[CANCER RESEARCH56, 5071-5078, November 1, 1996J

ABSTRACT

The integrin allbfi3 was initially believed to be expressed only in cellsfrom the megakaryocytic lineage, such as platelets or HEL cells. In thisstudy, we report for the first time that human prostate carcinoma PC-3and DU-145 cells express allbfi3. Reverse transcription-PCR from HEL(positive control), PC-3, and DU-145 cells amplified a predicted allb

fragment that hybridized to the full-length aIIb cDNA probe. DNA Sequencing of the PCR fragments revealed 100% sequence homology to thecorresponding extracellular domain ofplatelet aIIb but minimal sequence

homology to integrins av or aS. An RNase protection assay was used toconfirm the results from reverse transcriplion-PCR. An antisense riboprobe to nUb mRNA hybridized to total RNA from HEL, PC-3, and

DU-145 cells, suggesting that allb mRNA is transcribed in these tumorcells. In situ hybridization on surgical specimens from human prostatetumor tissue stained positive with an antisense riboprobe to aIIb mRNA.

The expression of allbfi3 protein in PC-3 and DU-145 cells was demonstrated by Western and dot blotting and flow cytometry with monoclonalantibodies (mAin) to allb (MAR 19%), fi3, and aflb@33 (AP-2). A proteinkinase C activator, phorbol 12-myristate 13-acetate, increased the adhesion of PC-3 cells to PAC-1, a mAb specific to the high-affinity state ofaIIbfi3, by more than 80-fold. The invasion of DU-145 cells through areconstituted basement membrane was blocked 40-50% by mAbs AP-2 orPAC-1. These data collectively suggest that: (a) prostate tumor cellsexpress a11b133;(b) surface expression of allbfi3 integrin is regulated byprotein kinase C; and (c) mAin to this receptor inhibit invasion of prostatecancer cells through a reconstituted basement membrane.

INTRODUCTION

Integnns are a/@3heterodimeric glycoproteins that are involved incell-cell and cell-matrix interactions during embryogenesis, thrombosis, wound healing, cell homing, growth regulation, apoptosis, immune reactions, and tumor progression (for reviews, see Refs. 1—3).Ingeneral, the @3land (33 subfamilies of integrins play an important rolein tumor invasion and dissemination (1, 4). The fibronectin receptor(a5@1) is involved in the proliferative response of quiescent humanmelanoma cells (2, 3), whereas the overexpression of f33 integrinsdirectly correlates with metastatic potential in melanoma (5).

Most integrins bind specifically to one or two ECM3 proteins,except two members from the @3integrin subfamily, aHb@3 andav@3. These two integrins demonstrate a wide affinity for multipleligands including fibrinogen, fibronectin, thrombospondin, and vonWillebrand factor (6, 7). The widespread expression of crvJ33 in

Received 4/3/96; accepted 9/4/96.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 with18 U.S.C. Section 1734 solely to indicate this fact.

I Supported by NIH Department of Health and Human Services Grants CA-47 I I5-08(K.V.H.), CA-29997-14 (K. V. H.), and TW-285-02 (K. V. H. and J. T.); NATOCRG950287 (K. V. H. and J. T.); Hungarian National Science Foundation T21 149 (J. T.);and Harper Developmental Fund (K. V. H. and A. T. P.).

2 To whom requests for reprints should be addressed, at 431 Chemistry Building,

Wayne State University, Detroit, MI 48202. Phone: (313) 577-1018; Fax: (313) 577-0798.3 The abbreviations used are: ECM, extracellular matrix; RPA, RNase protection

assay; ISH. in situ hybridization; RT, reverse transcription; ThE, Tris-borate EDTA; ECL,enhanced chemiluminescence; mAb, monoclonal antibody; pAb, polyclonal antibody;HRP, horseradish peroxidase; SFM, serum-free media; PMA, phorbol 12-myristate 13-acetate;CM, conditionedmedium;PKC, proteinkinaseC.

different tumors and its important role in cell adhesion (1, 4), angiogenesis (8), and survival (9) are well documented.

The other member of the @33integnn subfamily, aIIb/33, has beenextremely well characterized in platelets and is directly involved inplatelet aggregation and cell signaling ( 10). This integrin is expressedin two conformations, inactive and active, but only the latter iscapable of binding multiple ECM proteins (1 1—15).Stimulation ofplatelets with agonists such as thrombin or ADP results in a conformational change of aIIb@3 from an inactive to active form which thenbinds plasma fibrinogen and induces platelet aggregation. Severalinvestigators have reported aIIb@3 to function as a signaling moleculebecause its ligation or perturbation results in waves of tyrosine phosphorylation of cytoplasmic proteins (10). Therefore, this receptor isthought to be involved in “inside-out―and “outside-in―signaling (1 1).

