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NEOPLASIA
From the Department of Adult Oncology, Dana Farber
Cancer Institute, and Department of Medicine, Harvard Medical School,
Boston, MA.
Several tyrosine kinase oncogenes have been associated with
myeloproliferative diseases, including Bcr/Abl, Tel/Abl,
Tel/Jak2, and Tel/PDGFR. One target molecule shared by these oncogenes
is known to be STAT5. We generated sublines of Ba/F3 cells in which either wild-type STAT5 or a constitutively active mutant of STAT5 (STAT5-1*6) were expressed under the control of a
tetracycline-inducible promoter. These cell lines were compared with a
Ba/F3 cell line in which the expression of p210Bcr/Abl was
made inducible by a similar promoter. Before induction, all cells were
dependent on interleukin 3 (IL-3) for growth and survival. Both
STAT5-1*6 and Bcr/Abl enhanced viability and induced proliferation in
the absence of IL-3. We found that the proviability protein Bcl-XL, but not Bcl-2, was induced by both
p210Bcr/Abl and STAT5-1*6. Using a Bcl-X gene
promoter construct fused to a luciferase complementary DNA (cDNA), both
p210Bcr/Abl and STAT5-1*6 were shown to induce
transcription of Bcl-X. The increase in transcription of the Bcl-X
promoter and the increase in Bcl-X protein, due to
p210Bcr/Abl, were blocked by expression of a dominant
negative STAT5 mutant. Interestingly, however, STAT5-1*6 required the
continued presence of IL-3 to cause a significant increase in
Bcl-XL protein, whereas p210Bcr/Abl did not
need IL-3. Studies with enzyme inhibitors suggest that the extra signal
supplied by IL-3 may be supplied by the PI3K pathway. Overall, these
data suggest that constitutively activated STAT5 can increase viability
and proliferation of Ba/F3 cells. This may contribute to, but is not
likely sufficient for, the enhanced viability associated with Bcr/Abl transformation.
(Blood. 2000;96:2269-2276) Chronic myelogenous leukemia (CML) is a
myeloproliferative disorder associated with the t(9:22) translocation,
which fuses the BCR and ABL genes.1
The oncogene product, p210Bcr/Abl, displays enhanced
tyrosine kinase activity, transforms primary murine hematopoietic
cells,2 and converts interleukin 3 (IL-3)-dependent cell
lines to growth factor independence.3 A number of
signaling pathways are known to be activated by Bcr/Abl, including
p21Ras,4 PI3-kinase,5 c-jun and
c-myc.6,7 The STAT5 transcription factor is
also known to be constitutively phosphorylated and activated in Bcr/Abl
transformed cells,8,9 and is also known to be activated by
other tyrosine kinase oncogenes associated with myeloproliferative disorders, such as Tel/PDGFR. We have recently shown that
overexpression of a c-terminal truncated dominant negative form of
STAT5 reduces viability and growth of Bcr/Abl-transformed Ba/F3 murine
hematopoietic cells. These results suggested that STAT5 might play a
role in transformation by Bcr/Abl, but its significance and target
genes remain unclear.
STAT5 is a transcription factor that is activated in response to
multiple hematopoietic cytokines. STAT5A and STAT5B are the products of
2 different genes that may play different roles in signal
transduction.10 Tyrosine phosphorylation of STAT
transcription factors is believed to induce dimerization, translocation
to the nucleus, and binding to specific DNA motifs.11 Only
a small number of STAT5-regulated genes are known, including
In this study, we have attempted to dissect out the function of the
STAT5 pathway from the functions of the many other signaling pathways
activated by Bcr/Abl. A constitutively active mutant of STAT5,
termed STAT5-1*6, was recently described by Onishi et al.18 This mutant has 2 point mutations, at positions
H299R and S711F, and was discovered using a random mutagenesis
strategy, followed by selection for mutants conferring a growth
advantage. Recent studies suggest that the mutant is defective in
binding to a nuclear corepressor, explaining in part its mechanism of activation.19
Using the murine IL-3-dependent Ba/F3 cell line as a parent, we
generated stable sublines in which either wild-type STAT5, STAT5-1*6,
or p210Bcr/Abl were placed under the control of a
tetracycline-inducible promoter. The cell lines were then compared with
each other for biologic properties and gene induction. STAT5-1*6
enhanced viability and induced slow proliferation of Ba/F3 cells,
whereas overexpression of wild-type STAT5 had no effect. The effects of
STAT51*6 and Bcr/Abl on a proviability gene, Bcl-X, are
described in detail.
