International Journal of
HEMATOLOGY
The Protein Tyrosine Phosphatase CD45 Is Required for Interleukin 6 Signaling in U266 Myeloma Cells Qun Zhou, Yuan Yao, Solveig G. Ericson Blood and Marrow Transplant and Hematologic Malignancy Program, Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, West Virginia, USA Received March 31, 2003; received in revised form August 22, 2003; accepted August 26, 2003
Abstract The objective of this study was to examine whether CD45 mediates interleukin 6 (IL-6) signaling in human multiple myeloma (MM) cells. We chose U266 MM cells as a study model and isolated cells into CD45+ and CD45– subpopulations. CD45+ and CD45– U266 cells were cocultured with bone marrow stromal cells (BMSCs). IL-6–induced proliferation in CD45+ U266 cells was inhibited by vanadate, a potent protein tyrosine phosphatase inhibitor. However, IL-6–independent CD45– U266 cell growth was not affected by vanadate. CD45+ U266 cells, but not CD45– U266 cells, have the capability of cell adhesion concomitant with actin filament polymerization at the adherent cells. Adhesion of CD45+ U266 cells to BMSCs was impaired by vanadate. We clarified the signaling differences between CD45+ and CD45– U266 cells in response to IL-6. In CD45+ U266 cells, IL-6 increased tyrosine phosphorylation of gp130 and STAT3 and stimulated the level of Mcl-1 protein expression. An association between CD45 and the Src-family protein tyrosine kinase, Lyn, was maintained in the presence of IL-6; the formation of the CD45/Lyn complex was impaired by vanadate. Additionally, IL-6–induced Lyn kinase activity in CD45+ U266 cells was increased by the cross-linking of CD45, and this increase was due to the dephosphorylation of Tyr507 at Lyn. In conclusion, IL-6–dependent MM cells require CD45 to initiate IL-6 signaling and to maintain Lyn kinase activity, both of which are essential for cell proliferation and cell adhesion. Int J Hematol. 2004;79:63-73. ©2004 The Japanese Society of Hematology Key words: CD45; IL-6–dependent/independent myeloma cells; Lyn kinase
JAK/STAT pathway is thought to play an important role in MM development and has been elucidated in detail. Binding of IL-6 to its receptor component, the IL-6 receptor chain (IL-6R), triggers the formation of a signaling-competent receptor complex composed of IL-6, IL-6R, and the IL-6 receptor chain (gp130) and subsequently leads to activation of JAK and tyrosine phosphorylation of STATs [2]. Activated STATs homodimerize or heterodimerize and translocate into the nucleus, where they bind to DNA and mediate the function of key transcription factors and regulators, such as c-Myc, myeloid cell factor-1 (Mcl-1), and Bcl-2, that are involved in cell proliferation and antiapoptosis [2,9,10]. Mcl-1 is a member of the antiapoptotic Bcl-2 family [11]. Recent studies have reported that Mcl-1 is predominantly a survival factor for MM cells, because an Mcl-1 antisense oligonucleotide significantly inhibits the proliferative effect of IL-6 and increases MM cell sensitization to dexamethasone treatment [12,13].
1. Introduction Multiple myeloma (MM) is a cytokine-driven B-cell neoplasm. Interleukin 6 (IL-6) is a critical growth factor for MM cell growth and survival [1]. IL-6 is produced by bone marrow stromal cells (BMSCs) as well as by the MM cells themselves [2-5]. The current concept is that IL-6 can activate several signal transduction pathways, including the Janus kinase/ signal transducer and activator of transcription (JAK/STAT) pathway, Src-family kinases (Lyn, Fyn, and Hck), and the Ras/mitogen-activated protein kinase cascade [6-8]. The
Correspondence and reprint requests: Solveig G. Ericson, MD, PhD, Blood and Marrow Transplant and Hematologic Malignancy Program, Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, WV 26506, USA; 1-304-293-6859; fax: 1-304-293-2134 (e-mail:
[email protected]).
63
64
Zhou et al / International Journal of Hematology 79 (2004) 63-73
Although the JAK/STAT pathway has been well documented to initiate signaling of IL-6, the insight into the protein tyrosine phosphatases (PTPases) that regulate the IL-6 signaling pathway has not been completely elucidated. Signal transduction pathways depend on the balance between protein tyrosine kinases and PTPases. CD45 is a membrane-bound PTPase and is expressed on all nucleated leukocytes. Studies have shown that CD45 can not only be associated with the IL-6 receptor component gp130 but also be selectively associated with the Src-family kinases, such as Lyn in B-cells [7,14]. Investigators have reported that CD45 can dephosphorylate JAK [15], Srcfamily kinases (Lyn, Fyn, and Hck) [16], and STAT3 [17], indicating that CD45 plays a crucial role for B-cell antigen receptor (BCR) signaling [18,19]. Src-family kinases have been identified as primary molecular substrates for CD45 [20]. Src-family kinase activity is enhanced by the phosphorylation of tyrosine residues at the positive regulatory site located within the kinase domain and/or dephosphorylation of tyrosine residues at the carboxyl-terminal negative regulatory site [3,16]. Expression of CD45 is required for Src-family protein tyrosine kinase activation in BCR-mediated signal transduction [21]. Numerous studies have confirmed that the BCRmediated signaling pathway fails to activate Src-family kinases in CD45-deficient B-cells [18,21-23], suggesting that B-cell activation requires CD45 participation in the dephosphorylation of the Src-family kinases [14,24,25]. A considerable CD45+ cell population was found to exist in MM patients [26]. More recently, the role of CD45 in MM development has received increased attention. First, the expression of CD45 affects MM cells in response to IL-6 and chemotherapy. CD45– MM cells are greatly diminished in their capacity to respond to IL-6 but are more sensitive to chemotherapy. In contrast, the proliferation of CD45+ MM cells is IL-6 dependent and is protected from dexamethasone-induced apoptosis [23,27,28]. Second, CD45 is required for IL-6–induced activation of Lyn kinase in MM cells [23,29]. Activated Lyn kinase can strengthen cell-cell adhesion by remodeling actin filament polymerization [30]. Third, expression of CD45 is associated with MM invasive capacity. In the 5T2MM experimental mouse model, CD45+ MM cells represent an aggressive invasive phenotype compared with the CD45– MM cell [31]. The role of CD45 in MM cell proliferation remains unclear. In the present study, we used the U266 MM cell line as a study model to elucidate the function of CD45 in the IL-6 signaling pathway with emphasis on the molecular mechanisms of IL-6–dependent and IL-6–independent U266 cells.