allb/33 was initially identified in platelets and originally termed asGPIIbIIHa (7). It was considered to be expressed only in cells from themegakaryocytic lineage, such as platelets or human erythroleukemiacells (HEL; Refs. 7, 13, and 15). The cDNAs for aIIb and j33 fromhuman (16, 17) and other species have been cloned and sequenced.Genes of human megakaryocytic aIIb and (33 have been localized tochromosome 17 (18—22).We reported for the first time that aIIbf33 isexpressed in nonmegakaryocytic lineage murine metastatic B l6a melanoma cells (23, 24) and demonstrated its involvement in tumor cellinteractions with platelets, endothelial cells, and ECM (25—27).Besides the expression of aIIbJ33 in B 16a cells, there have been a fewreports in the literature documenting a protein immunologically related to GPIIbIHIa in other tumor cell lines (28—31);however, rigorous biochemical and/or molecular proof has been lacking. Therefore,we attempted to determine whether aHb@3 is expressed in humannonmegakaryocytic lineage tumor tissue and immortalized cell lines.In this study, we report for the first time the expression of the alIbintegrin at the mRNA and protein level in two human prostate carcinoma cell lines, PC-3 and DU-145, as well as three human prostatetumor tissues. In addition to demonstrating expression of alIb, wealso provide evidence that it is complexed to @3and that this integrincomplex plays a role in tumor cell invasion.

MATERIALS AND METHODS

Cell Lines. Humanprostatecancercell lines PC-3 (32) andDU-145 (33),isolated from metastatic lesions, human erythroleukemia cell line HEL, and

human fibrosarcoma cell line HT-1080 were obtained from American Type

Culture Collection (Rockville, MD). Tumor cells were cultured in RPMIsupplemented with 5% fetal bovine serum (Life Technologies, Inc., GrandIsland, NY) in a 5% CO2 humidified atmosphere at 37°C.All experimentswere performed with cells that were grown to approximately 80% confluence.DU-l45 and HT-1080 cells were passaged with 0.68 mM EDTA and I . 1 mrsi

glucose in PBS, PC-3 cells were harvested with the same buffer containing

0.1% ts-ypsin,and HEL cells were passaged as a suspension culture.RT-PCR. Total RNA from differenttumorcell lines was obtainedby the

acid-guanadinium isothiocyanate (34) method using the Tri-Reagent kit (Mo

lecular Research Center, Inc., Cincinnati, OH). Total RNA (2 @xg)from each

cell line was reverse-transcribed using aIThgene-specific primers as described(24). Briefly, for reverse transcription, 2 @igof tumor cell RNA and 0.5 @xgof

alit, antisense (5'-GCATAGGGGAGGGAGGACAC-3') primer were denatured at 68°C for 5 rain and incubated for an additional 10 mm at room

5071

Human Prostate Carcinoma Cells Express Functional aIIb@33Integrin1

Mohit Trikha, Jozsef Timar, Steven K. Lundy, Karoly Szekeres, Keqin Tang, David Grignon, Arthur T. Porter,

and Kenneth V. Honn2

Departments of Radiation Oncology [M. T., S. K. L, K. S., K. T., A. 1'. P., K. V. H.], Pathology ID. G., K. V. H.], and Chemistry [K. V. H.], Wayne State University. Detroit,Michigan 48202, and 1st institute ofPathology and Experimental Cancer Research, Semmeiweis University ofMedicine, Budapest H-1085, Hungary If. T.]

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EXPRESSION OF aIIb@3 IN HUMAN PROSTATE CARCINOMA

temperature. After incubation, the following reagents were added: RT buffer

[50 mittTris-HCI(pH 8.3), 75 mMKC1, 10 mn DTI', and 3 mrs@MgC12],0.5nlM deoxynucleotide triphosphates, 50 xg/ml BSA, 30 units RNasin (Promega,Madison, WI), and 200 units M-MLV reverse transcriptase (Life Technologies,Inc.) in a final volume of 20 p1 The reaction was incubated at 42°Cfor 60 mmand terminated by heating at 90°Cfor 5 mm. The RT mixture (1 pJ) wasamplified in 50 @tlof PCR buffer containing 30 mrviTris-HCI (pH 8.5), 4 misiMgC12, 100 jxg/ml BSA, 250 nM each of alib antisense (see above) and sense(5'-CTGGAAGAGGCTGGGGAGTC-3') primer, 0.2 mist deoxynucleotidetriphosphates, and 1 unit AmpliTaq (Perkin Elmer Cetus, Foster City, CA).PCR was performed in the GeneAmp PCR system 9600 (Perkin Elmer Cetus)by using the following cycle: 94°Cfor 30 s, 49°Cfor 30 s, and 72°Cfor 1 mmrepeated 30 times and followed by a 5-mm extension at 72°C. A portion of the

first-round PCR product (1 pA) was reamplified under the same conditions with250 ni@iof nested primers (sense, 5'-GCAGATACGGAGCAAGAACA-3';antisense, 5'-GATGTAGAGCAGGTCGGAGG-3'). The PCR product (15 pJ)was separated on a 2% agarose-TBE gel and stained with ethidium bromide tovisualize the amplified product. To prevent contamination by either genomicDNA or PCR product carry-over, the total RNA, RT mixture, and PCRsolutions were treated with 0.05 unit/@xlA/uI restriction enzyme (Stratagene,La Jolla, CA) for 1 h at 37°Cand heat-inactivated at 90°Cfor 10 mm. AluI waschosen because it has several restriction sites on the human aIIb gene.

Omission of tumor cell RNA from the reaction mixture served as a negativecontrol for RT-PCR.