Plasmid constructs
Cell lines and culture conditions
Antibodies and protein analysis Anti-Abl monoclonal antibody was a gift from R. Salgia (Dana Farber Cancer Institute). Anti-P-Tyr monoclonal antibody, clone 4G10, was a kind gift of B. J. Druker (Oregon Health Sciences University). Monoclonal antibodies against Bcl-XL (B22620) and Bcl-2 (B46620) and N-terminal STAT5 (S21520) were purchased from Transduction Laboratories (Pharmingen/Transduction Laboratories, San Diego, CA). Anti-p85 antiserum (06-195) was obtained from Upstate Biotechnology Inc (Lake Placid, NY). For protein analysis, cells were washed in phosphate-buffered saline (PBS) and lysed at 5 × 107 cells/mL in cold lysis buffer (50 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 0.5% Triton X100,10 mmol/L NaF, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L PMSF, 1 mmol/L NaVO3, 1 mg/mL leupeptin and aprotinin) for 30 minutes. Lysates were clarified by centrifugation at 15 000 × g for 20 minutes at 4°C. Protein amount was evaluated by Bradford assay before analysis. Equal amounts of lysates were loaded onto SDS-polyacrylamide gels, and transferred to a PVDF membrane (Millipore, Bedford, MA). Filters were blocked for 2 hours at room temperature with either 5% nonfat dry milk or 3% bovine serum albumin in Tris-buffered saline (TBS), 0.5% Tween (TBS-T). Filters were washed 3 times in TBS-T and incubated for 1 hour with optimal concentrations of primary antibodies, diluted in TBS-0.1% Tween. After 4 additional washes in TBS-T, filters were further incubated 45 minutes with horseradish peroxidase-conjugated secondary antibodies (Amersham Pharmacia Biotech Inc, Piscataway, NJ). Visualization was performed using NEN Renaissance system and Kodak X-Omat blue film (Eastman Kodak, Rochester, NY).Reporter gene assays Transcriptional activities were measured by using different luciferase-based reporter gene constructs. A pCMV- Gal plasmid was
used as a control for transfection efficiency. For each experiment, 25 µg of the indicated luciferase plasmid and the pCMV-Gal plasmid were electrotransfected into 10 × 106 cells. When using
the tetracycline-inducible cell lines, the cells were split into 2 equal cultures 8 hours after transfection and maintained in either the
presence or absence of 1 µg/mL doxycycline for 24 hours. After being
washed twice with cold PBS, cells were resuspended in 100 µL buffer-A
(25 mmol/L Tris PO4, pH 7.8; 2 mmol/L EDTA; 105 glycerol; 15 Triton
X100; 2 mmol/L DTT), and freeze-thawed 3 times. Lysates were clarified
by centrifugation at 15 000 × g for 10 minutes.
Immediately before measurement, 20 µL of lysates were incubated with
300 µL buffer-B (25 mmol/L glycylglycine, pH 7.8; 15 mmol/L potassium
phosphate, pH 7.8; 15 mmol/L MgSO4; 4 mmol/L EGTA; 2 mmol/L ATP; 1 mmol/L DTT) and 100 µL D-luciferin. Luciferase activity
was assessed with an automated luminometer (Lumat LB 9507, EG&G
Berthold, Gaithersburg, MD). The Gal activity was measured from
these lysates with a Gal assay kit (Invitrogen). In each case,
reporter gene activity was reported as a ratio of luciferase activity
to Gal activity.