2. Materials and Methods 2.1. Antibodies and Reagents Sodium orthovanadate was purchased from Fisher (Pittsburgh, PA, USA). Recombinant human IL-6 and goat F(ab)2 antimouse immunoglobulin G (IgG) plus IgM, which was used as cross-linker, were obtained from Biosource International (Camarillo, CA, USA). Fluorescein
isothiocyanate (FITC)-labeled phalloidin was obtained from Sigma Diagnostics (St. Louis, MO, USA). FITC-labeled anti–IL-6R mouse antibody, phycoerythrin (PE)-labeled anti-CD45 mouse antibody, and directly conjugated isotypespecific immunoglobulins were purchased from Caltag Laboratories (Burlingame, CA, USA).
2.2. Cell Culture and Cell Sorting U266 myeloma cells were purchased from the American Type Culture Collection (Rockville, MD, USA). The cells were maintained at 37C in a humidified atmosphere of 95% air and 5% carbon dioxide. After 4 or 5 days, cells were passaged at a 1:5 ratio. The CD45+ and CD45– subpopulations of U266 cells were separated with the CELLection Pan Mouse IgG kit (Dynal Biotech, Lake Success, NY, USA) according to the manufacturer’s instructions. In short, the Dynabeads (Dynal) were coated for direct selection with murine monoclonal antibody (MoAb) to human CD45 (Caltag Laboratories). The CD45– subpopulation was isolated by 4 sequential depletions with the murine antihuman CD45 antibody–coated beads. The purity of the CD45+ and CD45– U266 cells was greater than 95%, as confirmed by flow cytometry using PE-conjugated anti-CD45 antibody. Sorted CD45+ or CD45– U266 MM cells were cultured in RPMI-1640 medium supplemented with 15% fetal calf serum (FCS) and 2 mM L-glutamine (Gibco BRL, Gaithersburg, MD, USA).
2.3. Culture of BMSCs BMSCs were provided by Dr. Gibson (Department of Pediatrics, West Virginia University) and were cultured according to the method of Mudry et al [32] with minor modification. Briefly, 1 106 cells were plated in a 75-cm2 flask and cultured at 37C and with 5% carbon dioxide in –minimal essential medium (-MEM; Gibco BRL) supplemented with 5% FCS. All culture media were supplemented with 100 U/mL penicillin and 100 g/mL streptomycin. The nonadherent cells were removed after 3 days, and the cell culture medium was replaced as needed until a confluent monolayer had developed.
2.4. MTS Assay The confluent adherent BMSCs were subcultured into 96well microtiter plates (1 104 cells/well) and incubated in -MEM supplemented with 5% FCS. After 24 hours, sorted CD45+ or CD45– U266 cells (3 104 cells/well) were added to each well, and the medium was replaced with RPMI-1640 medium containing 15% FCS. Cocultured cells were incubated at 37C for 24 hours and then treated with IL-6, vanadate, or a combination of the two. Forty-eight hours after treatment, the CD45+ or CD45– U266 cells were collected from adherent stromal cells by vigorous pipetting. Cell proliferation was measured by a 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay kit (CellTiter 96 AQueous one-solution assay; Promega, Madison, WI, USA) in triplicate samples.
CD45 Required for IL-6 Signaling in U266 Myeloma Cells
2.5. Cross-Linking of CD45 on MM Cells Cross-linking of CD45 has been described previously [33]. Briefly, cells (1 106) were incubated for 10 minutes at room temperature with or without 10 g/mL F(ab)2 antiCD45 MoAb. Excess MoAb was removed by washing cells twice with phosphate-buffered saline (PBS), and 50 g/mL of goat F(ab)2 antimouse IgG and IgM then was added as the cross-linker. The time of addition of the cross-linker was considered the start of cell stimulation. Cells were incubated at room temperature with the cross-linker for the indicated times.
2.6. Western Blotting CD45+ or CD45– U266 cells (5 106 cells/mL) were incubated for 10 minutes with IL-6 alone, a combination of IL-6 and vanadate, or a combination of IL-6 and crosslinking of CD45. Cells were then lysed in the harvesting buffer (1% sodium dodecyl sulfate [SDS], 10 mM Tris-HCl, pH 7.4). The supernatants were collected after centrifugation at 14,000 rpm for 5 minutes at 4C in an Eppendorf microcentrifuge (Brinkmann, Westbury, NY, USA). Equal amounts of protein were loaded onto 10% SDS-polyacrylamide minigels. Proteins were transferred to polyvinylidene difluoride membranes (Invitrogen, Carlsbad, CA, USA) and blocked overnight at 4C with 3% nonfat milk blocking buffer (3 g nonfat dry milk per 100 mL of Trisbuffered saline [20 mM Tris-HCl, pH 7.5, 0.5 M NaCl]) and 0.05% (vol/vol) polysorbate 20 (Tween 20). Membranes were incubated for 3 hours at room temperature with the primary antibody anti–phospho-Lyn (Tyr507), anti-STAT3, anti–phospho-STAT3 (Tyr705) (Cell Signaling, Beverly, MA, USA), or Mcl-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The primary antibodies were diluted 1:500 in Western washing solution (0.1% nonfat dry milk, 0.1% chicken egg albumin, 1% [vol/vol] FCS, 10% [vol/vol] 10 PBS, pH 7.3, and 0.2% [vol/vol] Tween 20). After washing 3 times with Western washing solution and once with Tris-buffered saline, the antigen-antibody complexes were incubated for 1 hour at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibody (antimouse IgG–HRP or antirabbit IgG–HRP; Santa Cruz Biotechnology) diluted 1:3000 in Western washing solution. Antibody binding was visualized by means of enhanced chemiluminescence (SuperSignal West Pico; Pierce, Rockford, IL, USA) and autoradiography.
