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[Cancer Research 60, 457-466, January 15, 2000]
© 2000 American Association for Cancer Research

Tumor Biology

Thrombospondin-1 Promotes {alpha}3ß1 Integrin-mediated Adhesion and Neurite-like Outgrowth and Inhibits Proliferation of Small Cell Lung Carcinoma Cells

Neng-hua Guo, Nancy Smyth Templeton, Hebah Al-Barazi, JoAnne Cashel, John M. Sipes, Henry C. Krutzsch and David D. Roberts1

Laboratory of Pathology, National Cancer Institute, NIH, Bethesda, Maryland 20892 [N-h. G., H. A-B., J. C., J. M. S., H. C. K., D. D. R.], and Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas 77030 [N. S. T.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although human small cell lung carcinoma (SCLC) cell lines are typically anchorage-independent and do not attach on most extracellular matrix proteins, OH-1, and several other SCLC cell lines attached on substrates coated with thrombospondin-1 (TSP1). SCLC cells grew long-term as adherent cells on a TSP1-coated substrate. Adhesion of SCLC cells on TSP1 was inhibited by heparin, function-blocking antibodies recognizing {alpha}3 or ß1 integrin subunits, and by soluble {alpha}3ß1 integrin ligands. SCLC cells extended neurite-like processes on a TSP1 substrate, which was also mediated by {alpha}3ß1 integrin. Process formation on a TSP1 substrate was specifically stimulated by epidermal growth factor and somatostatin. Adhesion on TSP1 weakly inhibited SCLC cell proliferation, but this inhibition was strongly enhanced in the presence of epidermal growth factor. TSP1 and an {alpha}3ß1 integrin-binding peptide from TSP1 also inhibited proliferation when added in solution. High-affinity binding of 125I-labeled TSP1 to OH-1 cells was heparin-dependent and may be mediated by sulfated glycolipids, which are the major sulfated glycoconjugates synthesized by these cells. Synthesis or secretion of TSP1 by SCLC cells could not be detected. On the basis of these results, the {alpha}3ß1 integrin and sulfated glycolipids cooperate to mediate adhesion of SCLC cells on TSP1. Interaction with TSP1 through this integrin inhibits growth and induces neurotypic differentiation, which suggests that this response to TSP1 may be exploited to inhibit the progression of SCLC.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SCLC2 accounts for 20–25% of human lung cancers (reviewed in Refs. 1, 2, 3 ). SCLC commonly presents as a disseminated disease that is characterized by early metastasis to the lymph nodes, central nervous system, liver, and bone. SCLC cells have several properties characteristic of neuroendocrine cells, including production of dopa decarboxylase and neuron-specific enolase, secretion of various neuropeptides, and expression of neuronal surface markers such as the neural cell adhesion molecule and HNK-1 (1) . Although SCLC often responds well to chemotherapy when diagnosed at an early stage, disseminated SCLC responds poorly, and the overall 5-year survival rate is the lowest of all bronchogenic carcinomas. A better understanding of the molecular mechanisms for dissemination of SCLC is, therefore, needed to control this disease.

Many SCLC cell lines have been established that are typically nonadherent on tissue culture substrates and grow as tight aggregates in suspension (4 , 5) . Cell-cell adhesion is, therefore, the dominant interaction for SCLC cells and is mediated by E-cadherin (6) and neural cell adhesion molecules. Expression of the latter by SCLC correlates with poor prognosis (1 , 7) . Although SCLC cell lines generally fail to interact with the adhesive proteins in serum and with most extracellular matrix components, some SCLC lines can attach on laminin substrates (8, 9, 10) . SCLC cells express some ß1 integrins but not ß3, ß4, or ß5 integrins (11, 12, 13, 14, 15) and were reported to interact with laminins through {alpha}3ß1 and {alpha}6ß1 integrins (12 , 16) .

To better understand the role of cell-matrix interactions in the rapid dissemination of SCLC, we have examined the possible role of TSP1 to mediate interactions of SCLC cells with the extracellular matrix. TSP1 is a major component of the {alpha}-granules of platelets and is a member of the thrombospondin family of matricellular proteins that is synthesized by many cell types in response to growth factor stimulation (reviewed in Ref. 17 ).

In common with other extracellular matrix proteins such as fibronectin and laminins, TSP1 plays important roles in regulating growth, motility, survival, and adhesion of cells (reviewed in Ref. 18 ) and modulating tumor growth and metastasis (reviewed in Ref. 19 ). TSP1 can directly influence adhesion, growth, and motility of some tumor cell lines in vitro (reviewed in Ref. 19 ), but its major inhibitory effect on tumor growth in vivo is thought to result from the inhibition of angiogenesis (20, 21, 22, 23, 24, 25) .

TSP1 is recognized by several cell surface receptors including ß3 and ß1 integrins, CD47, syndecan-1, sulfatides, and CD36 (reviewed in Ref. 19 ). TSP1 also binds to several extracellular matrix components (26) , which may in turn mediate its binding to cells through additional receptors. Distinct signaling pathways may be induced by the binding of TSP1 to each class of receptor (27, 28, 29, 30) ; therefore, identification of the specific TSP1 receptors used by each cell type is important for understanding the responses that result from these interactions.

We recently observed that OH-1 cells and other nonadherent SCLC cell lines attached avidly to TSP1 substrates and grew as adherent cells on this substrate. We have characterized the receptors that mediate this response and demonstrate here that both sulfated glycolipids and {alpha}3ß1 integrin on SCLC cells function as TSP1 receptors. The activity of this integrin is stimulated by EGF and somatostatin. In addition to mediating adhesion, the {alpha}3ß1 integrin promotes neurite-like outgrowth on TSP1 substrates and modulates SCLC cell growth.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
TSP1 was purified from thrombin-activated human platelets as described previously (31) . TSP1 was labeled with Na125I (ICN Radiochemical, Irvine, CA) using Iodogen (Pierce Chemical Co., Rockford, IL) as described previously (32) . Monoclonal antibodies against TSP1 were provided by Dr. William Frazier (Washington University School of Medicine, St. Louis, MO). The integrin-binding peptides Gly-Arg-Gly-Asp-Ser and Gly-Arg-Gly-Glu-Ser were purchased from Sigma (St. Louis, MO). Synthetic peptides derived from TSP1 and inactive analogues were synthesized and characterized as described previously (32 , 33) . MBP-invasin 497 expressed in strain SB39 was purified as described (34) . Murine laminin-1 was provided by Dr. Lance Liotta (National Cancer Institute, Bethesda, MD). Type IV collagen was obtained from Collaborative Research, Inc. Recombinant human EGF was obtained from R&D Systems. Insulin was from Biofluids, and recombinant human IGF1, bombesin, and somatostatin-14 were from Bachem.

