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Stem Cells 2004;22:609-616 www.StemCells.com
© 2004 AlphaMed Press

Erythropoietin Overcomes Imatinib-Induced Apoptosis and Induces Erythroid Differentiation in TF-1/bcr-abl Cells

Mie Uchida, Tomoko Watanabe, Masae Kunitama, Masaki Mori, Satoru Kikuchi, Kozue Yoshida, Keita Kirito, Tadashi Nagai, Keiya Ozawa, Norio Komatsu

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{at}jichi.ac.jp


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Targeting BCR-ABL tyrosine kinase by treatment with the selective inhibitor imatinib (formerly STI571, Gleevec) has proved to be highly efficient for inhibiting leukemic growth in vitro. In addition, in clinical trials, imatinib has produced high response rates in patients with chronic myeloid leukemia (CML) in chronic phase and blastic crisis. However, episodes of severe cytopenia were also frequently observed, leading to discontinuation of therapy in some cases. Therefore, it is important to examine whether administration of cytokines overcomes the adverse effects of imatinib in in vitro systems. In this study, we examine the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (EPO) on TF-1/bcr-abl (which was generated by transduction of a bcr-abl fusion gene into the TF-1 cell line) as a model system for CML with blastic crisis. Imatinib induced apoptosis in TF-1/bcr-abl cells but not in the parental TF-1 cells. However, GM-CSF, a survival factor of the parental TF-1 cells, protected TF-1/bcr-abl cells from imatinib-induced apoptosis in a dose-dependent manner. Concomitantly, constitutive phosphorylation of Stat5 and FKHRL1 was significantly inhibited by imatinib, and the inhibition was canceled by the addition of GM-CSF, accompanied by upregulation of Bcl-xL and downregulation of p27/Kip1. In addition, although untreated TF-1/bcr-abl cells had lost responsiveness to both GM-CSF and EPO and showed autonomous growth, GM-CSF enhanced phosphorylation of Stat5 and FKHRL1 in these cells. Importantly, imatinib-treated TF-1/bcr-abl cells differentiated into hemoglobin-positive cells in the presence of EPO, as in the case for the parental TF-1 cells. Taken together, imatinib-treated CML cells may differentiate into mature cells in the presence of differentiation-inducing cytokines such as EPO.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
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 [1]. 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 [2]. 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 [3–7]. Imatinib specifically suppresses ABL tyrosine kinase activity by binding competitively to the ATP-binding sites of the kinase [8]. Indeed, imatinib selectively inhibits the growth of BCR-ABL–positive leukemia cells and induces apoptosis in these leukemia cells in vitro [8,9]. A phase II study revealed that oral administration of imatinib to patients with CML results in clinical responses in more than 90% of patients [3]. 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 [7]. 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 [10,11]. 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 [10]. On the other hand, BCR-ABL–expressing TF-1/bcr-abl cells grow without any growth factors and rapidly undergo apoptosis after imatinib treatment [11]. 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
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and Antibodies
Fetal calf serum (FCS) was purchased from Sigma (St. Louis). Polyclonal antibodies against BCR and p27/Kip1 were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Antibodies against phospho-FKHRL1 (Thr32) and phosphoc-Abl (Tyr245) were purchased from Cell Signaling Technology Inc. (Beverly, MA). The 2-phenylamino-pyrimidine derivative imatinib (molecular weight, 590 da) was developed and kindly provided by Novartis (Basel, Switzerland). The stock solutions of this compound were prepared at 1 mM with dimethylsulfoxide and stored at –20°C.

Cell Culture of CML-Derived Cell Lines and Generation of Transfectants
TF-1/bcr-abl cell lines were maintained in liquid culture with Iscove’s modified Dulbecco’s medium (IMDM) containing 10% FCS [10,11]. TF-1 cells were maintained in liquid culture with IMDM containing 10% FCS with GM-CSF (1 ng/ml).