2.7. Immunoprecipitation and In Vitro Kinase Assay Sorted CD45+ or CD45– U266 cells were incubated with medium alone, IL-6, a combination of IL-6 and vanadate, or a combination of IL-6 and cross-linking of CD45 for 10 minutes. The cells were collected and washed twice with ice-cold PBS. Pellets were incubated in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM -glycerophosphate, 1 mM sodium vanadate, 1 mg/mL leupeptin, and 1 mM phenylmethyl sulfonyl fluoride) for 30 minutes on ice and then homogenized with a Dounce
65
homogenizer (T type, 10 strokes). Lysates were centrifuged in an Eppendorf microcentrifuge (14,000 rpm, 15 minutes) at 4C to remove nuclei and cell debris. The supernatants were precleared by incubation with protein G–Sepharose for 1 hour at 4C to minimize nonspecific protein binding. Lyn proteins were immunoprecipitated from precleared supernatants by the addition of a polyclonal rabbit anti-Lyn antibody (SC-15; Santa Cruz Biotechnology) for 6 hours at 4C, and conjugates of antibody and Lyn proteins were coupled to protein G–Sepharose beads (Santa Cruz Biotechnology) by incubating the rotating suspension overnight at 4C. Lyn kinase activity in the immune complex was measured by either SDS–polyacrylamide gel electrophoresis (SDS-PAGE) or a tyrosine kinase enzyme-linked immunosorbent assay (ELISA) kit (Chemicon International, Temecula, CA, USA) as described by the manufacturer. For the SDSPAGE kinase assay, the immune complex–coated beads were washed twice with lysis buffer and 3 times with kinase buffer (25 mM Tris, pH 7.5, 5 mM -glycerophosphate, 2 mM dithiothreitol, 0.1 mM sodium orthovanadate, and 10 mM MgCl2) and then incubated with kinase buffer containing 200 M adenosine triphosphate and aciddenatured rabbit muscle enolase (Sigma Chemical Company, St. Louis, MO, USA) at room temperature for 30 minutes. The reactions were terminated by adding SDS sample buffer. The denatured samples were subjected to 12% SDS-PAGE analysis. Phosphorylated Lyn was detected by a monoclonal anti–phosphorylated tyrosine antibody (4G10; Upstate Biotechnology, Lake Placid, NY, USA) and quantified by means of Eagle Eye II analysis (Stratagene, La Jolla, CA, USA).
2.8. Flow Cytometry Analysis Flow cytometry analysis for the presence of cell surface receptors was done as described by Ericson et al [34]. Direct immunofluorescence staining was performed for CD45 expression. Cells (1 106) were incubated with PE-labeled anti-CD45 antibody for 60 minutes at 4C. Following staining, cells were washed with 1 mL PBA (10 mM PBS containing 1% of a 10% bovine serum albumin solution and 0.2% sodium azide) and fixed with 400 L 1% paraformaldehyde. Surface immunofluorescence was then detected by the Becton Dickinson FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Indirect immunofluorescence staining was used to detect expression of the IL-6R receptor. The mouse antihuman antibody to IL-6R (CD126) was incubated with cell samples for 60 minutes. Cells were washed with 1 PBA and incubated with FITC-conjugated antimouse IgG antibody (Biosource International) on ice for 30 minutes. The cells were then washed with PBA and fixed as described above.
2.9. Immunofluorescence Staining and Detection of F-Actin Localization The confluent adherent BMSCs were plated on 95% ethanol–washed glass coverslips (5 5 mm2) in 35-cm2
66
Zhou et al / International Journal of Hematology 79 (2004) 63-73
dishes (1 104 cells/dish) and incubated for 24 hours in -MEM containing 5% FCS before coculture was started. Sorted CD45+ or CD45– U266 cells (5 105 cells/dish) were added to each dish, and the medium was replaced with RPMI-1640 medium containing 15% FCS. Prior to stimulation, the cells were incubated at 37C for 48 hours. The effects of CD45 on F-actin filament polymerization were evaluated by treating cocultured cells with or without vanadate for 48 hours. Cells on coverslips were fixed in 3.7% formaldehyde, rinsed in PBS, and then treated briefly with 0.4% Triton X-100 in 1 PBS. After 3 rinses in 1 PBS, the coverslips were incubated for 30 minutes with FITC-phalloidin (5 g/0.1 mL) in the dark. All coverslips were rinsed in 1 PBS and mounted with Fluoromount-G (Southern Biotechnology Associates, Birmingham, AL, USA) containing 2.5% N-propyl gallate. Images were obtained with a Zeiss Axiovert 100 M confocal microscope equipped with a 63 objective lens (Carl Zeiss Microimaging, Thornwood, NY, USA).
3. Results 3.1. Isolation of CD45+ and CD45– Subpopulations from U266 Cells Mahmoud et al reported that U266 cells show a remarkable heterogeneous expression of CD45 and that most of the cells (approximately 70%) are CD45– [27]. U266 cells were separated into CD45+ and CD45– subpopulations by immunomagnetic cell sorting. Flow cytometric analysis confirmed that sorted CD45+ and CD45– U266 cells were >98% and 94% pure, respectively (Figure 1A). Fluorescence microscopy also revealed membrane-associated CD45 staining in CD45+ U266 cells but not in CD45– U266 cells (Figure 1B). Furthermore, CD45+ U266 cells showed strong IL-6R (gp80) expression. However, CD45– U266 cells had no expression of IL-6R greater than that of the isotype control.
3.2. CD45+ U266 Cells Are IL-6 Dependent in the Presence of BMSCs To evaluate whether the expression of CD45 on U266 cells affects the proliferative effect of IL-6, we used an MTS assay to monitor cell proliferation in cocultures of BMSCs with CD45+ or CD45– U266 cells. As shown in Figure 2, IL-6 (10 ng/mL) by 48 hours had stimulated the cellular proliferation of CD45+ U266 cells, whereas IL-6 did not stimulate proliferation in CD45– U266 cells. Similar results were observed with CD45– RPMI-8226 MM cells (data not shown). Moreover, vanadate (10 M), a potent PTPase inhibitor, was used to block CD45 PTPase. As seen in Figure 2, IL-6–induced CD45+ U266 cell proliferation was inhibited by vanadate. Interestingly, vanadate alone also inhibited CD45+ U266 cell growth. In contrast, CD45– U266 cell proliferation was not affected by vanadate. In addition, BMSC proliferation was not influenced by vanadate (data not shown). Taken together, these data demonstrate that CD45+ U266 cells showed an IL-6–dependent response and that CD45– U266 cells were independent of IL-6 stimulation, sug-
gesting that CD45 PTPase plays a key role in the MM response to IL-6.
