STAT5 is constitutively phosphorylated in
p210Bcr/Abl-transformed cells and binds to DNA in gel-shift assays.7 Our current study extends these observations by
showing that Bcr/Abl was associated with increased STAT5-dependent gene transcription with use of a STAT5 reporter gene assay and constitutive expression of a STAT5-regulated gene, CIS. These activities
were due directly to Bcr/Abl, since they could be inhibited by
treatment of cells with the Abl tyrosine kinase inhibitor STI571.
Furthermore, we found that STAT5 activity is higher in Ba/F3 cells
transformed with p210Bcr/Abl than in untransformed Ba/F3 cells, even
when the latter cells were maintained in the presence of IL-3.
A tetracycline-inducible promoter was used to regulate expression of a
truncated form of STAT5B in Ba/F3p210 cells. This mutant was truncated
at aa 683 and lacked the transcriptional activation domain and the
major tyrosine phosphorylation site of STAT5B at aa 699. STAT5
functioned as a dominant negative inhibitor of wild-type STAT5
function, since it inhibited GAS-luciferase activity and also reduced
expression of CIS, a STAT5-regulated gene. Induction of
STAT5 resulted in a slower growth rate but did not induce cell-cycle
arrest. This finding suggested that STAT5 might reduce viability,
and when viability was analyzed by the sensitive technique of annexin V
staining, expression of STAT5 resulted in a reduction in viability
sufficient to account for the slower growth rate. Moreover, induction
of STAT5 activity was associated with increased sensitivity to the
apoptotic effects of hydroxyurea and cytarabine, 2 chemotherapeutic
drugs commonly used to treat CML. A reduction in viability with
STAT5 expression in Bcr/Abl-transformed 32Dcl3 cells was observed by
Nieborowska-Skorska et al.31 However, in
contrast to what we observed in Ba/F3p210 cells, STAT5 induction in
the 32Dcl3p210 cells in their study led to inhibition of cell-cycle progression. The different results in the 2 studies could possibly be
attributed to phenotypic differences between the cell lines used.
The Bcr/Abl oncogene exerts several biologic effects on hematopoietic
cells that are partly dependent on the model system being studied. In
cell-line models and in vivo studies in mice, Bcr/Abl is mitogenic,
induces factor independence,22 inhibits apoptosis,32 and alters integrin-mediated adhesion and
motility.33 In primary CML cells, the major defects
associated with Bcr/Abl are aberrant regulation of
adhesion33 and possibly enhanced motility34 and
viability,32 although not all investigators agree on the
latter.35,36 Studies have suggested, however, that very immature primary myeloid cells from patients in the stable
phase of CML are more likely to show enhanced viability and
growth-factor independence.37 Despite the identification of
many different signaling pathways activated by Bcr/Abl, it has been
difficult to link any specific signaling event to a specific biologic
effect. Thus, our results showing a link between constitutive activation of STAT5 and enhanced viability of Bcr/Abl-transformed cells
are especially interesting.
Enhanced viability of myeloid lineage cells would be expected to result
in an accumulation of cells with relatively normal function, in
accordance with the known clinical phenotype of CML. In fact, available
data suggest that Bcr/Abl activates several biochemical pathways that
enhance viability. For example, Bcr/Abl is known to activate p21ras
through binding of GRB2/SOS to tyrosine 177 of Bcr3 and
probably also through SHP2, which is tyrosine phosphorylated by
Bcr/Abl.38 Ras has been linked to viability signaling in a
number of different cell types.39,40 Also, Bcr/Abl is known
to activate PI3K, which initiates a pathway that enhances viability,
possibly through activation of Akt and subsequent phosphorylation of
Bad.5 Finally, Bcr/Abl induces expression of the
antiapoptotic mitochondrial protein, BclX.41
Because the BclX promotor has STAT5 binding
sites24 and we and others28 have shown that a
constitutive active mutant of STAT5 can induce BclX, we
wondered whether Bcr/Abl induction of STAT5 was required for increased BclX expression. Although we found that increased CIS
expression was dependent on STAT5, expression of BclX
was not.
These results suggest that the decreased viability induced by STAT5
is not due to decreased levels of BclX and that the
increased expression of BclX induced by Bcr/Abl is not
mediated only by STAT5. Overall, the finding of multiple signaling
pathways to viability suggests that this is likely to be an important
aspect of Bcr/Abl function and that inhibition of any single pathway is
unlikely to eliminate the viability effect of this oncogene, as we
showed here. This hypothesis can be tested more formally when mice with
targeted disruption of genes involved in viability signaling pathways,
including STAT5, are tested for sensitivity to Bcr/Abl transformation.
Our data suggest that the defect will be small unless hematopoietic
cells defective in multiple viability signaling pathways are used.