The function-blocking CD36 antibody OKM-5 was purchased from Ortho-mune (Raritan, NJ). The integrin {alpha}vß3 antibody LM609 was the gift of Dr. David Cheresh (Scripps Research Institute, La Jolla, CA; Ref. 35 ). Rat monoclonal antibodies to the human ß1 integrin (mAb 13) and {alpha}5 subunits (mAb 16) were provided by Dr. Kenneth Yamada (National Institute for Dental and Craniofacial Research, Bethesda, MD; Ref. 36 ). Integrin function-blocking antibodies P1B5 ({alpha}3ß1), P4C2 ({alpha}4ß1), and P1D6 ({alpha}5ß1) were obtained from Life Technologies, Inc. The ß1 integrin-activating antibody TS2/16 (37) and the CD98 antibody 4F2 were prepared from hybridomas obtained from the American Type Culture Collection (Rockville, MD).

The following oligonucleotides were synthesized (The Midland Certified Reagent Co., Midland, TX) and used as primers for the RT-PCR: upstream primer for human THBS1: 5'-CAA CCA CAA TGG AGA GCA CCG-3'; downstream primer sequence for THBS1: 5'-TAG TTG CAC TTG GCG TTC TTG TTG-3'; upstream primer for human THBS2: 5'-CTC CAC CAG CAA GGT GCC TCG CTG-3'; downstream primer for THBS2: 5'-CCG TCG CCC GCG TAG CCT GTC TGG-3'; upstream primer sequence for human THBS3: 5'-GAC ACA GTG CCT GAG GAC TTT GAG-3'; downstream primer for THBS3: 5'-TGG CAA TGT GCT GTC ATC TTT CC-3'; upstream primer for glyceraldehyde-3-phosphate dehydrogenase: 5'-GCT CTC CAG AAC ATC ATC CCT GCC-3'; downstream primer sequence of human glyceraldehyde-3-phosphate dehydrogenase: 5'-TCC TTG GAG GCC ATG TGG GCC ATG-3'.

Cell Culture.
The OH-1 cell line (38) was provided by Dr. Joel Shaper (Johns Hopkins University, Baltimore, MD). Variant OH-1 arose after prolonged culture of OH-1 and had lost the tight aggregate morphology. The H128, H69, H82, and H209 cell lines were purchased from the American Type Culture Collection. These cell lines were established from pleural fluids of SCLC patients (5) . N417 and H345 cell lines (39) were provided by Dr. A. Gazdar (University of Texas Southwestern Medical Center, Dallas, TX). N417 originated from a lung metastasis and H345 from a bone marrow metastasis. All of the cell lines were cultured suspended in RPMI 1640 with 15% FCS (Biofluids Inc., Rockville, MD) at 37°C in a 5% CO2 incubator. The medium was changed every 5 days.

Cells were passaged every 9- 11 days. In brief, cells were centrifuged at 400 x g for 2 min, and the medium was aspirated. Cell pellets were washed once with RPMI 1640 containing 5 mM MgCl2 and treated for 5 min with 50 µg/ml DNase-1 (Biofluids Inc.) in RPMI 1640 containing 5 mM magnesium chloride. The cells were triturated three times, and 0.1 volume of trypsin (2.5%; Life Technologies, Inc.) was added for 5 min and triturated as above. The single cell suspension was washed once with the same medium, centrifuged, and suspended in fresh medium.

For adhesion assays, cell aggregates were washed once with RPMI 1640 and centrifuged at 200 x g for 2 min. The pellet was suspended in PBS (pH 7.4), containing 2.5 mM EDTA and incubated for 10 min at 37°C. After trituration three times and centrifugation for 2 min at 400 x g, the cells were resuspended in RPMI 1640 containing 0.1% BSA (Sigma). Trypan blue staining showed greater than 90% cell viability.

Adhesion of SCLC Cells to Extracellular Matrix Proteins.
Extracellular matrix proteins or peptides in Dulbecco’s PBS were adsorbed onto polystyrene by incubating overnight at 4°C. Adsorption isotherms of TSP1 on plastic have been reported previously (40) . The supernatant fluid was removed, and the dishes were incubated with Dulbecco’s PBS with 1% BSA for 30 min to minimize nonspecific adhesion. The dishes were washed twice with cold PBS (pH 7.2) and overlaid with dissociated SCLC cells, prepared as described above, at a density of 5 x 104/cm2. For inhibition, inhibitors or antibodies were added and incubated with SCLC cells at the indicated concentrations. After incubation for 60–90 min at 37°C, the dishes were washed three times with PBS (pH 7.2), fixed with 1% glutaraldehyde in PBS (pH 7.2), and stained with Diff-Quik. Attached cells were counted microscopically.

Neurite outgrowth was assessed in SCLC cells after incubation for 75–90 min on a TSP1 substrate. Neurites extending more than one-cell-diameter from the central cell body were counted microscopically in four adjacent 0.25-mm2 fields for each triplicate analysis.

Immunoprecipitation and Western Analysis.
OH-1 cells were surface-labeled using EZ-Link Sulfo-NHS-LC-Biotin (Pierce) as suggested by the manufacturer. The cells were then lysed in 50 mM Tris (pH 7.5), 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 1 mM EGTA, 1 mM NaF supplemented with 10 µg/ml each of the following protease inhibitors: antipain, pepstatin A, chymostatin, leupeptin, aprotinin, soybean trypsin inhibitor, and 1 mM phenylmethylsulfonyl fluoride. A total of 90 µg of proteins were immunoprecipitated using 0.75 µg mouse monoclonal anti-EGF receptor antibody (Transduction Laboratories) prebound to antimouse IgG agarose (Sigma). After washing, the immune complex was eluted with sample buffer. Immunoprecipitated proteins were fractionated on a 10% SDS gel along with 5 µg of A431 carcinoma total proteins as a control. The proteins were transferred to polyvinylidene difluoride membrane and blotted with mouse anti-EGF receptor antibody. The membrane was washed and incubated with a mixture of horseradish peroxidase-conjugated goat antimouse IgG antibody (Kirkegaard and Perry, Gaithersburg, MD) and horseradish peroxidase-streptavidin (Pierce) and was visualized using chemiluminescent substrate (Pierce).

