Erythropoietin Overcomes Imatinib-Induced Apoptosis and Induces Erythroid Differentiation in TF-1/bcr-abl Cells
http://www.100md.com
《干细胞学杂志》
Division of Hematology, Department of Medicine, Jichi Medical School, Tochigi, Japan
Key Words. CML ? Erythropoietin ? Erythroid differentiation ? Imatinib ? FKHRL1
Correspondence: Norio Komatsu, M.D., Ph.D., Division of Hematology, Department of Medicine, Jichi Medical School, Minamikawachi-machi, Kawachi-gun, Tochigi-ken 329-0498, Japan. Telephone: 81-285-58-7353; Fax: 81-285-44-5258; e-mail: nkomatsu@jichi.ac.jp
ABSTRACT
The bcr-abl fusion gene originates from a reciprocal translocation between the long arms of chromosomes 9 and 22, resulting in the formation of the Philadelphia chromosome . The resultant bcr-abl fusion gene encodes chimeric BCR-ABL proteins p210 and p190 (210 and 190 kDa, respectively). These fusion proteins possess constitutively active tyrosine kinase activities and are sufficient to produce chronic myeloid leukemia (CML)–like myeloproliferative disease in murine models . Therefore, a specific inhibitor of BCR-ABL would be one of the promising therapeutic agents for CML.
Recently, the introduction of imatinib (formerly STI571, Gleevec) has revolutionized the treatment of CML . Imatinib specifically suppresses ABL tyrosine kinase activity by binding competitively to the ATP-binding sites of the kinase . Indeed, imatinib selectively inhibits the growth of BCR-ABL–positive leukemia cells and induces apoptosis in these leukemia cells in vitro . A phase II study revealed that oral administration of imatinib to patients with CML results in clinical responses in more than 90% of patients . However, grade 3 or 4 neutropenia, thrombocytopenia, or anemia was frequently observed in these patients. In addition, episodes of severe cytopenia were frequent in patients with chronic myelogenous leukemia in myeloid blast crisis . Thus, cytopenia seems to be a common adverse effect in imatinib treatment. Therefore, it is important to overcome imatinib-induced cytopenia to enable the continuous administration of imatinib.
In this study, we examined the effects of cytokines on the cell growth and survival of BCR-ABL–expressing cells after imatinib treatment in vitro. To this end, we used TF-1 and TF-1/bcr-abl cell lines . TF-1 cells grow well in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) and differentiate into hemoglobin-positive cells in the presence of erythropoietin (EPO) alone . On the other hand, BCR-ABL–expressing TF-1/bcr-abl cells grow without any growth factors and rapidly undergo apoptosis after imatinib treatment . Therefore, these cell lines should be good models for examining the effects of cytokines on imatinib-treated CML cells. We show here that TF-1/bcr-abl cells can survive in the presence of GM-CSF after exposure to imatinib. In addition, imatinib-treated TF-1/bcr-abl, but not untreated TF-1/bcr-abl cells, can differentiate into hemoglobin-positive cells in the presence of EPO. Therefore, EPO may induce erythroid differentiation of not only normal erythroid progenitors but also imatinib-treated CML-derived erythroid cells via protecting apoptosis.
MATERIALS AND METHODS
Proliferative Response to Cytokines after Imatinib Treatment in TF-1/bcr-abl Cell Line
The TF-1/bcr-abl cell line was established as a model system for CML with blastic crisis by retrovirus transfection of the parental TF-1 cell line, a human leukemia cell line dependent on GM-CSF for growth and survival . Therefore, the TF-1/bcr-abl cell line should be a good model for analyzing the effects of BCR-ABL expression on cytokine response by comparison with the parental TF-1 cell line. As shown in Figure 1A, TF-1/bcr-abl cells grew well without GM-CSF and thus had lost the dependence on GM-CSF displayed by the parental TF-1 cells. Imatinib completely inhibited cell growth of this cell line in the absence of GM-CSF. However, GM-CSF stimulated the cell growth of imatinib-treated TF-1/bcr-abl cells in a dose-dependent manner (Fig. 1A). The parental TF-1 cells grew well in the presence of GM-CSF, and imatinib did not affect the cell growth or viability of this cell line with or without GM-CSF (Fig. 1B). Because the TF-1 cells proliferate slightly in response to EPO, we examined the effect of EPO on the cell growth of TF-1/bcr-abl cells in the presence or absence of imatinib. As shown in Figure 2A, imatinib inhibited the cell growth of TF-1/bcr-abl cells without EPO. However, like GM-CSF, EPO stimulated the cell growth of imatinib-treated cells in a dose-dependent manner (Fig. 2A). A high dose of EPO slightly inhibited the cell growth of TF-1/bcr-abl cells in the absence of imatinib (Fig. 2A). Imatinib did not affect the cell growth of the parental TF-1 cells in the presence or absence of EPO (Fig. 2B). Taken together, these results suggest that imatinib-treated TF-1/bcr-abl cells have reacquired responsiveness to GM-CSF and EPO after imatinib treatment, resembling the responsiveness of the parental TF-1 cells to these cytokines.