In normal hematopoiesis, the role of STAT5 remains unclear, since adult
mice in which both alleles of STAT5A and STAT5B are inactivated by gene
targeting have largely normal blood counts.17 However,
STAT5 activation still seems to be required to support immature
hematopoiesis, since the STAT5A and STAT5B double-knockout mice had a
reduction in myeloid progenitor numbers in the bone marrow.
Furthermore, a study has suggested that STAT5 is required for normal
fetal erythropoiesis.16 Even if STAT5 turns out not to have
a major role in postnatal hematopoiesis, constitutive activation of
STAT5, as described here for CML cells, may confer an important
phenotype. This idea is supported by a previous study showing that
point mutations of the STAT5 molecule leading to a constitutively
active form of STAT5 can support growth of Ba/F3 cells in the absence
of murine IL-3.42 An analogous situation is that with IL-3
or GM-CSF: gene-targeting studies indicated that neither cytokine is
required for normal hematopoiesis,43 but when the cytokines
are expressed constitutively in transgenic mice, both cause a
myeloproliferative disorder.44-46
The mechanism whereby STAT5 functions as a dominant negative
inhibitor of STAT5 function in Bcr/Abl-transformed cells is of
interest. Mui and colleagues18 showed that
STAT5 inhibits tyrosine phosphorylation of wild-type STAT5, and our
current findings confirm those results. The mechanism of reduced
tyrosine phosphorylation is unclear, but it is possible that STAT5
binds to the kinase (Bcr/Abl may or may not be the kinase in this case)
and then fails to disengage because it lacks the major tyrosine
phosphorylation site and is therefore not phosphorylated. STAT5
would thereby block binding and subsequent phosphorylation of wild-type
STAT5. We also found here that a small amount of STAT5 can
coprecipitate with STAT5, suggesting that heterodimers are formed
either in vivo or in vitro. Because the transcriptional activation
domain of STAT5 has been deleted, the resulting heterodimer would be expected to have reduced ability to transactivate
STAT5-dependent genes. The formation of heterodimers
presumably depends on the binding of the SH2 domain of STAT5 to
phosphotyrosine SH2-binding sites on wild-type STAT5.
Overall, our data suggest a model in which STAT5 activation by Bcr/Abl
helps maintain viability in hemopoietic cells, thereby contributing to
myeloproliferative disease. It will be of interest to determine the
exact mechanisms involved, particularly the role of
BclX and other viability genes that may be
regulated by STAT5, and to use dominant negative mutants of STAT5 in
the transformation of primary fetal and adult primary cells by
activated Abl oncogenes. Such studies should complement studies in
STAT5 knockout mice and provide a better understanding of the role of this pathway in CML. Finally, it will be important to determine the
contribution of the STAT5 pathway relative to that of other viability
signaling pathways, including p21ras and PI3K/Akt.
| |
Footnotes |
Submitted July 20, 1999; accepted November 17, 1999.
Supported by grants J1458-MED and J1707-MED from the Austrian
Fonds zur Förderung der wissenschaftlichen Forschung in
Österreich (C.S.), fellowship grant FIJC-95/INT from the
José Carreras International Leukemia Foundation (M.S.), the
Association pour la Recherche sur le Cancer (F.G.), and grants
CA36167, CA66996, and DK50654 from the National Institutes of Health
(J.D.G.).
Reprints: James D. Griffin, Dana Farber Cancer Institute,
Department of Adult Oncology, 44 Binney Street, Boston, MA 02115;
email: james_griffin{at}dfci.harvard.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
1.
Nowell PC, Hungerford DA.
A minute chromosome in human chronic granulocytic leukemia.
Science.
1960;132:1497.
2.
Konopka JB, Witte ON.
Detection of c-abl tyrosine kinase activity in vitro permits direct comparison of normal and altered abl products.
Mol Cell Biol.
1985;5:3116[Abstract/Free Full Text].
3.
Pendergast AM, Quilliam LA, Cripe LD, et al.
BCR-ABL-induced oncogenesis is mediated by direct interaction with the SH2 domain of the GRB-2 adaptor protein.
Cell.
1993;75:175[Medline]
[Order article via Infotrieve].
4.
Mandanas RA, Leibowitz DS, Gharehbaghi K, et al.
Role of p21 RAS in p210 bcr-abl transformation of murine myeloid cells.
Blood.
1993;82:1838[Abstract/Free Full Text].
5.
Skorski T, Bellacosa A, Nieborowska-Skorska M, et al.
Transformation of hematopoietic cells by BCR/ABL requires activation of a PI-3k/Akt-dependent pathway.
EMBO J.
1997;16:6151[Medline]
[Order article via Infotrieve].
6.
Skorski T, Nieborowska-Skorska M, Wlodarski P, et al.