Southern Blotting. PCR-amplifiedaIThcDNA (15 pA)was separatedon a1% agarose-TBE gel and transferred to GeneScreenPlus nylon membrane(DuPont NEN Research Products, Boston, MA) using a PosiBlot pressureblotter (Stratagene), and the transferred DNA was cross-linked in a UVStratalinker (Stratagene). Full-length human integrin allb (23) and av cDNA

(35) were labeledwith fluoresceinby the ECL randomprime labelingkit(Amersham, Rockford, IL) and purified by size-exclusion Chroma-spin col

umns (Clontech, Palo Alto, CA). The fluorescein-labeled probes, in conjunction with the antibody to fluorescein and the ECL detection kit, were used

according to the manufacturer's instructions for Southern hybridization. BothaIIb and av Southern blots were exposed to autoradiographic film for at least30 mm. alib and av probes were adequately labeled with fluorescein becausethey demonstrated positive staining when dot-blotted to unlabeled aHb or av

probe, respectively.DNA Sequencing. PCRproductsfromHEL,PC-3,andDU-145 cells were

separated on a 2% agarose-TBE gel and purified by gel elution. The primerswere designed to amplify a 283-bp fragment of the human alib cDNA basedon the published sequence (18, 36). Sequencing was performed by the Taq dyedeoxynucleotide Terminator Cycle Sequencing kit from Applied Biosystems(Foster City, CA) by using the standard protocol on a Perkin Elmer Cetusthermal cycler (model 480). Sequencing reactions were run on a DNA sequencer (Applied Biosystems, Inc., model 373), and the sequence was alignedto allb, aS, and av by using MacVectorTM4,1 software on a MacintoshQuadra 630 computer.

RPA. To confirm the results obtained from RT-PCR, Southern blot, and

DNA sequencing, we performed RPA. Human integrin alib (3.2 kb) wascloned in the sense orientation into the pBluescript SK vector, and this wasconfirmed by restriction mapping and partial sequencing of the plasmid with

T3 and T7 primers (23). The alIb plasmid was linearized with variousrestriction enzymes, and the sense and antisense riboprobes were preparedusing T3 and T7 RNA polymerase, respectively. Specifically, alIb plasmid

was digested with AflhIIor BSSHIIrestriction enzymes (New England Biolabs,Beverly, MA) and transcribed in vitro by Ti polymerase, which generated thetemplates for antisense riboprobes of 325 and 387 bp, respectively. Senseriboprobes of 552 and 238 bp were generated by T3 polymerase transcription

of the alIb plasmid digested with ScaI or Nan restriction enzymes (NewEngland Biolabs), respectively. Sequencing of the sense and antisense linearized DNA templates with T3 and 17 primers, respectively, revealed complete

homology to aIIb but less than 20% homology to av or aS. To determine thequality of total RNA, a human @-actinDNA template (Ambion, Inc., Austin,TX) was used to generate an antisense RNA probe (T7 polymerase) thatresulted in a protected length of 125 bp. The linearized DNA templates werepurified with proteinase K/phenol-chloroform extraction before transcription.In vitro transcription was performed using the Maxiscript transcription kit(Ambion) with final concentrations of 500 @xMeach of AlP, GTP, and CTP;

3.15 @LM[a-32PIUTP(800 Ci/mmol); and 5 .LMofcold UTP. Transcription wasinitiated by the addition of 1 pARNA polymerase, and the reaction mixture (20pJ) was incubated for 1 h at 37°C.Termination was achieved by the additionof RNase-free DNase I, and the runoff transcripts were purified by Chromaspin size-exclusion columns (Clontech). The quality of the transcribed probewas determined by running an aliquot (10% of total sample) on a denaturing

5% acrylamide-urea gel followed by autoradiography, and only transcriptsresulting in a single band were used for the experiment. Hybridization andnuclease digestion were performed using the RPA II RNase protection kit(Ambion) according to the manufacturer's instructions, except for minormodifications. Tumor cell RNA, purified by the Tri-Reagent kit, from HEL (20

,.Lg), PC-3 (100 @.Lg),DU 145 (100 @xg),and a negative control torulla yeastRNA (100 @xg;Ambion) were dried in a Speedvac and dissolved in 20 jxl ofhybridization buffer containing 2 X i0@ cpm of purified probe. Hybridization

was performed at 42°Cfor 14 h, and the mixture was digested with RNasefollowed by inactivation and RNA precipitation. The precipitated protected

fragments were separated on a 5% acrylamide-8 M urea gel, and the bands werevisualized by autoradiography.

ISH. We performedISH to determinewhetheralib mRNA was expressedin human prostate tumor tissue. The samples were obtained from radical

prostatectomy specimens obtained from male patients undergoing surgery forclinically localized prostatic adenocarcinoma. Digoxigenin-labeled alP, senseand antisense riboprobes were prepared essentially as described for RPA, withslight modifications. The alib plasmid, which was digested with AflIII or

BSSHII (antisense) and NarI (sense), was used, and transcription was performed with the Riboprobe Gemini II transcription kit (Boehringer Mannheim,Indianapolis, IN).

ISH was carried out in the Microprobe System staining station (FisherScientific) according to the method described previously (37). Briefly, forma

lin-fixed and paraffin-embedded surgical sections from human prostate tumorwere dewaxed with xylene, permeabilized with Triton X-lOO (0.5%) andproteinase K (50 p@g/m1)treatment, acetylated (0.25% acetic anhydride in 0.1M triethanolamine), and washed with buffer I (100 mt@Tris-HC1 and 150 mMNaC1, pH 7.5) containing 0.075% Brij at room temperature. The tumor specimens were prehybridized at 55°C for 2 h in prehybridization buffer [50%

formamide, 10% dextran sulfate, 1 X Denhart's solution, 20 mM Tris-HC1, 300

mM NaCl, and 5 mM EDTA (pH 8.0)] containing 0.1 mg/ml salmon spermDNA. Hybridization was performed for 2 h at 55°C in prehybridization buffer

containing100ng/ml of digoxigenin-labeledprobe.After hybridization,thespecimens were washed at room temperature with buffer I-Brij at roomtemperature (30 5 X 3) and with 2 x SSC and 0.1% SDS-Brij (3 mm X 4).Then a high-temperature wash at 55°C was performed with 0.1 X SSC and

0.1% SDS-Brij (2 mm X 5). The tumor slides were subsequently washed withantibody dilution buffer (buffer I-Brij and 1%BSA; 5 mm X 3) and incubatedwith anti-digoxigenin Fab conjugated to alkaline phosphatase (1:1000 dilution;

Boehnnger Mannheim) in the antibody dilution buffer at 37°C for 1 h. After

incubation, excess antibody was washed off with buffer I-Brij and 1% BSA (2mm X 5), and the specimens were washed with 100 mr@iNaC1, 50 mM MgC12,

and 100 mM Tns (pH 9.5; 5 mm X 2). Color development was carried outusing the stable mix of nitro blue tetrazoliuml5-bromo-4-chloro-3-indoyl phos

phate (Life Technologies, Inc.) at 37°Cfor 5—30mm in the dark. No colordevelopment was observed after 3 h in the absence of probe. Tumor tissue fromthe same patient was used for staining with the antisense and sense probes, and

similar results were obtained from three different patient specimens.

Antibodies. The mouse mAb PAC-1 (1gM),which specifically recognizesthe active conformation of aIIb@3, was purchased from the University of

Pennsylvania (Philadelphia, PA). A rabbit pAb that recognizes denaturedaIIb@3 and mouse mAbs AP-2 (subtype IgG), AP-4 (subtype IgA), andanti-@33 antibody (subtype IgG) that recognize aIIb@3, alIb, and @3,respec

lively, were generous gifts from Dr. Thomas Kunicki (The Scripps Research

Institute, La Jolla, CA). PAC-l (38) and AP-2 (39) inhibit platelet aggregationby binding to aIIb@3 and do not cross-react with avf33 or other knownintegrins. MAB 1990, an IgG mAb that recognizes allb in Western blotting(15), was purchased from Chemicon (Temecula, CA). Normal mouse IgG(MOPC-21) and 1gM (TEPC) were purchased from Sigma (St. Louis, MO).

Western and Dot Blotting. Tumorcells in exponentialgrowthphasewerelysed in cell lysis buffer [20 mM Tris (pH 7.5), 150 mM NaC1, 5 jxg/ml BSA,0.5% NP4O,0.5% Tween 20, 1 mMphenylmethylsulfonyl fluoride, 100 mMleupeptin, 10 mM aprotinin, and 20 mM EDTA] at an approximate concentra

5072

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EXPRESSION OF aIIb@3 IN HUMAN PROSTATE CARCINOMA

tion of 50 X 106cells/ml. Freshly lysed cell solution was sonicated (15 s X 3)and used for Western or dot blotting. Protein concentration was determined byBio-Rad's DC protein assay kit (Bio-Rad, Richmond, CA).

For Western blotting, total protein (50 @g)was mixed with an equal volumeof 2 X sample buffer [0.125 MTris (pH 6.8), 4% SDS, 20% glycerol, and 20%2-mercaptoethanoll, boiled for 5 mm, separated on 8% SDS-PAGE, and

electrophoretically transferred to a nylon membrane. The alib integrin subunitwas detected by MAB 1990(10 ng/ml) or AP-4 (I ,xg/ml), and the f33integrinsubunit was probed with anti-j33 mAb (1:1000 dilution). Bound primaryantibody was detected by anti-mouse IgG conjugated to HRP (1:1000 dilution;

Bio-Rad) followed by ECL detection (Amersham). Gel electrophoresis andWestern blot were performed in the Bio-Rad Miniprotean II apparatus according to the manufacturer's instructions.

For protein dot blotting, tumor cells were lysed in the same lysis buffer asdescribed above, with the exception of EDTA. Dot blotting of total protein

(1—20xg) was performed in Bio-Rad's Bio-Dot Microfiltration apparatusaccording to the manufacturer's instructions. aIIb/33 was detected by AP-2 (1p.g/ml) followed by the addition of anti-mouse IgG conjugated to HRP (1:1000dilution) and detected by the ECL method. As a negative control, the membrane was probed with MOPC-2l (1 @xg/ml)and/or anti-mouse IgG conjugatedto HRP (1:1000 dilution).

Flow Cytometry. DU-l45 and PC-3 cells were harvested, washed three

times with HBSS, and allowed to recover for 1 h in a tissue culture incubator.