Figure 1. Proliferative response to GM-CSF in imatinib-treated TF-1/bcr-abl cell line. TF-1/bcr-abl (A) or TF-1 (B) cells were plated at a density of 10,000 cells/well in IMDM supplemented with 5% fetal calf serum and cultured with various concentrations of GM-CSF (0.01–10 ng/mL) in the absence or presence of imatinib (1 μM). The MTT reduction assay was performed after 3 days of culture. The values represent the mean ± standard deviation from triplicate cultures. Abbreviation: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
Figure 2. Proliferative response to EPO in imatinib-treated TF-1/bcr-abl cell line. TF-1/bcr-abl (A) or TF-1 (B) cells were plated at a density of 10,000 cells/well in IMDM supplemented with 5% fetal calf serum and cultured with various concentrations of EPO (0.01–10 U/mL) in the absence or presence of imatinib (1 μM). The MTT reduction assay was performed after 3 days of culture. The values represent the mean ± standard deviation from triplicate cultures. Abbreviations: EPO, erythropoietin; MTT, 3-(4,5-dimethylthi-azol-2-yl)-2,5-diphenyltetrazolium bromide.
GM-CSF and EPO Protect TF-1/bcr-abl Cells from Imatinib-Induced Apoptosis
Next, we examined whether GM-CSF and EPO protected the TF-1/bcr-abl cells from cell death. Flow cytometric analysis with Annexin V staining revealed that imatinib increased the ratio of Annexin V–positive cells, and this increase was drastically decreased in the presence of GM-CSF or EPO (Fig. 3), indicating that the imatinib-induced cell death of the TF-1/bcr-abl cells was attributable to apoptosis and that GM-CSF and EPO blocked the imatinib-induced apoptosis. The apoptotic events were confirmed by morphological changes such as nuclear fragmentation and chromatin condensation (data not shown).
Figure 3. GM-CSF and EPO protect imatinib-treated TF-1/bcr-abl cells from apoptosis. TF-1/bcr-abl cells were cultured with imatinib (1 μM) in the presence or absence of GM-CSF (1 ng/mL) or EPO (1 U/mL). Three days later, the cells were harvested for Annexin-V analysis. Abbreviation: EPO, erythropoietin.
Imatinib-Treated TF-1/bcr-abl Cells Differentiate into Hemoglobin-Positive Cells in the Presence of EPO
It was originally reported that TF-1 cells differentiate into erythroid lineage cells after exposure to EPO . As shown in Table 1, more than 20% of EPO-treated TF-1 cells became hemoglobin positive. However, imatinib alone had no effect on the erythroid differentiation of TF-1 cells. Next, we examined whether TF-1/bcr-abl cells regain the capacity to differentiate into erythroid lineage cells after imatinib treatment. Neither EPO nor GM-CSF induced erythroid differentiation of TF-1/bcr-abl cells without imatinib. However, most imatinib-treated TF-1/bcr-abl cells became hemoglobin positive in the presence of EPO (Table 1), indicating that imatinib-treated TF-1/bcr-abl cells reacquire the capacity to differentiate into erythroid cells in the presence of EPO.
Table 1. The percentage of hemoglobin-positive cells in TF-1 and TF-1/bcr-abl cells under several conditions
Mechanism of Imatinib-Induced Reacquisition of Responsiveness to Cytokines in TF-1/bcr-abl Cells
To clarify the molecular mechanism involved in imatinib-induced reacquisition of responsiveness to cytokines, we performed Western blotting analysis with anti-phosphotyrosine antibody. As shown in Figure 4A, the tyrosine phoshorylation of a 90-kDa protein was suppressed by imatinib treatment and recovered to the basal level in response to the addition of GM-CSF (Fig. 4A). We focused on Stat5 because this molecule is a 90-kDa protein that is tyrosine phosphorylated by GM-CSF in several GM-CSF–dependent cell lines. Cell lysates from untreated TF-1/bcr-abl cells or those treated with imatinib for 6 hours were immunoprecipitated with anti-phosphotyrosine antibody (PY20). Western blotting analysis with PY20 demonstrated that a 90-kDa protein was detected in the immunoprecipitates. To confirm that one of the 90-kDa proteins is identical to Stat5, we performed Western blotting analysis with anti-Stat5 antibody. As shown in Figure 4B, the immunoprecipitated 90-kDa proteins contained Stat5. Therefore, we identified one of the 90-kDa proteins as Stat5. In addition, Stat5 was constitutively phosphorylated in TF-1/bcr-abl cells in the absence of GM-CSF, and the phosphorylation of Stat5 was completely inhibited by imatinib treatment (Fig. 4C). However, GM-CSF induced tyrosine phosphorylation of Stat5 in imatinib-treated TF-1/bcr-abl cells as it did in TF-1 cells (Fig. 4C). In addition, tyrosine phosphorylation of Stat5 was also observed after EPO stimulation in imatinib-treated TF-1/bcr-abl cells (Fig. 4C; upper panel). These findings suggest that activation of Stat5 is consistent with responsiveness to cytokines in imatinib-treated TF-1/bcr-abl cells. Because Stat5 protects normal hematopoietic cells and CML cells from apoptosis via induction of the Bcl-xL gene , we examined the expression of Bcl-xL protein in imatinib-treated TF-1/bcr-abl cells. As shown in Figure 4C (lower panel), untreated TF- 1/bcr-abl cells expressed Bcl-xL at the protein level. However, Bcl-xL expression was inhibited by imatinib treatment, and the expression level was restored almost to the basal level by the addition of GM-CSF and EPO.