The SH3 domain contributes to BCR/ABL-dependent leukemogenesis in vivo: role in adhesion, invasion, and homing.
Blood.
1998;91:406[Abstract/Free Full Text].
7.
Carlesso N, Frank DA, Griffin JD.
Tyrosyl phosphorylation and DNA binding activity of signal transducers and activators of transcription (STAT) proteins in hematopoietic cell lines transformed by Bcr/Abl.
J Exp Med.
1996;183:811[Abstract/Free Full Text].
8.
Ilaria RL Jr, Van Etten RA.
P210 and P190 (BCR/ABL) induce the tyrosine phosphorylation and DNA binding activity of multiple specific STAT family members.
J Biol Chem.
1996;271:31,704[Abstract/Free Full Text].
9.
Mui AL, Wakao H, O'Farrell AM, Harada N, Miyajima A.
Interleukin-3, granulocyte-macrophage colony stimulating factor and interleukin-5 transduce signals through two STAT5 homologs.
EMBO J.
1995;14:1166[Medline]
[Order article via Infotrieve].
10.
Schwaller J, Frantsve J, Aster J, et al.
Transformation of hematopoietic cell lines to growth-factor independence and induction of a fatal myelo- and lymphoproliferative disease in mice by retrovirally transduced TEL/JAK2 fusion genes.
EMBO J.
1998;17:5321[Medline]
[Order article via Infotrieve].
11.
Okuda K, Golub TR, Gilliland DG, Griffin JD.
p210BCR/ABL, p190BCR/ABL, and TEL/ABL activate similar signal transduction pathways in hematopoietic cell lines.
Oncogene.
1996;13:1147[Medline]
[Order article via Infotrieve].
12.
Shuai K, Horvath CM, Huang LH, et al.
Interferon activation of the transcription factor Stat91 involves dimerization through SH2-phosphotyrosyl peptide interactions.
Cell.
1994;76:821[Medline]
[Order article via Infotrieve].
13.
Morrigl R, Gouilleux-Gruart V, Jahne R, et al.
Deletion of the carboxy-terminal transactivation domain of MGF-STAT5 results in sustained DNA-binding and a dominant negative phenotype.
Mol Cell Biol.
1996;16:5691[Abstract].
14.
Matsumoto A, Masuhara M, Mitsui K, et al.
CIS, a cytokine inducible SH2 protein, is a target of the JAK-STAT5 pathway and modulates STAT5 activation.
Blood.
1997;89:3148[Abstract/Free Full Text].
15.
Yoshimura A, Ichihara M, Kinjyo I, et al.
Mouse oncostatin M: an immediate early gene induced by multiple cytokines through the JAK-STAT5 pathway.
EMBO J.
1996;15:1055[Medline]
[Order article via Infotrieve].
16.
Socolovsky M, Fallon AEJ, Brugnara C, Lodish HF.
Fetal anemia and apoptosis of red-cell progenitors in Stat5a /5b/ mice: a direct role for Stat5 in Bcl-X(L) induction.
Cell.
1999;98:181[Medline]
[Order article via Infotrieve].
17.
Teglund S, McKay C, Schuetz E, et al.
Stat5a and Stat5b proteins have essential and nonessential, or redundant, roles in cytokine responses.
Cell.
1998;93:841[Medline]
[Order article via Infotrieve].
18.
Mui AL, Wakao H, Kinoshita T, Kitamura T, Miyajima A.
Suppression of interleukin-3-induced gene expression by a C-terminal truncated Stat5: role of Stat5 in proliferation.
EMBO J.
1996;15:2425[Medline]
[Order article via Infotrieve].
19.
Klucher KM, Lopez DV, Daley GQ.
Secondary mutation maintains the transformed state in BaF3 cells with inducible BCR/ABL expression.
Blood.
1998;91:3927[Abstract/Free Full Text].
20.
Daley GQ, Van Etten RA, Baltimore D.
Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chromosome.
Science.
1990;247:824[Abstract/Free Full Text].
21.
Jaster R, Zhu Y, Pless M, Bhattacharya S, Mathey-Prevot B, D'Andrea AD.
Jak2 is required for induction of the murine DUB-1 gene.
Mol Cell Biol.
1997;17:3364[Abstract].
22.
Daley GQ, Baltimore D.
Transformation of an interleukin 3-dependent hematopoietic cell line by the chronic myelogenous leukemia-specific P210bcr/abl protein.
Proc Natl Acad Sci U S A.
1988;85:9312[Abstract/Free Full Text].
23.
Druker BJ, Tamura S, Buchdunger E, et al.
Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells.
Nat Med.
1996;2:561[Medline]
[Order article via Infotrieve].
24.
Boucheron C, Dumon S, Santos SCR, et al.
