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The Journal of Immunology, 2000, 164: 1169-1174.
Copyright © 2000 by The American Association of Immunologists

c-Src Mediates Mitogenic Signals and Associates with Cytoskeletal Proteins upon Vascular Endothelial Growth Factor Stimulation in Kaposi’s Sarcoma Cells1

Neru Munshi*, Jerome E. Groopman*, Parkash S. Gill{dagger} and Ramesh K. Ganju2,*

* Robert Mapplethorpe Laboratory, Divisions of Experimental Medicine and Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115; and {dagger} Division of Hematology/Oncology, Norris Cancer Center, University of Southern California, Los Angeles, CA 90033


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Vascular endothelial growth factor (VEGF) appears to be a critical cytokine modulating the growth and spread of Kaposi’s sarcoma (KS). Furthermore, infection with the KS herpes virus results in up-regulation of VEGF and triggering of VEGF receptor activation. The molecular mechanisms regulating such cytokine-driven proliferation of KS cells are not well characterized. We investigated the role of Src-related tyrosine kinases in VEGF-mediated signaling in model KS 38 tumor cells. VEGF stimulation specifically activated c-Src kinase activity but not that of other related Src kinases such as Lyn, Fyn, or Hck in KS cells. Pyrazolopyrimidine, a selective inhibitor of Src family tyrosine kinases, significantly blocked the VEGF-induced growth of KS cells. Further studies using mutants of c-Src kinase revealed that Src mediates mitogen-activated protein kinase activation induced by VEGF. We also observed that VEGF stimulation resulted in increased tyrosine phosphorylation of the focal adhesion components paxillin and p130cas. Furthermore, VEGF induction enhanced the complex formation between Src kinase and paxillin. Src kinase appears to play an important functional role in VEGF-induced signaling in KS cells and may act to link pathways from the VEGF receptor to mitogen-activated protein kinase and cytoskeletal components, thereby effecting tumor proliferation and migration.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Kaposi’s sarcoma (KS)3 is the most common neoplasm arising in the HIV-1-infected host (1, 2, 3, 4). Its pathogenesis has been attributed to HIV-1 TAT protein, to cytokines, particularly vascular endothelial growth factor (VEGF), platelet-derived growth factor, oncostatin M (OSM), and IL-6, and most recently to KS herpes virus (KSHV), also known as human herpes virus 8 (HHV-8) (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17). KSHV has been shown to encode sequences for various cellular homologues that may stimulate angiogenesis in KS (16, 17, 18, 19, 20).

VEGF and its cognate receptors, VEGF-receptor-1 (VEGFR-1) or FMS-like tyrosine kinase-1 (Flt-1) and VEGF-receptor-2 (VEGFR-2) or fetal liver kinase-1 (Flk-1/KDR), appear to play central roles in mediating effects of the various proposed causative agents of KS (10, 21, 22, 23, 24, 25). KS cells have been shown to secrete large amounts of VEGF protein (23). In vitro and in vivo studies using antisense constructs to VEGF or VEGF chimeric toxin demonstrated a significant reduction in KS spindle cell proliferation when the growth factor was inhibited (10, 26). HIV-1 TAT, via its basic domain, can also bind to and activate the Flk-1/KDR receptor in KS cells (27, 28). KSHV/HHV-8 infection causes paracrine induction of Flk-1/KDR expression (20). Furthermore, KSHV/HHV-8 encodes a constitutively active G-protein coupled receptor (GPCR), which appears to induce expression of VEGF (19, 20).

Despite the emerging role of VEGF and its receptor in the pathogenesis of KS, relatively little is known about the various signaling molecules that mediate its effects in KS cells. Previously, we have shown that VEGF induces phosphorylation of the related adhesion focal tyrosine kinase (RAFTK; also known as Pyk2 or Cak-ß) in KS cells (29). In the present study, we show that VEGF differentially stimulates c-Src kinase activity and induces phosphorylation of components of the focal adhesion complex, including paxillin and p130cas. Src kinase appears to be an early mediator that links VEGF signals from the cell surface to cytoskeletal signaling complexes and downstream to transcriptional activators in KS cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and cell culture

