[CANCER RESEARCH 51, 1065-1070, February 15, 1991]

0i Integrin Expression on Human Small Cell Lung Cancer Cells1

Lawrence E. Feldman,2 Kye C. Shin, Ronald B. Natale, and Robert F. Todd III

Simpson Memorial Research Institute, Department of Internal Medicine, Division of Hematology and Oncology, The University of Michigan Medical School, Ann Arbor,Michigan 48109

ABSTRACT

The integrins are a supergene family of cell surface glycoproteins thatpromote cellular adhesion. Each member of the family is an a/ßhetero-dimer composed of a distinct a subunit noncovalently linked to one of atleast six common ßsubunits. These include the six ß,integrins («,„¿�/,;,)which represent receptors for extracellular matrix proteins and the threej82integrins (aL, «MI«x//92)that are expressed by leukocytes and whichbind to C3bi and/or endothelial ligands. Recently, it was reported thatcertain human tumor cells express the 0, integrins and that small celllung cancer (SCLC) cell lines express the /32integrin Mol (a^/ßi).Toextend these initial observations, we examined SCLC cell lines forintegrin expression at the glycoprotein and mRNA levels and assessedthe potential function of these integrins in promoting SCLC adhesion.An indirect immunofluorescence analysis of five SCLC cell lines (NCI-HI 87, H345, H146, H 209, and N417) using a and ßsubunit-specificmonoclonal antibodies demonstrated the uniform expression of ß,(/<, •¿�-/i2 > /ii = fit). Among the #,-associated a subunits, <n was uniformlyexpressed at high surface density by all five cell lines (as confirmed inH345 cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresisanalysis of ami-/;, and :uiii-<r, immunoprecipitates), while a5 was notdetected. The leukocyte (/32-associated) <*Mand aL subunits were alsovariably expressed by the five lines. Consistent with the surface expression of /i, integrin gene products, /A (but not /•(.•)mRNA was detected in

SCLC cells by Northern blot analysis. That ßtintegrin expression wasinvolved in SCLC adhesion was suggested by the adherence of H345cells to laminili, a known ligand for the a^ß,integrin. Moreover, anantibody specific for the ß,subunit inhibited this adhesion, indicatingthat the ß,subunit promotes adhesion to laminin. We conclude that ßtintegrin molecules are expressed by human SCLC cells (with uniformexpression of «,//)'i)and promote their adhesion to laminin.

INTRODUCTION

Integrins are a supergene family of cell surface glycoproteinswhich function in cell adhesion (1). Each integrin receptor isexpressed as a heterodimer composed of a single ßsubunitnoncovalently linked to one of several possible a subunits (2).At present, three ßsubunit families (/8i-j) have been extensivelystudied and reviewed (3-5). The ßiintegrins include six receptors (CDw49a-f/CD29) for ECM3 proteins such as laminin,

collagen, and, fibronectin (3). The 02 integrins consist of threeleukocyte adhesion molecules (CDlla-c/CD18) that bind to avariety of ligands such as ICAM-1 (CD54), lipopolysaccharide,factor X, fibrinogen, and C3bi (4). The j33integrins include areceptor for vitronectin (CD51/CD61) and platelet glycoprotein Ilb/IIIa (CD41/CD61) that recognizes ligands such as

Received 7/10/90; accepted 12/3/90.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported by N1H Grants R01CA39064 (R. F. T.) and National Research Service Award IF32CA08873-01 (L. E. F.).

2To whom requests for reprints should be addressed, at Simpson MemorialResearch Institute, The University of Michigan, 102 Observatory Street, AnnArbor. MI 48109.

3The abbreviations used are: ECM. extracellular matrix; BSA, bovine serumalbumin; LPO, lactoperoxidase; MoAb, monoclonal antibody; PBS, phosphate-buffered saline; PLL, poly-L-lysine; SCLC, small cell lung cancer; SDS, sodiumdodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; cDNA, complementary DNA.

fibronectin, fibrinogen, and Von Willebrand factor (5). Other ßintegrin families (184,185,ßp)have recently been identified andare currently under investigation (6-8).