Combined annexin V-propidium iodide staining Viability was assessed using annexin V staining (annexinV-FLUOS, Boehringer Mannheim) following the manufacturer's specifications. Binding of fluorescein-conjugated annexin V and propidium iodide (PI) was assessed by fluorescence-activated cell separation (Coulter EPICS XL, Miami, FL).In vivo studies Ton.B.STAT5 or Ton.B.1*6 cells were resuspended at a cell density of 50 × 106 cells/mL in PBS and 100 mL per mouse were injected in the tail vein of nude mice (Taconic, Germantown, NY). One week before injection, the mice were separated into 2 groups, one group receiving water plus 1% sucrose and one group receiving water plus 1% sucrose plus 500 µg/mL doxycyline. Water containing doxycycline was changed every 2 days. For each cell line used, 5 mice were injected in each group. At the indicated time point (6 weeks after injection), one mouse of each group was killed. Mice were killed after 6 weeks, blood smears were prepared and stained with Wright-Giemsa stain, and spleen and lymph nodes were sectioned and smears stained with hematoxylin and eosin.Polymerase chain reaction analysis Bcl-XL RNA levels were assessed by semiquantitative polymerase chain reaction (PCR). G3PDH RNA levels were used as an internal control. Cells were treated as indicated and total RNA was recovered by Trizol (GIBCO, Gaithersburg, MD) extraction. Reverse transcription was performed on 100 ng total RNA, with 50 pmol oligo-dT, 2.5 mmol/L dNTP and 2 units of AMV-RT (Promega, Madison, WI), in a total volume of 20 µL for 90 minutes at 42°C. PCR (30 cycles) was performed on 5 µL of the reverse transcription products, in presence of 30 pmol of the specific primers, 2 units of Taq polymerase (Perkin Elmer, Norwalk, CT), and 125 µmol/L dNTP, in a final volume of 100 µL. The following primers were used: Bcl-XL -1: 5'-CCG GAG AGC GTT CAG TGA TC-3'; Bcl-XL: 5'-TCA GGA ACC AGC GGT TGA AG-3'; GAPDH-1: 5'-TGA AGG TCG GTG TGA ACG GAT TTG GC-3' and GAPDH-2 : 5'-CTC CTT GGA GGC CAT GTA GGC CAT GAG G-3'. Linearity of the PCR was assessed by titration of the sample.
Inducible expression of STAT5, STAT5-1*6, and p210Bcr/Abl in Ba/F3 cells Onishi et al18 recently described a constitutively active mutant of STAT5, named STAT5-1*6, obtained by random mutagenesis. The STAT5-1*6 cDNA and a wild-type STAT5 cDNA were cloned into the pTRE plasmid, under the control of a tetracycline-inducible promoter. These constructs were then individually cotransfected, as described in "Materials and methods," with a pTK-hygro plasmid in the Ton.B.1 cell line, already expressing the reverse tet-transactivator. The transfected cells were selected for 2 to 3 weeks in the presence of 400 µg/mL hygromycin, in medium containing IL-3 to avoid selecting for factor-independence. Individual clones were obtained by limiting dilution and screened for increased STAT5 expression in response to doxycycline. Several clones were obtained for each construct. The Ton.B.210 Ba/F3 cell line has been previously described.22 Each of the cell lines were normally cultured in the absence of doxycycline and in the presence of IL-3 to avoid selection of sublines with growth advantages. Cell lysates prepared 24 hours after the addition of doxycycline demonstrated more than a 10-fold increase in Bcr/Abl, STAT5, or STAT5-1*6, respectively (Figure 1A). Gene expression was reversible after withdrawal of doxycycline (not shown).