Figure 4. Cytokine-induced activation of Stat5 and phosphorylation of FKHRL1 in imatinib-treated TF-1/bcr-abl cells. (A): The cells were treated with imatinib (1 μM) for 1–9 hours, then stimulated with GM-CSF (10 ng/mL) for 10 minutes. After solubilization, cell extracts were resolved by 7.5% SDS-PAGE and immunoblotted with antiphosphotyrosine antibody (PY20). As a control, growth factor–deprived TF-1 cells were stimulated with GM-CSF (10 ng/mL) for 10 minutes. (B): The cells were treated with imatinib (1 μM) for 6 hours, then stimulated with GM-CSF (1 ng/mL) for 10 minutes. After solubilization, cell extracts were immunoprecipitated with PY20, and the immunoprecipitates were resolved by 7.5% SDS-PAGE. The membrane was immunoblotted with anti-PY20 or anti-Stat5 antibody. As a control, growth factor–deprived TF-1 cells were stimulated with GM-CSF (10 ng/mL) for 10 minutes. (C): The cells were treated with imatinib (1 μM) for the indicated times (6 or 24 hours), then stimulated with GM-CSF (10 ng/mL) or EPO (10 U/mL) for 10 minutes. After solubilization of the cells, the cell extracts were resolved by 7.5% or 12% SDS-PAGE and immunoblotted with antibody against phosphoStat5, Bcl-xL, phosphoFKHRL1, or p27/Kip protein. The blot was reprobed with anti-Stat5, anti-FKHRL1 antibody, or ?-actin to confirm equal loading of protein. As a control, growth factor–deprived TF-1 cells were stimulated with GM-CSF (10 ng/mL) or EPO (10 U/mL) for 10 minutes. Abbreviation: EPO, erythropoietin.
We previously demonstrated that FKHRL1, a member of the Forkhead subfamily, lies downstream of the BCR-ABL signaling pathway ; FKHRL1 is constitutively phosphorylated and inactivated by BCR-ABL tyrosine kinase, and imatinib activates FKHRL1 by suppression of BCR-ABL tyrosine kinase activity, leading to cell-cycle arrest at the G0/G1 phase and subsequent apoptosis. Moreover, we found that overexpression of active FKHRL1 induced apoptosis in BCR-ABL–expressing cell lines. Therefore, we hypothesized that GM-CSF protects the imatinib-induced TF-1/bcr-abl cells from apoptosis in part via inactivation of FKHRL1. As shown in Figure 4C (lower panel), FKHRL1 was constitutively phosphorylated in TF-1/bcr-abl cells, and exposure to imatinib resulted in the dephosphorylation of FKHRL1. However, GM-CSF induced the phosphorylation of FKHRL1 in imatinib-treated TF-1/bcr-abl cells as in GM-CSF–starved TF-1 cells (Fig. 4C; lower panel).
To confirm whether FKHRL1 had reacquired transcriptional regulatory activity after imatinib treatment, we examined the expression of the target molecule, p27/Kip1, by Western blotting analysis. As shown in Figure 4C (lower panel), the expression of p27/Kip1 protein was upregulated after imatinib treatment. By contrast, the upregulation of this molecule was canceled by the addition of GM-CSF and EPO even in the presence of imatinib, suggesting that these cytokines protected TF-1/bcr-abl cells from imatinib-induced apoptosis in part via downregulation of p27/Kip1.
DISCUSSION
We thank Novartis Pharmaceuticals (Basel, Switzerland) for the generous gift of imatinib. This work was supported by Grants-in-Aid for Cancer Research and Scientific Research from the Ministry of Education, Science and Culture of Japan and by grants from the Japan Leukemia Research Foundation and the Pharmacological Research Foundation.
REFERENCES
McLaughlin J, Chianese E, Witte ON. In vitro transformation of immature hematopoietic cells by the P210 BCR/ABL oncogene product of the Philadelphia chromosome. Proc Natl Acad Sci U S A 1987;84:6558–6562.
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;85:9312–9316.
Kantarjian H, Sawyers C, Hochhaus A et al. Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia. N Engl J Med 2002;346:645–652.
O’Brien SG, Guilhot F, Larson RA et al. Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med 2003;348:994–1004.
Talpaz M, Silver RT, Druker BJ et al. Imatinib induces durable hematologic and cytogenetic responses in patients with accelerated phase chronic myeloid leukemia: results of a phase 2 study. Blood 2002;99:1928–1937.