Colorimetric 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Assay for Cell Proliferation
Cell growth was examined by a colorimetric assay according to Mosmann [12] with some modifications. Briefly, cells were incubated at a density of 1 x104/100 µL in 96-well plates in IMDM containing 10% FCS. After 72 hours of culture at 37°C, 20 µL of sterilized 5-mg/ml MTT (Sigma) was added to each well. After 2 hours of incubation at 37°C, 100 µL of 10% sodium dodecyl sulfate (SDS) was added to each well to dissolve the dark-blue crystal product. The optical density was measured at a wavelength of 595 nm using a microplate reader (model 3550; Bio-Rad, Richmond, CA).

Preparation of Cell Lysates, Immunoprecipitation, and Western Blotting
The cells were washed and suspended in lysis buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1.7 ng/ml aprotinin, 50 µg/ml leupeptin, 2 mM sodium orthovanadate, and 20 mM sodium fluoride. After 20 minutes of incubation on ice, insoluble materials were removed by centrifugation at 15,000 g for 20 minutes. In some experiments, the supernatants were immunoprecipitated with anti-PY20 attached to protein G sepharose for 4 hours at 4°C in an Eppendorf shaker. Immunoprecipitates were collected by a brief centrifugation and washed four times with 1 ml of lysis buffer. The supernatants or immunoprecipitated proteins were boiled for 5 minutes in SDS–polyacrylamide gel electrophoresis (PAGE) sample buffer, resolved by SDS-PAGE, and electroblotted onto polyvinylidene difluoride membranes (Bio-Rad). The blots were blocked with 5% skim milk in Tris-buffered saline (TBS) for 1 hour at room temperature and then incubated with the appropriate concentration of primary antibody overnight at 4°C or for 1–2 hours at RT. After the blots were washed with TBS containing Tween 20 (1:2,000), they were probed with a 1:10,000 dilution of anti-rabbit or anti-mouse horseradish peroxidase–conjugated second antibodies for 90 minutes at RT. After a second washing, the blots were incubated with an enhanced chemiluminescence substrate (ECL Western blot detection system; Amersham Pharmacia Biotech, Buckinghamshire, England) and exposed to Hyperfilm ECL (Amersham Pharmacia Biotech) to visualize the immunoreactive bands. The blots were stripped with 62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM 2-mercaptoethanol at 50°C for 30 minutes, washed, blocked, and reprobed.

Dianisidine Staining
Hemoglobin concentrations were examined by incubating cells in serum-free IMDM medium containing 0.2% 3,3'-dimethoxybenzidine, fast blue B (dianisidine; Sigma), 0.3% acetic acid, and 0.3% H2O2 for 30 minutes at RT.

Detection of Apoptotic Cells
Apoptotic cells were detected according to the manufacturer’s instructions. In brief, the cells were washed with phosphate-buffered saline and resuspended in binding buffer. After 15 minutes of incubation with Annexin V–fluorescein isothiocyanate, the cell samples were analyzed by flow cytometry (FACScan, Becton Dickinson, Franklin Lakes, NJ) using a single laser-emitting excitation light at 488 nm.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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 [10,11]. 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 1AGo, 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. 1AGo). 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. 1BGo). 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 2AGo, 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. 2AGo). A high dose of EPO slightly inhibited the cell growth of TF-1/bcr-abl cells in the absence of imatinib (Fig. 2AGo). Imatinib did not affect the cell growth of the parental TF-1 cells in the presence or absence of EPO (Fig. 2BGo). 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.



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  		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.

 


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  		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. 3Go), 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).



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  		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 [10]. As shown in Table 1Go, 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 1Go), indicating that imatinib-treated TF-1/bcr-abl cells reacquire the capacity to differentiate into erythroid cells in the presence of EPO.