Bcr/Abl has previously been shown to induce tyrosine phosphorylation
and DNA binding of STAT5.8 In the current studies, STAT5
activity was measured by a reporter gene assay using a construct containing 4 tandem The effects of STAT5-1*6, STAT5, and Bcr/Abl on growth and viability of
the IL-3-dependent Ba/F3 cells were then examined (Figure
2). In the absence of doxycycline, all 3 lines were indistinguishable from untransfected Ba/F3 cells, and
rapidly underwent apoptosis when deprived of IL-3. After adding
doxycycline for 24 hours, Ba/F3 cells inducibly expressing STAT5-1*6
(Ton.B.1*6) proliferated slowly in the absence of added IL-3, whereas
Ba/F3 cells expressing the wild-type STAT5 (Ton.B.WT) could not
proliferate and died rapidly. Thus, the observed effect of STAT5-1*6
was related to enhanced STAT5 activity, and not to overexpression of
STAT5 protein. However, the Ton.B.1*6 cells grew much more slowly than
doxycyline-induced Ton.B.210 cells (Figure 2A). These observations
suggest that constitutive activation of STAT5 alone is sufficient to
induce growth in the absence of IL-3 in Ba/F3 cells, but also that
Bcr/Abl is more significant as a mitogen than STAT5-1*6.
In addition to proliferation, induction of STAT5-1*6 and Bcr/Abl, but not STAT5, prevented cell death after IL-3 withdrawal. Viability was directly examined in these cell lines using a combined annexin V-PI staining method (Figure 2B). Thirty hours after IL-3 withdrawal, approximately 60% and 40% of noninduced Ton.B.1*6 and Ton.B.210 cells, respectively, stained with annexin-V-FITC, whereas less than 30% of cells induced with doxycycline to express STAT5-1*6 and less than 20% of cells induced to express Bcr/Abl were annexin-V positive. These results indicate that STAT5 activity alone is sufficient to rescue most cells from apoptosis after abrupt withdrawal of IL-3, and is approximately as effective as Bcr/Abl in this biologic effect. The effects of STAT5-1*6 observed in tissue culture could also be replicated in an in vivo murine model. Injection of Ton.B.210 cells has been shown to induce leukemia within 4 weeks in nude mice that were provided with drinking water containing doxycycline.22 Following this protocol, the Ton.B.1*6 and Ton.B.WT cells (5 × 106 cells) were injected intravenously into nude mice. Mice not given doxycycline remained healthy for the 6-week observation period. Also, mice given Ton.B.WT cells with doxycycline remained healthy for the observation period. However, mice injected with Ton.B.1*6 cells and provided with doxycycline, showed a dramatic weight loss (about 50% total weight) and developed visibly enlarged lymph nodes and at 6 weeks were found to have high numbers of circulating immature hematopoietic cells in the blood, as well as replacement of marrow and spleen with immature hematopoietic cells with the morphology of Ba/F3 cells (Figure 2C). Thus, the ability of STAT5-1*5 to induce factor-dependent growth and viability in culture is reflected in the generation of doxycyline-dependent leukemia in this nude mouse model. We did not attempt to directly compare the effects of Bcr/Abl and STAT5-1*6 in vivo, but limited experience would suggest that Bcr/Abl is a more virulent oncogene in this system than is STAT51*6. Together, these data show that constitutive activation of STAT5 alone, at least in the form of STAT5-1*6, is sufficient to induce IL-3-independent growth and survival of Ba/F3 cells, in vitro and in vivo, although to a lesser extent than p210Bcr/Abl. Effects of Bcr/Abl and STAT5-1*6 on Bcl-XL protein levels Although the increase in viability associated with Bcr/Abl transformation is widely appreciated, the signaling pathways responsible for viability have not been clearly defined. One source of variability is that cell lines containing Bcr/Abl have a tendency to acquire additional mutations, and thus reflect both the effects of Bcr/Abl and any accumulated mutations in other genes. We took advantage of the unique properties of these inducible cell lines to study which of the Bcl-2 family members are specifically induced by Bcr/Abl. Through the use of STAT5 A and B double-knockout mice, it has recently been demonstrated that STAT5 deficiency leads to a fetal anemia, possibly because of a lack of Bcl-XL protein expression in EPO signaling.15 Furthermore, it has been shown that the Bcl-X gene promoter contains several STAT binding sites.23Ton.B.210 cells were deprived of IL-3 and induced with doxycycline for
0 to 72 hours. In the Ton.B.210 cells, Bcr/Abl protein was expressed as
early as 6 hours after induction and maximum levels obtained at 24 hours (Figure 3A). Bcl-2 expression was not affected by increased Bcr/Abl, whereas the levels of
Bcl-XL increased steadily starting at 24 hours of
induction. Levels of the p85 subunit of PI3K were used to demonstrate
approximately equal loading of the gel.