Sawyers CL, Hochhaus A, Feldman E et al. Imatinib induces hematologic and cytogenetic responses in patients with chronic myelogenous leukemia in myeloid blast crisis: results of a phase II study. Blood 2002;99:3530–3539.
Druker BJ, Sawyers CL, Kantarjian H et al. Activity of a specific inhibitor of the BCRABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome. N Engl J Med 2001;344:1038–1042.
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;2:561–566.
Deininger MW, Goldman JM, Lydon N et al . The tyrosine kinase inhibitor CGP57148B selectively inhibits the growth of BCR-ABL-positive cells. Blood 1997;90:3691–3698.
Kitamura T, Tange T, Terasawa T et al. Establishment and characterization of a unique human cell line that proliferates dependently on GM-CSF, IL-3, or erythropoietin. J Cell Physiol 1989;140:323–334.
Nakajima A, Tauchi T, Ohyashiki K. ABL-specific tyrosine kinase inhibitor, STI571 in vitro, affects Ph-positive acute lymphoblastic leukemia and chronic myelogenous leukemia in blastic crisis. Leukemia 2001;15:989–990.
Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55–63.
Dumon S, Santos SC, Debierre-Grockiego F et al. IL-3 dependent regulation of Bcl-xL gene expression by STAT5 in a bone marrow derived cell line. Oncogene 1999;18: 4191–4199.
Horita M, Andreu EJ, Benito A et al. Blockade of the Bcr-Abl kinase activity induces apoptosis of chronic myelogenous leukemia cells by suppressing signal transducer and activator of transcription 5-dependent expression of Bcl-xL. J Exp Med 2000;191:977–984.
Komatsu N, Watanabe T, Uchida M et al. A member of Fork-head transcription factor FKHRL1 is a downstream effector of STI571-induced cell cycle arrest in BCR-ABL–expressing cells. J Biol Chem 2003;278:6411–6419.
Wakao H, Harada N, Kitamura T et al. Interleukin 2 and erythropoietin activate STAT5/MGF via distinct pathways. EMBO J 1995;14:2527–2535.
Gouilleux F, Pallard C, Dusanter-Fourt I et al. Prolactin, growth hormone, erythropoietin and granulocyte-macrophage colony stimulating factor induce MGFStat5 DNA binding activity. EMBO J 1995;14:2005–2013.
Socolovsky M, Fallon AE, Wang S et al. Fetal anemia and apoptosis of red cell progenitors in Stat5a–/–5b–/–mice: a direct role for Stat5 in Bcl-X(L) induction. Cell 1999; 98:181–191.
Packham G, White EL, Eischen CM et al. Selective regulation of Bcl-XL by a Jak kinase-dependent pathway is bypassed in murine hematopoietic malignancies. Genes Dev 1998;12:2475–2487.
Silva M, Benito A, Sanz C et al. Erythropoietin can induce the expression of bcl-x(L) through Stat5 in erythropoietin-dependent progenitor cell lines. J Biol Chem 1999;274: 22165–22169.
Anderson MJ, Viars CS, Czekay S et al. Cloning and characterization of three human forkhead genes that comprise an FKHR-like gene subfamily. Genomics 1998;47:187–199.
Kaufmann E, Knochel W. Five years on the wings of fork head. Mech Dev 1996;57:3–20.
Lin K, Dorman JB, Rodan A et al. daf-16: an HNF-3/fork-head family member that can function to double the lifespan of Caenorhabditis elegans. Science 1997;278:1319–1322.
Ogg S, Paradis S, Gottlieb S et al. The Fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature 1997;389:994–999.
Brunet A, Bonni A, Zigmond MJ et al. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 1999;96:857–868.
Kashii Y, Uchida M, Kirito K et al. A member of Forkhead family transcription factor, FKHRL1, is one of the downstream molecules of phosphatidylinositol 3-kinase-Akt activation pathway in erythropoietin signal transduction. Blood 2000;96:941–949.
Tanaka M, Kirito K, Kashii Y et al. Forkhead family transcription factor FKHRL1 is expressed in human megakary-ocytes: regulation of cell cycling as a downstream molecule of thrombopoietin signaling. J Biol Chem 2001;276: 15082–15089.
Dijkers PF, Medema RH, Pals C et al. Forkhead transcription factor FKHRL-1 modulates cytokine-dependent transcriptional regulation of p27(KIP1). Mol Cell Biol 2000;20:9138–9148.
Dijkers PF, Medema RH, Lammers JW et al. Expression of the pro-apoptotic Bcl-2 family member Bim is regulated by the forkhead transcription factor FKHR-L1. Curr Biol 2000;10:1201–1204.
Fang G, Kim CN, Perkins CL et al. CGP57148B (STI-571) induces differentiation and apoptosis and sensitizes Bcr-Abl-positive human leukemia cells to apoptosis due to antileukemic drugs. Blood 2000;96:2246–2253.
Bartolovic K, Balabanov S, Hartmann U et al. Inhibitory effect of imatinib on normal progenitor cells in vitro. Blood 2004;103:523–529.