The surface expression of integrin molecules by tumors suchas melanoma (9), carcinoma of the bladder (10), neuroblastoma(3), head and neck cancer (11, 12), and osteosarcoma (13) haspreviously been reported, and it has been suggested that thesemolecules may provide a mechanism for tumor cell adhesionduring métastases(2, 14). Since SCLC represents a particularlymalignant neoplasm characterized by early and widespreadmétastases(15), we have examined functional integrin expression by these cells.

Consistent with our hypothesis that integrin receptors mightplay a role in SCLC tumor metastasis, we found that humanSCLC cells express high levels of cell surface ßtintegrin molecules, particularly arf,, and also constitutively express the ß\mRNA. Moreover, a functional role in SCLC cell adhesion foraj/3, was strongly suggested by our finding that SCLC cells bindto the ECM protein laminin and that this binding is inhibitableby a MoAb against the ß\subunit.

MATERIALS AND METHODS

Reagents. All chemical reagents used were purchased from SigmaChemical Co. (St. Louis, MO) unless otherwise noted.

Cell Lines. The human SCLC cell lines used were originally developed by Carney et al. (16). Lines NCI-H345, NCI-HI87, and NCI-N417 were a generous gift of Dr. Adi Gazdar, National Cancer Institute-Navy Medical Oncology Branch. Lines NCI-HI46 and NCI-H209were obtained from the American Type Culture Collection (Rockville,MD). Cell lines H345, H187, H146, and H209 are considered to beclassic SCLC cell lines and N417 is considered a variant SCLC cellline by previously published criteria (16, 17). Each of these SCLC celllines grow in suspension as floating aggregates. The human invelimicell line U-937 was obtained from Dr. Thomas Huard (Simpson Memorial Research Institute, Ann Arbor, MI). All cell lines were maintained in culture in RPMI 1640 medium supplemented with 10% calfbovine serum (Hyclone Lab, Logan, TX), 2 miviL-glutamine, 50 units/ml penicillin, and 50 mg/ml streptomycin, under sterile conditions inan atmosphere of 5% CO2 in air at 37°C.In order to obtain single cell

suspensions, SCLC cells were disassociated prior to each experimentby vortexing cells in media containing 0.2 g/liter of EDTA for 60 s,followed by incubation at 4°Cfor 30 min, and then washed in Ca+2-and Mg+2-free PBS (145 miviNaCl, 5.9 IHMKH2PO„,2.4 miviKH2PO„).

Cell viability was determined by the trypan blue exclusion test.Flow Cytometric Analysis. Anti-integrin MoAbs were used in our

study to characterize integrin surface expression on SCLC cells. Theantibodies used are listed in Table 1. Flow cytometric analysis wascarried out as previously reported (32). Viable cells were selected by bitmap gating after staining with 0.5 Mg/ml of propidium iodide (33).

Immunoprecipitation/SDS-PAGE Analysis. Surface protein labelingwas performed by the LPO method (34). Briefly, 60 x 10* cells werewashed twice in Dulbecco's PBS and then incubated with 2 mCi of I25I

(Amersham, Arlington Heights, IL) in the presence of 5 units of LPOand 0.03% H2O2 for 11 min (with additional amounts of LPO andH2O2 introduced after 5 min). The cells were then washed once in highglucose Dulbecco's minimal essential medium (Gibco, Grand Island,NY), 3 times in Dulbecco's PBS containing 5 mM KI, and once in Tris-

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Table 1 Anti-integrin MoAbs used

Subunit/3i182ft04«1«l«3«4«30«OL«M«XMoAbnameA1A5;

AIIB2IOF12;1B4GPIlIaA9TS2/712F1J143B5G10BIIG2GoH32F12904L29SourceM.Hemler; C.DamskyJ.Ritz; S.WrightDako

Corp. (Carpintería,CA)T.CareyM.

HemlerV.WoodsL.OldM.HemlerC.DamskyA.SonnenbergJ.

RitzJ.GriffinL.