Similarly, Ton.B.STAT5-1*6 cells were treated with doxycycline. STAT5-1*6 levels were detectable at 6 hours, and were again maximum at about 24 hours (Figure 3B). Interestingly, in the absence of IL-3, no significant accumulation of Bcl-XL was detected. In contrast, when IL-3 was left in the medium, the induction of STAT51*6 was associated with a steady accumulation of Bcl-XL protein. This required induction of STAT5-1*6 because no increase was seen without doxycycline (Time 0 with IL-3, Figure 3B, and not shown). Also, no change in Bcl-2 levels was detected (not shown). These results suggest that constitutive activation of STAT5 can contribute to Bcl-X induction, but STAT5 by itself is not sufficient. A second pathway, activated through the IL-3 receptor or Bcr/Abl, is required to increase Bcl-XL protein expression. Bcr/Abl increases Bcl-X gene transcription and protein levels through STAT5 To better understand the role of STAT5 in Bcl-X gene expression, we compared the effects of Bcr/Abl and STAT5-1*6 on the activity of a Bcl-X promoter (in the absence of IL-3). Induction of p210Bcr/Abl by doxycyline resulted in a greater than 5-fold induction of Bcl-X promoter activity in all promoter constructs tested (Figure 4B). This increase in transcriptional activity was confirmed by semiquantitative PCR on Bcl-XL transcripts compared with GAPDH transcripts (Figure 4B insert), demonstrating a clear increase of Bcl-XL RNA in Bcr/Abl expressing cells (ie, Ba/F3-Bcr/Abl, lane 2, or doxycycline-induced Ton.B.210 cells, lane 4) compared with nontransformed cells (ie, Ba/F3 cells, lane 1, or noninduced Ton.B.210 cells, lane 4). The GAS-luc construct was used in these experiments as a positive control for STAT5 activity in Ton.B.p210 cells.
Various Bcl-XL promoter constructs were used in this study. All these constructs contain the STAT5 binding site, as described by Dumon et al24 (Figure 4A). Induction of STAT5-1*6 induced a greater than 5-fold increase in Bcl-X promoter activity (Figure 4C). Similar results were obtained in presence of IL-3; however, there was a higher level of promoter activity in the noninduced cells (not shown). Bcr/Abl-transformed cells were not studied in the presence and absence of IL-3. Because both Bcr/Abl and STAT51*6 induced Bcl-X promoter activity, we
asked if STAT5 was required for the activity of Bcr/Abl. A dominant
negative STAT5 mutant,
Together, these data indicate that STAT5 activity contributes to the increased expression of Bcl-X induced by Bcr/Abl. However, other pathways appear to be important because STAT5-1*6 requires a small amount of IL-3 to obtain optimal increases in Bcl-X protein accumulation. Cooperation between STAT5 and the PI3K pathway in the induction of Bcl-XL The results shown in Figures 3B and 4B indicate that, although STAT5 can activate Bcl-X promoter activity, it is insufficient by itself to cause maximum accumulation of Bcl-XL protein. An additional signal, supplied by IL-3, is required. In an initial effort to identify the second pathway(s), Ton.B.1*6 cells cultured in the presence of IL-3 were exposed to several drugs believed to specifically inhibit various signaling pathways (Figure 6). Bcl-X expression induced by STAT5-1*6 expression was not affected by the addition of the diluent, DMSO, or PD98059, an inhibitor of MAPK, indicating that the ERK/MAPK pathway is not involved in this mechanism. However, 2 drugs known to act at different levels in the PI3K/Akt pathway did have a reproducible effect. The PI3K inhibitor, LY294002, significantly decreased Bcl-XL expression, suggesting the requirement of PI3K in Bcl-XL regulation. Second, and to a lessor extent, Bcl-XL expression was reduced in response to the p70 S6 kinase inhibitor, rapamycin. These results suggest that signals provided potentially by the PI3K pathway can synergize with STAT5 to enhance Bcl-X expression, possibly by decreasing protein degradation, enhancing translation, or through another mechanism.