Heim D, Ebnother M, Meyer-Monard S et al. G-CSF for imatinib-induced neutropenia. Leukemia 2003;17:805–807.(Mie Uchida, Tomoko Watana)
Key Words. CML ? Erythropoietin ? Erythroid differentiation ? Imatinib ? FKHRL1
Correspondence: Norio Komatsu, M.D., Ph.D., Division of Hematology, Department of Medicine, Jichi Medical School, Minamikawachi-machi, Kawachi-gun, Tochigi-ken 329-0498, Japan. Telephone: 81-285-58-7353; Fax: 81-285-44-5258; e-mail: nkomatsu@jichi.ac.jp
ABSTRACT
The bcr-abl fusion gene originates from a reciprocal translocation between the long arms of chromosomes 9 and 22, resulting in the formation of the Philadelphia chromosome . The resultant bcr-abl fusion gene encodes chimeric BCR-ABL proteins p210 and p190 (210 and 190 kDa, respectively). These fusion proteins possess constitutively active tyrosine kinase activities and are sufficient to produce chronic myeloid leukemia (CML)–like myeloproliferative disease in murine models . Therefore, a specific inhibitor of BCR-ABL would be one of the promising therapeutic agents for CML.
Recently, the introduction of imatinib (formerly STI571, Gleevec) has revolutionized the treatment of CML . Imatinib specifically suppresses ABL tyrosine kinase activity by binding competitively to the ATP-binding sites of the kinase . Indeed, imatinib selectively inhibits the growth of BCR-ABL–positive leukemia cells and induces apoptosis in these leukemia cells in vitro . A phase II study revealed that oral administration of imatinib to patients with CML results in clinical responses in more than 90% of patients . However, grade 3 or 4 neutropenia, thrombocytopenia, or anemia was frequently observed in these patients. In addition, episodes of severe cytopenia were frequent in patients with chronic myelogenous leukemia in myeloid blast crisis . Thus, cytopenia seems to be a common adverse effect in imatinib treatment. Therefore, it is important to overcome imatinib-induced cytopenia to enable the continuous administration of imatinib.
In this study, we examined the effects of cytokines on the cell growth and survival of BCR-ABL–expressing cells after imatinib treatment in vitro. To this end, we used TF-1 and TF-1/bcr-abl cell lines . TF-1 cells grow well in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) and differentiate into hemoglobin-positive cells in the presence of erythropoietin (EPO) alone . On the other hand, BCR-ABL–expressing TF-1/bcr-abl cells grow without any growth factors and rapidly undergo apoptosis after imatinib treatment . Therefore, these cell lines should be good models for examining the effects of cytokines on imatinib-treated CML cells. We show here that TF-1/bcr-abl cells can survive in the presence of GM-CSF after exposure to imatinib. In addition, imatinib-treated TF-1/bcr-abl, but not untreated TF-1/bcr-abl cells, can differentiate into hemoglobin-positive cells in the presence of EPO. Therefore, EPO may induce erythroid differentiation of not only normal erythroid progenitors but also imatinib-treated CML-derived erythroid cells via protecting apoptosis.
MATERIALS AND METHODS
Proliferative Response to Cytokines after Imatinib Treatment in TF-1/bcr-abl Cell Line
The TF-1/bcr-abl cell line was established as a model system for CML with blastic crisis by retrovirus transfection of the parental TF-1 cell line, a human leukemia cell line dependent on GM-CSF for growth and survival . Therefore, the TF-1/bcr-abl cell line should be a good model for analyzing the effects of BCR-ABL expression on cytokine response by comparison with the parental TF-1 cell line. As shown in Figure 1A, TF-1/bcr-abl cells grew well without GM-CSF and thus had lost the dependence on GM-CSF displayed by the parental TF-1 cells. Imatinib completely inhibited cell growth of this cell line in the absence of GM-CSF. However, GM-CSF stimulated the cell growth of imatinib-treated TF-1/bcr-abl cells in a dose-dependent manner (Fig. 1A). The parental TF-1 cells grew well in the presence of GM-CSF, and imatinib did not affect the cell growth or viability of this cell line with or without GM-CSF (Fig. 1B). Because the TF-1 cells proliferate slightly in response to EPO, we examined the effect of EPO on the cell growth of TF-1/bcr-abl cells in the presence or absence of imatinib. As shown in Figure 2A, imatinib inhibited the cell growth of TF-1/bcr-abl cells without EPO. However, like GM-CSF, EPO stimulated the cell growth of imatinib-treated cells in a dose-dependent manner (Fig. 2A). A high dose of EPO slightly inhibited the cell growth of TF-1/bcr-abl cells in the absence of imatinib (Fig. 2A). Imatinib did not affect the cell growth of the parental TF-1 cells in the presence or absence of EPO (Fig. 2B). Taken together, these results suggest that imatinib-treated TF-1/bcr-abl cells have reacquired responsiveness to GM-CSF and EPO after imatinib treatment, resembling the responsiveness of the parental TF-1 cells to these cytokines.