3.3. CD45+ U266 Cells Show F-Actin Filament Polymerization in the Presence of BMSCs It has been reported that CD45– MM cells have lower migration and homing capacities than CD45+ MM cells in the BMSC microenvironment [35]. Cocultures of BMSCs and MM cells result in a considerable increase in IL-6 secretion [36]. The interaction between MM cells and BMSCs stimulates the expression of a variety of adhesion molecules, such as VCAM-1 for MM homing [37]. To determine whether CD45 affects the adhesion of MM cells to BMSCs, we used immunofluorescence microscopy to assess cell adhesion and actin filament organization. Cocultures of BMSCs with CD45+ or CD45– U266 cells were treated with either medium alone (control) or vanadate for 48 hours. Under these conditions, CD45+ U266 cells clearly appeared to adhere to BMSCs, and actin filament polymerization was seen in the adherent CD45+ U266 cells (Figure 3A). After 48 hours of treatment with vanadate, the adhesion of CD45+ U266 cells to BMSCs was impaired, and CD45+ U266 cells showed no actin filament polymerization in the cytoplasm. In contrast, CD45– U266 cells were unable to attach to BMSCs. These data indicate that CD45 can regulate cell adhesion. It is necessary to determine whether IL-6 secretion by BMSCs is regulated by phosphatase activity. Cocultures of BMSCs and CD45+ U266 cells were incubated with 10 M vanadate for 48 hours, and the supernatants were collected for IL-6 ELISA and VCAM-1 ELISA. Compared with controls, vanadate incubation did not change the levels of IL-6 and VCAM-1 protein (data not shown). These observations suggest that the production of IL-6 and VCAM-1 protein by BMSCs is unlikely to be regulated by signaling that depends on PTPase.
3.4. IL-6 Induces Tyrosine Phosphorylation of gp130 in CD45+ U266 Cells A model of IL-6–induced signal transduction maintains that gp130 is phosphorylated at tyrosine residues and predominantly activates STAT3 [38,39]. Dimerized STAT3 translocates into the nucleus and leads to the transcriptional activation of target genes such as Mcl-1 [40]. To determine if the IL-6/gp130/STAT3 pathway contributes to IL-6–dependent MM cell proliferation, we compared the signaling pathway differences of IL-6–dependent CD45+ U266 cells and IL-6–independent CD45– U266 cells. Cells were treated with or without IL-6 for 10 minutes. Cell lysates were immunoprecipitated with gp130 and immunoblotted with an antibody that specifically recognizes phosphorylated tyrosine. Figure 4A shows that IL-6 strongly induced tyrosine phosphorylation of gp130 in CD45+ U266 cells, whereas IL-6 was not able to induce tyrosine phosphorylation of gp130 in CD45– U266 cells. The activation of STAT3 in CD45+ and CD45– U266 cells was measured by comparing the tyrosine-phosphorylated (Tyr705) levels of STAT3 in the presence and absence of
CD45 Required for IL-6 Signaling in U266 Myeloma Cells
67
Figure 1. Identification of sorted CD45+ (CD45-expressing) and CD45– (CD45-nonexpressing) U266 cells. A, Expression of CD45 was determined by flow cytometry in sorted CD45+ and CD45– U266 cells. The cells were immunolabeled with the phycoerythrin (PE)-conjugated or fluorescein isothiocyanate (FITC)-conjugated anti-CD45 monoclonal antibody (green line). The x-axis indicates the fluorescence intensity. Isotype-specific controls are indicated with the blue color. B, Localization of CD45 was confirmed by confocal microscopy. The cells were fixed and incubated to PEconjugated anti-CD45 antibody. CD45 is expressed only on sorted CD45+ U266 cells (original magnification 40). C, Expression of CD126 (interleukin-6R) in sorted CD45+ and CD45– U266 cells was analyzed by flow cytometry using FITC-conjugated anti-CD126 antibody. Data shown are from one of 3 independent experiments.
IL-6.Western blotting was performed with antibodies against STAT3 and tyrosine-phosphorylated (Tyr705) STAT3. IL-6 induced phosphorylation of STAT3 in CD45+ as well as in CD45– U266 cells (Figure 4C). These data suggest that activation of STAT3 by IL-6 can occur in IL-6–independent CD45– MM cells, although these cells showed no gp130 phosphorylation in response to IL-6. We also examined whether the activation of STAT3 by IL-6 was correlated with the expression of Mcl-1 in CD45+ and CD45– U266 cells. Equal amounts of protein were separated by SDS-PAGE, and Western blotting was performed with an antibody against Mcl-1. IL-6 caused a 2.5-fold eleva-
tion of Mcl-1 levels in CD45+ U266 cells but not in CD45– U266 cells (Figure 4E). IL-6 treatment did not alter Bcl-2 protein expression in either CD45+ or CD45– U266 cells (data not shown).
3.5. Phosphorylation of STAT3 by IL-6 Is Not Targeted by CD45 PTPase To explore if CD45 PTPase affected the activation of STAT3, we pretreated CD45+ and CD45– U266 cells with either vanadate or cross-linking of CD45 for 15 minutes and subsequently exposed the cells to IL-6 for 10 minutes. Cell
68
Zhou et al / International Journal of Hematology 79 (2004) 63-73
oligonucleotide significantly reduces IL-6–induced cell proliferation [23]. Previous work with U266 MM cells demonstrated that CD45 is the critical modulator for IL-6–induced activation of Lyn kinase [23,29]. It is essential to define whether CD45 PTPase affects the association of Lyn and CD45 in MM cells. CD45+ U266 cells and CD45– U266 cells were pretreated with vanadate for 15 minutes, and this treatment was followed by 10 minutes of IL-6 stimulation. Cell lysates were immunoprecipitated with an antibody against CD45, and the immune complexes were analyzed by Western blotting using an anti-Lyn antibody. As shown in Figure 5, the association of CD45 with Lyn was seen in resting CD45+ U266 cells. On IL-6 stimulation, Lyn protein became preferentially associated with CD45, but vanadate blocked the formation of the CD45/Lyn complex. The data indicate that CD45 PTPase activity is required for stabilizing the formation of the CD45/Lyn complex.