A single amino acid in the DNA binding regions of STAT5a and STAT5b confers distinct DNA binding specificities.
J Biol Chem.
1998;273:33,936[Abstract/Free Full Text].
25.
Yoshimura A, Ohkubo T, Kiguchi T, et al.
A novel cytokine-inducible gene encodes an SH2-containing protein that binds to tyrosine-phosphorylated interleukin 3 and erythropoietin receptors.
EMBO J.
1995;14:2816[Medline]
[Order article via Infotrieve].
26.
Verdier F, Rabionet R, Gouilleux F, et al.
A sequence of the CIS gene promoter interacts preferentially with two associated STAT5A dimers: a distinct biochemical difference between STAT5A and STAT5B.
Mol Cell Biol.
1998;18:5852[Abstract/Free Full Text].
27.
Verdier F, Chretien S, Muller O, et al.
Proteasomes regulate erythropoietin receptor and signal transducer and activator of transcription 5 (STAT5) activation: possible involvement of the ubiquitinated Cis protein.
J Biol Chem.
1998;273:28,185[Abstract/Free Full Text].
28.
Nosaka T, Kawashima T, Misawa K, Ikuta K, Mui ALF, Kitamura T.
STAT5 as a molecular regulator of proliferation, differentiation and apoptosis in hematopoietic cells.
EMBO J.
1999;18:4754[Medline]
[Order article via Infotrieve].
29.
Verhoven B, Schlegel R, Williamson P.
Mechanism of phosphatidylserine exposure, a phagocyte recognition signal, on apoptotic T lymphocytes.
J Exp Med.
1995;182:1597[Abstract/Free Full Text].
30.
Klampfer L, Gammenga J, Wisniewski HG, Nimer SD.
Sodium salicylate activates caspases and induces apoptosis of myeloid leukemia cell lines.
Blood.
1999;93:2386[Abstract/Free Full Text].
31.
Nieborowska-Skorska M, Wasik MA, Slupianek A, et al.
Signal transducer and activator of transcription (STAT)5 activation by BCR/ABL is dependent on intact Src homology (SH)3 and SH2 domains of BCR/ABL and is required for leukemogenesis.
J Exp Med.
1999;189:1229[Abstract/Free Full Text].
32.
Bedi A, Zehnbauer BA, Barber JP, Sharkis SJ, Jones RJ.
Inhibition of apoptosis by BCR-ABL in chronic myeloid leukemia.
Blood.
1994;83:2038[Abstract/Free Full Text].
33.
Gordon MY, Dowding CR, Riley GP, Goldman JM, Greaves MF.
Altered adhesive interactions with marrow stroma of haematopoietic progenitor cells in chronic myeloid leukaemia.
Nature.
1987;328:342[Medline]
[Order article via Infotrieve].
34.
Salgia R, Li JL, Ewaniuk DS, et al.
BCR/ABL induces multiple abnormalities of cytoskeletal function.
J Clin Invest.
1997;100:46[Medline]
[Order article via Infotrieve].
35.
Albrecht T, Schwab R, Henkes M, Peschel C, Huber C, Aulitzky WE.
Primary proliferating immature myeloid cells from CML patients are not resistant to induction of apoptosis by DNA damage and growth factor withdrawal.
Br J Haematol.
1996;95:501[Medline]
[Order article via Infotrieve].
36.
Amos TA, Lewis JL, Grand FH, Gooding RP, Goldman JM, Gordon MY.
Apoptosis in chronic myeloid leukaemia: normal responses by progenitor cells to growth factor deprivation, X-irradiation and glucocorticoids.
Br J Haematol.
1995;91:387[Medline]
[Order article via Infotrieve].
37.
Jiang X, Fujisaki T, Berger M, Eaves A, Eaves C.
Autonomous multi-lineage differentiation in vitro of primitive PH+ CD34+ cells from patients with chronic myelogenous leukemia (CML) [abstract].
Blood.
1998;92:1041a.
38.
Tauchi T, Feng GS, Shen R, et al.
SH2-containing phosphotyrosine phosphatase Syp is a target of p210bcr-abl tyrosine kinase.
J Biol Chem.
1994;269:15,381[Abstract/Free Full Text].
39.
Chen CY, Faller DV.
Direction of p21ras-generated signals towards cell growth or apoptosis is determined by protein kinase C and Bcl-2.
Oncogene.
1995;11:1487[Medline]
[Order article via Infotrieve].
40.
Kinoshita T, Yokota T, Arai K, Miyajima A.
Regulation of Bcl-2 expression by oncogenic Ras protein in hematopoietic cells.
Oncogene.
1995;10:2207[Medline]
[Order article via Infotrieve].
41.