The KS spindle cell line, KS 38, was derived from the biopsy of a cutaneous lesion from an AIDS patient (30, 31, 32, 33). The cell line possesses many characteristics of primary KS spindle cells, including endothelial markers and smooth muscle markers, and has been used as a model for cytokine-mediated signaling studies. Like most spindle cells in primary tumor lesions, it is negative for KSHV/HHV-8 sequences. KS 38 cells were grown on 1.5% gelatin-coated flasks as described and were carried in RPMI 1640 with 15% FCS, 2 mM glutamine, 1 mM MEM sodium pyruvate, 0.05 mM MEM nonessential amino acids, 1x MEM amino acids, 1% Nutridoma-HU (Boehringer Mannheim, Indianapolis, IN), and 50 µg/ml penicillin and 50 µg/ml streptomycin. Cells were grown to confluence before the signaling studies.

Reagents and Abs

Abs to the various Src family members were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and Upstate Biotechnology (Lake Placid, NY). Monoclonal Abs against p130cas and paxillin were obtained from Transduction Laboratories (Lexington, KY). Anti-phosphotyrosine Ab (PY99) was obtained from Santa Cruz Biotechnology. Recombinant VEGF was obtained from Genentech (South San Francisco, CA). Electrophoresis reagents and the nitrocellulose membrane were obtained from Bio-Rad (Hercules, CA). The protease inhibitors leupeptin, aprotinin, and all other reagents were obtained from Sigma (St. Louis, MO). Tritiated [3H]thymidine was obtained from DuPont NEN (Boston, MA). Pyrazolopyrimidine (PP1), a selective inhibitor of Src family tyrosine kinases, was obtained from Calbiochem-Novabiochem (La Jolla, CA). The lipofectamine PLUS reagent package was obtained from Life Technologies (Gaithersburg, MD). Src mutant cDNA was obtained from Upstate Biotechnology.

Transfections

KS 38 cells were transfected with dominant-negative Src (K297R) in which lysine has been replaced by arginine at position 297, Src-activated mutant (Y529F) in which tyrosine has been replaced by phenylalanine (Y529F), wild-type Src (WT), or with control vector. The transfections were performed using the Lipofectamine method. Briefly, the cells were plated on a 100-mm dish and grown to 50–80% confluence. For each plate, 4 µg of transfection-grade eukaryotic expression vector alone or containing Src cDNA were precomplexed with 20 µl of PLUS reagent and then mixed with 30 µl lipofectamine reagent. These DNA-PLUS lipofectamine reagent complexes were gently added to the cells and incubated at 37°C at 5% CO2 for 3 h. After 3 h, the medium containing the complexes was removed and fresh medium added. Expression of Src mutants was detected after 48 h by immunoblotting transfected cells with 1 µg/ml of anti-Src Ab.

Stimulation of cells

KS 38 cells, grown to confluence, were serum-starved for 16–18 h and washed twice with HBSS (Life Technologies) before VEGF treatment. The cells were then treated with 100 ng/ml of VEGF plus 10 IU/ml of heparin. The growth factor was added to cell cultures singly for different time periods in vitro. Controls included media with or without 10 IU/ml of heparin in the absence of growth factor. The cell lysates were prepared directly within the culture dish by lysis in 500 µl modified RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM PMSF, 10 µg/ml aprotinin, leupeptin, and pepstatin, 10 mM sodium vanadate, 10 mM sodium fluoride, and 10 mM sodium pyrophosphate). Total cell lysates (TCL) were clarified by centrifugation at 10,000 x g for 15 min. Protein concentrations were determined by protein assay (Bio-Rad).

Immunoprecipitation and Western blot analysis

Immunoprecipitation was conducted as described (34). Briefly, identical amounts of protein from each sample were clarified by incubation with protein A Sepharose or {gamma}-bind Sepharose beads (Amersham Pharmacia Biotech, Piscataway, NJ) for 1 h at 4°C, followed by brief centrifugation. The solution was then incubated with different Abs for each experiment for 4 h or clarified overnight at 4°C. The immunoprecipitations of the Ab-Ag complexes were performed by incubation for 2 h at 4°C with 50 µl of the protein A Sepharose or {gamma}-bind Sepharose (10% suspension). Nonspecific proteins were removed by washing the Sepharose beads three times with the modified RIPA buffer and one time with PBS. Bound proteins were solubilized in 40 µl of 2x Laemmli buffer and further analyzed by immunoblotting. Samples were separated on 8% SDS-PAGE and then transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat milk protein and probed with primary Ab for 2 h at room temperature or 4°C overnight. Immunoreactive bands were visualized using HRP-conjugated secondary Ab and the enhanced chemiluminescent (ECL) system (Amersham Pharmacia Biotech).