Proliferation.
Effects of soluble and substrate-bound TSP1 or TSP1 peptides on cell proliferation were quantified using a tetrazolium proliferation assay (CellTiter Assay, Promega). Treatment with soluble TSP1 was performed in 96-well tissue culture plates, and proliferation was determined after 72 h in RPMI containing 15% FCS. Proteins and peptides were immobilized on Nunc Maxisorp 96-well plates by overnight incubation with the proteins or peptides dissolved in 50 µl of sterile Dulbecco’s PBS. The supernatant fluid was removed, and the wells were incubated for 30 min. in Dulbecco’s PBS containing 1% BSA. OH-1 cells (1 x 104/well) were added in RPMI containing 15% FCS and incubated for 72 h at 37° in 5% CO2. For assessing inhibition by soluble proteins or peptides, OH-1 cells were grown in suspension in Nunclon 96-well tissue culture plates using the same medium supplemented with the indicated inhibitors and growth factors.

Cell-binding Assay.
A 0.2-ml cell suspension, dissociated using EDTA as above, was transferred into 12 x 75-mm polypropylene tubes (PGC Scientific Inc., Gaithersburg, MD). Iodinated proteins (final concentration of 0.2 µg/ml) were added and incubated for 1 h on ice with rotary shaking. Bound radioactivity was quantified after centrifugation of the cells through oil as described previously (32) . For inhibition studies, inhibitors were added first and incubated with the cells for 15 min. Iodinated proteins, peptides, or fragments were then added and incubated as above. To test the effect of divalent cations on binding of TSP1 to OH-1 cells, the cells were treated and suspended in HBSS or HBSS containing EDTA or divalent cations. After incubation for 1 h at 4°C, cells were centrifuged and separated from the unbound ligand by centrifugation through oil and counted in a gamma counter. Using the same procedure, other media (including DMEM, RPMI 1640, or RPMI 1640 without phosphate) were tested but had no effect on the binding of TSP1 to the cells.

Extraction of Sulfatide and Interaction with TSP1.
Sulfatides were extracted from SCLC cells, desalted by Sephadex G-25, and separated into neutral and acidic fractions by DEAE-Sepharose ion-exchange chromatography according to the previously described method (41) . Fractions were evaporated to dryness and dissolved in chloroform-methanol (1:1) for analysis. The extracts were chromatographed on aluminum-backed high-performance TLC plates and incubated with 125I-labeled TSP1 according to the previously described method (41) .

RT/PCR.
Total RNAs from SCLC cell lines were extracted using RNAzol B according to the supplier’s protocol. RNA pellets were washed with 75% ethanol, briefly dried under vacuum for 2 min, and stored in -70°C. RT-PCR was performed according to the manufacturer’s procedure (Perkin-Elmer Cetus). Aliquots (2 µl) containing 250 ng of human SCLC total RNA were brought to a final volume of 20 µl with reverse transcription buffer containing rTth polymerase and downstream primer. The samples were heated to 70° for 15 min. PCR buffer with the same amount of sense primers was added to a final volume of 100 µl. The reaction mixture was heated to 95° for 1 min and amplified for 30–50 cycles: at 95° for 10 s and at 60° for 15 s. Finally, the sample was incubated at 60° for 5 min. The PCR products were analyzed by agarose gel electrophoresis.

Metabolic Labeling of Sulfatides and Proteoglycans.
SCLC cells were cultured in 5 ml of RPMI 1640 without methionine containing 4% Ultroser HY (IBF Biotechnics) for 12 h in 25-cm2 culture flask before the addition of 125 µCi of [35S]sulfate (ICN Radiochemicals). After 48 h, the cells were harvested, and glycolipids or proteoglycans were extracted according to the previously described method (41) . For some experiments, cells were grown in sulfate-depleted medium supplemented with sodium chlorate to inhibit sulfation as described previously (42) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TSP1 Specifically Promotes Adhesion of SCLC Cells.
Several SCLC lines were tested for adhesion on substrates coated with TSP1, laminin, or fibronectin (Fig. 1Citation ; Table 1Citation ). All of the SCLC lines tested grew as aggregates in suspension with no adhesion to the substratum when cultured in serum-based media. The cell lines H128 (Fig. 1ACitation ), OH-1 (Fig. 1CCitation ), and a variant of OH-1 (Fig. 1DCitation ) that had lost the classic tight aggregate morphology (38) , all attached avidly on TSP1 but failed to attach on murine laminin-1 or human plasma fibronectin. All three of these proteins were functional to promote comparable levels of adhesion for A2058 melanoma cells (Fig. 1BCitation ). OH-1 SCLC cells also failed to attach on substrates coated with vitronectin, fibrinogen, type IV collagen, or gelatin (data not shown). Thus, the OH-1 SCLC line lacks adhesion receptors for all of the matrix proteins tested except TSP1. Several additional SCLC cell lines attached on TSP1 but also exhibited some adhesion to laminin-1 or fibronectin (Table 1)Citation . H345 and H69 cells attached on TSP1 and laminin-1 at higher levels than on fibronectin substrates, whereas N417 cells adhered preferentially on laminin-1.



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  		Fig. 1. TSP1 specifically promotes SCLC cell adhesion. Bacteriological polystyrene was coated with the indicated concentrations of TSP1 (•), murine laminin-1 ({circ}), or human plasma fibronectin ({blacktriangleup}). SCLC lines H128 (A), OH-1 (C), OH-1 variant (D), and the A2058 melanoma (B) cell lines were allowed to attach on each substrate for 60 min. Adherent cells were counted microscopically and are presented as the mean of triplicate determinations.

 

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  		Table 1 SCLC cell adhesion to extracellular matrix proteins

Adhesion of SCLC cell lines (4 x 105 cells/well) was determined using substrates coated with TSP1, fibronectin, or laminin-1 at the indicated concentrations. Adhesion was quantified microscopically and is presented as cells/mm2, mean ± SD (n = 3).