Flow cytometry was performed as described previously (40), with minormodifications. Briefly, for surface or total labeling, cells were fixed for 10 mm

at 4°Cwith either 2% paraformaldehyde-HBSS solution or acetone/methanol(1:1), respectively. After fixation, nonspecific sites were blocked with normalgoat serum (1:3 dilution; Caltag Laboratories, South San Francisco, CA) for 30rain at room temperature and incubated with primary antibody (10 @xWml)for1 h at room temperature. Bound primary antibody was detected with goatanti-mouse IgG conjugated to FITC (1:300 dilution; Organon Teknika,Durham, NC). The fluorescence oflabeled tumor cells (1 X l0@)was measuredin a Coulter Epics Profile II flow cytometer (Coulter, Hialeah, FL), and themean fluorescence was determined. Background fluorescence was determinedby incubating tumor cells with either MOPC-21 (10 @xg/ml)or secondaryantibody in the absence of primary antibody. The data are reported as thepercentage increase in mean fluorescence obtained by using the following

formula: [(mean fluorescence with primary + secondary antibody)/(meanfluorescence of secondary antibody alone)] X 100.

Cell Adhesion Assay. Tumor cells in exponential growth phase wereharvested and washed extensively with SFM and incubated at 37°Cfor 1 hbefore plating on antibody-coated Immunolon II 96-well plates (DynatechLaboratories, Chantilly, VA). Antibody coating was performed by immobiliz

ing 100 p1 of 20 pg/mi ofeither PAC-l or TEPC in PBS at 4°Covernight. The

cell adhesion assay was performed as described previously (41), with modifications. Briefly, after washing off excess antibody from the wells, nonspecificsiteswereblockedwith200@.dof2mg/mlBSAat37°Cforatleast2h.Tumorcells (5 X l0@ cells/ml) were treated with various concentrations of PMA (Life

Technologies, Inc.) for 15 mm at 37°C,and 100 x1of cell suspension wereadded to antibody-coated 96-well plates. Adhesion was allowed to proceed for30—60 mm, then unbound cells were washed with SFM (3 X 200 pi/well). The

extent of cell adhesion was determined by the addition of SFM (100 pA) and20 p@lof Cell Titer@'@ nonradioactive cell proliferation dye (Promega),and the absorbance at 490 nm was recorded by a Bio-Rad plate reader (model3550). The assay is based on the cellular conversion of the tetrazolium salt intoformazan. The absorbance at 490 nm is directly proportional to the number ofliving cells (42). To demonstrate that cell adhesion to immobilized PAC-loccurred via the integrin binding domain but not through the Fc receptor of this

domain, tumor cells were treated with 500 p@g/m1 of GRGDS (inhibitor of

integrin-mediated cell adhesion) or GRGES (negative control) peptide beforecell adhesion. The GRGDS peptide completely blocked adhesion, therebydemonstrating that immobilized PAC-1 was binding to tumor cell-associated

aIIb@3. As a negative control, the assay was performed in TEPC- or BSAcoated wells, and in both cases, the absorbance was comparable to backgroundlevels.

Tumor Cell Invasion Assay. This assay was performedin a 48-wellChemotaxis chamber (Neuroprobe, Inc., Cabin John, MD) according to themanufacturer's instructions, with slight modifications. Briefly, CM preparedby growing HT-l080 cells in SFM for 48 h was added to the bottom part of the

chamber. Next, a polycarbonate filter (8-@xmpore size) was coated withreconstituted basement membrane (Matrigel; 3 mg/ml; Collaborative Biomedical, Bedford, MA), air-dried, and placed on the chamber with the coated side

facing up. Tumor cells (1 x l06/ml) in SFM were incubated with variousantibodies for 15 mm before the addition of 50 p1 of the cell suspension to the

upper well of the chamber, and the assay was allowed to proceed for 12—16hin a 5% CO2 humidified atmosphere at 37°C. After incubation, cells in the

upper chamber of the filter were scraped off, and the bottom part of the filterwas stained with DiffQuick solution (American Scientific Products, McGrawPark, IL). The number of cells that invaded the Matrigel-coated filter wascounted under a phase-contrast microscope at a magnification of X200. Eachdata point was performed in quadruplicate, and every experiment was repeated

at least twice.

RESULTS

Detection of aIIb mRNA in Human Prostate Tumor. Severallines of experimental evidence demonstrate that prostate cancer cellsexpress alib mRNA. RT-PCR (Fig. 1A) amplified a predicted alib

fragment of 283 bp from PC-3, DU-145, and HEL (positive control)

cells. Amplified cDNA from all three cell lines comigrated to thesame molecular weight in agarose gel electrophoresis and hybridizedto full-length aIIb-cDNA probe but not full-length av-cDNA probe.DNA sequencing of these PCR fragments revealed a 100% sequencehomology to the corresponding extracellular domain of alib that mapsto 2.288—2.570kb (36). The PCR fragments have approximately 20%sequence homology to integrins av (43) or aS (44), thereby explaining the negative result obtained with the av Southern blot. The

RT-PCR cDNA product was a direct result from tumor cell mRNAand not due to contamination from genomic DNA or PCR carry-overbecause total RNA and all PCR buffers were treated with AluI. As anadditional negative control, the omission of RNA from the mixtureresulted in no amplification.

A significantly less sensitive technique, RPA, was used to confirmthe results from RT-PCR. An antisense riboprobe to ctllb was developed that mapped to the 3' region of alib mRNA. This probehybridized to total RNA from HEL (positive control), PC-3, andDU-l45 cells (Fig. 1B), suggesting that allb mRNA is transcribed inthese tumor cell lines. The detection of the protected fragment wasspecific because the antisense riboprobe was not recognized by yeastRNA (Fig. 1B, Lane 4), and additionally, no bands were detectedwhen a sense riboprobe to aITh was used (data not shown).