c-Src kinase assay

Kinase assays with different Src family members such as Lyn, Fyn, Src, or Hck were conducted as described (28). Briefly, the complexes obtained by immunoprecipitating the cell lysates with Abs to various Src family members were washed twice with RIPA buffer and once with kinase buffer (10 mM HEPES, pH 7.4, 5 mM MnCl2, 10 µM Na3VO4). For the in vitro kinase assay, the immune complex was incubated in kinase buffer containing acid-denatured rabbit muscle enolase (Sigma) and 5 µCi [{gamma}-32P]ATP at room temperature for 30 min. The reaction was stopped by adding 2x SDS sample buffer and boiling the samples for 5 min. The samples were run on 10% SDS-PAGE and detected by autoradiography.

Flk-1 assay

The Flk-1 autophosphorylation assay was done as described (28). Briefly, the Flk-1/KDR immunoprecipitates were incubated in kinase buffer (50 mM HEPES, pH 7.4, 10 mM MnCl2, 10 mM MgCl2, 1 mM DTT, 20 µM ATP) plus 5 µCi [{gamma}-32P]ATP for 30 min at 25°C. The samples were then subjected to SDS-PAGE (8% polyacrylamide), and the proteins were visualized by autoradiography.

Cell growth

KS cells were plated in complete medium at 1 x 104 cells/well in a 96-well assay plate. The cells were left to adhere in the incubator overnight and were treated with various concentrations of VEGF for 72 h in RPMI 1640 medium containing 0.5% FCS, then analyzed for cell growth as described below. To assess the functional importance of Src kinase family activation in KS cells, we tested VEGF-induced cell growth under different conditions. The cells were treated with various concentrations of PP1 (Src kinase inhibitor) or diluent control (DMSO) in the presence of VEGF (100 ng/ml) in RPMI 1640 medium containing 0.5% FCS and incubated for 72 h at 37°C. Cell growth was assessed by the addition of 1 µCi/well [3H]thymidine for 8 h at 37°C (after 72 h). The radioactive labeling was terminated by washing the cells three times with 1x PBS. The cells were then lysed with 1% SDS solution, and the amount of incorporated [3H]thymidine was determined using a Packard Microplate Scintillation counter (Meriden, CT).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
VEGF induced tyrosine phosphorylation of cellular proteins in KS cells

VEGF has been shown to induce tyrosine phosphorylation of a number of intracellular proteins in endothelial cells. Because KS cells express VEGF receptors, we first examined KS cells for changes in tyrosine phosphorylation in response to VEGF. As shown in Fig. 1Go, VEGF stimulation resulted in the increased tyrosine phosphorylation of several cellular proteins.



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  		FIGURE 1. Tyrosine phosphorylation of cellular proteins in KS cells after VEGF stimulation. Serum-starved KS cells were untreated (0) or treated with VEGF (100 ng/ml) for the indicated times. Total cell lysates (50 µg) obtained after cell lysis were size-fractionated on 8% SDS-PAGE and subjected to Western blot analysis with the anti-phosphotyrosine Ab 4G10. The arrows indicate the protein bands that show increased tyrosine phosphorylation after VEGF treatment. MW, molecular weight in thousands.

 
Src kinases are differentially activated in response to VEGF in KS cells

Because we observed an increase in tyrosine phosphorylation of intracellular proteins around 55–60 kDa, we were interested in determining if Src kinases are activated upon VEGF stimulation. The Src family kinases have been shown to play important roles in mediating the effects of cognate ligands binding to various growth factor receptors, integrins, and GPCRs (35, 36, 37, 38). These kinases transmit receptor signals downstream via RAFTK/Pyk2, adaptor molecules, and other substrates (39, 40, 41). We observed that KS cells express several members of the Src family kinases, including c-Src, Lyn, Fyn, and Hck (data not shown). Treatment of KS cells with VEGF resulted in no detectable activation of Lyn, Fyn, or Hck kinase activity (Fig. 2Go, A–C) using enolase as substrate. However, VEGF did increase the kinase activity of c-Src under these conditions (Fig. 2GoD). This Src kinase activation was delayed, and maximum activity was achieved by 20 min, whereas VEGF-induced tyrosine phosphorylation of cellular proteins was rapid, occurring within 2 min. Equivalent amounts of c-Src protein were present in each sample as confirmed by Western blotting with c-Src Ab (Fig. 2GoE).