 
Adhesion of SCLC on TSP1 is Mediated by {alpha}3ß1 Integrin.
Because TSP1 was the only extracellular matrix protein recognized by OH-1 cells, we used this cell line to identify the specific TSP1 receptor expressed on SCLC cells. Several function-blocking antibodies that recognize known TSP1 receptors were examined. Antibodies against the TSP1 receptors, {alpha}vß3 integrin (LM609) and CD36 (OKM5), had no effect on adhesion to TSP1 (results not shown). A function-blocking antibody recognizing ß1 integrin was a dose-dependent inhibitor of OH-1 cell adhesion on TSP1 but inhibited adhesion only by half at saturating concentrations (Fig. 2ACitation and data not shown). The residual integrin-independent adhesion of OH-1 cells on TSP1 may be mediated by the heparin-binding sites of TSP1 because heparin also partially inhibited adhesion of OH-1 cells on TSP1, and a combination of heparin with either the ß1-blocking antibody or the ß1 integrin ligand invasin (43) completely inhibited adhesion (Fig. 2ACitation ).



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  		Fig. 2. SCLC cell adhesion on TSP1 is mediated by the {alpha}3ß1 integrin. A, OH-1 cell adhesion on a substrate coated with 40 µg/ml TSP1 (mean ± SD; n = 3) was determined in RPMI containing 1 mg/ml BSA (control) or the same medium containing 25 µg/ml heparin, 5 µg/ml mAb13 (anti-ß1), mAb13 and heparin (anti-ß1 + heparin), or 40 µg/ml MBP-invasin fusion protein and 25 µg/ml heparin (invasin + heparin). B, OH-1 SCLC cell adhesion on substrates coated using 40 µg/ml TSP1 ({blacksquare}), 5 µM TSP1 peptide 678 (), or 0.2 µg/ml MBP-invasin ({square}) was determined in the presence of 5 µg/ml antibody P1B5 (anti-{alpha}3), 5 µg/ml antibody P4C2 (anti-{alpha}4), 5 µg/ml antibody P1D6 (anti-{alpha}5), 5 µg/ml antibody mAb13 (anti-ß1), 20 µM TSP1 peptide 678 (p678), or 40 µg/ml MBP-invasin (invasin). Results are presented as a percent of control adhesion determined for each protein without inhibitors (mean ± SD; n = 3). C, OH-1 cell adhesion to substrates coated with 25 µg/ml TSP1 or 5 µM of TSP1 peptides that bind to {alpha}3ß1 integrin (p678), CD36 (Mal II), or heparin (p246) was determined in the absence ({blacksquare}) or presence of the ß1 integrin-activating antibody TS2/16 at 5 µg/ml (). Results are presented as mean ± SD (n = 3)

 
Function-blocking integrin {alpha} subunit antibodies were used to define the specific ß1 integrin that recognized TSP1 (Fig. 2BCitation ). An {alpha}3ß1 integrin function-blocking antibody (P1B5) but not anti-{alpha}4 or anti-{alpha}5 integrin antibodies, which have been reported to recognize TSP1 in other cell types (44 , 45) , partially inhibited adhesion on TSP1. The {alpha}3 and ß1 function-blocking antibodies also partially inhibited adhesion of OH-1 cells on an immobilized TSP1 peptide (peptide 678), which is recognized by the {alpha}3ß1 integrin on breast carcinoma cells (33 , 45) , and on immobilized invasin (Fig. 2BCitation ). Invasin binds to several ß1 integrins, including {alpha}3ß1, {alpha}4ß1, and {alpha}5ß1 (43) ; therefore. the failure of the {alpha}4ß1 and {alpha}5ß1 antibodies to significantly inhibit adhesion to immobilized invasin combined with their lack of adhesion on the well-characterized ligand for these integrins, fibronectin (Fig. 1Citation ), indicates that OH-1 cells do not express functional {alpha}4ß1 nor {alpha}5ß1 integrins. The consistently greater inhibition by the integrin antagonists of adhesion on invasin relative to that on TSP1 is also consistent with involvement of a heparin-sensitive receptor in adhesion to TSP1.

The ß1 integrin-activating antibody TS2/16 enhanced adhesion on TSP1 and on the TSP1 peptide 678 but not on a CD36-binding peptide (Mal II) or a heparin-binding peptide (p246) from TSP1 (Fig. 2CCitation ). This further confirmed that the recognition of TSP1 peptide 678 by OH-1 cells is ß1 integrin-mediated and suggested that this integrin exists in a partially inactive state on OH-1 cells.

TSP1 Promotes Neurite-like Outgrowth of SCLC.
The attached OH-1 cells generally retained rounded cell bodies, but many cells rapidly extended neurite-like processes on the TSP1 substrate (Fig. 3 ACitation ). {alpha}3ß1 integrin binding was necessary for neurite outgrowth on TSP1 because function-blocking {alpha}3 and ß1 integrin antibodies inhibited the response (Fig. 3, B and DCitation ). In contrast, function-blocking antibodies that recognized {alpha}4ß1 or {alpha}5ß1 integrins did not inhibit neurite outgrowth (Fig. 3DCitation ). The {alpha}3ß1 integrin-binding peptide 678 from TSP1 (Fig. 3, C and DCitation ) and the {alpha}3ß1 ligand invasin also inhibited neurite outgrowth on a TSP1 substrate (Fig. 3DCitation ). Ligation of {alpha}3ß1 integrin also was sufficient to promote formation of neurite-like processes, in that substrates coated with immobilized TSP1 peptide 678 or invasin both promoted neurite formation (Fig. 3ECitation ). The control peptide 690, in which the essential Arg residue was replaced by Ala, was inactive, and an analogue with decreased integrin binding, peptide 686 (33) , was a weaker stimulator of neurite outgrowth. The TSP1 heparin-binding peptide 246 only weakly promoted neurite outgrowth, which indicated that the heparin-binding activity of TSP1 is not sufficient to stimulate the neurite outgrowth response. Activation of the {alpha}3ß1 integrin by antibody TS2/16 increased neurite formation on TSP1 (Fig. 4ACitation ).