Amarante-Mendes GP, McGahon AJ, Nishioka WK, Afar DE, Witte ON, Green DR.
Bcl-2-independent Bcr-Abl-mediated resistance to apoptosis: protection is correlated with up regulation of Bcl-XL.
Oncogene.
1998;16:1383[Medline]
[Order article via Infotrieve].
42.
Onishi M, Nosaka T, Misawa K, et al.
Identification and characterization of a constitutively active STAT5 mutant that promotes cell proliferation.
Mol Cell Biol.
1998;18:3871[Abstract/Free Full Text].
43.
Nishinakamura R, Miyajima A, Mee PJ, Tybulewicz VL, Murray R.
Hematopoiesis in mice lacking the entire granulocyte-macrophage colony-stimulating factor/interleukin-3/interleukin-5 functions.
Blood.
1996;88:2458[Abstract/Free Full Text].
44.
Wong PM, Chung SW, Dunbar CE, Bodine DM, Ruscetti S, Nienhuis AW.
Retrovirus-mediated transfer and expression of the interleukin-3 gene in mouse hematopoietic cells result in a myeloproliferative disorder.
Mol Cell Biol.
1989;9:798[Abstract/Free Full Text].
45.
Lang RA, Metcalf D, Cuthbertson RA, et al.
Transgenic mice expressing a hemopoietic growth factor gene (GM-CSF) develop accumulations of macrophages, blindness, and a fatal syndrome of tissue damage.
Cell.
1987;51:675[Medline]
[Order article via Infotrieve].
46.
D'Andrea RJ, Harrison-Findik D, Butcher CM, et al.
Dysregulated hematopoiesis and a progressive neurological disorder induced by expression of an activated form of the human common beta chain in transgenic mice.
J Clin Invest.
1998;102:1951[Medline]
[Order article via Infotrieve].
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
C. Baumgartner, S. Cerny-Reiterer, K. Sonneck, M. Mayerhofer, K. V. Gleixner, R. Fritz, M. Kerenyi, C. Boudot, F. Gouilleux, J.-W. Kornfeld, et al.
Expression of Activated STAT5 in Neoplastic Mast Cells in Systemic Mastocytosis: Subcellular Distribution and Role of the Transforming Oncoprotein KIT D816V
Am. J. Pathol.,
December 1, 2009;
175(6):
2416 - 2429.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Motiwala, S. Majumder, K. Ghoshal, H. Kutay, J. Datta, S. Roy, D. M. Lucas, and S. T. Jacob
PTPROt Inactivates the Oncogenic Fusion Protein BCR/ABL and Suppresses Transformation of K562 Cells
J. Biol. Chem.,
January 2, 2009;
284(1):
455 - 464.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Huguet, N. Giocanti, C. Hennequin, M. Croisy, E. Touboul, and V. Favaudon
Growth inhibition by STI571 in combination with radiation in human chronic myelogenous leukemia K562 cells
Mol. Cancer Ther.,
February 1, 2008;
7(2):
398 - 406.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. A. Mallette, M.-F. Gaumont-Leclerc, G. Huot, and G. Ferbeyre
Myc Down-regulation as a Mechanism to Activate the Rb Pathway in STAT5A-induced Senescence
J. Biol. Chem.,
November 30, 2007;
282(48):
34938 - 34944.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. V. Gleixner, M. Mayerhofer, K. Sonneck, A. Gruze, P. Samorapoompichit, C. Baumgartner, F. Y. Lee, K. J. Aichberger, P. W. Manley, D. Fabbro, et al.
Synergistic growth-inhibitory effects of two tyrosine kinase inhibitors, dasatinib and PKC412, on neoplastic mast cells expressing the D816V-mutated oncogenic variant of KIT
Haematologica,
November 1, 2007;
92(11):
1451 - 1459.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Schepers, D. van Gosliga, A. T. J. Wierenga, B. J. L. Eggen, J. J. Schuringa, and E. Vellenga
STAT5 is required for long-term maintenance of normal and leukemic human stem/progenitor cells
Blood,
October 15, 2007;
110(8):
2880 - 2888.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Kondo, K. V. Gleixner, M. Mayerhofer, A. Vales, A. Gruze, P. Samorapoompichit, K. Greish, M.-T. Krauth, K. J. Aichberger, W. F. Pickl, et al.