LanierRéf.18,

1920,212223242510261927282930

147 Telios Pharmaceuticals, Inc. 31

buffered saline (150 mM NaCl-10 HIMTris-base, pH 7.8). Cell pelletswere lysed in 0.4 ml Nonidet P-40 lysis buffer containing 1% NonidetP-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 1% stocksolution containing 100 Mg/ml each of leupeptin, pepstatin A, antipain,and chymostatin, and 10 mM iodoacetamide in Tris-buffered saline aspreviously described (35). The supernatant was collected after centrif-ugation at 11,600 x g (SUREspin, Helena Laboratory Corp., Beaumont, TX; 12,900 rpm, 15 min) and then at 96,000 x g (BeckmanAirfuge Ultracentrifuge; A-95 rotor, 16 psi, 30 min) and preclearedwith formalin-fixed Siaphylococcus aureus cell walls as described previously (34). Immunoprecipitation was carried out using anti-integrinmonoclonal antibodies by the solid phase immunoisolation techniqueas previously reported (36). Immunoabsorbed integrin proteins weresuspended in sample buffer and subjected to SDS-PAGE (5.5% acryl-amide gels) (Bio-Rad, Rockville Center, NY) under both reducing andnonreducing conditions using a mini-gel Bio-Rad apparatus and auto-

radiography (35).Northern Blot Analysis. Total cellular RNA was isolated and quan

tified as previously described (37). RNA electrophoresis (10 Mg/'ane)was performed on a denaturing formaldehyde gel and then transferredto a nylon membrane (Hybond-N, Amersham) as previously reported(38). Efficiency of transfer of RNA was assessed by staining the membrane with méthylèneblue (39). The following cDNA probes were used:(a) ß,cDNA (40), a 2.5-kilobase EcoRl fragment (pGEMl-P32) (graciously provided by E. Ruoslahti, La Jolla, CA); (¿>)ft cDNA (41), a3-kilobase Pvull fragment (J-8) (a gift of A. Law, Oxford, England);and (c) /3-actin cDNA (42), a 2.0-kilobase fragment (generously provided by L. Keiles, Palo Alto, CA). cDNA inserts were radiolabeleddirectly in low melting agarose by random hexamer priming (43) usingthe Multiprime DNA labeling system (Amersham). Hybridization andautoradiography of Northern membranes were carried out according tothe manufacturer's recommendations (44).

Cell Adhesion and Inhibition Assays. Wells in flat-bottom 96-wellplates (Costar Corp., Cambridge, MA), were precoated (30 ng/ml) withBSA or one of five ECM proteins (45): type I collagen (Sigma),fibronectin (Telios Pharmaceuticals, Inc., San Diego, CA), laminin(graciously provided by Dr. Mary Zeigler, University of Michigan, AnnArbor, MI), type IV collagen (Calbiochem Corp., La Jolla, CA), andvitronectin (Telios). Positive control wells were precoated with PLL(M, 30,000-50,000), 30 Mg/ml in PBS for 30 min at 25°C,as previouslydescribed (46). To each well was added a 1.5 x IO5 H345 cells (in 0.2ml of Dulbecco's minimal essential medium). Following incubation ofthe cells for l h at 37°C,the wells were washed to remove nonadherent

cells and then the remaining adherent cells were stained with toluidineblue as previously reported (45). The stained cells were quantitatedfollowing cell lysis with 2% SDS by measuring the absorbance at 630nm in an enzyme-linked immunosorbent assay microtiter plate reader(Dynatech Laboratories, Inc., Chantilly, VA). To assess the effect ofanti-integrin MoAb on SCLC substrate adhesion, H345 cells werepretreated for 10 min at 4°Cwith a saturating concentration of either

anti-/3i MoAb AIIB2 (19) (1:3 dilution of culture supernatant kindlyprovided by C. Damsky, San Francisco, CA), the anti-«3MoAb J143

(10) (a 1:30 dilution of ascites that was a gift from L. Old, New York,NY), or one of a series of isotype-identical control antibodies. Followingpreincubation with antibody, the cells were added directly to the protein-coated wells (without washing) and the adhesion assay was carriedout as described above.