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  		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 4AGo, 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. 4AGo). 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 4BGo, 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. 4CGo). However, GM-CSF induced tyrosine phosphorylation of Stat5 in imatinib-treated TF-1/bcr-abl cells as it did in TF-1 cells (Fig. 4CGo). In addition, tyrosine phosphorylation of Stat5 was also observed after EPO stimulation in imatinib-treated TF-1/bcr-abl cells (Fig. 4CGo; 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 [13,14], we examined the expression of Bcl-xL protein in imatinib-treated TF-1/bcr-abl cells. As shown in Figure 4CGo (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.



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  		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 ß-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 [15]; 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 4CGo (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. 4CGo; 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 4CGo (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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we examined the effect of cytokines on the cell growth and survival of imatinib-treated CML cells. We showed here that imatinib-induced apoptosis was drastically reduced by GM-CSF and EPO in TF-1/bcr-abl cells. Both GM-CSF and EPO induced the expression of an antiapoptotic molecule, Bcl-xL, and downregulated that of a proapoptotic molecule, p27/Kip1, leading to the reduction of the fraction of Annexin V–positive cells. In addition, we found that GM-CSF stimulated cell growth of imatinib-treated TF-1/bcr-abl cells without affecting their maturation, whereas EPO slightly stimulated cell growth and induced erythroid differentiation in these cells. Therefore, it is conceivable that these cytokines exert their intrinsic actions via protection of the cells from imatinib-induced apoptosis, leading to cell growth in the case of GM-CSF and erythroid differentiation in the case of EPO treatment of TF-1/bcr-abl cells.

It is noteworthy that imatinib-treated TF-1/bcr-abl cells survived and grew in response to GM-CSF and EPO, which are survival and growth factors of the parental TF-1 cells, whereas untreated TF-1/bcr-abl cells had lost responsiveness to both GM-CSF and EPO and showed autonomous growth. Considering that phosphorylation of Stat5 and FKHRL1 was enhanced by GM-CSF in untreated TF-1/bcr-abl cells, BCR-ABL tyrosine kinase–induced and cytokine-induced signals may be redundant rather than one set of signals blocking the other. In addition and most important, EPO induced ery-throid differentiation of imatinib-treated TF-1/bcr-abl cells, as it did in the parental TF-1 cells [10]. This finding suggests that normal EPO signaling pathways are reopened by the inhibition of BCR-ABL tyrosine kinase activity in BCR-ABL–expressing erythroid progenitor cells.

Among phosphorylated proteins, a 90-kDa protein was strongly tyrosine phosphorylated by GM-CSF in imatinib-treated TF-1/bcr-abl cells, and this molecular size was almost identical to that of Stat5. In addition, we showed that Stat5 is also activated by EPO in imatinib-treated TF-1/bcr-abl cells, accompanied by erythroid differentiation. Several studies have shown that Stat5 is rapidly activated after EPO receptor stimulation [16,17]. Recently it was reported that Stat5a–/–5b–/–embryos are severely anemic because of the low number of erythroid progenitors, higher levels of apoptosis, and lower responsiveness to EPO [18]. Considering the fact that GM-CSF activated Stat5 but did not induce erythroid differentiation of TF-1/bcr-abl cells, activation of Stat5 may be required but not sufficient for EPO-induced erythroid differentiation in these cells. Alternatively, a third molecule may be required for EPO-induced erythroid differentiation.

Both GM-CSF and EPO protected imatinib-treated TF-1/bcr-abl cells from apoptosis. It was previously shown that Bcl-xL functions as a key cytokine that regulates antiapoptotic proteins in myelopoiesis and contributes to leukemia cell survival [19]. In addition, EPO induced the binding of Stat5 to the consensus Stat-binding sites on the Bcl-x gene promoter [20]. As shown in Figure 4CGo, imatinib downregulated the expression of Bcl-xL in TF-1/bcr-abl cells. However, GM-CSF and, to a lesser degree, EPO upregulated the expression of Bcl-xL in imatinib-treated TF-1/bcr-abl cells. Taken together, these findings suggest that GM-CSF and EPO rescued the imatinib-treated TF-1/bcr-abl cells from apoptosis in part via the activation of Stat5 and subsequent induction of the Bcl-xL gene.

FKHRL1 is a mammalian orthologue of DAF-16 and belongs to the Forkhead transcription factor family [21]. This family is characterized by the presence of a highly conserved Forkhead domain having a winged-helix motif and DNA-binding activity and is involved in embryogenesis, differentiation, and tumorigenesis [22]. Because DAF-16 plays an important role in the longevity of Caenorhabditis elegans [23,24], it is possible that FKHRL1 plays an important role in mammalian biology. FKHRL1 has been identified as a substrate of Akt, which has been identified as a downstream target of PI3K necessary for cell survival, and the phosphorylation by Akt is essential for suppressing the transcription activity of FKHRL1. In other words, Akt negatively regulates the transcription activity of FKHRL1 by phosphorylation [25]. Previously, we reported that FKHRL1 is directly phosphorylated by activated Akt and functions as one of the downstream molecules in the PI3K/Akt activation pathway for cytokine signaling [26,27]. In addition, recently we demonstrated that FKHRL1 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, over-expression of active FKHRL1 induced cell-cycle arrest at the G0/G1 phase and apoptosis in BCR-ABL–expressing cell lines. Therefore, we hypothesized that GM-CSF protected imatinib-induced TF-1/bcr-abl cells from apoptosis in part via inactivation of FKHRL1. As expected, imatinib-induced upregulation of p27/Kip1, which is a possible target molecule for FKHRL1 [28,29], was canceled after the addition of GM-CSF or EPO. Taken together, these results suggest that loss of function of FKHRL is in part involved in the antiapoptotic effects of GM-CSF and EPO.

The percentage of hemoglobin-positive cells was much higher in imatinib-treated TF-1/bcr-abl cells than in TF-1 cells in the presence of EPO (88% versus 23%). It has been reported that imatinib induced hemoglobin synthesis in K562 and HL-60/bcr-abl cells [30]. These findings may reflect non–kinase-dependent effects of BCR-ABL or compensatory response to the BCR-ABL–dependent block in erythroid differentiation. Alternatively, although the concept is speculative at present, imatinib itself may have a stimulatory effect on hemoglobinization. In our study, imatinib alone did not increase the percentage of hemoglobin-positive cells, presumably because of induction of apoptosis. This finding suggests that a survival factor—in this study, GM-CSF or EPO—is required for the imatinib-induced hemoglobin synthesis in TF-1/bcr-abl cells. Indeed, treatment with GM-CSF plus imatinib, but not GM-CSF alone, slightly induced hemoglobin synthesis in TF-1/bcr-abl cells (7.7% versus 1%).

Imatinib has dose-dependent hematological side effects, including neutropenia, thrombocytopenia, and anemia, presumably because of its significant inhibitory effect on normal CD34+ progenitor cells [31]. Therefore, it is clinically important to rescue the normal progenitor cells from apoptosis by administration of cytokines, although attention should always be paid to the possibility that cytokines adversely stimulate cell growth of CML cells. Recently, Heim et al. [32] reported that G-CSF overcame imatinib-induced neutropenia in CML via stimulation of myelopoiesis and allowed for continued administration of imatinib. In this study, we showed that EPO overcame imatinib-induced apoptosis and induced erythroid differentiation in TF-1/bcr-abl cells. Therefore, although in vivo administration of cytokines such as EPO and G-CSF mainly acts on normal progenitor cells, these cytokines may in part induce differentiation of imatinib-treated CML-derived progenitor cells into lineage-restricted mature cells by protecting against imatinib-induced apoptosis.


   ACKNOWLEDGMENTS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received September 10, 2003; accepted for publication December 16, 2003.



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Y.-Y. Kuo and Z.-F. Chang
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