Figure 1. Proliferative response to GM-CSF in imatinib-treated TF-1/bcr-abl cell line. TF-1/bcr-abl (A) or TF-1 (B) cells were plated at a density of 10,000 cells/well in IMDM supplemented with 5% fetal calf serum and cultured with various concentrations of GM-CSF (0.01–10 ng/mL) in the absence or presence of imatinib (1 μM). The MTT reduction assay was performed after 3 days of culture. The values represent the mean ± standard deviation from triplicate cultures. Abbreviation: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
Figure 2. Proliferative response to EPO in imatinib-treated TF-1/bcr-abl cell line. TF-1/bcr-abl (A) or TF-1 (B) cells were plated at a density of 10,000 cells/well in IMDM supplemented with 5% fetal calf serum and cultured with various concentrations of EPO (0.01–10 U/mL) in the absence or presence of imatinib (1 μM). The MTT reduction assay was performed after 3 days of culture. The values represent the mean ± standard deviation from triplicate cultures. Abbreviations: EPO, erythropoietin; MTT, 3-(4,5-dimethylthi-azol-2-yl)-2,5-diphenyltetrazolium bromide.
GM-CSF and EPO Protect TF-1/bcr-abl Cells from Imatinib-Induced Apoptosis
Next, we examined whether GM-CSF and EPO protected the TF-1/bcr-abl cells from cell death. Flow cytometric analysis with Annexin V staining revealed that imatinib increased the ratio of Annexin V–positive cells, and this increase was drastically decreased in the presence of GM-CSF or EPO (Fig. 3), indicating that the imatinib-induced cell death of the TF-1/bcr-abl cells was attributable to apoptosis and that GM-CSF and EPO blocked the imatinib-induced apoptosis. The apoptotic events were confirmed by morphological changes such as nuclear fragmentation and chromatin condensation (data not shown).
Figure 3. GM-CSF and EPO protect imatinib-treated TF-1/bcr-abl cells from apoptosis. TF-1/bcr-abl cells were cultured with imatinib (1 μM) in the presence or absence of GM-CSF (1 ng/mL) or EPO (1 U/mL). Three days later, the cells were harvested for Annexin-V analysis. Abbreviation: EPO, erythropoietin.
Imatinib-Treated TF-1/bcr-abl Cells Differentiate into Hemoglobin-Positive Cells in the Presence of EPO
It was originally reported that TF-1 cells differentiate into erythroid lineage cells after exposure to EPO . As shown in Table 1, more than 20% of EPO-treated TF-1 cells became hemoglobin positive. However, imatinib alone had no effect on the erythroid differentiation of TF-1 cells. Next, we examined whether TF-1/bcr-abl cells regain the capacity to differentiate into erythroid lineage cells after imatinib treatment. Neither EPO nor GM-CSF induced erythroid differentiation of TF-1/bcr-abl cells without imatinib. However, most imatinib-treated TF-1/bcr-abl cells became hemoglobin positive in the presence of EPO (Table 1), indicating that imatinib-treated TF-1/bcr-abl cells reacquire the capacity to differentiate into erythroid cells in the presence of EPO.
Table 1. The percentage of hemoglobin-positive cells in TF-1 and TF-1/bcr-abl cells under several conditions
Mechanism of Imatinib-Induced Reacquisition of Responsiveness to Cytokines in TF-1/bcr-abl Cells
To clarify the molecular mechanism involved in imatinib-induced reacquisition of responsiveness to cytokines, we performed Western blotting analysis with anti-phosphotyrosine antibody. As shown in Figure 4A, the tyrosine phoshorylation of a 90-kDa protein was suppressed by imatinib treatment and recovered to the basal level in response to the addition of GM-CSF (Fig. 4A). We focused on Stat5 because this molecule is a 90-kDa protein that is tyrosine phosphorylated by GM-CSF in several GM-CSF–dependent cell lines. Cell lysates from untreated TF-1/bcr-abl cells or those treated with imatinib for 6 hours were immunoprecipitated with anti-phosphotyrosine antibody (PY20). Western blotting analysis with PY20 demonstrated that a 90-kDa protein was detected in the immunoprecipitates. To confirm that one of the 90-kDa proteins is identical to Stat5, we performed Western blotting analysis with anti-Stat5 antibody. As shown in Figure 4B, the immunoprecipitated 90-kDa proteins contained Stat5. Therefore, we identified one of the 90-kDa proteins as Stat5. In addition, Stat5 was constitutively phosphorylated in TF-1/bcr-abl cells in the absence of GM-CSF, and the phosphorylation of Stat5 was completely inhibited by imatinib treatment (Fig. 4C). However, GM-CSF induced tyrosine phosphorylation of Stat5 in imatinib-treated TF-1/bcr-abl cells as it did in TF-1 cells (Fig. 4C). In addition, tyrosine phosphorylation of Stat5 was also observed after EPO stimulation in imatinib-treated TF-1/bcr-abl cells (Fig. 4C; upper panel). These findings suggest that activation of Stat5 is consistent with responsiveness to cytokines in imatinib-treated TF-1/bcr-abl cells. Because Stat5 protects normal hematopoietic cells and CML cells from apoptosis via induction of the Bcl-xL gene , we examined the expression of Bcl-xL protein in imatinib-treated TF-1/bcr-abl cells. As shown in Figure 4C (lower panel), untreated TF- 1/bcr-abl cells expressed Bcl-xL at the protein level. However, Bcl-xL expression was inhibited by imatinib treatment, and the expression level was restored almost to the basal level by the addition of GM-CSF and EPO.