RESULTS

Integrin Expression on SCLC Cells. To determine whetherintegrin molecules are expressed on the surface of SCLC cells,an indirect immunofluorescence flow cytometric analysis wasperformed using MoAbs specific for the a and ßintegrinsubunits (Table 2). The results demonstrate that, of the 4 ßsubunits tested, ß\was expressed in the highest relative surfacedensity on all 5 cell lines. While the surface ß2expression wasgenerally detectable above background, its density was at least1 log lower than that of ß,;there was relatively little, if any, ß3or (34expressed by the 5 cell lines. There was greater heterogeneity in the ß\-associatedoi_6 subunit expression with «3demonstrating a uniformly high surface density on all 5 cell lines;«swas undetectable. Whereas a} was the predominant a subunitexpressed by H345 and H187, «6demonstrated a comparablelevel of surface density on H146, H209, and N417. Among the02-associated a subunits, aM was variably expressed by all 5lines [consistent with previous reports (47-49)]. t*Lwas alsodetectable but «xwas consistently absent. Also barely detectablewas the /^-associated subunit, av- A representative experimentdemonstrating uniform distribution of ß\integrin subunitexpression by the cell line H345 is shown in Fig. 1.

Immunoprecipitation of a.,/3, from H345 SCLC Cell Line. Todocument the surface expression of ß\integrin proteins bySCLC cells (Table 2), detergent lysates of I25I surface-labeledH345 cells were subjected to immunoprecipitation/SDS-PAGEanalysis using MoAbs specific for the ß\and a3 integrin subunits(or isotype-identical negative control MoAbs ). Under nonreducing conditions of SDS-PAGE, the anti-/3, immunoprecipi-tated material appeared as two major bands of M, ~ 150,000and 110,000 (Fig. 2). When the same material was run underreducing conditions a broad band of M, ~ 130,000 was seen.These findings are consistent with the previously reported elec-trophoretic mobilities of the a} (Mr ~ 150,000 nonreducing;-130,000 reducing) and ß,(M, 110,000 nonreducing; 130,000reducing) integrin subunits (50). The same two species of M,-150,000 and 110,000 (nonreducing) were seen after immu-noprecipitation by a MoAb specific for the ce}subunit, confirming the surface expression of a3ßiby these cells. Also of note isa lighter band of M, —¿�200,000seen in the ß\nonreducing lane.This band likely represents the a¡integrin subunit which has areported M, 200,000 and, as shown in Table 2 and Fig. 1, isexpressed by H345 cells.

ß,Integrin Gene Expression by SCLC Cells. Fig. 3 shows aNorthern blot analysis in which total cellular RNA from H345cells was probed with 32P-labeled cDNAs specific for the ß\and$2 integrin subunits. A band of —¿�4.2kilobases was seen to

hybridize with the ßtprobe (40), consistent with the surfaceexpression of ß\integrin gene products by this cell line. Alsoconsistent with the flow cytometric data was the absence ofdetectable 02 mRNA expression. As a control, RNA from U937cells which strongly express both ßiand ßiintegrin subunits(data not shown) contained transcripts for both ßmolecules(Fig. 3).

Adhesion of SCLC Cells to ECM Proteins. Since the ß,inte-grins are known to function as adhesion receptors for ECM

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Table 2 Integrin expression on SCLC cell linesIntegriti cell surface expression was assessed by flow cytometric analysis as indicated in "Materials and Methods." Briefly, indirect immunofluorescence staining

of SCLC cells (1 x 10' cells/determination) was carried out with an excess of anti-integrin MoAb for 30 min at 4'C. A second incubation with a fluorescein-conjugatedgoat anti-mouse immunoglobulin (Tago, Inc., Burlingame, CA) or anti-rat immunoglobulin (Diagnostic Concepts, Inc., Niles, IL) was performed. Fluorescencestaining was quantitated on an EPICS C flow cytometer (Coulter Electronics, Hialeah. FL).