3.7. Cross-Linking of CD45 Enhances Lyn Kinase Activity in CD45+ U266 Cells
Figure 2. Vanadate inhibits interleukin 6 (IL-6)-induced cellular proliferation in CD45+ U266 cells but not in CD45– U266 cells. CD45+ U266 cells (A) and CD45– U266 cells (B) were cocultured with an adherent layer of bone marrow stromal cells for 48 hours in medium alone (control), 10 ng/mL IL-6, 10 M vanadate, or a combined treatment of IL-6 and 10 M vanadate.The cells were then collected by vigorous pipetting, and cellular proliferation was measured in triplicate samples with an MTS assay. Data shown are the mean SEM and are representative of 2 independent experiments. lysates were analyzed by Western blotting using antibodies against STAT3 and phosphorylated STAT3. Activation of STAT3 by IL-6 was not affected by either vanadate or the cross-linking of CD45 in CD45+ and CD45– U266 cells (Figure 4F). These results indicate that activation of STAT3 by IL-6 is not directly targeted by CD45 PTPase. In addition, phosphorylation of STAT3 by IL-6 does not correlate with the overexpression of Mcl-1 in CD45– U266 cells, suggesting that activation of STAT3 is not sufficient for cell proliferation in IL-6–independent MM cells [23].
3.6. The Association of CD45 and Lyn Is Found in CD45+ U266 Cells Of the Src-family kinases, Lyn exhibits the most restricted expression in MM cells [23]. Recent work with CD45+ U266 cells has revealed that a Lyn antisense
We explored the mechanism of how CD45 regulates Lyn kinase activity. CD45+ and CD45– U266 cells were pretreated with either vanadate or cross-linking of CD45 for 15 minutes and then incubated with IL-6 for 10 minutes. Cell lysates were immunoprecipitated with anti-Lyn antibody and analyzed for kinase activity by using enolase as the substrate. In CD45+ U266 cells, activation of Lyn kinase was markedly increased after IL-6 treatment, as demonstrated by the phosphorylation of enolase and of Lyn itself. This increase in Lyn kinase activity was suppressed by vanadate and enhanced by cross-linking of CD45 (Figure 6A). The same cell samples were quantified for Lyn kinase activity by means of a tyrosine kinase ELISA, and the results are illustrated in Figure 6B. The results presented in Figures 6A and 6B show a similar pattern. Western blot analysis confirmed an equal loading of Lyn (Figure 6C). In contrast, IL-6 had no effect on Lyn kinase activity in CD45– U266 cells, compared with that of the control. Neither vanadate nor cross-linking of CD45 altered Lyn kinase activity in IL-6–treated CD45– U266 cells. These data support the hypothesis that IL-6 activation of Lyn kinase requires CD45 PTPase activity.
3.8. CD45 Dephosphorylates a Tyrosine Residue of Lyn at the Carboxyl-Terminal Negative Regulatory Site Two tyrosine residues (Tyr396 and Tyr507) have been reported to regulate Lyn kinase activity [41]. Phosphorylation of Tyr396 (equivalent to Tyr416 of c-Src) in the positive regulatory site of Lyn maintains Lyn kinase activation, whereas phosphorylation of Tyr507 (equivalent to Tyr527 of c-Src) at the carboxyl-terminal negative regulatory site of Lyn inactivates Lyn kinase. We used the same cell lysates as described in the Lyn kinase assay and examined whether CD45 can dephosphorylate Lyn at Tyr507. Western blot analysis was performed with antibody against Tyr507phosphorylated Lyn (Figure 6D). In CD45+ U266 cells, IL-6 slightly decreased the level of Tyr507-phosphorylated Lyn
CD45 Required for IL-6 Signaling in U266 Myeloma Cells
69
Figure 3. Vanadate inhibits adhesion of CD45+ U266 cells to bone marrow stromal cells (BMSCs) and blocks actin filament polymerization of the nonadherent CD45+ U266 cells. A, CD45+ U266 cells were cocultured with an adherent layer of BMSCs for 48 hours in medium alone (control) or 10 M vanadate for 48 hours. Fluorescein isothiocyanate–conjugated phalloidin was visualized with an immunofluorescence microscope (original magnification 63). B, In contrast to CD45+ U266 cells, CD45– U266 cells were unable to attach to BMSCs when incubated with either medium or vanadate (original magnification 63). compared with the control. Cross-linking of CD45 markedly decreased the level of Tyr507-phosphorylated Lyn in IL-6–treated CD45+ U266 cells. No change in Tyr507phosphorylated Lyn protein expression was observed when CD45– U266 cells were treated with IL-6 alone, a combination of IL-6 and vanadate, or a combination of IL-6 and cross-linking of CD45. Although an antibody for phosphoLyn (Tyr396) is not available, immunoblotting with an antibody that specifically recognizes the phospho-Src family (Tyr416) showed that it was not detectable (data not shown). Thus, CD45 increases Lyn kinase activity by dephosphorylating the tyrosine residue located in the carboxyl-terminal negative regulatory site.
4. Discussion We have shown two central roles for the membranebound PTPase CD45 in U266 MM cells. First, CD45 is
required for U266 cell proliferation as well as for cell adhesion in the bone marrow microenvironment. Second, CD45 participates in IL-6 signaling pathway initiation and positively regulates Lyn kinase activity. Our data extend the work of other investigators by confirming that CD45 is one of the determinant factors for the proliferation of IL-6–dependent MM cells. Several studies have suggested that CD45 is involved in the proliferation of cultured MM cell lines [42,43]. We considered that CD45 might affect MM proliferation in the bone marrow microenvironment. Our results demonstrated that the proliferation of IL-6–dependent CD45+ U266 cells is accomplished by the activation of CD45 PTPase, because the addition of vanadate inhibited IL-6–induced cell growth. Because changes in CD45 phosphatase activity are thought to play a role in the regulation of adhesion [44-46], it is important to identify whether CD45 affects MM cell adherence to BMSCs. CD45 is essential for cell adhesion, as evi-
70
Zhou et al / International Journal of Hematology 79 (2004) 63-73
Figure 4. Effect of interleukin 6 (IL-6) on expression of gp130, STAT3, and Mcl-1 in CD45+ and CD45– U266 cells. Cells (5 106 cells per sample) were treated in medium as control or with 10 ng/mL IL-6 for 10 minutes. A, Cell lysates were immunoprecipitated (IP) with anti-gp130 antibody and then immunoblotted with anti–tyrosine-phosphorylated (anti-pY) antibody. B, Blots were stripped and reprobed with anti-gp130 antibody. C-E, Cell lysates were analyzed by Western blotting with antibodies against Tyr705-phosphorylated STAT3 (pY-STAT3), STAT3, and Mcl-1. F and G, CD45 protein tyrosine phosphatase does not affect the phosphorylated STAT3 in CD45+ and CD45– U266 cells. Cells were pretreated with either vanadate or cross-linking of CD45 (CD45CL) for 15 minutes and then incubated with IL-6 for 10 minutes. Cell lysates (50 g/lane) were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and analyzed by immunoblotting with antibodies against phosphorylated STAT3 (STAT3-p) and STAT3.