CML is a clonal disorder of hematopoietic stem cells caused by the Bcr/Abl oncogene and characterized by expansion of myeloid cells in the blood, spleen, and bone marrow.25 In contrast to acute myeloid leukemias, differentiation of CML myeloid cells appears to be normal, resulting in the overproduction of mature, functional, neutrophils and other hematopoietic elements. Although the exact biologic effects of Bcr/Abl on hematopoietic cells have been controversial, most investigators agree that the oncogene enhances proliferation,26 prolongs viability,27 and alters adhesion and mobility.28 These effects require the constitutively active tyrosine kinase activity of Bcr/Abl, which presumably functions by activating cellular signaling pathways. Although a number of target molecules downstream of Bcr/Abl have been identified, few have so far been linked to specific aspects of the transformed phenotype. STAT5 is phosphorylated and activated in Bcr/Abl-transformed
cells, and also in leukemia cells containing other activated tyrosine
kinase oncogenes, including v-abl, Tel/Jak2, Tel/Abl, and
Tel/PDGFR.29,30 In these leukemias, STAT5 may be a direct phosphorylation target of the oncogenes, but this has not been proven.
Interestingly, STAT5 is phosphorylated in cells from many patients with
acute myeloid leukemias, suggesting that secondary activation of
endogenous tyrosine kinases may also be sufficient to activate
STAT5.31 In both AML and CML, the contribution of STAT5 to
transformation is currently unknown. In a previous study, we showed
that expression of a truncation mutant of STAT5 lacking the
transcriptional activation domain ( In an effort to better define the potential contribution of STAT5 to transformation, we generated several comparable cell lines in which either wild-type STAT5, STAT5-1*6, or Bcr/Abl were placed under the control of a tetracycline inducible promoter. The parent cell line, Ba/F3, undergoes cell cycle arrest and apoptosis in the absence of IL-3. Treatment of the inducible cell lines with doxycycline resulted in rapid, high-level, expression of the respective genes, and allowed for very precise determination of biologic effects and signal pathway activation. These inducible cell lines have the advantage that any observed effects on biology or signaling are necessarily due to the induced gene and cannot occur because of a mutation in an unknown gene. Induction of wild-type STAT5 had no discernable effect on growth or viability, and was not further studied in any detail. In contrast, both Bcr/Abl and STAT5-1*6 resulted in factor-independent proliferation and enhanced viability. STAT51*6 was not as potent as Bcr/Abl, and would only support slow proliferation by itself, but maintained high cell viability for a prolonged period. Interestingly, these tissue culture effects were also observed in vivo. The Ba/F3 cell line does not form tumors in nude mice, whereas Bcr/Abl-transformed Ba/F3 cells proliferate rapidly in nude mice, accumulating particularly in marrow, spleen, nodes, and blood (our results and Klucher et al22). Ba/F3 cells expressing the doxycycline-inducible STAT51*6 were injected into nude mice, which were then given either regular drinking water or water containing doxycycline. Mice given doxycycline, but not mice given regular water, developed enlarged lymph nodes, leukocytosis, and a marrow infiltrated with cells identical in morphology to Ba/F3 cells. Thus, the signaling pathways activated by STAT5-1*6 appear to have some overlap with those of Bcr/Abl, at least at a functional level. Studies looking for downstream targets of STAT5 and Bcr/Abl that might contribute to transformation are therefore of interest. In this study, we focused on genes involved in regulating viability, because both Bcr/Abl and STAT5-1*6 had a prominent effect on the viability of Ba/F3 cells. The increase in viability associated with Bcr/Abl is likely to be an important aspect of transformation. Bcr/Abl can rapidly convert IL-3-dependent cell lines to complete factor independence.3 This effect is a direct effect of Bcr/Abl as shown by studies with inducible Bcr/Abl and with the Abl kinase inhibitor, STI571.22,33,34 In addition, Bcr/Abl has been reported to improve viability after exposure to chemotherapy drugs and to ionizing radiation, suggesting that viability is enhanced in the setting of several different types of apoptotic stimuli.27,35 Here, we report that both Bcr/Abl and STAT5-1*6 induce expression of