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  		Fig. 3. TSP1 promotes {alpha}3ß1-dependent neurite-like process outgrowth by OH-1 SCLC cells. OH-1 SCLC cells were incubated for 75 min on a substrate coated with 40 µg/ml TSP1 alone (A) or in the presence of 5 µg/ml mAb13 (B) or 20 µM TSP1 peptide 678 (C). Scale bar (A), 10 µm for A-C. D, OH-1 cells were plated on TSP1 alone (control), in the presence of 5 µg/ml indicated integrin function-blocking antibodies, in the presence of 20 µM of peptide 678, or in the presence of 40 µg/ml MBP-invasin. Neurite outgrowth was quantified by counting four 0.25-mm2 grids for each triplicate determination (mean ± SD). E, neurite outgrowth was determined as in D on substrates coated with 40 µg/ml TSP1, 5 µM TSP1 peptide 678 or the control peptides 690 or 686, 5 µM TSP1 heparin-binding peptide 246, or 1 µg/ml MBP invasin. F and G, OH-1 cells attached on TSP1 in the presence of 10 ng/ml EGF were stained for F-actin using BODIPY TR-X phallacidin and for ß1 integrin using TS2/16 and BODIPY FL antimouse IgG. Scale bar (F), 20 µm for F and G.

 


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  		Fig. 4. EGF and somatostatin stimulate adhesion and neurite outgrowth on TSP1. A, neurite outgrowth on a substrate coated with 40 µg/ml TSP1 was determined as in Fig. 3Citation . Cells were plated in RPMI containing 1 mg/ml BSA (control) or the same medium containing 5 µg/ml antibody TS2/16, 3 ng/ml EGF, 10 nM IGF1, 100 ng/ml basic FGF2, 1 µM insulin, 10 µM TSP1 peptide 7N3, 0.5 µM bombesin, or 1 µM somatostatin. A, inset, OH-1 cells were surface labeled with biotin and immunoprecipitated using an EGF receptor antibody. After SDS gel electrophoresis and transfer to polyvinylidene difluoride membrane, immunoprecipitated EGF receptor was detected using streptavidin-peroxidase and chemiluminescent detection. A lysate of A431 epidermoid carcinoma cells was used as a positive control. B,: EGF-stimulated SCLC outgrowth on TSP1 is mediated by the {alpha}3ß1 integrin. OH-1 cells were plated on a substrate coated with 40 µg/ml TSP1 in medium without EGF (-EGF) or in medium containing 10 ng/ml EGF alone or with 5 µg/ml of the integrin function-blocking antibodies P1B5 (EGF+anti-{alpha}3) or mAb13 (EGF+anti-ß1) or with 10 µg/ml heparin to inhibit the heparin-binding sites of TSP1. C, neurite outgrowth on a substrate coated with 0.2 µg/ml of MBP-invasin was determined as in A for untreated OH-1 cells (control) or in the presence of 5 µg/ml antibody TS2/16, 3 ng/ml EGF, 10 nM IGF1, or 100 ng/ml basic FGF2.

 
The neurites induced by a TSP1 substrate contained F-actin, as visualized by phallacidin staining, but no organization of actin was observed in the rounded cell bodies (Fig. 3FCitation ). ß1 Integrin staining was also prominent in the neurites formed on TSP1 (Fig. 3GCitation ). The integrin antibody also visualized long processes extending from the neurites that lacked detectable actin staining. This localization of the ß1 integrin supports the evidence from antibody blocking studies which indicates that the {alpha}3ß1 integrin binding mediates neurite-like outgrowth on TSP1.

EGF and Somatostatin Stimulate Integrin-mediated Outgrowth of SCLC on TSP1.
Although we previously demonstrated that insulin and insulin-like growth factor-1 specifically stimulated the {alpha}3ß1 integrin-mediated spreading of breast carcinoma cells on TSP1 (45) , these growth factors had no significant effect on the function of the same integrin in SCLC cells to promote adhesion on TSP1 or neurite outgrowth (Fig. 4ACitation and data not shown). Basic FGF also had no effect, but EGF was a potent inducer of OH-1 neurite outgrowth on TSP1 and moderately increased cell adhesion on TSP1 or peptide 678 (Fig. 4ACitation and data not shown). Expression of EGF receptor by OH-1 cells was verified by immunoprecipitation using an EGF receptor antibody, which comigrated with the EGF receptor from A431 epidermoid carcinoma cells (inset in Fig. 4ACitation ).

EGF-stimulated outgrowth of neurite-like processes on a TSP1 substrate required the {alpha}3ß1 integrin because both {alpha}3- and ß1-specific function-blocking antibodies reversed the stimulation by EGF (Fig. 4BCitation ). Blocking the heparin-binding sites of TSP1 using soluble heparin, in contrast, had no effect on EGF-stimulated outgrowth (Fig. 4BCitation ). EGF also specifically induced neurite outgrowth on the {alpha}3ß1 ligand invasin, confirming that the stimulation of neurite outgrowth by EGF was ß1 integrin-dependent (Fig. 4CCitation ).

Several neuropeptides have also been reported to promote neurite outgrowth in other neurectoderm-derived cell lines (46 , 47) . Representatives of two major neuropeptide families were tested. Somatostatin-14, a member of the somatostatin family, stimulated outgrowth on TSP1 (Fig. 4ACitation ) and slightly stimulated adhesion on TSP1 (results not shown). In contrast, bombesin, a member of the bombesin/gastrin-releasing peptide family that are secreted by many SCLC cell lines, was inactive (Fig. 4ACitation ).

CD47-binding peptides from the carboxyl-terminal domain of TSP1 activate the function of several integrins in other cell types (27 , 29 , 48) but did not stimulate {alpha}3ß1 integrin function in breast carcinoma cells (45) . Consistent with the latter results, the CD47-binding peptide 7N3 did not significantly stimulate neurite outgrowth or adhesion of OH-1 cells on TSP1 (Fig. 4ACitation and results not shown). Therefore, TSP1 cannot stimulate a neurite-like outgrowth response of SCLC cells to itself by binding to CD47.