Specific mean linear fluorescence"

SubunitA&a,ß.«i«Z«J«4«3at«L«M«X«Vn"64224444624442H34513.0±4.6f0.8

±1.0-0.8±0.10.6

±1.42.8

±0.61.2±1.25.9±2.90.6±0.6-0.2±0.41.1

±0.11.4

±1.01.6±0.9-0.7

±0.30.5

±1.6n42222222422210H1879.6

±3.71.0+2.0-0.3±0.6-0.4±0.71.0

±1.2-0.3±0.08.4±4.81.8±

2.9-0.6±0.50.4±0.32.8

±2.54.1±2.1-1.5NDn52244444332444H

146I0.4±

4.00.3±0.80.4±0.3-0.3±1.52.0

±1.02.1±1.44.2±1.20.7±0.40.0±0.45.7±1.81.0

±0.71.1±0.40.0

±0.50.2

±0.5n32122222112222H2095.0

±1.00.5±0.40.50.7

±1.00.8

±0.70.5±0.52.8+0.20.7±1.00.23.41.3+

1.10.6±0.60.3±0.50.4

±0.6n64224444224420N41710.7

+2.20.7±1.5-0.9±0.50.1

±0.80.9+

1.01.1±0.82.2+1.62.0

±1.7-0.2±0.52.0±1.01.9

±2.32.4+2.3-1.3

±0.2ND

" Each value is expressedas the specificmean linear fluorescencewhichis a relativequantitativemeasureof receptorexpression.*n, numberof separateexperiments;ND. not determined.cMean ±SD.

a i a 2 a-} a4

- FLUORESCENCE INTENSITY (LOG 10 SCALE) —¿�

Fig. 1. Integrin expression by the SCLC cell line NCI-H345. Results of arepresentative experiment demonstrating the profile of integrin subunit expression by the classic SCLC cell line NCI-H345 are shown. Aliquots (100 /J)containing 0.5-2.0 x 10' cells were subjected to indirect immunofluorescencestaining for the expression of surface integrins relative to background stainingusing isotype-identical negative control MoAb. The shaded histograms indicatethe fluorescence profile of 5,000 viable cells (excluding propidium iodide) stainedby anti-integrin MoAbs as compared to the background fluorescence shown inthe open histograms.

proteins (3), we sought to determine the adhesive properties ofthe SCLC cells in a cell attachment assay (45). When H345cells were introduced into the wells of polystyrene tissue cultureplates, either uncoated (not shown) or precoated with BSA,collagen type I, fibronectin, laminin, collagen type IV, or vitro-nectin, avid binding was restricted to laminin-coated wells (P <0.001 relative to BSA-coated wells) as shown in Fig. 4. Theproportion of H345 cells bound to laminin was in the samerange as that adherent to wells pretreated with PLL which wasused as a positive control. Qualitatively, attachment of H345cells to laminin resulted in a confluent monolayer when viewedunder an inverted microscope (not shown). This finding suggests that H345 SCLC cells possess a high affinity receptor forlaminin.

Inhibition of Adherence to Laminin by Anti-integrin Monoclonal Antibody. To determine whether the adherence of H345cells to laminin is mediated by a ß\integrin, a MoAb inhibition

NR/R:

MoAb:

200 -

116 -95 -

NR

ßl <x3 NC

*

R

ßl N3

Fig. 2. Immunoprecipitation of a}ß,from H345 SCLC cell line. HCI-H345cell surface proteins were labeled with '"I by the lactoperoxidase method andthen cell lysates were prepared as described in "Materials and Methods." Following immunoprecipitation with an anti-integrin MoAb (13, or a3) or isotype-identical negative control MoAb, proteins were subjected to SDS/PAGE andautoradiography under both nonreducing (NR) and reducing (R) conditions.

actm-ß2

18 s

Fig. 3. 0, gene expression by Northern blot analysis. Total cellular RNA wasextracted from the cell lines NCI-H345 and U937, and Northern blot analysiswas carried out as described in "Materials and Methods." Hybridization wasperformed using fi¡and /j2 integrin cDNA probes. A tf-actin cDNA probe wasused to ensure that approximately equivalent amounts of RNA were loaded ontoeach lane.

assay was performed. As shown in Fig. 5, pretreatment of H 345cells with a MoAb specific for the ßtsubunit significantlyblocked the attachment of these cells to laminin as comparedto preincubation with a series of isotype-identical control antibodies (P < 0.001). The anti-a, antibody (J143) used in thisstudy did not inhibit cell adherence.