Identification of heat shock protein 32 (Hsp32) as a novel survival factor and therapeutic target in neoplastic mast cells
Blood,
July 15, 2007;
110(2):
661 - 669.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y.-Y. Kuo and Z.-F. Chang
GATA-1 and Gfi-1B Interplay To Regulate Bcl-xL Transcription
Mol. Cell. Biol.,
June 15, 2007;
27(12):
4261 - 4272.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Walz, B. J. Crowley, H. E. Hudon, J. L. Gramlich, D. S. Neuberg, K. Podar, J. D. Griffin, and M. Sattler
Activated Jak2 with the V617F Point Mutation Promotes G1/S Phase Transition
J. Biol. Chem.,
June 30, 2006;
281(26):
18177 - 18183.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Hoelbl, B. Kovacic, M. A. Kerenyi, O. Simma, W. Warsch, Y. Cui, H. Beug, L. Hennighausen, R. Moriggl, and V. Sexl
Clarifying the role of Stat5 in lymphoid development and Abelson-induced transformation
Blood,
June 15, 2006;
107(12):
4898 - 4906.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Ye, N. Wolff, L. Li, S. Zhang, and R. L. Ilaria Jr
STAT5 signaling is required for the efficient induction and maintenance of CML in mice
Blood,
June 15, 2006;
107(12):
4917 - 4925.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Nieborowska-Skorska, G. Hoser, L. Rink, M. Malecki, P. Kossev, M. A. Wasik, and T. Skorski
Id1 transcription inhibitor-matrix metalloproteinase 9 axis enhances invasiveness of the breakpoint cluster region/abelson tyrosine kinase-transformed leukemia cells.
Cancer Res.,
April 15, 2006;
66(8):
4108 - 4116.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Scherr, A. Chaturvedi, K. Battmer, I. Dallmann, B. Schultheis, A. Ganser, and M. Eder
Enhanced sensitivity to inhibition of SHP2, STAT5, and Gab2 expression in chronic myeloid leukemia (CML)
Blood,
April 15, 2006;
107(8):
3279 - 3287.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. V. Gleixner, M. Mayerhofer, K. J. Aichberger, S. Derdak, K. Sonneck, A. Bohm, A. Gruze, P. Samorapoompichit, P. W. Manley, D. Fabbro, et al.
PKC412 inhibits in vitro growth of neoplastic human mast cells expressing the D816V-mutated variant of KIT: comparison with AMN107, imatinib, and cladribine (2CdA) and evaluation of cooperative drug effects
Blood,
January 15, 2006;
107(2):
752 - 759.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. J. Aichberger, M. Mayerhofer, M.-T. Krauth, A. Vales, R. Kondo, S. Derdak, W. F. Pickl, E. Selzer, M. Deininger, B. J. Druker, et al.
Low-Level Expression of Proapoptotic Bcl-2-Interacting Mediator in Leukemic Cells in Patients with Chronic Myeloid Leukemia: Role of BCR/ABL, Characterization of Underlying Signaling Pathways, and Reexpression by Novel Pharmacologic Compounds
Cancer Res.,
October 15, 2005;
65(20):
9436 - 9444.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Zhuang, Y. Qiu, S. J. Haque, and F. Dong
Tyrosine 729 of the G-CSF receptor controls the duration of receptor signaling: involvement of SOCS3 and SOCS1
J. Leukoc. Biol.,
October 1, 2005;
78(4):
1008 - 1015.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Feldhahn, F. Klein, J. L. Mooster, P. Hadweh, M. Sprangers, M. Wartenberg, M. M. Bekhite, W.-K. Hofmann, S. Herzog, H. Jumaa, et al.
Mimicry of a constitutively active pre-B cell receptor in acute lymphoblastic leukemia cells
J. Exp. Med.,
June 6, 2005;
201(11):
1837 - 1852.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. J. Gu, L. Santiago, and B. S. Mitchell
Synergy between imatinib and mycophenolic acid in inducing apoptosis in cell lines expressing Bcr-Abl
Blood,
April 15, 2005;
105(8):
3270 - 3277.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. J. Aichberger, M. Mayerhofer, M.-T. Krauth, H. Skvara, S. Florian, K. Sonneck, C. Akgul, S. Derdak, W. F. Pickl, V. Wacheck, et al.
Identification of mcl-1 as a BCR/ABL-dependent target in chronic myeloid leukemia (CML): evidence for cooperative antileukemic effects of imatinib and mcl-1 antisense oligonucleotides
Blood,
April 15, 2005;
105(8):
3303 - 3311.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Rahmani, E. Reese, Y. Dai, C. Bauer, L. B. Kramer, M. Huang, R. Jove, P. Dent, and S. Grant
Cotreatment with Suberanoylanilide Hydroxamic Acid and 17-Allylamino 17-demethoxygeldanamycin Synergistically Induces Apoptosis in Bcr-Abl+ Cells Sensitive and Resistant to STI571 (Imatinib Mesylate) in Association with Down-Regulation of Bcr-Abl, Abrogation of Signal Transducer and Activator of Transcription 5 Activity, and Bax Conformational Change
Mol. Pharmacol.,
April 1, 2005;
67(4):
1166 - 1176.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Guo, C. Sigua, P. Bali, P. George, W. Fiskus, A. Scuto, S. Annavarapu, A. Mouttaki, G. Sondarva, S. Wei, et al.