TSP1 and an {alpha}3ß1 Integrin-binding Peptide from TSP1 Inhibit SCLC Proliferation.
TSP1 is known to modulate the growth of several cell types (reviewed in Ref. 19 ). The addition of soluble TSP1 to nonadherent OH-1 cells markedly inhibited their proliferation, with an IC50 of 40 nM (Fig. 5ACitation ). This inhibition may result from ligation of the {alpha}3ß1 integrin because two additional ligands for this integrin, MBP-invasin (IC50 = 80 nM) and the TSP1 peptide 678 (IC50 = 6 µM), also inhibited OH-1 cell proliferation (Fig. 5ACitation ). The activity of peptide 678 was specific in that the analogue 686, in which the essential Asn residue was replaced by Ala (33) , was inactive. A heparin-binding peptide from the type 1 repeats (peptide 246) only weakly inhibited OH-1 cell proliferation at the same concentrations (data not shown), further indicating that the inhibition by the integrin-binding peptide from TSP1 is specific.



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  		Fig. 5. TSP1 inhibits SCLC cell proliferation. A, soluble TSP1 and {alpha}3ß1 integrin ligands inhibit SCLC cell proliferation. OH-1 cells (1 x 104/well) were incubated for 72 h in growth medium containing the indicated concentrations of TSP1 (•), MBP-invasin ({circ}), TSP1 peptide 678 ({blacktriangleup}), or the inactive peptide analogue 686 ({triangleup}). Net proliferation was determined by the CellTiter assay (Promega) and is presented as mean ± SD (n = 3). B, growth on immobilized TSP1 inhibits proliferation in the presence of EGF. OH-1 cell proliferation in growth medium (•) or medium supplemented with 10 ng/ml EGF ({circ}) was determined after 72 h on substrates coated with the indicated concentrations of TSP1, mean ± SD (n = 3). C, cell proliferation was determined in the presence of the indicated concentrations of EGF in wells coated with BSA (•) or with 50 µg/ml TSP1 ({circ}). D, {alpha}3ß1 integrin ligands cooperate with EGF to inhibit OH-1 cell proliferation. Proliferation in the absence ({blacksquare}) or presence of 10 ng/ml EGF () was determined in wells coated with 10 µM TSP1 peptides 678 ({alpha}3ß1 ligand), 246 (heparin-binding peptide), 7N3 (CD47 ligand), or 1 µg/ml MBP-invasin ({alpha}3ß1 ligand). Net proliferation is presented as a percent of the -EGF control (mean ± SD; n = 3 for treated groups; n = 6 for control groups).

 
When OH-1 cells were plated on a TSP1 substrate, the attached SCLC cells continued to grow and formed extended flattened colonies on the TSP1 substrate. In the absence of TSP1, the cells remained as floating aggregates with no substrate adhesion. Adhesion of OH-1 cells on a substrate coated with TSP1 only weakly inhibited the proliferation of OH-1 cells in their growth medium (Fig. 5BCitation ). On the basis of the observation that EGF stimulated the neurite outgrowth response to TSP1 (Fig. 4Citation ), we also examined the effect of immobilized TSP1 on the proliferation of OH-1 cells in the presence of this growth factor.

Surprisingly, OH-1 cell proliferation was much more sensitive to inhibition by immobilized TSP1 in the presence of EGF (Fig. 5BCitation ). The addition of EGF alone had no significant effect on the proliferation of OH-1 cells, but, in the presence of immobilized TSP1, it produced a dose-dependent inhibition of proliferation (Fig. 5CCitation ). The inhibition of proliferation on a TSP1 substrate by EGF was specific in that IGF1 and bombesin did not display synergism with TSP1 to inhibit proliferation (data not shown). The inhibition of proliferation by a TSP1 substrate in the presence of EGF may also be mediated by the {alpha}3ß1 integrin because substrates coated with TSP1 peptide 678 or MBP-invasin showed similar cooperative effects with EGF to inhibit OH-1 cell proliferation (Fig. 5DCitation ). TSP1 peptides that bind to CD47 (7N3) or heparin (p246) did not synergize with EGF, which indicated that the activity of TSP1 peptide 678 is specific (Fig. 5DCitation ). Thus, EGF specifically and synergistically suppressed proliferation of SCLC cells attached on TSP1 or an {alpha}3ß1-binding sequence from TSP1.

Sulfatides Mediate High-affinity Binding of Soluble TSP1 to SCLC Cells.
Although integrins mediate these biological responses of OH-1 cells to TSP1, heparin-inhibitable binding accounted for most of the high-affinity binding of soluble TSP1 to OH-1 cells. TSP1 bound saturably to OH-1 cells with a dissociation constant of 72 ± 16 nM and 2.6 x 105 sites/cell (Fig. 6ACitation ). This is comparable to the binding constant of 50 nM for TSP1 reported for TSP1 binding to resting platelets (49) and of 22 nM for keratinocytes (50) . The dissociation constant for H128 cells was slightly higher than for OH-1 cells (Kd = 92 ± 21 nM) with 2.1 x 105 binding sites/cell (data not shown). Additional low-affinity sites may be present on both cell lines but were not detected using the accessible TSP1 concentrations.



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  		Fig. 6. Sulfated glycoconjugates mediate high-affinity binding of TSP1 to SCLC cells. A, scatchard plot for binding of 125I-labeled TSP1 to OH-1 cells. Each point is the mean of triplicate determinations. B, inhibition of binding of thrombospondin to OH-1 cells by antibodies and heparin. Binding of 125I-labeled TSP1 to untreated OH-1 cells (2 x 106/ml) in the presence of 20 µg/ml TSP1 antibodies A2.5 (NH2-terminal heparin-binding domain), C6.7 (COOH-terminal CD47-binding domain), D4.6 (calcium-dependent epitope), A4.1 (central stalk of TSP1), or 25 µg/ml heparin, and to OH-1 cells grown in the presence of 10 mM chlorate to inhibit sulfation (86 ± 4% inhibition of sulfation assessed by 35SO4 incorporation) is presented as a percent of binding for untreated controls (mean ± SD; n = 3).

 
The binding of soluble TSP1 was almost completely inhibited by heparin (Fig. 6BCitation ). Of the TSP1 antibodies tested, only A2.5, which blocks heparin and sulfatide binding to TSP1 (51) , significantly inhibited high-affinity TSP1 binding to OH-1 cells (Fig. 6BCitation ). To confirm the role of sulfated glycoconjugates in high-affinity binding of soluble TSP1 to OH-1 cells, the cells were cultured in medium containing chlorate to inhibit sulfation. Incorporation of [35S]sulfate in macromolecules was inhibited by 86% at 10 mM chlorate, and binding of TSP1 was inhibited by 55% for these cells (Fig. 6BCitation ). Binding of soluble TSP1 was not significantly inhibited in the presence of 2.5 mM EDTA (67.4 ± 25.0 of control; P, 0.19 by a 2-tailed t test), which provided further evidence that most high-affinity binding of TSP1 to OH-1 cells was not mediated by the {alpha}3ß1 integrin.