denced by the findings of vanadate inhibition on actin filament polymerization in CD45+ U266 cells and the blocking of CD45+ U266 cell adhesion to BMSCs. However, vanadate was not found to inhibit IL-6 and VCAM-1 protein secretion in cocultures of CD45+ U266 cells with BMSCs. Thus, it is likely that CD45 participates in adhesion through the regulation of actin filament assembly. It is possible that CD45 mediates the activation of tyrosine kinase cascades, such as Src-family kinases, that are important in the formation of actin filament polymerization. The mechanism underlying IL-6–dependent proliferation observed in CD45+ MM cells has been elucidated. The phosphorylation of gp130 is a prerequisite for the initiation of IL-6 signaling, and Mcl-1 expression is the result of a downstream transcriptional event [13,47]. We focused on the gp130/STAT3/Mcl-1 signaling pathway and determined that
differences in intracellular signaling can exist in IL-6–dependent CD45+ and IL-6–independent CD45– U266 cells. In CD45+ U266 cells, IL-6 stimulated the phosphorylation of gp130 and STAT3 with a subsequent induction of Mcl-1 protein expression. In contrast, IL-6 was unable to activate gp130 and increase Mcl-1 protein expression in CD45– U266 cells. These data suggest that IL-6–dependent and IL-6–independent cell responses can be discriminated, based on the status of CD45 and IL-6R. CD45+ MM cells have a bright IL-6R expression, whereas CD45– MM cells display very little IL-6R expression. These differences may determine the response of MM cells to IL-6 [28,48]. Activation of gp130 in these cells requires IL-6 binding to IL-6R [49], and CD45 may participate in maintaining the stability of the IL-6/IL-6R complex. Therefore, in the presence of CD45 and IL-6R, IL-6 may bind to IL-6R sufficiently to
CD45 Required for IL-6 Signaling in U266 Myeloma Cells
71
Figure 5. The association of CD45 and Lyn is affected by CD45 protein tyrosine phosphatase in CD45+ U266 cells. CD45+ and CD45– U266 cells (1 107/sample) were pretreated with vanadate for 15 minutes and then incubated for 10 minutes with 10 ng/mL interleukin 6 (IL-6). Cell lysates were immunoprecipitated (IP) with an anti-CD45 polyclonal antibody coupled to protein A–agarose beads. The immune complex proteins were analyzed on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and probed with anti-Lyn monoclonal antibody. Blots were stripped and reprobed with anti-CD45 antibody.
activate gp130 with the subsequent activation of the downstream IL-6 signaling in CD45+ U266 cells. Although CD45– U266 cells express gp130, IL-6 signaling is not initiated in the absence of CD45 and IL-6R. It is not clear why mature CD45– MM cells simultaneously lose IL-6R and CD45 expression. Tyr705-phosphorylated STAT3 is up-regulated by IL-6 in both CD45+ and CD45– U266 cells, but such activation of STAT3 is independent of CD45 phosphatase activity, because neither vanadate nor cross-linking of CD45 affects IL-6–induced Tyr705 phosphorylation of STAT3. This finding presumably reflects that STAT3 is not a substrate for CD45 PTPase. Several signaling pathways, including the JAK-1, PI3K (phosphatidylinositol 3-kinase), and ERK1/2 pathways, have previously been reported to be activated by IL-6 in IL-6–independent human myeloma cells [23,50,51]. These pathways may contribute to STAT3 activation in IL-6–independent myeloma cells. However, activation of STAT3 has distinct transactivating capabilities in IL-6–dependent and IL-6–independent MM cells. IL-6–dependent STAT3 signaling confers resistance to apoptosis in MM cells through STAT3-mediated up-regulation of Mcl-1 protein expression [52], whereas IL-6–independent STAT3 signaling is committed to the process of IL-6 independence and transformation [50,53]. In IL-6–independent MM cells, the expression of Mcl-1 protein is independent of the activated STAT3 pathway, because the transcriptional activity of the regulatory region 5 to the Mcl-1 gene serves as an IL-6–independent indicator gene to maintain steady-state levels of Mcl-1 messenger RNA and protein [51]. The detailed mechanism of how STAT3 activation contributes to intracellular signaling in IL-6–independent MM cells remains to be elucidated. The Src-family kinases are involved in tumor cell proliferation, migration, and invasion [29,54,55]. However, the mechanism by which CD45 regulates Lyn kinase activity in
IL-6–dependent MM cells has not been identified. In the present study, we have demonstrated that CD45 positively regulates Lyn kinase activity. This conclusion is supported by the results of the following experiments. First, when CD45+ U266 cells were treated with IL-6, the association of CD45 and Lyn was preferentially formed; however, the formation of CD45/Lyn complex was impaired in the presence of vanadate. Second, tyrosine phosphorylation of Lyn by IL-6 was enhanced by the cross-linking of CD45 and attenuated by the presence of vanadate. In accord with these observations, cross-linking of CD45 selectively dephosphorylated Tyr507 at Lyn. These data are consistent with previous reports that tyrosine dephosphorylation at the carboxyl-terminal negative regulatory site of Lyn is an essential step for the maintenance of the kinase activity. The future goal will be to determine how the activation of Lyn kinase elicited by IL-6 regulates cell adhesion via actin filament polymerization. In summary, the IL-6–induced signaling pathway is mediated by CD45 in MM cells. Our results highlight the importance of CD45 in the initiation of IL-6 signaling and the positive regulation of Lyn kinase activity in MM cells. Although CD45+ MM cells may represent a small fraction of the total number of myeloma cells in patients with MM, CD45+ MM cells can significantly impact growth factor–mediated cell survival and the chemotherapeutic response. Understanding the mechanism of how CD45 regulates the IL-6 signaling pathway in MM cells will allow us to develop new strategies for the treatment of patients with MM.
Acknowledgments This study was supported by grant P20 RR16440 from the National Institutes of Health. The authors are grateful to Ms. Kelly Kavanaugh for cell isolation. Our thanks are extended
72
Zhou et al / International Journal of Hematology 79 (2004) 63-73
Figure 6. A, Cross-linking of CD45 (CD45CL) enhances interleukin 6 (IL-6)-induced activation of Lyn kinase in CD45+ U266 cells. CD45+ and CD45– U266 cells were incubated with the indicated treatments for 10 minutes. Lyn kinase activity was measured as described in “Materials and Methods.” pY (Lyn) indicates tyrosine phosphorylated Lyn kinase; Enolase-p, phosphorylated enolase protein. B, Lyn kinase activity was quantified by a tyrosine kinase assay kit. OD indicates optical density; VD, vanadate. C, Equal amounts of Lyn protein were present in each sample. Lyn protein was detected by Western blotting with an anti-Lyn antibody. D, Cross-linking of CD45 dephosphorylates the Lyn Tyr507 at the carboxyl-terminal negative regulatory site. Cell lysates (50 g/lane) were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and analyzed by immunoblotting with an antibody specific for Lyn phosphorylated at the Tyr507 residue (pY507Lyn).
to Dr. H. Ishikawa for providing technical assistance with the Lyn kinase assay.
References 1. Klein B, Zhang XG, Jourdan M, et al. Paracrine rather than autocrine regulation of myeloma-cell growth and differentiation by interleukin-6. Blood. 1989;73:517-526. 2. Hallek M, Bergsagel PL, Anderson KC. Multiple myeloma: increasing evidence for a multistep transformation process. Blood. 1998;91:3-21. 3. Thomas X, Anglaret B, Magaud JP, Epstein J, Archimbaud E. Interdependence between cytokines and cell adhesion molecules to induce interleukin-6 production by stromal cells in myeloma. Leuk Lymphoma. 1998;32:107-119. 4. Schwab G, Siegall CB, Aarden LA, Neckers LM, Nordan RP. Characterization of an interleukin-6-mediated autocrine growth loop in the human multiple myeloma cell line, U266. Blood. 1991;77: 587-593. 5. Bisping G, Leo R, Wenning D, et al. Paracrine interactions of basic fibroblast growth factor and interleukin-6 in multiple myeloma. Blood. 2003;101:2775-2783. 6. Kishimoto T, Akira S, Narazaki M, Taga T. Interleukin-6 family of cytokines and gp130. Blood. 1995;86:1243-1254. 7. Hallek M, Neumann C, Schaffer M, et al. Signal transduction of
8.
9.
10.
11. 12.
13. 14.
15.
interleukin-6 involves tyrosine phosphorylation of multiple cytosolic proteins and activation of Src-family kinases Fyn, Hck, and Lyn in multiple myeloma cell lines. Exp Hematol. 1997;25:1367-1377. Ogata A, Chauhan D, Teoh G, et al. IL-6 triggers cell growth via the Ras-dependent mitogen-activated protein kinase cascade. J Immunol. 1997;159:2212-2221. Schwarze MM, Hawley RG. Prevention of myeloma cell apoptosis by ectopic bcl-2 expression or interleukin 6-mediated up-regulation of bcl-xL. Cancer Res. 1995;55:2262-2265. Puthier D, Bataille R, Amiot M. IL-6 up-regulates mcl-1 in human myeloma cells through JAK/STAT rather than ras/MAP kinase pathway. Eur J Immunol. 1999;29:3945-3950. Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell survival. Science. 1998;281:1322-1326. Derenne S, Monia B, Dean NM, et al. Antisense strategy shows that Mcl-1 rather than Bcl-2 or Bcl-x(L) is an essential survival protein of human myeloma cells. Blood. 2002;100:194-199. Zhang B, Gojo I, Fenton RG. Myeloid cell factor-1 is a critical survival factor for multiple myeloma. Blood. 2002;99:1885-1893. Brown VK, Ogle EW, Burkhardt AL, Rowley RB, Bolen JB, Justement LB. Multiple components of the B-cell antigen receptor complex associate with the protein tyrosine phosphatase, CD45. J Biol Chem. 1994;269:17238-17244. Irie-Sasaki J, Sasaki T, Matsumoto W, et al. CD45 is a JAK phos-
CD45 Required for IL-6 Signaling in U266 Myeloma Cells
16.
17.
18.
19.
20.
21.
22.
23.
24.
25. 26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
phatase and negatively regulates cytokine receptor signalling. Nature. 2001;409:349-354. Thomas ML, Brown EJ. Positive and negative regulation of Srcfamily membrane kinases by CD45. Immunol Today. 1999;20: 406-411. Blank N, Kriegel M, Hieronymus T, et al. CD45 tyrosine phosphatase controls common gamma-chain cytokine-mediated STAT and extracellular signal-related kinase phosphorylation in activated human lymphoblasts: inhibition of proliferation without induction of apoptosis. J Immunol. 2001;166:6034-6040. Yanagi S, Sugawara H, Kurosaki M, Sabe H, Yamamura H, Kurosaki T. CD45 modulates phosphorylation of both autophosphorylation and negative regulatory tyrosines of Lyn in B-cells. J Biol Chem. 1996;271:30487-30492. D’Oro U, Ashwell JD. Cutting edge: the CD45 tyrosine phosphatase is an inhibitor of Lck activity in thymocytes. J Immunol. 1999;162:1879-1883. Trowbridge IS, Thomas ML. CD45: an emerging role as a protein tyrosine phosphatase required for lymphocyte activation and development. Annu Rev Immunol. 1994;12:85-116. Greer SF, Lin J, Clarke CH, Justement LB. Major histocompatibility class II-mediated signal transduction is regulated by the proteintyrosine phosphatase CD45. J Biol Chem. 1998;273:11970-11979. Pao LI, Cambier JC. Syk, but not Lyn, recruitment to B-cell antigen receptor and activation following stimulation of CD45– B-cells. J Immunol. 1997;158:2663-2669. Ishikawa H, Tsuyama N, Abroun S, et al. Requirements of src family kinase activity associated with CD45 for myeloma cell proliferation by interleukin-6. Blood. 2002;99:2172-2178. Mustelin T, Altman A. Dephosphorylation and activation of the T cell tyrosine kinase pp56lck by the leukocyte common antigen (CD45). Oncogene. 