Bcl-XL, a known proviability protein. Further, we
demonstrate that this occurs in part at the level of transcription of
the Bcl-X gene, which enhanced Bcl-X gene
transcription because Bcr/Abl can be significantly inhibited by the
dominant negative inhibitor of STAT5, However, we found that the regulation of Bcl-XL protein expression also requires signaling pathways other than STAT5. Although both Bcr/Abl and STAT5-1*6 could induce transcription of the Bcl-X gene by themselves in Ba/F3 cells, only Bcr/Abl could induce an increase in the level of Bcl-XL protein. STAT5-1*6 required the continued presence of IL-3 to induce accumulation of Bcl-XL. Thus, in these cells, Bcl-XL protein accumulation is likely to be regulated by 2 or more pathways, one involving STAT5 and Bcl-X transcription, and a second unknown signal, possibly affecting Bcl-X translation or protein metabolism. In contrast to STAT1*6, Bcr/Abl supplies both signals. One possibility for the second pathway involves the PI3K pathway, because the addition of an inhibitor of PI3K reduced STAT5-1*6/IL-3-induced expression of Bcl-XL protein. Bcr/Abl is known to activate PI3K, and therefore could supply this second signal independently of IL-3. However, there may be other redundant pathways, because the addition of the PI3K inhibitor LY294002 did not effectively block Bcr/Abl-induced accumulation of Bcl-XL. The PI3K pathway has been repeatedly linked to viability signaling, and in one popular model, Bcr/Abl activates PI3K, leading to activation of Akt, which then phosphorylates and inactivates Bad, a pro-death molecule.5,37 Overall, our studies suggest that STAT5 activation can have significant effects on the biological behavior of Ba/F3 hematopoietic cells, and can mimic some, but not all, of the activities of Bcr/Abl. Identification of other genes regulated by STAT5, and of other pathways cooperating with STAT5, will be important to understand the survival and proliferative function of Bcr/Abl and related tyrosine kinase oncogenes in hematopoietic cells.
Submitted February 9, 2000; accepted May 27, 2000.
Supported by a National Institutes of Health grant CA66996 (J.D.G.) and a fellowship from the Association pour la Recherche sur le Cancer (F.G.).
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.
Reprints: James D. Griffin, Dana Farber Cancer Institute, Department of Adult Oncology, 44 Binney St, Boston MA 02115; e-mail: james_griffin{at}dfci.harvard.edu.
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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]
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C. Yu, G. Krystal, L. Varticovksi, R. McKinstry, M. Rahmani, P. Dent, and S. Grant Pharmacologic Mitogen-activated Protein/Extracellular Signal-regulated Kinase Kinase/Mitogen-activated Protein Kinase Inhibitors Interact Synergistically with STI571 to Induce Apoptosis in Bcr/Abl-expressing Human Leukemia Cells Cancer Res., January 1, 2002; 62(1): 188 - 199. [Abstract] [Full Text] [PDF]
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A. D. Schimmer, D. W. Hedley, L. Z. Penn, and M. D. Minden Receptor- and mitochondrial-mediated apoptosis in acute leukemia: a translational view Blood, December 15, 2001; 98(13): 3541 - 3553. [Full Text] [PDF]
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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]
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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]
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F. Gesbert, W. R. Sellers, S. Signoretti, M. Loda, and J. D. Griffin BCR/ABL Regulates Expression of the Cyclin-dependent Kinase Inhibitor p27Kip1 through the Phosphatidylinositol 3-Kinase/AKT Pathway J. Biol. Chem., December 8, 2000; 275(50): 39223 - 39230. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
-STAT5 plasmid was constructed as previously
described.
) doxycyline. Cells were harvested and lysed, and the
lysates were separated by SDS-PAGE. After transfer to a PVDF filter,
the inducible expression was evaluated by immunoblotting with the
specified antibodies. (B) 1 × 107 Ton.B.210,
Ton.B.STAT5, or Ton.B.STAT51*6 cells were cotransfected with GAS-luc
and pCMV-