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20-

T

BSA Col I Lm Col IV

Substrate

'p < 0.001

Fig. 4. H345 SCLC cell adhesion to laminin. H345 cells (1.5 x IO5) wereincubated for 60 min at 37"C in quintuplicate microtiter wells coated with each

of the indicated ECM proteins or with either BSA (negative control) or PLL(positive control). After the remaining cells bound to the wells were washed toremove nonadherent cells, they were quantitated spectrophotometrically aftertoluidine blue staining as described in "Materials and Methods." Column, meanabsorbance (630 n.vi)of 2-7 separate experiments; bar, ±SD.Significant bindingof H345 cells to laminin (P < 0.001 relative to binding to BSA) was observed.Col I, collagen type I; Fn, fibronectin; Lm, laminin: Col IV, collagen type IV;Vn, vitronectin.

1.0-

0.8-

MoAb: -C1 -C2 -C3 03 -C4 -C5 -C6(BSA)

p < 0.001

Fig. 5. Inhibition of H345 cell adherence to laminin by anti-fii MoAb. H345cells were preincubated with saturating concentrations of MoAbs specific for f}\(AIIB2) and a, (J143), with a series of isotype-identical rat (-Cl, -C2, -C3) ormouse (-C4, -C5, -C6) negative control MoAbs or no MoAb ( —¿�).Without beingwashed, these MoAb-pretreated (or sham-pretreated) cells were added to microtiter wells coated with laminin or BSA with cell adherence quantitated accordingto the protocol described in Fig. 4. Column, mean absorbance (630 nM) ofquintuplicate determinations in a single experiment (with similar observationsseen in 3 other experiments); bar, ±SD.Pretreatment of H345 cells with anti-/3iMoAb significantly inhibited their adherence to laminin (P < 0.001 as comparedto anti-«3,negative control MoAb, and no MoAb controls).

DISCUSSION

ß,integrin expression has previously been observed on celllines derived from several human solid tumors (Table 3) including osteosarcoma, melanoma, neuroblastoma, rhabdo-myosarcoma, hepatoma, fibrosarcoma, teratocarcinoma, andcarcinomas of the bladder, breast, and lung (non-small cell) (26,51-61). To this list of |8i integrin-bearing tumors, we now addSCLC. We selected this tumor type for our analysis becauseSCLC exhibits a highly aggressive and metastatic phenotype invivo and has been reported to express an integrin of the /32(leukocyte) family, specifically «M(CD1 Ib) (47-49). Therefore,SCLC might be a good model system for studying the role ofintegrins in tumor cell invasion. By flow cytometric analyses of

five SCLC cell lines (including four classic and one variantline), we observed a uniformly high level of a3ßiexpression,which, in the case of classic line H345, was confirmed byimmunoprecipitation/SDS-PAGE analyses. With the exception of «5which was not detectable on any of the SCLC lines,the other /^-associated a subunits were variably expressed withthe level of <*6approximating that of a} on H146, H209, andN417. Moreover, steady-state ß,mRNA expression by H345SCLC cells was also readily detectable by Northern blot analysis.

Since previous reports suggested that SCLC cells also express«M(47-49), we assayed for the expression of the leukocyte ß2integrins on SCLC cells. We found variable degrees of c*Land«Msubunit expression by all five lines examined (particularlyH187), while ax was undetectable. However, the relative magnitude of ß2subunit expression was somewhat less than expected for the corresponding density of <*Mand aL. This apparent discrepancy may be due to a relative lack of recognition ofthe 02 subunit as expressed by SCLC cells [which is unlikelysince an additional anti-ft MoAb demonstrated a similar staining profile (not shown)] or an association of the leukocyte asubunits with a different ßsubunit, such as ß,(not heretoforereported). The minimal surface expression of the ß2subunit isconsistent with our failure to detect constitutive ß2transcriptsin the H345 line.

The functional role that the ß,integrins may play in tumorcell adhesion was recently examined. To date, osteosarcoma(56-58) and melanoma (51, 59-61) cell lines have been mostextensively studied. Osteosarcoma cells express 03/81and adhereto the ECM protein laminin and to a lesser extent to fibronectinand collagen as compared to BSA or untreated plastic. Moreover, inhibition of cell adhesion to these ECM proteins wasseen in the presence of anti-ßisera, suggesting that cell adhesionto ECM proteins is mediated by this receptor, perhaps by arecognition site on the /3, subunit (58). We report here thatSCLC cells also most consistently express the a3|8r integrinreceptor and adhere most avidly to laminin and that an ami-.i,antibody blocks this adherence.

It was recently shown (62) that the ct}ß,receptor on humanosteosarcoma cells binds to laminin at the putative cell attachment and neurite outgrowth site of laminin near the COOHterminus of the long arm of the laminin molecule. Similarly,we have found that human SCLC cells bind strongly to this siteon laminin in the form of the peptide fragment PA-22 but notto another laminin fragment, PA-21.4

The hypothesis that integrins play an important role inmelanoma cell adhesion to ECM proteins was supported by theobservation that polyclonal anti-0i antisera inhibited attachment of B16-BL6 murine melanoma cells to laminin, fibronectin, and collagen type IV (59, 60). Subsequently, human melanoma cells were shown to bind to laminin (via a novel aß,heterodimer), fibronectin (via ce5ßt),and collagen types I andIV (via «201and atß,,respectively) (51, 61). B16-F10 murinemelanoma cells have also been found to bind to a PA-22 lamininfragment thereby stimulating the release of the enzyme colla-genase IV and enhancing the development of métastasesin anin vivo mouse model (63).

In conclusion, ß\integrin molecules are strongly expressedby SCLC cell lines, as detected by indirect immunofluorescenceflow cytometry. Overall, the a?,ß\integrin is invariably expressed on all five SCLC cell lines tested. Immunoprecipita-

4 L. E. Feldman, M. Zeigler, R. F. Todd, unpublished data.

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Table 3 Summary o//3, integrin expression by tumorcellsTumor

type (celllines)OsteosarcomaMelanomaNeuroblastomaBladder

carcinomaRhabdomyosarcomaHepatomaBreast

carcinomaFibrosarcomaTeratocarcinomaNon-small

cell lungcarcinomaSmallcell lung carcinomaß,

inlegrinexpression"«3/3,,

«ißi(ï/3|,

Cfißii Ct20ltí*3Pl»''

j.i|. ítjp),ri,,.'|«10l«3/3,«401«201,

«101«201«301,

«101«501«201«301ECM

binding inhibitablebyanti-01Lm,*Fn, ColIVLm,

Fn, Col I, ColIVLmNDNDNDNDLm.

Fn, Col I, ColIVFnNDLmRef.26,

56-5850,51,59-612626262626525254,55This

report" Predominant /}, integrin expressed.* ND, not determined; Lm, laminin; Fn, fibronectin; Col I. collagen type I; Col IV, collagen type IV.' A novel a/3,, heterodimer (Ref. 51).

tion/SDS-PAGE analysis confirms the expression of a3ßthet

erodimer on the surface of H345 cells. Consistent with theknown ligand-binding properties of the a3/3, integrin (57),SCLC cells adhere most avidly to laminin. An antibody specificfor the 01 subunit blocks the adherence of SCLC cells tolaminin, suggesting that a -1,integrin promotes adhesion to thisECM substrate.

Our data with cell lines derived from SCLC supports thenotion that ß,integrin expression by SCLC cells could contribute to tumor metastasis in vivo by promoting the adherence ofthese malignant cells to the vascular basement membrane andmay thus facilitate the development of metastatic foci. Thelimited availability of fresh SCLC tumor tissue has hamperedour ability to extend these observations beyond the establishedcell lines tested, but integrin expression by SCLC taken fromsurgical or autopsy specimens will be a focus for future investigations.

ACKNOWLEDGMENTS

We wish to thank Clare E. Rogers and Kristine L. Weber for help inflow cytometry and Tracy L. Lockhart for her assistance in the preparation of the manuscript.

REFERENCES

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7. Cheresh, D. A., Smith, J. W., Cooper, H. M., and Quaranta, V. A novelvitronectin receptor integrin («A) is responsible for distinct adhesive properties of carcinoma cells. Cell, 57: 59-69, 1989.

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1991;51:1065-1070. Cancer Res   Lawrence E. Feldman, Kye C. Shin, Ronald B. Natale, et al.  

Integrin Expression on Human Small Cell Lung Cancer Cells1β

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