Figure 4. Cytokine-induced activation of Stat5 and phosphorylation of FKHRL1 in imatinib-treated TF-1/bcr-abl cells. (A): The cells were treated with imatinib (1 μM) for 1–9 hours, then stimulated with GM-CSF (10 ng/mL) for 10 minutes. After solubilization, cell extracts were resolved by 7.5% SDS-PAGE and immunoblotted with antiphosphotyrosine antibody (PY20). As a control, growth factor–deprived TF-1 cells were stimulated with GM-CSF (10 ng/mL) for 10 minutes. (B): The cells were treated with imatinib (1 μM) for 6 hours, then stimulated with GM-CSF (1 ng/mL) for 10 minutes. After solubilization, cell extracts were immunoprecipitated with PY20, and the immunoprecipitates were resolved by 7.5% SDS-PAGE. The membrane was immunoblotted with anti-PY20 or anti-Stat5 antibody. As a control, growth factor–deprived TF-1 cells were stimulated with GM-CSF (10 ng/mL) for 10 minutes. (C): The cells were treated with imatinib (1 μM) for the indicated times (6 or 24 hours), then stimulated with GM-CSF (10 ng/mL) or EPO (10 U/mL) for 10 minutes. After solubilization of the cells, the cell extracts were resolved by 7.5% or 12% SDS-PAGE and immunoblotted with antibody against phosphoStat5, Bcl-xL, phosphoFKHRL1, or p27/Kip protein. The blot was reprobed with anti-Stat5, anti-FKHRL1 antibody, or ?-actin to confirm equal loading of protein. As a control, growth factor–deprived TF-1 cells were stimulated with GM-CSF (10 ng/mL) or EPO (10 U/mL) for 10 minutes. Abbreviation: EPO, erythropoietin.
We previously demonstrated that FKHRL1, a member of the Forkhead subfamily, lies downstream of the BCR-ABL signaling pathway ; FKHRL1 is constitutively phosphorylated and inactivated by BCR-ABL tyrosine kinase, and imatinib activates FKHRL1 by suppression of BCR-ABL tyrosine kinase activity, leading to cell-cycle arrest at the G0/G1 phase and subsequent apoptosis. Moreover, we found that overexpression of active FKHRL1 induced apoptosis in BCR-ABL–expressing cell lines. Therefore, we hypothesized that GM-CSF protects the imatinib-induced TF-1/bcr-abl cells from apoptosis in part via inactivation of FKHRL1. As shown in Figure 4C (lower panel), FKHRL1 was constitutively phosphorylated in TF-1/bcr-abl cells, and exposure to imatinib resulted in the dephosphorylation of FKHRL1. However, GM-CSF induced the phosphorylation of FKHRL1 in imatinib-treated TF-1/bcr-abl cells as in GM-CSF–starved TF-1 cells (Fig. 4C; lower panel).
To confirm whether FKHRL1 had reacquired transcriptional regulatory activity after imatinib treatment, we examined the expression of the target molecule, p27/Kip1, by Western blotting analysis. As shown in Figure 4C (lower panel), the expression of p27/Kip1 protein was upregulated after imatinib treatment. By contrast, the upregulation of this molecule was canceled by the addition of GM-CSF and EPO even in the presence of imatinib, suggesting that these cytokines protected TF-1/bcr-abl cells from imatinib-induced apoptosis in part via downregulation of p27/Kip1.
DISCUSSION
We thank Novartis Pharmaceuticals (Basel, Switzerland) for the generous gift of imatinib. This work was supported by Grants-in-Aid for Cancer Research and Scientific Research from the Ministry of Education, Science and Culture of Japan and by grants from the Japan Leukemia Research Foundation and the Pharmacological Research Foundation.
REFERENCES
McLaughlin J, Chianese E, Witte ON. In vitro transformation of immature hematopoietic cells by the P210 BCR/ABL oncogene product of the Philadelphia chromosome. Proc Natl Acad Sci U S A 1987;84:6558–6562.
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;85:9312–9316.
Kantarjian H, Sawyers C, Hochhaus A et al. Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia. N Engl J Med 2002;346:645–652.
O’Brien SG, Guilhot F, Larson RA et al. Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med 2003;348:994–1004.
Talpaz M, Silver RT, Druker BJ et al. Imatinib induces durable hematologic and cytogenetic responses in patients with accelerated phase chronic myeloid leukemia: results of a phase 2 study. Blood 2002;99:1928–1937.
Sawyers CL, Hochhaus A, Feldman E et al. Imatinib induces hematologic and cytogenetic responses in patients with chronic myelogenous leukemia in myeloid blast crisis: results of a phase II study. Blood 2002;99:3530–3539.