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  		FIGURE 2. Differential activation of Src family kinases in response to VEGF. KS 38 cells were serum-starved overnight and then treated with VEGF (100 ng/ml + 10 IU/ml heparin) for the indicated time periods. Total cell lysates (500 µg) from unstimulated (treated with 10 IU/ml of heparin alone) or VEGF-stimulated (100 ng/ml + 10 IU/ml heparin) cells were immunoprecipitated with anti-Lyn (A), -Fyn (B), -Hck (C), or -Src (D) Ab. The immune complexes were subjected to in vitro kinase assay using enolase as a substrate. The 32P-incorporated proteins were resolved on 12% SDS-PAGE, followed by autoradiography (E). Total cell lysates (50 µg) from each sample were fractionated on 8% SDS-PAGE and subjected to Western blot analysis with the c-Src Ab. Control lane represents immunoprecipitation with control Ab.

 
Src family tyrosine kinase inhibitor PP1 inhibits VEGF-induced growth

We determined the growth of KS cells in response to VEGF over a concentration range. As shown in Fig. 3GoA, VEGF induced cell growth in a dose-dependent manner with a plateau achieved at ~100 ng/ml.



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  		FIGURE 3. Inhibition of Src kinase (with PP1) in VEGF-stimulated KS cells leads to reduced cell growth. KS cells were cultured in RPMI 1640 medium at 1 x 104 cells/well in a 96-well assay plate. The cells were then treated with various VEGF concentrations (A) or VEGF (100 ng/ml) along with varying concentrations of the Src kinase family inhibitor PP1 (B). At 72 h, cell growth was assessed by the addition of 1 µCi/well [3H]thymidine for 8 h at 37°C. The radioactive labeling was terminated by washing the cells three times with 1x PBS. The cells were then lysed with 1% SDS solution, and the amount of incorporated [3H]thymidine was correlated with the number of live cells. The data are averages of four wells, and the experiment was repeated three times.

 
To address the functional importance of Src kinases in this response, we treated cells with various concentrations of PP1, a selective inhibitor of Src family tyrosine kinases. As shown in Fig. 3GoB, PP1, at a concentration of 100 nM, caused a 50% reduction in KS cell growth in response to VEGF. At these concentrations, PP1 by itself had no toxic effects on KS cell growth (data not shown).

Src kinase mediates VEGF-stimulated MAP kinase activation in KS cells

The Src kinase inhibitor PP1 was shown to inhibit the VEGF-stimulated growth of KS cells. We further examined the effect of PP1 on VEGF-induced signal transduction. As shown in Fig. 4GoA, PP1 inhibited the tyrosine phosphorylation of some of the proteins induced by VEGF after 20 min of stimulation. Further analysis revealed that PP1 did not inhibit VEGF-stimulated Flk-1/KDR autophosphorylation (Fig. 4GoB). However, PP1 was shown to inhibit VEGF-induced MAP kinase activity as compared with the diluent control (DMSO) (Fig. 4GoC). Equivalent amounts of MAP kinase protein were present in each sample as analyzed by Western blotting of the cell lysates with anti-extracellular signal-related kinase (ERK)-1/MAP kinase Ab (Fig. 4GoD).



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  		FIGURE 4. Src kinase inhibition in KS cells leads to inhibition of VEGF-induced MAP kinase activation. KS cells were pretreated with DMSO or PP1 (100 nM) dissolved in DMSO for 45 min and stimulated with VEGF for the indicated time periods. Cell lysates (50 µg) from unstimulated (0) or VEGF-stimulated cells were run on 8% SDS-PAGE and subjected to Western blot analysis with anti-phosphotyrosine Ab (A). Cell lysates (500 µg) from unstimulated or VEGF-stimulated cells were immunoprecipitated with anti-Flk-1 Ab (B) or with anti-p44/42 MAP kinase (ERK-1/ERK-2) (C). The immune complexes were subjected to in vitro kinase reaction. For MAP kinase, myelin basic protein (MBP) (7 µg) was used as a substrate. The 32P-labeled proteins were run on 8% or 15% SDS-PAGE, followed by autoradiography. Cell lysates (50 µg) were subjected to Western blot analysis with ERK-1/MAP kinase Ab (D).