Mechanistic role of heat shock protein 70 in Bcr-Abl-mediated resistance to apoptosis in human acute leukemia cells
Blood,
February 1, 2005;
105(3):
1246 - 1255.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. W. Sternberg and D. G. Gilliland
The Role of Signal Transducer and Activator of Transcription Factors in Leukemogenesis
J. Clin. Oncol.,
January 15, 2004;
22(2):
361 - 371.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Mohapatra, B. Chu, S. Wei, J. Djeu, P. K. Epling-Burnette, T. Loughran, R. Jove, and W. J. Pledger
Roscovitine Inhibits STAT5 Activity and Induces Apoptosis in the Human Leukemia Virus Type 1-Transformed Cell Line MT-2
Cancer Res.,
December 1, 2003;
63(23):
8523 - 8530.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. J. Irvin, L. D. Wood, L. Wang, R. Fenrick, C. G. Sansam, G. Packham, M. Kinch, E. Yang, and S. W. Hiebert
TEL, a Putative Tumor Suppressor, Induces Apoptosis and Represses Transcription of Bcl-XL
J. Biol. Chem.,
November 21, 2003;
278(47):
46378 - 46386.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Laurent, D. L. Mitchell, Z. Estrov, M. Lowery, S. L. Tucker, M. Talpaz, and R. Kurzrock
Impact of p210Bcr-Abl on Ultraviolet C Wavelength-induced DNA Damage and Repair
Clin. Cancer Res.,
September 1, 2003;
9(10):
3722 - 3730.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. W. N. Deininger and B. J. Druker
Specific Targeted Therapy of Chronic Myelogenous Leukemia with Imatinib
Pharmacol. Rev.,
September 1, 2003;
55(3):
401 - 423.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Nimmanapalli, L. Fuino, P. Bali, M. Gasparetto, M. Glozak, J. Tao, L. Moscinski, C. Smith, J. Wu, R. Jove, et al.
Histone Deacetylase Inhibitor LAQ824 Both Lowers Expression and Promotes Proteasomal Degradation of Bcr-Abl and Induces Apoptosis of Imatinib Mesylate-sensitive or -refractory Chronic Myelogenous Leukemia-Blast Crisis Cells
Cancer Res.,
August 15, 2003;
63(16):
5126 - 5135.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. G. Chiorean, S. J. Dylla, K. Olsen, T. Lenvik, Y. Soignier, and J. S. Miller
BCR/ABL alters the function of NK cells and the acquisition of killer immunoglobulin-like receptors (KIRs)
Blood,
May 1, 2003;
101(9):
3527 - 3533.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Benekli, M. R. Baer, H. Baumann, and M. Wetzler
Signal transducer and activator of transcription proteins in leukemias
Blood,
April 15, 2003;
101(8):
2940 - 2954.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F.-X. Mahon, F. Belloc, V. Lagarde, C. Chollet, F. Moreau-Gaudry, J. Reiffers, J. M. Goldman, and J. V. Melo
MDR1 gene overexpression confers resistance to imatinib mesylate in leukemia cell line models
Blood,
March 15, 2003;
101(6):
2368 - 2373.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Ravandi, M. Talpaz, and Z. Estrov
Modulation of Cellular Signaling Pathways: Prospects for Targeted Therapy in Hematological Malignancies
Clin. Cancer Res.,
February 1, 2003;
9(2):
535 - 550.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. M. Golas, K. Arndt, C. Etienne, J. Lucas, D. Nardin, J. Gibbons, P. Frost, F. Ye, D. H. Boschelli, and F. Boschelli
SKI-606, a 4-Anilino-3-quinolinecarbonitrile Dual Inhibitor of Src and Abl Kinases, Is a Potent Antiproliferative Agent against Chronic Myelogenous Leukemia Cells in Culture and Causes Regression of K562 Xenografts in Nude Mice
Cancer Res.,
January 15, 2003;
63(2):
375 - 381.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. L. Bradley, T. S. Hawley, and K. D. Bunting
Cell intrinsic defects in cytokine responsiveness of STAT5-deficient hematopoietic stem cells
Blood,
December 1, 2002;
100(12):
3983 - 3989.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Mayerhofer, P. Valent, W. R. Sperr, J. D. Griffin, and C. Sillaber
BCR/ABL induces expression of vascular endothelial growth factor and its transcriptional activator, hypoxia inducible factor-1alpha , through a pathway involving phosphoinositide 3-kinase and the mammalian target of rapamycin
Blood,
November 15, 2002;
100(10):
3767 - 3775.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Nimmanapalli, E. O'Bryan, M. Huang, P. Bali, P. K. Burnette, T. Loughran, J. Tepperberg, R. Jove, and K. Bhalla