OH-1 cells incorporated 35SO4 into acidic glycolipids, glycoproteins, and proteoglycans. In contrast to the predominant labeling of proteoglycans in most cell types examined previously (Ref. 41 and unpublished results),3 the majority (89%) of [35S]-incorporation was recovered in the lipid fraction extracted using chloroform/methanol. No binding of 125I-labeled TSP1 was detected to the glycoprotein or proteoglycan fractions (results not shown); therefore, these fractions were not further characterized. The sulfated glycolipids identified by [35S]-labeling in extracts of OH-1 cells are shown in Fig. 7Citation , Lane a. Galactosyl sulfatide was the major sulfated lipid based on comigration of the labeled glycolipid with authentic bovine brain sulfatide in two developing solvents. When the total acidic glycolipids separated on TLC were incubated with 125I-labeled TSP1 (Lane b), a major band comigrating with authentic galactosyl sulfatide from bovine brain (Lane c) was strongly labeled. Several more complex glycolipids also incorporated [35S]sulfate (Lane a) but were not present in sufficient concentration to detect TSP1 binding (Lane b).



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  		Fig. 7. Sulfated glycolipids synthesized by OH-1 cells bind TSP1. Glycolipids extracts from OH-1 cells were resolved on silica gel high-performance TLC plates developed in chloroform:ethanol:0.25% aqueous CaCl2 (60:35:7). Lane a, acidic lipids from OH-1 cells metabolically labeled with [35S]sulfate (20 mg wet weight of cells) were separated by high-performance TLC and detected by autoradiography. Lanes b and c, acidic glycolipids from OH-1 cells (Lane b) and purified bovine brain sulfatide (50 ng, Lane c) were resolved by high-performance TLC and incubated with 0.2 µg/ml 125I-labeled TSP1. TSP1 binding was detected by autoradiography.

 
SCLC Cells Do Not Express TSP1.
Initial attempts to detect synthesis of TSP1 by SCLC cell lines using a TSP1 RIA or metabolic labeling and immunoprecipitation were unsuccessful (data not shown). On the basis of RIA of conditioned medium from OH-1 cells grown in 4% Ultroser HY, which lacked detectable TSP1 immunoreactivity, the cells secreted less than 100 ng of TSP1/106 cells/72 h. Total RNAs were extracted from the SCLC cell lines, and mRNA for three thrombospondin genes were detected using the RT-PCR. THBS1 mRNA expression was not detected in any SCLC cell lines (Fig. 8Citation , Lane a, and data not shown), but the predicted PCR product was readily detected using endothelial cell RNA (Lane e). THBS2 expression was detected at low levels in some SCLC cell lines but only after 50 cycles of amplification (data not shown). However, all six of the human SCLC cell lines examined expressed significant levels of THBS3 mRNA (Fig. 8Citation , Lane c, and data not shown).



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  		Fig. 8. RT-PCR analysis of THBS mRNA expression. Total mRNA was prepared from OH-1 SCLC (Lanes a-d) and aortic endothelial cells (Lane e). Specific primers were used to reverse transcribe and amplify 192-, 258-, and 291-bp fragments of THBS1 (Lanes a, e), THBS2 (Lane b), and THBS3 mRNA (Lanes c), respectively. Control primers amplified a 400-bp fragment of glyceraldehyde 3-phosphate dehydrogenase cDNA from positions 664 to 1064 (Lane d). Numbers in margins indicate migration of {phi}{chi}174 HaeIII digested markers. All of the primers amplified the expected size products using RNA from the human breast carcinoma cell line MDA-MB-435, which was used as a positive control (not shown).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
TSP1 is an adhesive protein for all of the SCLC lines we examined. Although some SCLC lines attached on other extracellular matrix components, the two classic SCLC lines, OH-1 and H128, attached only on substrates coated with TSP1. SCLC cell adhesion on TSP1 is mediated by interactions of TSP1 with both {alpha}3ß1 integrin and sulfated glycolipids. Interaction of the {alpha}3ß1 integrin on SCLC cells with immobilized TSP1 stimulates formation of neurite-like processes and inhibits SCLC cell growth. Both the induction of neurite-like processes and the inhibition of proliferation by TSP1 are enhanced in the presence of EGF, which suggests that TSP1 and EGF cooperate to reduce SCLC growth by inducing neurotypic differentiation.

These results demonstrate that the {alpha}3ß1 integrin is an important receptor in SCLC cells for transducing signals from TSP1. On the basis of the activity of a TSP1 peptide, these signals are initiated by the binding of residues 190–201 in the NH2-terminal pentraxin module of TSP1 to this integrin. The {alpha}3ß1 integrin also plays a major role in the adhesion of breast carcinoma and endothelial cells to TSP1 (45) ,4 promotes neurite outgrowth in rat neurons on TSP1 (52) , and was recently shown to mediate interactions of neuroblastoma cells with TSP1 (53) . Although the {alpha}4ß1 and {alpha}5ß1 integrins are TSP1 receptors on other cell types (44 , 45 , 53) , these integrins do not play a significant role in SCLC cell adhesion on TSP1. The high-affinity binding of TSP1 to SCLC cells, however, is not mediated by this integrin. This observation is consistent with previous reports that the affinity of TSP1 binding to {alpha}3ß1 integrin is relatively low (34) and that high-affinity binding of TSP1 to both breast carcinoma cells and SCLC cells is mediated by sulfated glycoconjugates rather than the {alpha}3ß1 integrin (45) .

Sulfated glycolipids on cell membranes play a role in the interactions of several cell types with TSP1 (54) . In melanoma (41) , carcinoma, and endothelial cells,5 sulfated glycolipids typically account for only 0.5–10% of the total 35SO4 incorporation at steady state, but these are the major sulfated glycoconjugate on OH-1 cells. These glycolipids mediate most high-affinity binding of soluble TSP1 to OH-1 cells and significantly contribute to their adhesion on immobilized TSP1 but do not mediate neurite outgrowth.

Formation of neurite-like processes has been observed previously when SCLC cells were plated on substrates coated with laminin-1 (10) , polyethyleneimine or the extracellular matrix produced by PC-9 lung carcinoma cells (55) . The present results identify TSP1 as a matrix protein that also induces this response in SCLC cells and is mediated by the {alpha}3ß1 integrin. This is consistent with the observations that the {alpha}3ß1 integrin mediated neurite outgrowth of rat sympathetic neurons on TSP1 (52) and that central and peripheral neurons (56) and neuroblastoma cells (53) formed neurites when plated on TSP1. EGF-induced neurotypic differentiation of thymic epithelial cells induced TSP1 expression, and TSP1 also induced neurite outgrowth in these cells (57) . Therefore, TSP1 may inhibit the growth of SCLC cells by triggering signaling through the {alpha}3ß1 integrins that induce the cells to differentiate along a neuronal pathway. Enhancement of this differentiation signal by EGF may explain the synergism of these two proteins to inhibit SCLC cell proliferation. EGF is known to have both growth-stimulating and -inhibitory activities in other cell types (58) , but inhibition by EGF has not been observed in SCLC.

As was observed in breast carcinoma and endothelial cells (45) ,4 the activation state of {alpha}3ß1 integrin in SCLC cells to recognize TSP1 is regulated. The signals that mediate this regulation, however, seem to be different for each cell type. The {alpha}3ß1 integrin in SCLC cells is activated by EGF and somatostatin but not by CD47 ligation or by FGF or IGF1 receptor ligands, whereas IGF1 but not EGF activates that same integrin in breast carcinoma cells. The specificity for EGF receptor signaling versus that from two other tyrosine kinase receptors in SCLC cells is interesting in light of the extensive overlap in signaling pathways regulated by these receptors (reviewed in Ref. 59 ). One established outcome of EGF signaling is modulation of integrin activation (60) . Additional work is needed to determine how the activation state of the {alpha}3ß1 integrin is differentially regulated by IGF1 and EGF receptor signaling in SCLC and breast carcinoma cells.

Extracellular matrix can be an important regulator of the malignant phenotype. Blocking of ß1 integrin signaling in breast carcinoma cells induced differentiation of the tumor cells with loss of their malignant phenotype (61) . TSP1 has been implicated in suppressing growth or inducing differentiation of several tumor cell types. TSP1 inhibits proliferation of melanoma cells (28) and breast carcinoma cells (62) . It also inhibits proliferation of HL60 cells while inducing their differentiation (63) . Increased TSP1 expression is also associated with retinoic acid-induced differentiation of neuroblastoma cells (64) . On the basis of the present data, TSP1 may coordinately induce differentiation of SCLC and suppress its growth. Attachment on murine laminin-1 induced increased expression of differentiation markers on SCLC, although proliferation was not inhibited (10) . The laminin-1 effect may also be mediated by {alpha}3ß1, although laminin-1 is not a high-affinity ligand for this integrin (65) .

SCLC cell lines fail to express TSP1 at the protein or mRNA level but consistently express TSP3 mRNA. p53 mutations, which are common in SCLC, may suppress TSP1 expression in these cells (23) . A minor population of lung cells was identified as a site of THBS3 gene expression in the mouse (66 , 67) . The cells that expressed TSP3 were not identified, but the present data suggest that neuroendocrine cells, from which SCLC may derive, are a source of TSP3 expression in lung.

Expression of receptors for TSP1 but not TSP1 may contribute to the tumor biology of SCLC. Because TSP1 is an inhibitor of angiogenesis (20, 21, 22, 23, 24, 25) , lack of TSP1 expression may increase tumor growth by permitting neovascularization in response to angiogenic signals. Loss of TSP1 expression also may release the SCLC cells from the autocrine differentiating and antiproliferative activities of this matrix component and, therefore, may create an additional selective pressure to suppress TSP1 expression in SCLC cells.

Signals from the extracellular matrix may provide both positive (68) and, as demonstrated here, negative signals to control SCLC growth and survival. We have identified direct effects of soluble TSP1 and a synergistic interaction between a TSP1 matrix and EGF to suppress growth and increase neurotypic differentiation of SCLC. Inducing neuroendocrine differentiation of SCLC by a combination of TSP1 (or the integrin-binding peptide from TSP1) and EGF may facilitate treatment of this cancer because neuroendocrine differentiation is correlated with increased sensitivity of SCLC to radiotherapy (38) .

ACKNOWLEDGMENTS
We thank Drs. Steven Akiyama (NIH, Research Triangle Park, NC), David Cheresh, William Frazier, Adi Gazdar, Ralph Isberg (Tufts University School of Medicine, Boston, MA), Lance Liotta, Joel Shaper, and Kenneth Yamada for providing reagents.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at NIH, National Cancer Institute, Building 10, Room 2A33, 10 Center Drive, MSC 1500, Bethesda, MD 20892-1500. Phone: (301) 496-6264; Fax: (301) 402-0043; E-mail: droberts{at}helix.nih.gov Back

2 The abbreviations used are: SCLC, small cell lung carcinoma; EGF, epidermal growth factor; IGF1, insulin-like growth factor-1; mAb, monoclonal antibody; FGF, fibroblast growth factor; MBP, maltose-binding protein; peptide 678, FQGVLQNVRFVF (TSP1 residues 190–201); peptide 686, FQGVLQAVRFVF; peptide 690, FQGVLQNVAFVF; peptide 246, KYRFKQDGGWSHWSPWSS (TSP1 residues 412–428); peptide 7N3, FIRVVMYEGKK (TSP1 residues 1102–1112); TSP1, thrombospondin-1; RT-PCR, reverse transcription-PCR. Back

3 Roberts, D. D., unpublished observations. Back

4 Chandrasekaran, L., He, C-Z., Krutzsch, H. C., Iruela-Arispe, M. L., and Roberts, D. D. Modulation of endothelial cell behavior and angiogenesis by an {alpha}3ß1 integrin-binding peptide from thrombospondin-1, submitted for publication. Back

5 Roberts, D. D., unpublished observations. Back

Received 7/15/99. Accepted 11/11/99.


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 RESULTS
 DISCUSSION
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