 
The role of c-Src in MAP kinase activation was further investigated by transfecting KS cells with a control vector, dominant-negative (K297R) Src, WT Src, or constitutively active (Y529F) Src using the lipofectamine method. Higher Src expression was observed in the K297R, Y529F, or WT transfectants as compared with the cells transfected with the control vector (Fig. 5GoA). Furthermore, equivalent amounts of Src protein expression were observed in cells transfected with the various Src constructs (Fig. 5GoA). As shown in Fig. 5GoB, overexpression of constitutively active Src or WT Src led to higher VEGF-induced tyrosine phosphorylation of intracellular proteins as compared with overexpression of the dominant-negative Src. Further studies revealed that overexpression of dominant-negative, constitutively inactive Src markedly inhibited MAP kinase activity, whereas overexpression of the constitutively active Src induced higher MAP kinase activity as compared with cells transfected with the control vector (Fig. 5GoC). Equivalent amounts of ERK-1/MAP kinase protein were present in each sample as detected by Western blot analysis (Fig. 5GoD). However, overexpression of constitutively active Src (Y529F), WT Src, or dominant-negative Src (K297R) had no effect on Flk-1/KDR autophosphorylation activity (data not shown).



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  		FIGURE 5. Overexpression of constitutively active or WT Src induces MAP kinase activation in VEGF-stimulated KS cells. KS cells were transiently transfected using the lipofectamine method with control vector, dominant-negative Src (K297R), constitutively active Src (Y529F), or WT Src. The transfectants were stimulated with VEGF for 20 min, and the cell lysates were subjected to SDS-PAGE and then blotted with either anti-Src (A) or anti-phosphotyrosine Ab (B). Unstimulated or VEGF-stimulated cell lysates (500 µg) were immunoprecipitated with anti-p44/42 MAP kinase Ab (C) and subjected to in vitro kinase assay as described in Materials and Methods. Then, 50 µg of cell lysates were fractionated on SDS-PAGE and blotted with anti-ERK-1/MAP kinase Ab (D).

 
VEGF stimulates phosphorylation of the components of focal adhesions and their association with Src kinases

Paxillin and p130cas are cytoskeletal proteins that are components of focal adhesions (42, 43, 44, 45, 46, 47). These proteins are regulated during various cellular functions, such as migration and adhesion (48, 49). As shown in Fig. 6Go, VEGF treatment resulted in the enhanced tyrosine phosphorylation of paxillin (Fig. 6GoA) and p130cas (ß-Cas) (Fig. 6GoB). Equal amounts of protein were present in each lane as shown in Fig. 6Go, A and B (bottom panels). Furthermore, we observed that there was an enhanced association of Src with paxillin (Fig. 7Go) upon VEGF stimulation. These results suggest an enhanced focal adhesion complex formation, related to Src kinase activation upon VEGF treatment in KS cells.



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  		FIGURE 6. VEGF induces phosphorylation of the cytoskeletal proteins paxillin and p130cas. KS cells were stimulated with VEGF (100 ng/ml) for the indicated time periods, and then stimulated or unstimulated (0) cell lysates (500 µg) were immunoprecipitated with anti-paxillin (A) or anti-p130cas (ß-Cas) (B) Ab (top panels). The immunoprecipitates were run on SDS-PAGE and subjected to Western blotting with anti-phosphotyrosine Ab (top panels) and anti-paxillin (A) or anti-p130cas (ß-Cas) (B) Ab (bottom panels). TCL represents 50 µg of total cell lysates.