Molecular Characterization and Sensitivity of STI-571 (Imatinib Mesylate, Gleevec)-resistant, Bcr-Abl-positive, Human Acute Leukemia Cells to SRC Kinase Inhibitor PD180970 and 17-Allylamino-17-demethoxygeldanamycin
Cancer Res.,
October 15, 2002;
62(20):
5761 - 5769.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Slupianek, G. Hoser, I. Majsterek, A. Bronisz, M. Malecki, J. Blasiak, R. Fishel, and T. Skorski
Fusion Tyrosine Kinases Induce Drug Resistance by Stimulation of Homology-Dependent Recombination Repair, Prolongation of G2/M Phase, and Protection from Apoptosis
Mol. Cell. Biol.,
June 15, 2002;
22(12):
4189 - 4201.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Nieborowska-Skorska, G. Hoser, P. Kossev, M. A. Wasik, and T. Skorski
Complementary functions of the antiapoptotic protein A1 and serine/threonine kinase pim-1 in the BCR/ABL-mediated leukemogenesis
Blood,
May 29, 2002;
99(12):
4531 - 4539.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Buettner, L. B. Mora, and R. Jove
Activated STAT Signaling in Human Tumors Provides Novel Molecular Targets for Therapeutic Intervention
Clin. Cancer Res.,
April 1, 2002;
8(4):
945 - 954.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Sonoyama, I. Matsumura, S. Ezoe, Y. Satoh, X. Zhang, Y. Kataoka, E. Takai, M. Mizuki, T. Machii, H. Wakao, et al.
Functional Cooperation among Ras, STAT5, and Phosphatidylinositol 3-Kinase Is Required for Full Oncogenic Activities of BCR/ABL in K562 Cells
J. Biol. Chem.,
March 1, 2002;
277(10):
8076 - 8082.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. F. Dorsey, J. M. Cunnick, S. M. Mane, and J. Wu
Regulation of the Erk2-Elk1 signaling pathway and megakaryocytic differentiation of Bcr-Abl+ K562 leukemic cells by Gab2
Blood,
February 15, 2002;
99(4):
1388 - 1397.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Ghaffari, C. Kitidis, M. D. Fleming, H. Neubauer, K. Pfeffer, and H. F. Lodish
Erythropoiesis in the absence of janus-kinase 2: BCR-ABL induces red cell formation in JAK2-/- hematopoietic progenitors
Blood,
November 15, 2001;
98(10):
2948 - 2957.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. C. Wolff and R. L. Ilaria Jr
Establishment of a murine model for therapy-treated chronic myelogenous leukemia using the tyrosine kinase inhibitor STI571
Blood,
November 1, 2001;
98(9):
2808 - 2816.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. M. Gelfanov, G. S. Burgess, S. Litz-Jackson, A. J. King, M. S. Marshall, H. Nakshatri, and H. S. Boswell
Transformation of interleukin-3-dependent cells without participation of Stat5/bcl-xL: cooperation of akt with raf/erk leads to p65 nuclear factor {kappa}B-mediated antiapoptosis involving c-IAP2
Blood,
October 15, 2001;
98(8):
2508 - 2517.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. S. Boosalis, R. Bandyopadhyay, E. H. Bresnick, B. S. Pace, K. Van DeMark, B. Zhang, D. V. Faller, and S. P. Perrine
Short-chain fatty acid derivatives stimulate cell proliferation and induce STAT-5 activation
Blood,
May 15, 2001;
97(10):
3259 - 3267.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. J. Donato, J. Y. Wu, L. Zhang, H. Kantarjian, and M. Talpaz
Down-regulation of interleukin-3/granulocyte-macrophage colony-stimulating factor receptor {beta}-chain in BCR-ABL+ human leukemic cells: association with loss of cytokine-mediated Stat-5 activation and protection from apoptosis after BCR-ABL inhibition
Blood,
May 1, 2001;
97(9):
2846 - 2853.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
X. Zhang, R. Wong, S. X. Hao, W. S. Pear, and R. Ren
The SH2 domain of Bcr-Abl is not required to induce a murine myeloproliferative disease; however, SH2 signaling influences disease latency and phenotype
Blood,
January 1, 2001;
97(1):
277 - 287.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. W. N. Deininger, J. M. Goldman, and J. V. Melo
The molecular biology of chronic myeloid leukemia
Blood,
November 15, 2000;
96(10):
3343 - 3356.
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Gesbert and J. D. Griffin
Bcr/Abl activates transcription of the Bcl-X gene through STAT5
Blood,
September 15, 2000;
96(6):
2269 - 2276.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction