Introduction

Protein tyrosine kinase plays an important role in cell growth and oncogenesis. Receptor tyrosine kinases and tyrosine kinase-associated receptors are activated by ligand binding with their extracellular domain and transduce an extracellular signal to the inside. Some tyrosine kinase-associated receptors have no inherent kinase activity but can regulate the hematopoietic system by activating nonreceptor protein tyrosine kinases. The deregulation of their signaling plays a significant role in the malignant transformation of hematological disorders.^1,2

Syk is a nonreceptor tyrosine kinase that is widely expressed in a variety of hematopoietic cells. In the B-lineage lymphoid cell, Syk is a key regulator of signal transduction and differentiation. It has two amino-terminal Src-homology 2 domains and a carboxy-terminal kinase domain, binds to the immunoreceptor tyrosine-based activation motif (ITAM) and initiates signaling through the Ras, phosphatidyl inositol 3 kinase (PI3-K) and phospholipase C- (PLC-) signal pathways.^3,4,5,6 Syk-deficient mice show a maturational block at the pro-B to pre-B cell transition.^7,8 Aberrant splicing and protein deficiency of Syk was found in childhood pro-B cell acute lymphoblastic leukemia (ALL).⁹ The TEL-Syk fusion observed in myeloid cells resulted in myeloid dysplasia and transformation in the presented case.¹⁰ Syk is also reported to be an important suppressor of breast cancer cell growth and metastasis.¹¹

Chromosomal translocation is one of the mechanisms that leads to the generation of activated tyrosine kinase fusions. A significant number of chromosomal rearrangements fuse the ETS transcriptional repressor TEL with receptor or nonreceptor tyrosine kinase in many hematological malignancies.^12,13 The TEL pointed self-association motif (PNT)-mediated oligomerization leads to the constitutive activation of tyrosine kinase fusion that is essential for cell transformation. The reported fusion genes of TEL with tyrosine kinases in hematological disorders are TEL-PDGFR in chronic myelomonocytic leukemia,¹⁴ TEL-ABL in ALL,¹⁵ TEL-Janus kinase 2 (JAK2) in ALL and chronic myeloid leukemia (CML),^16,17 TEL-TRKC in acute myeloid leukemia (AML),¹⁸ TEL-ARG in AML¹⁹ and TEL-Syk in myelodysplastic syndrome (MDS).²⁰

The JAK-STAT pathway is widely used by members of the cytokine receptor superfamily.^21,22 Dysregulation of the JAK-STAT pathway has been observed in a large number of hematopoietic malignancies.²³ TEL-JAK2 fusions in lymphoid and myeloid leukemia patients led to the constitutive activation of the tyrosine kinase and conferred cytokine-independent proliferation to the interleukin-3 (IL-3)-dependent BaF3 hematopoietic cell line.^16,17 The activation of STAT5 plays a critical role in myelo- and lymphoproliferative TEL-JAK2 diseases;²⁴ however, serine/threonine phosphorylation is also important in intracellular signal transduction.²⁵ PI3-K/Akt signal pathway is also constitutively activated in the BaF3 cell transformed by TEL-JAK2.²⁶ Mitogen-activated protein kinase (MAPK) is constitutively activated in a majority of acute leukemia.²⁷ Aberrant expression of Raf oncoproteins can abrogate cytokine dependency of the hematopoietic cell lines TF-1 and FDC-P1.^28,29

We previously reported the fusion of the TEL gene to Syk gene in a patient with MDS with t(9;12)(q22;p12). TEL-Syk fusion transformed IL-3-dependent murine hematopoietic cell line BaF3 to growth factor independence. TEL-Syk was constitutively tyrosine autophosphorylated.^10,20 We here show intracellular signal components specifically activated in the TEL-Syk-transformed BaF3 cell.

Materials and methods

DNA constructs and expression plasmids

Full-length TEL-Syk and TELPNT-Syk (deletion of nt 222–461in the TEL gene) cDNA were constructed by PCR as described previously.²⁰ PCR products were confirmed by sequencing to be devoid of mutations. We used retroviral expression vector pBabeNeo and Flag epitope-tagged expression vector pFlag (Invitrogen, San Diego, CA, USA). Retroviral expression vector pBabeNeo and pFlag have been described, respectively.^30,31 We cloned the TEL-Syk and TELPNT-Syk constructs into pBabeNeo (pB-TS and pB-TPS) and cloned the TEL-Syk into pFlag (pF-TS) (Figure 1).

Figure 1.

Schematic representation of constructed TEL-Syk and deletion mutant TELPNT-Syk (deletion of nt 222–461). For high expression in BaF3 cells, TEL-Syk and TELPNT-Syk were cloned into the pBabeNeo construct, a retroviral vector based on an MMLV-expressing neomycin-resistant gene. For immunofluorescence microscopy, TEL-Syk cDNA cloned into pFlag (the Flag epitope tagged) was used. LTR, long-terminal repeat; SV40, SV40 promoter; SV40pA, SV40 polyadenylation signal; CMV, CMV promoter.

Full figure and legend (15K)

Cell transfection assays

The pCMV-G-containing vesicular stomatitis virus envelope glycoprotein was cotransfected with pB-TS and pB-TPS or vector alone as a control into 293GP cells, a retrovirus packaging cell line expressing MMLV gag and pol genes.^32,33 The IL-3-dependent murine leukemia BaF3 cells were infected with these supernatants in the presence of 5 g/ml polybrene. G418-resistant BaF3 expressing the indicated proteins and control cells were grown in RPMI 1640 supplemented with 10% fetal calf serum (FCS) and 5 ng/ml recombinant murine IL-3.

COS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% FCS. pF-TS was transfected into subconfluent COS-7 cells by Lipofectamin (Invitrogen).

Antibody

The antibodies used are as follows: anti-Syk 4D–10 monoclonal antibody (mAb) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-PI3-K p85 antisera (Upstate Biotechnology, Lake Placid, NY, USA), rabbit anti-phospho-Akt antisera (Cell Signaling Technology, Beverly, MA, USA), rabbit anti-Akt antisera (Cell Signaling Technology), rabbit anti-Vav antisera (Santa Cruz Biotechnology), rabbit anti-PLC-1 antisera (Santa Cruz Biotechnology), rabbit anti-PLC-2 antisera (Santa Cruz Biotechnology), rabbit anti-phospho-p44/42 MAPK antisera (Cell Signaling Technology), rabbit anti-p44/42 MAPK antisera (Cell Signaling Technology), rabbit anti-phospho-MEK 1/2 antisera (Cell Signaling Technology), rabbit anti-MEK 1/2 antisera (Cell Signaling Technology), rabbit anti-STAT1 antisera (Santa Cruz Biotechnology), rabbit anti-STAT3 antisera (Santa Cruz Biotechnology), rabbit anti-STAT5 antisera (kind gift from Dr JN Ihle, St Jude Children's Research Hospital, Memphis, TN, USA), rabbit anti-phospho-STAT5 antisera (Cell Signaling Technology), rabbit anti-STAT5A antisera (R&D Systems, Minneapolis, MN, USA), rabbit anti-phospho-JAK2 antisera (Upstate Biotechnology), rabbit anti-JAK2 antisera (Upstate Biotechnology), anti-phosphotyrosine mAb PY-20 (BD Biosciences, San Jose, CA, USA), HRP-linked whole anti-mouse IgG antibody (Amersham Pharmacia Biotech, Tokyo, Japan), HRP-linked whole anti-rabbit IgG antibody (Amersham Pharmacia), anti-Flag mAb (Eastman-Kodak Company, New Haven, CT, USA) and goat anti-mouse IgG (FITC) (Santa Cruz Biotechnology).

Immunoprecipitation

The BaF3 cells were grown to a density of approximately 1 10⁶/ml. The cells were washed in phosphate-buffered saline (PBS) at 4°C and lysed in a lysis buffer: 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10% glycerol, 0.5% NP-40, 1 mM Na₃VO₄, 50 mM NaF and protease inhibitor cocktail (Roche, Mannheim, Germany). The cell lysates were centrifuged and the supernatants were incubated with the indicated antibodies for 1 h followed by protein G-agarose (Invitrogen) for 2 h at 4°C. The immunoprecipitates were washed four times in the lysis buffer.

Immunoblotting

Whole-cell lysates or immunoprecipitates were boiled with the electrophoresis SDS sample buffer for 3 min. The samples were separated by SDS-PAGE (polyacrylamide gel electrophoresis) and transferred onto polyvinylidine difluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membrane blots were blocked with 5% skimmed milk in Tris-buffered saline (TBS) containing 0.05% Tween 20 (TBS-T), or 1% blocking reagent (pH 7.5) (Roche) for 1 h at 37°C followed by incubation with primary antibodies in TBS-T for 2 h at room temperature or overnight at 4°C. After being washed, the membranes were incubated with HRP-linked whole anti-mouse or anti-rabbit IgG antibody in TBS-T for 2 h at room temperature. After washing, an enhanced chemiluminescence assay was performed and positive bands were identified on the X-ray films.

Cell proliferation assay

Tetracolor One cell proliferation assay (Seikagaku Co., Tokyo, Japan) was performed as described.³⁴ The cells (1 10⁴/well) were added to 96-well plates in 100 l RPMI containing 10% FCS in the presence or absence of IL-3 with varying concentrations of LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benopyran-4-one] (Promega, Madison, WI, USA), the inhibitor of PI3-K or MEK inhibitor U0126 (Promega). The plates were incubated at 37°C for 72 h before the addition of a mixture of tetrazolium [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] and electron carrier (1-methoxy-5-methylphenazium methylsulfate) (final volume 110 l/well). The cells were incubated for an additional hour at 37°C, and the absorption of 450 nm was measured using an ELISA plate reader.

Immunofluorescence microscopy

COS-7 cells were transfected with pF-TS using Lipofectamin (Invitrogen). The cells were grown on chamber slides. The transfected COS-7 cells were subjected to indirect immunofluorescence antibody staining 48 h after transfection. The cells were fixed with ice-cold methanol:acetone (1:1) and blocked in 1% BSA in PBS, and then anti-Flag mAb was added for 45 min. After washing the cells, goat anti-mouse IgG (FITC) was added for 30 min. The cells were then washed and incubated with 0.5 g/ml propidium iodide (Sigma Aldrich, St Louis, MO, USA), which was used for fluorescent staining of the nucleic acid, for 5 min, washed again and coverslips were then mounted on chamber slides. The cells were visualized with a fluorescence microscope and photographed.

Results

TEL-Syk fusion protein activated the PI3-K/Akt signal pathway

We used the stable transfectants to unveil the intracellular signal transduction of TEL-Syk. Akt is a protein serine/threonine kinase and the major effector of the PI3-K pathway, which is activated in downstream signaling of Syk.^6,35,36 First, BaF3 cell lysates were immunoblotted with phospho-Akt antibody to assess the activation of the PI-3K/Akt signal pathway. Only in the TEL-Syk-transformed BaF3 cell was Akt activated in the absence of IL-3 (Figure 2a). Next, the BaF3 cell lysates were immunoprecipitated with antibody against p85 PI3-K and immunoblotted with the human-specific anti-Syk mAb to assess the interaction between TEL-Syk and PI3-K. Interestingly, TEL-Syk fusion products were co-immunoprecipitated with the p85 subunit of PI3-K and this association was not observed in the BaF3 cell with TELPNT-Syk in which the oligomerization domain was deleted (Figure 2b).

Figure 2.

TEL-Syk fusion proteins were associated with PI3-K and constitutively activated Akt. (a) BaF3 cells were deprived of IL-3 for 12 h and then restimulated with 5 ng/ml of IL-3 for 30 min. Cell lysates were immunoblotted with antibody against phospho-Akt and reprobed with antibody against Akt. Akt was activated in TEL-Syk-transformed BaF3 cells in the absence of IL-3. (b) Cell lysates were immunoprecipitated with antibody against the 85K subunit of PI3-K and immunoblotted with human-specific Syk mAb. The same membrane was stripped and reprobed with antibody against the 85K subunit of PI3-K. TEL-Syk fusion products (lane 2) were co-precipitated with 85K subunit of PI3-K, and BaF3 cell lysates (lane 1) and TELPNT-Syk lysates (lane 3) were not. Lane 4 consisted of whole-cell lysates of TEL-Syk and lane 5 consisted of TELPNT-Syk.

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Vav, PLC-2 and MAPK signal pathways were constitutively activated in TEL-Syk-transformed BaF3 cells

Vav, the proto-oncogene product, is known to bind to nonreceptor protein tyrosine kinase. Nuclear phosphorylated Vav is associated to the p85 subunit of PI3-K and to PLC-1 in HL-60 cells differentiated by all-trans retinoic acid.³⁷ BaF3 cell lysates were immunoprecipitated with antibody against Vav, PLC- 1 and PLC- 2, and immunoblotted with the mAb against phosphotyrosine. Vav and PLC-2 were tyrosine phosphorylated in TEL-Syk-transformed BaF3 cells in the absence of IL-3; however, PLC-1 was not activated (Figure 3a).

Figure 3.

Constitutive kinase activation in TEL-Syk-transformed BaF3 cell. (a) BaF3 cell lysates were immunoprecipitated with antibody against Vav, PLC-1 and PLC-2, and immunoblotted with the mAb against phosphotyrosine. Vav and PLC-2 were tyrosine phosphorylated in TEL-Syk-transformed BaF3 cells in the absence of IL-3. These tyrosine kinase activations were not observed in the BaF3 cells with TELPNT-Syk. (b) BaF3 cells were deprived of IL-3 for 12 h and then restimulated with 5 ng/ml of IL-3 for 30 min. Cell lysates were immunoblotted with the antibody against phospho-ERK1/2 (p42/44 MAPK) and phospho-MEK1/2 (ERK kinases), and reprobed with antibody against ERK1/2, MEK1/2, respectively. ERK and MEK were activated in TEL-Syk-transformed BaF3 cells in the absence of IL-3.

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MAPK, also known as extracellular signal-regulated kinase (ERK), is serine/threonine kinase that appears to be one of the key regulators of cell proliferation and differentiation.^25,38,39 BaF3 cell lysates were immunoblotted with the antibody against phospho-ERK1/2 (p42/44 MAPK) and phospho-MEK1/2 (ERK kinase). Both ERK1/2 and MEK1/2 were constitutively activated in TEL-Syk-transformed BaF3 cells (Figure 3b).

Constitutive tyrosine phosphorylation of STAT5 in the absence of JAK2 activation

STAT plays an important role in growth factor-mediated intracellular signal transduction.^40,41 Dysregulation of the JAK-STAT pathway has been observed in many hematopoietic malignancies.²³ STATs were constitutively activated in some acute leukemia blasts.^42,43,44,45 To assess the potential role of STATs phosphorylation in signaling by the TEL-Syk, BaF3 cell lysates were immunoprecipitated with antibody against STAT1, STAT3 and STAT5, and immunoblotted with the mAb against phosphotyrosine. STAT5 was tyrosine phosphorylated in TEL-Syk-transformed BaF3 cells in the absence of IL-3. STAT1 and STAT3 activation were not detected because of the low expression in the BaF3 cells in the presence and absence of IL-3 (Figure 4a). JAK2 phosphorylation was not detected in the absence of IL-3 and was inhibited in the presence of IL-3 in TEL-Syk-transformed BaF3 cells (Figure 4b). The fact that the TEL oligomerization domain played a critical role in the activation of the kinases examined also strengthens our proposal that these signals are closely involved in tumor progression in TEL-Syk-induced leukemia.

Figure 4.

TEL-Syk fusion protein constitutively activates STAT5 in the absence of JAK2 activation. (a) BaF3 cell lysates were immunoprecipitated with antibody against STAT1, STAT3 and STAT5, and immunoblotted with the mAb against phosphotyrosine. STAT5 was tyrosine phosphorylated in TEL-Syk-transformed BaF3 cells in the absence of IL-3. (b) BaF3 cells were deprived of IL-3 for 12 h and then restimulated with 5 ng/ml of IL-3 for 30 min. The cell lysates were immunoblotted with the antibody against phospho-STAT5 and phospho-JAK2, and reprobed with antibody against STAT5A, JAK2, respectively. The BaF3 cell transformed by TEL-Syk was constitutively tyrosine phosphorylated of STAT5 in the absence of JAK2 activation.

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MAPK signal pathway was required for TEL-Syk-mediated cell proliferation

To determine which signal pathway is important in factor-independent cell growth, we performed cell proliferation assays with several concentrations of the PI3-K inhibitor, LY294002 and MEK inhibitor U0126.

A reduction in the number of BaF3 cells and TEL-Syk-transformed BaF3 cells was observed with increasing LY294002 concentrations, even in the presence of IL-3. LY294002 suppressed both TEL-Syk-mediated and IL-3-induced BaF3 cell proliferation (Figure 5a). To determine whether the activation of Akt is dependent on PI3-K, BaF3 cells were pretreated with LY294002 and immunoblotted with the antibody against phospho-Akt. The activation of Akt by TEL-Syk and IL-3 was blocked by LY294002, although phosphorylation of ERK1/2 and STAT5 was not blocked by LY294002 (Figure 5b). These data suggest that PI3-K/Akt activation is important for both TEL-Syk-mediated and IL-3-induced cell proliferation.

Figure 5.

LY294002 inhibited the growth of TEL-Syk-transformed BaF3 cells and phosphorylation of Akt. (a) BaF3 cells and TEL-Syk-transformed BaF3 cells were added to 96-well plates with varying concentrations of LY294002 or carrier (DMSO) alone in the presence or absence of IL-3. The plates were incubated 37°C for 72 h before the addition of a mixture of tetrazolium and electron carrier. Cell proliferation assay was performed in triplicate, and mean absorption values with standard deviation were graphed. (b) BaF3 cells were deprived of IL-3 for 12 h, pretreated with 20 M of LY294002 (+) or without LY294002 (-) (DMSO alone) for 30 min and then stimulated in the presence (+) or absence (-) of IL-3. The cell lysates were immunoblotted with the antibody against phospho-Akt, phospho-ERK1/2 and phospho-STAT5, and reprobed with antibody against Akt, ERK1/2, STAT5A, respectively. Phosphorylation of Akt by TEL-Syk and IL-3 are PI3-K dependent.

Full figure and legend (127K)

TEL-Syk-mediated BaF3 cell growth was selectively inhibited by MEK inhibitor U0126. TEL-Syk-mediated cell proliferation was suppressed more strongly than IL-3-mediated cell proliferation with 10–20 M of U0126 (Figure 6a). To determine whether growth inhibition is dependent on the activation of MAPK, BaF3 cells were pretreated with U0126 and immunoblotted with the antibody against phospho-ERK1/2. The activation of ERK1/2 by TEL-Syk was inhibited by U0126, although phosphorylation of Akt and STAT5 were not inhibited by U0126 (Figure 6b). These data suggest that MAPK signal pathway is required for cytokine-independent cell proliferation.

Figure 6.

The MAPK signal pathway is required for TEL-Syk-mediated cell proliferation. (a) BaF3 cells and TEL-Syk-transformed BaF3 cells were added to 96-well plates with varying concentrations of U0126 or carrier (DMSO) alone in the presence or absence of IL-3. Cell proliferation assay was performed in triplicate, and mean absorption values with standard deviation were graphed. TEL-Syk-mediated cell proliferation was inhibited by U0126. (b) BaF3 cells were deprived of IL-3 for 12 h, pretreated with 20 M of U0126 (+) or without U0126 (-) (DMSO alone) for 4 h and then stimulated in the presence (+) or absence (-) of IL-3. Cell lysates were immunoblotted with the antibody against phospho-ERK1/2, phospho-Akt and phospho-STAT5, and reprobed with antibody against ERK1/2, Akt, STAT5A, respectively. Phosphorylation of ERK1/2 by TEL-Syk was inhibited by U0126.

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TEL-Syk fusion protein was located in cytoplasm

TEL is a nuclear phosphoprotein that is widely expressed in normal tissues.^46,47 Syk is distributed in cytosol and initiates signal transduction cascades in lymphoid cells.^4,48 To determine the localization of TEL-Syk fusion protein, COS-7 cells transfected with pF-TS were stained with anti-Flag mAb and visualized with goat anti-mouse IgG (FITC). The immunofluorescence technique revealed the TEL-Syk fusion protein to be located in the cytoplasm (Figure 7a and b). TEL-Syk fusion products, not in the nucleus but in the cytoplasm, led to the constitutive activation of PI3-kinase, MAPK and STAT5 signal pathways, which are important in the IL-3-independent cell proliferation of BaF3 cells.

Figure 7.

Immunofluorescence localization of TEL-Syk fusion protein. (a) COS-7 cells transfected with pF-TS was stained with anti-Flag mAb and visualized with goat anti-mouse IgG (FITC). (b) COS-7 cells transfected with pF-TS was stained with goat anti-mouse IgG (FITC) (secondary antibody alone). Propidium iodide (Sigma Aldrich) was used for the fluorescent staining of the nucleic acid.

Full figure and legend (330K)

Discussion

We investigated the intracellular signal transduction in TEL-Syk-transformed BaF3 cells. Syk binds to ITAM and initiates signaling through the Ras, PI3-K and PLC- signal pathways.^5,6 We showed the association between the p85 subunit of PI3-K and TEL-Syk fusion protein followed by the constitutive activation of Akt. Akt is the serine/threonine kinase and an important component of cell survival signaling. Akt phosphorylates the BCL family member BAD and suppresses apoptosis.⁴⁹ Subsequently, apoptosis is inhibited by phosphorylation and inactivation of caspase-9.⁵⁰ TEL-Syk may play a critical role in antiapoptosis and cell survival of IL-3-depleted BaF3 through the suppression of the caspase pathway. Beitz et al⁶ demonstrated that Syk plays a major role in linking the B-cell receptor to PI3-K activation; however, the Syk-dependent pathways for PI3-K activation is not shown. Our data suggest that Syk and the p85 subunit of PI3-K may form a protein complex.

Deregulation of the JAK-STAT pathway is observed in many hematopoietic malignancies.²³ STAT5 is phosphorylated and activated in a variety of leukemia cells containing tyrosine kinase oncogenes, including BCR-ABL, TEL-JAK2, TEL-PDGFR and FLT3 with internal tandem duplication.^24,51,52,53 However, the constitutive JAK activation is not necessarily required for transformation.⁵⁴ A constitutively active mutant of STAT5 is sufficient to induce factor independence of BaF3 cells.⁵⁵ We have previously reported the constitutive kinase activation of Syk and cytokine-independent proliferation in TEL-Syk-transformed BaF3 cells.²⁰ We here show the constitutive tyrosine phosphorylation of STAT5 in the absence of JAK2 activation and the suppression of IL-3-induced JAK2 phosphorylation in TEL-Syk-transformed BaF3 cells. The expression of JAB (also called SOCS-1) was unable to inhibit the growth of TEL-Syk-transformed BaF3 cells and tyrosine phosphorylation of STAT5 (data not shown), although JAB can inhibit JAK signaling.^56,57 The constitutive tyrosine kinase activation of TEL-Syk led to the activation of STAT5, not through the JAK-family members, while the exact transduction mechanism from TEL-Syk to STAT5 is not known. Klejman et al⁵⁸ recently reported hematopoietic cell kinase-coupled BCR-ABL to STAT5 activation. In vitro phosphorylation assay and in vitro interaction assay between STAT5 and TEL-Syk may reveal whether the interaction is direct. We do not know the reasons behind the downregulation of IL-3-induced JAK2 phosphorylation in TEL-Syk-transformed BaF3 cells, but hope to examine the functional activity of IL-3 receptor in TEL-Syk-transformed BaF3 cells in the future.

MAPK, also known as ERK, is serine/threonine kinase that appears to be one of a key regulator of cell proliferation and differentiation, and is constitutively activated in a majority of acute leukemia.^25,27,38 The upstream components of the MAPK pathway contain the Ras signaling cascade. ERK1/2 and MEK1/2, which were constitutively activated in the TEL-Syk-transformed BaF3 cells, might be activated through the Ras/Raf/MEK/ERK cascade downstream of TEL-Syk signaling. MEK inhibitor U0126 selectively inhibited the TEL-Syk-mediated BaF3 cell growth and activation of MAPK. Constitutively activated form of MEK1 can transform NIH 3T3 and Chinese hamster fibroblast,^59,60 and conditionally active form of MEK1 can also abrogate cytokine dependency of hematopoietic cell lines TF-1, FDC-P1 and FL5.12.^61,62 These data suggest that the MAPK signal pathway is important in TEL-Syk-mediated BaF3 cell proliferation and might be one of the targets for chemotherapy in TEL-Syk disease.

Tyrosine kinase inhibitors are being explored in leukemia therapy.² The BCR-ABL tyrosine kinase inhibitor STI571 (Imatinib mesylate) has significant antileukemic activity in patients with CML.⁶³ Piceatannol, which is known as a Syk-selective tyrosine kinase inhibitor,^64,65 suppressed the survival and proliferation of TEL-Syk-transformed BaF3 cells and inhibited the phosphorylation of STAT5, but piceatannol also suppressed IL-3-induced BaF3 cell growth (data not shown). Piceatannol not only inhibits protein tyrosine kinase Syk but also interferes with other kinases and induces apoptosis in leukemic lymphoblasts.^66,67 TEL-Syk fusion is a useful tool in the search for another Syk-specific inhibitor and for future investigation of Syk.

Recent reports suggest that Syk is a tumor suppressor. Goodman et al⁹ reported that childhood pro-B-cell ALL is caused by Syk deficiency, and Coopman et al¹¹ showed that Syk suppresses breast cancer cell growth and metastasis. However, the TEL-Syk fusion gene was discovered in the presented case of myeloid dysplasia with unique clinical features, including skin involvement, dry cough and eosinophilia, and Syk was constitutively activated in hematopoietic cells.^10,20 The presented evidence reveals another function of Syk constitutively activated by oligomerization through TEL, that of a stimulator of tumor progression.

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Acknowledgements

We thank Dr JN Ihle for providing anti-STAT5 antisera, Dr Kazuhito Yamamoto and Hitoshi Kiyoi for helpful discussions, and Satoshi Suzuki and Chika Wakamatsu for their excellent technical assistance. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Osaka Foundation for Promotion of Clinical Immunology, the Akiyama Foundation, the Suhara Memorial Foundation, the Mochida Memorial Foundation for Medical and Pharmaceutical Research and the Uehara Memorial Foundation.