 


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  		FIGURE 7. VEGF treatment of KS cells leads to the enhanced association of Src kinase with paxillin. Unstimulated (0) or VEGF-stimulated KS cell lysates (500 µg) were immunoprecipitated with Src Ab and then immunoblotted with anti-paxillin Ab. The blots were then stripped and reblotted with anti-Src Ab. TCL represents 50 µg of total cell lysates.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we characterized VEGF-induced signaling pathways that may regulate cell growth, angiogenesis, and migration in KS cells. VEGF is a multifunctional cytokine that induces angiogenesis, growth, vascular permeability, and cell migration (50, 51, 52, 53, 54, 55, 56, 57). It also is an important mediator of tumor-related angiogenesis and is secreted by various cancers including prostate, lung, and breast (58, 59, 60, 61). VEGF is emerging as an important growth factor in KS pathogenesis (21, 22, 23, 24, 25, 26). KSHV/HHV-8, which has been implicated as a primary etiological factor in KS, induces paracrine expression of the Flk-1/KDR receptor (VEGFR-2) and secretion of the VEGF protein (19, 20). Dissecting these signaling pathways used by VEGF in KS cells affords the opportunity to understand KS pathogenesis at a molecular level and to identify new targets for its therapy.

Recently, VEGF was shown to act as an autocrine growth factor for KS cells (10). VEGF stimulation was found to induce tyrosine phosphorylation of several proteins and to activate Flk-1/KDR, c-Src kinase, and p44/42 MAP (ERK-1/ERK-2) kinase in KS 38 cells. The activation of p44/42 MAP kinase has been shown to be important for VEGF-induced cell proliferation in different cell types (62, 63). We also observed that VEGF-induced cell growth was inhibited by the c-Src kinase inhibitor PP1. Inhibition of Src kinase led to a significant reduction in MAP kinase activation, but had no effect on Flk-1/KDR. Furthermore, overexpression of the constitutively active Src mutant or WT Src enhanced this activation, whereas overexpression of the constitutively inactive Src mutant markedly inhibited MAP kinase activation. Therefore, it appears that Src activation contributes to VEGF mitogenic signaling by increasing MAP kinase activity in KS cells. The reduced growth of KS 38 cells in the presence of the Src kinase inhibitor, PP1, is likely due in part to blunted MAP kinase activation.

The process of angiogenesis involves both proliferation and migration of cells and alteration in cytoskeletal proteins (57, 59). VEGF appears to be an important angiogenic factor in KS and induces migration in other cells (21, 22, 23, 24, 25, 64, 65). In our studies, VEGF stimulation of KS cells caused the enhanced tyrosine phosphorylation of two major cytoskeletal proteins, p130cas and paxillin. These proteins are components of focal adhesions, important structures that participate in chemotaxis and cell adhesion (48, 49). p130cas is phosphorylated upon activation by various stimuli, including integrins and the B cell receptor, and may act as docking molecules for Src homology 2 and Src homology 3 domain-containing proteins (42, 43, 45, 46). Paxillin, a major focal adhesion assembly protein, has been shown to be phosphorylated upon VEGF stimulation of endothelial cells (66). We also observed that VEGF stimulation enhanced the association of Src to paxillin.

Taken together, these studies suggest that Src kinase is a key component of VEGF signaling pathways in KS cells. Src kinases mediate signals downstream to MAP kinase and thereby effect proliferation. Src kinases also regulate the cytoskeletal apparatus via changes in association of focal adhesion components. The role of c-Src in VEGF mitogenic signaling provides a differential target in the design of therapies to inhibit KS tumor growth and spread.


   Acknowledgments
 
We thank Janet Delahanty for editing and preparation of the figures and Simone Jadusingh for typing this manuscript.


   Footnotes
 
1 This research was funded by National Institutes of Health Grant CA76950 (to R.K.G.). Back

2 Address correspondence and reprint requests to Dr. Ramesh K. Ganju, Divisions of Experimental Medicine and Hematology/Oncology, Harvard Institutes of Medicine-Beth Israel Deaconess Medical Center, 4 Blackfan Circle, Boston, MA 02115. E-mail address: Back

3 Abbreviations used in this paper: KS, Kaposi’s sarcoma; ECL, enhanced chemiluminescent; Flk-1/KDR, fetal liver kinase-1; Flt-1, FMS-like tyrosine kinase-1; GPCR, G-protein coupled receptor; HHV-8, human herpes virus 8; KSHV, KS herpes virus; MAP, mitogen-activated protein; MBP, myelin basic protein; OSM, oncostatin M; ERK, extracellular signal-related kinase; PP1, pyrazolopyrimidine; RAFTK, related adhesion focal tyrosine kinase; TCL, total cell lysates; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; WT, wild type. Back

Received for publication March 26, 1999. Accepted for publication November 10, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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