1990;5:809-813. Justement LB. The role of CD45 in signal transduction. Adv Immunol. 1997;66:1-65. Jensen GS, Mant MJ, Belch AJ, Berenson JR, Ruether BA, Pilarski LM. Selective expression of CD45 isoforms defines CALLA+ monoclonal B-lineage cells in peripheral blood from myeloma patients as late stage B-cells. Blood. 1991;78:711-719. Mahmoud MS, Ishikawa H, Fujii R, Kawano MM. Induction of CD45 expression and proliferation in U-266 myeloma cell line by interleukin-6. Blood. 1998;92:3887-3897. Grigorieva I, Thomas X, Epstein J. The bone marrow stromal environment is a major factor in myeloma cell resistance to dexamethasone. Exp Hematol. 1998;26:597-603. Kawano MM, Ishikawa H, Tsuyama N, et al. Growth mechanism of human myeloma cells by interleukin-6. Int J Hematol. 2002; 76(suppl 1):329-333. Piccardoni P, Sideri R, Manarini S, et al. Platelet/polymorphonuclear leukocyte adhesion: a new role for SRC kinases in Mac-1 adhesive function triggered by P-selectin. Blood. 2001;98:108-116. Asosingh K, Menu E, Van Valckenborgh E, et al. Mechanisms involved in the differential bone marrow homing of CD45 subsets in 5T murine models of myeloma. Clin Exp Metastasis. 2002;19:583-591. Mudry RE, Fortney JE, York T, Hall BM, Gibson LF. Stromal cells regulate survival of B-lineage leukemic cells during chemotherapy. Blood. 2000;96:1926-1932. Gao H, Henderson A, Flynn DC, Landreth KS, Ericson SG. Effects of the protein tyrosine phosphatase CD45 on FcRIIa signaling and neutrophil function. Exp Hematol. 2000;28:1062-1070. Ericson SG, Zhao Y, Gao H, et al. Interleukin-6 production by human neutrophils after Fc-receptor cross-linking or exposure to granulocyte colony-stimulating factor. Blood. 1998;91:2099-2107. Vanderkerken K, Asosingh K, Braet F, Van Riet I, Van Camp B. Insulin-like growth factor-1 acts as a chemoattractant factor for 5T2 multiple myeloma cells. Blood. 1999;93:235-241.
73
36. Frassanito MA, Cusmai A, Iodice G, Dammacco F. Autocrine interleukin-6 production and highly malignant multiple myeloma: relation with resistance to drug-induced apoptosis. Blood. 2001;97:483-489. 37. Okado T, Hawley RG. Adhesion molecules involved in the binding of murine myeloma cells to bone marrow stromal elements. Int J Cancer. 1995;63:823-830. 38. Darnell JE Jr. STATs and gene regulation. Science. 1997;277: 1630-1635. 39. Darnell JE Jr, Kerr IM, Stark GR. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science. 1994;264:1415-1421. 40. Shuai K, Schindler C, Prezioso VR, Darnell JE Jr. Activation of transcription by IFN-gamma: tyrosine phosphorylation of a 91-kD DNA binding protein. Science. 1992;258:1808-1812. 41. Donella-Deana A, Cesaro L, Ruzzene M, Brunati AM, Marin O, Pinna LA. Spontaneous autophosphorylation of Lyn tyrosine kinase at both its activation segment and C-terminal tail confers altered substrate specificity. Biochemistry. 1998;37:1438-1446. 42. Hata H, Matsuzaki H, Sonoki T, et al. Establishment of a CD45positive immature plasma cell line from an aggressive multiple myeloma with high serum lactate dehydrogenase. Leukemia. 1994; 8:1768-1773. 43. Ishikawa H, Mahmoud MS, Fujii R, Abroun S, Kawano MM. Proliferation of immature myeloma cells by interleukin-6 is associated with CD45 expression in human multiple myeloma. Leuk Lymphoma. 2000;39:51-55. 44. Asosingh K, De Raeve H,Van Riet I,Van Camp B,Vanderkerken K. Multiple myeloma tumor progression in the 5T2 murine model is a multi-stage and dynamic process of differentiation, proliferation, invasion and apoptosis. Blood. 2003;101:3136-3141. 45. Asosingh K, De Raeve H, Croucher P, et al. In vivo homing and differentiation characteristics of mature (CD45–) and immature (CD45+) 5T multiple myeloma cells. Exp Hematol. 2001;29:77-84. 46. Shenoi H, Seavitt J, Zheleznyak A, Thomas ML, Brown EJ. Regulation of integrin-mediated T cell adhesion by the transmembrane protein tyrosine phosphatase CD45. J Immunol. 1999;162:7120-7127. 47. Spets H, Stromberg T, Georgii-Hemming P, Siljason J, Nilsson K, Jernberg-Wiklund H. Expression of the bcl-2 family of pro- and anti-apoptotic genes in multiple myeloma and normal plasma cells: regulation during interleukin-6 (IL-6)-induced growth and survival. Eur J Haematol. 2002;69:76-89. 48. Hata H, Xiao H, Petrucci MT, Woodliff J, Chang R, Epstein J. Interleukin-6 gene expression in multiple myeloma: a characteristic of immature tumor cells. Blood. 1993;81:3357-3364. 49. Ancey C, Kuster A, Haan S, Herrmann A, Heinrich PC, MullerNewen G. A fusion protein of the gp130 and interleukin-6R ligand-binding domains acts as a potent interleukin-6 inhibitor. J Biol Chem. 2003;278:16968-16972. 50. Rawat R, Rainey GJ, Thompson CD, Frazier-Jessen MR, Brown RT, Nordan RP. Constitutive activation of STAT3 is associated with the acquisition of an interleukin 6-independent phenotype by murine plasmacytomas and hybridomas. Blood. 2000;96:3514-3521. 51. Zhang B, Potyagaylo V, Fenton RG. IL-6-independent expression of Mcl-1 in human multiple myeloma. Oncogene. 2003;22:1848-1859. 52. Puthier D,Thabard W, Rapp M, et al. Interferon alpha extends the survival of human myeloma cells through an upregulation of the Mcl-1 anti-apoptotic molecule. Br J Haematol. 2001;112: 358-363. 53. Chakraborty A, Tweardy DJ. Stat3 and G-CSF-induced myeloid differentiation. Leuk Lymphoma. 1998;30:433-442. 54. Irby RB, Yeatman TJ. Role of Src expression and activation in human cancer. Oncogene. 2000;19:5636-5642. 55. Roach T, Slater S, Koval M, et al. CD45 regulates Src family member kinase activity associated with macrophage integrin-mediated adhesion. Curr Biol. 1997;7:408-417.