Druker BJ, Sawyers CL, Kantarjian H et al. Activity of a specific inhibitor of the BCRABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome. N Engl J Med 2001;344:1038–1042.
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;2:561–566.
Deininger MW, Goldman JM, Lydon N et al . The tyrosine kinase inhibitor CGP57148B selectively inhibits the growth of BCR-ABL-positive cells. Blood 1997;90:3691–3698.
Kitamura T, Tange T, Terasawa T et al. Establishment and characterization of a unique human cell line that proliferates dependently on GM-CSF, IL-3, or erythropoietin. J Cell Physiol 1989;140:323–334.
Nakajima A, Tauchi T, Ohyashiki K. ABL-specific tyrosine kinase inhibitor, STI571 in vitro, affects Ph-positive acute lymphoblastic leukemia and chronic myelogenous leukemia in blastic crisis. Leukemia 2001;15:989–990.
Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55–63.
Dumon S, Santos SC, Debierre-Grockiego F et al. IL-3 dependent regulation of Bcl-xL gene expression by STAT5 in a bone marrow derived cell line. Oncogene 1999;18: 4191–4199.
Horita M, Andreu EJ, Benito A et al. Blockade of the Bcr-Abl kinase activity induces apoptosis of chronic myelogenous leukemia cells by suppressing signal transducer and activator of transcription 5-dependent expression of Bcl-xL. J Exp Med 2000;191:977–984.
Komatsu N, Watanabe T, Uchida M et al. A member of Fork-head transcription factor FKHRL1 is a downstream effector of STI571-induced cell cycle arrest in BCR-ABL–expressing cells. J Biol Chem 2003;278:6411–6419.
Wakao H, Harada N, Kitamura T et al. Interleukin 2 and erythropoietin activate STAT5/MGF via distinct pathways. EMBO J 1995;14:2527–2535.
Gouilleux F, Pallard C, Dusanter-Fourt I et al. Prolactin, growth hormone, erythropoietin and granulocyte-macrophage colony stimulating factor induce MGFStat5 DNA binding activity. EMBO J 1995;14:2005–2013.
Socolovsky M, Fallon AE, Wang S et al. Fetal anemia and apoptosis of red cell progenitors in Stat5a–/–5b–/–mice: a direct role for Stat5 in Bcl-X(L) induction. Cell 1999; 98:181–191.
Packham G, White EL, Eischen CM et al. Selective regulation of Bcl-XL by a Jak kinase-dependent pathway is bypassed in murine hematopoietic malignancies. Genes Dev 1998;12:2475–2487.
Silva M, Benito A, Sanz C et al. Erythropoietin can induce the expression of bcl-x(L) through Stat5 in erythropoietin-dependent progenitor cell lines. J Biol Chem 1999;274: 22165–22169.
Anderson MJ, Viars CS, Czekay S et al. Cloning and characterization of three human forkhead genes that comprise an FKHR-like gene subfamily. Genomics 1998;47:187–199.
Kaufmann E, Knochel W. Five years on the wings of fork head. Mech Dev 1996;57:3–20.
Lin K, Dorman JB, Rodan A et al. daf-16: an HNF-3/fork-head family member that can function to double the lifespan of Caenorhabditis elegans. Science 1997;278:1319–1322.
Ogg S, Paradis S, Gottlieb S et al. The Fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature 1997;389:994–999.
Brunet A, Bonni A, Zigmond MJ et al. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 1999;96:857–868.
Kashii Y, Uchida M, Kirito K et al. A member of Forkhead family transcription factor, FKHRL1, is one of the downstream molecules of phosphatidylinositol 3-kinase-Akt activation pathway in erythropoietin signal transduction. Blood 2000;96:941–949.
Tanaka M, Kirito K, Kashii Y et al. Forkhead family transcription factor FKHRL1 is expressed in human megakary-ocytes: regulation of cell cycling as a downstream molecule of thrombopoietin signaling. J Biol Chem 2001;276: 15082–15089.
Dijkers PF, Medema RH, Pals C et al. Forkhead transcription factor FKHRL-1 modulates cytokine-dependent transcriptional regulation of p27(KIP1). Mol Cell Biol 2000;20:9138–9148.
Dijkers PF, Medema RH, Lammers JW et al. Expression of the pro-apoptotic Bcl-2 family member Bim is regulated by the forkhead transcription factor FKHR-L1. Curr Biol 2000;10:1201–1204.
Fang G, Kim CN, Perkins CL et al. CGP57148B (STI-571) induces differentiation and apoptosis and sensitizes Bcr-Abl-positive human leukemia cells to apoptosis due to antileukemic drugs. Blood 2000;96:2246–2253.
Bartolovic K, Balabanov S, Hartmann U et al. Inhibitory effect of imatinib on normal progenitor cells in vitro. Blood 2004;103:523–529.
Heim D, Ebnother M, Meyer-Monard S et al. G-CSF for imatinib-induced neutropenia. Leukemia 2003;17:805–807.(Mie Uchida, Tomoko Watana)