We next questioned whether alib mRNA was also expressed inprostate tumor tissue, and ISH was performed to address this question(Fig. 1C). Results from ISH revealed that prostatic adenocarcinomafrom three different patients stained positively with a digoxigeninlabeled antisense riboprobe to aIIb and no staining was observed witha sense probe or no probe.

Detection of aIIb@33 Protein in PC-3 and DU-145 Cells. Theresults described above demonstrate that alIb mRNA is transcribed inprostate tumor cells. Hence, we questioned whether aIIbj33 proteinwas also expressed. The mAbs to alIb, MAB 1990 (Fig. 14) and AP-4(data not shown), recognized a single band migrating at approximately120 kDa (the approximate molecular mass for reduced alib; Ref. 12).A mAb to f33 (Fig. 2B) recognized a single band at 100 kDa (theapproximate molecular mass for nonreduced @33integrin; Ref. 12),indicating that both aIIb and f33 integrin subunits are expressed inPC-3 and DU-145 cells.

To ascertain whether aITh was complexed to @3in prostate tumorcells, three experimental approaches were used: protein dot blotting,flow cytometry, and cell adhesion assay. Dot blotting (Fig. 2C)revealed that an anti-aIIb@3 complex mAb, AP-2, recognized anantigen in total cell lysates from PC-3 and DU- 145 cells. Flow

cytometry on PC-3 and DU-l45 cells indicated higher total staining5073

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EXPRESSION OF aIIb@3 IN HUMAN PROSTATE CARCINOMA

A M(bp) 12 34 1 234 1 23 4 B

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Fig. I. Detection of afib mRNA. A, allb mRNA detection by RT-PCR/Southem blotting. Total RNA from PC-3 (L.ane 1), DU-145 (Lane 2), HEL (Lane 3), and negative control(water substituted for RNA; Lane 4) was reverse-transcribed and amplified as described in “Materialsand Methods.―The amplified products (283 bp) were Southern-blotted withfluorescein-labeled alIb or av probes. The origin of the higher band in HEL is not known. B, detection of alib mRNA by RPA. Total RNA was hybridized with the alib antisenseriboprobe.digestedwith RNase,separatedon a 5% denaturingacrylamidegel, andvisualizedby autoradiographyasdescribedin “MaterialsandMethods.―Lanes1—3,thepresenceofthe predicted325-bpprotectedfragmentwhenhybridizedto totalRNAfromHEL(20 @xg),PC-3(100pg), andDU-145(100 @xg),respectively.Lane4, thenegativecontrolin whichtorulla yeast RNA (100 @g)was used instead of tumor cell RNA. The lOO-bp 32P-labeled RNA markers are shown in Lane 5. C, detection of alIb mRNA by ISH. aIIb plasmid digestedwith BSSHII (antisense) and Nan (sense) was transcribed. ISH was performed on tumor specimens from the same patient (photomicrographs are from a Gleason score 7 prostaticadenocarcinoma) with digoxigenin-labeled alib sense (a) and antisense (b) probe as described in “Materialsand Methods.―Color was developed for 20 mm at 37'C in the dark, andafter mounting, the slides were photographed at a magnification of X400. The data represent staining from three different pairs of matched tumor specimens. In each case, no stainingwas obtained in the absence of probe.

RT-PCR allb-Southem av- Southern

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for AP-2 than surface labeling (Fig. 3, A and B), suggesting that asignificant proportion of a11bf33 resided intracellularly. These findings were confirmed by the adhesion assay because a PKC activator,PMA (100 nM), increased the adhesion of PC-3 cells to PAC-l bymore than 80-fold (Fig. 4). The data collectively demonstrate that inprostate cancer cell lines DU-l45 and PC-3, allb is complexed to 133,of which the majority resides intracellularly as an aIIb133 pool, andthat it can translocate to the cell surface upon PKC activation.

Effect of Antibodies to aITh@33on Invasion of Prostate TumorCells. Results described above indicate that allbf33 is expressed inprostate tumor cells. Therefore, we questioned whether it plays a rolein tumor cell invasion. Antibodies to aIIb133, AP-2 and PAC-l,inhibited CM-stimulated invasion of DU-145 cells through a reconstituted basement membrane (Fig. 5). Both antibodies blocked invasion by approximately 40%. In contrast, antibodies specific to the allbsubunit, AP-4 and MAB 1990 (data not shown), or the pAb to allbfJ3that recognizes the denatured form of aIIbf33 (45), or an isotypematched nonimmune IgG, MOPC-21, did not affect invasion of DU145 cells (Fig. SA). The results suggest that intact aIIbf33 integrincomplex is involved in tumor cell invasion.

DISCUSSION

The integrin aIth is believed to be uniquely expressed in cells ofmegakaryocytic lineage, and its expression in nonmegakaryocyticlineage cells has long been an issue of debate (7, 13). By using variousgenetic and immunological approaches, we identified for the first time

the expression of aHb133 in nonmegakaryocytic murine melanomaBl6a cells (23, 24). In this study, our aim was to test the hypothesisthat the integrin ceIIb133is expressed in human prostate tumor cellsand determine whether it plays a role in invasion. The novel findingsfrom this work are as follows: (a) in PC-3 and DU-l45 cell lines, aIIbmRNA can be detected by RT-PCR/Southem hybridization, partialcDNA sequencing, and RPA; (b) in a limited number of humanprostate adenocarcinoma specimens, aITh mRNA can be detected byISH; (c) the allbf33 protein can be detected by Western and dotblotting, flow cytometry, and cell adhesion assay; (e) activation ofPKC stimulates adhesion of PC-3 cells to PAC-l; and (f) antibodiesspecific to the a11bj33 complex block invasion of DU-145 cellsthrough a reconstituted basement membrane.

The results from RT-PCR indicate that human prostate tumor cells

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AP-2 MOPC-21 2@b

I @Hfl

•-@

S.

EXPRESSION OF allbfi3 IN HUMAN PROSTATE CARCINOMA

B

M(kDa)@

AIn

f'•)

M(kDa)

204—

121 —

82 —

50.7 —

34.2—

28.1—

204 —

@,,

Fig. 2. Detectionof aIIb@3proteinby Westens and dot blotting. A, total protein (50 @.tg)fromPC-3 and DU-145 cells was diluted in reducingsample buffer, boiled, separated on an 8% SDSPAGE gel, and transferred to a nylon membrane.and the aIIb integrin subunit was detected byWestern blotting with MAB 1990 (10 ng/nil) asdescribedin “MaterialsandMethods.―B, in another experiment, 50 @xgof total protein weredissolved in nonreducing sample buffer, boiled,separated on an 8% SDS-PAGE gel, transferred,and probed with an anti-@3integrin mAb (1 @tg/ml). *, a nonreproducibleband that might resultfrom proteolytic degradation. Left, the molecularweight markers are indicated. C, the allb@3 integrincomplexin HEL,PC-3,andDU-l45cellswas detected by dot blotting 20 ,xg (1), 5 @.tg(2),and 1 @&g(3) of total protein and probing themembrane with AP-2 (1 pg/al) as described in“Materialsand Methods.―Negative control wasperformed by using a subtype-matched nonimmune antibody, MOPC-2l (1 @tg/m1),or by removal of primary antibody during detection(2°Ab).Negative control for DU-l45 was similarto PC-3 and HEL (data not shown).

*

28.1

@rn,i:@:@—@@ cxlIb 121

82

50.7 —

34.2—

C

2

3

express aHb mRNA. However, RT-PCR is a sensitive technique andmay lead to misinterpretation of data; therefore, we took severalprecautions to prevent false positives. Primers for aIIb were designedto frame sequences that spanned three introns on the human aHb gene,and the amplified product from genomic DNA would have a predictedsize of 840 bp. Therefore, product obtained from genomic DNA couldbe easily differentiated from the cDNA product, which has a predictedsize of 283 bp. Additionally, treatment of reaction mixtures with A!uI,which has several restriction sites on the aHb gene, prevented con

tamination from genomic DNA. Finally, the authenticity of the amplified cDNA was confirmed by Southern blotting and DNA sequencing.

Nested RT-PCR is an extremely sensitive technique, and positiveresults can be obtained due to leaky transcription. Therefore, wequestioned the biological relevance of allb mRNA detection by thistechnique. RPA, a significantly less sensitive technique, was used to

confirm the results from RT-PCR. An allb antisense riboprobe thatmapped to a different region than the amplified PCR product hybrid

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EXPRESSION OF aIIbf33 IN HUMAN PROSTATE CARCINOMA

cell surface (40). We next questioned whether aIIb133 was involved ininvasion of prostate tumor cells. Interestingly, only AP-2 and PAC-l,mAbs that block platelet aggregation (presumably by binding to theactive form of aIIb133), partially inhibited invasion of prostate tumorcells through a reconstituted basement membrane. In contrast, mAbsthat preferentially recognize the inactive state of aIIb/33 did notinhibit invasion. The results collectively suggest that the active stateof a11b133(but not the inactive conformation) is partially required forinvasion of tumor cells.

Formation of the active-state aIIbj33 complex may have importantconsequences on the development of the metastatic phenotype inprostate cancer. For example, as described for Bl6a cells (1), aHb133may mediate interactions of prostate tumor cells with host cells suchas platelets and endothelial cells or with the ECM. Studies using ISHare in progress to elucidate whether the staining of prostate tumorcells with an antisense riboprobe to aIIb correlates with the pathological stage and grade of prostate cancer specimens. Results fromthese studies would indicate whether the aIIb gene may serve as apotential prognostic marker for prostate cancer.

It was recently reported that PC-3 cells induce GPIIbIIIIadependent platelet aggregation; however, pretreatment of thesecells with mAbs to GPIIbIIIIa did not block tumor cell-inducedplatelet aggregation (47). This report corroborates the results fromthis study, which suggests that in PC-3 cells, the majority of theaIIbf33 pool resides intracellularly, and activation of PKC modulates surface expression of the active-state a11b133. Prostate tumorcells express a number of other integrin receptors (48, 49) including a513l and av133, but little is known about their contribution tocell adhesion, spreading, or invasion. Immunohistochemical studies on prostate carcinoma tissues reveal a complete loss of the 134integrin subunit and redistribution of the a6 and 131subunits (50).How these integrin receptors influence prostate tumor progressionand invasion is largely unclear. Matrigel is rich in collagen andlaminin but also contains vitronectin (51). Thus, it is possible thatantibodies to aIIb133 block tumor cell interactions with these

proteins and thereby inhibit invasion. Immunofluorescence studies

indicate that DU-l45 cells adhered to Matrigel express aIIbf33 on

C0r1@4?

.@

PMA (nM) 0 25 100

Fig.4. PMA-stimulatedadhesionof PC-3cellsto immobilizedPAC-l. PC-3cellsweretreated with SFM or PMA for 15 mm before plating on immobilized antibodies (20

@g/ml),and the cell adhesion assay was performed as described in “MaterialsandMethods.― The results are indicated as PMA-stimulated percentage increase in celladhesion with respect to adhesion in the absenceof PMA. Each data point represents themean of at least triplicate determinations ±SD and is representative of three differentexperiments.

Surface Total

L

.0

.@

e@

A 1400

@1200a,

C@ 1000

a,I.

@ 800Ia.

@600

.@ 400

@200

0

B 300

a, 250Ca,C.,

: 200I.0C

@ 150Cc8

!@@ 50

0

Surface Total

Fig. 3. Detection of aIIb@3 protein in PC-3 and DU-145 cells by flow cytometry. PC-3cells (A) and DU-145 cells (B) were either fixed with paraformaldehyde or acetone/methanol for surface or total labeling, respectively, as described in “MaterialsandMethods.―The allb@3 integrin was detected by AP-2 (10 @tg/ml)followed by the additionof goat anti-mouse IgG conjugated to FITC (1:300 dilution), and mean fluorescence wasdetected by an Epics flow cytometer. Background fluorescence was determined byincubating tumor cells with either an isotype-matched nonimmune antibody (MOPC-21;10 @g/ml)or secondaryantibodyin theabsenceof primaryantibody(2-Ab).Thedataarereported as the percentage increase in mean fluorescence ±SD by using the formuladescribedin “MaterialsandMethods.―Eachdatapoint wasperformedin triplicateandisrepresentative of three different experiments.

ized to RNA from HEL, PC-3, and DU-145 cells. The data from RPAand RT-PCR suggest that full-length aIIb mRNA is transcribed inprostate tumor cell lines. The amount of aIIb mRNA in PC-3 andDU-145 cells is significantly less than that in HEL. This finding is inaccordance with published reports demonstrating a decrease in expression of a11b133with long-term in vitro culture of B16a cells (27,46). Althoughtheexpressionof alib mRNAis weakerin prostatetumor cells as compared to HEL, the protein is translated in thesecells. The expression of aIIb133 protein in the prostate tumor cellscould be detected by several mAbs to allb and/or 133in Western anddot blotting and flow cytometry. The allbf33 receptor expressed inprostate tumor cells seems to function as platelet-type GPllbIIlla.PAC-l, a mAb that specifically recognizes the active state of aIIb!33,bound primarily to PC-3 cells that were stimulated with PMA, suggesting that PKC regulates the surface expression of active-stateaIIb133. This observation confirms reports documenting involvementof PKC in the translocation of aIIbf33 from an intracellular pool to the

5076

.0

e@

C.1@ Ci@@a, @. a. t@' @.

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EXPRESSION OF a1Ib@3IN HUMAN PROSTATE CARCINOMA

F

Ua@@t)@ @..

1 2 3

Fig. 5. Role of aIIb@3 in HT-1080 CM-stimulated invasion of prostate tumor cells. DU-l45 cells were treated with various antibodies, and the invasion assay was performed asdescribed in “Materialsand Methods.―A, the number of tumor cells that invaded in the absence of antibody. B, tumor cell invasion in the presence of 100 @WmlMOPC-2l. C, invasionof tumor cells in the presence of pAb to allb@3 (75 @.sg/ml).D, invasion of tumor cells in the presence of AP-2 (100 @sg/ml).E, invasion of tumor cells in the presence of PAC-l (100@.tg/ml).F, graphical representation of tumor cell invasion. Control, the number of tumor cells that invaded in the absence of antibody. 1, 2, and 3, tumor cell invasion in the presence

of 50 @tg/ml,100 @tg/ml,and 75 @tg/miof each antibody, respectively. The results are indicated as the change in percentage invasion of tumor cells in the presence of various antibodieswhen compared to control. At least 50 cells were counted for each data point, and the results are the mean of quadruplicate determinations ±SD.

their surface and it seems to be associated with adhesion sites (datanot shown), suggesting that surface expression of this integrinmight be important for invasion. Recent evidence in the literaturereveals that aIIbj33 transfected into Chinese hamster ovary cellsparticipates in fibrillogenesis (52). Therefore, it is conceivable thatthe role of a11b133 in invasion could be to participate in theformation of a fibronectin matrix on Matrigel, or as mentionedabove, it might be using the proteins present in Matrigel as ligandsthat in turn could modulate invasion.

In conclusion, we have demonstrated that allb/33 is functionally expressed in human prostate carcinoma cells; thus, its expression in nonmegakaryocyticlineage tumor cells raises interestingquestions: why andhow is the allb gene transcribed in prostate tumor cells? Transcriptionalactivation of the allb gene in tumor cells could be due to several factorssuch as alterations in the promoter(s) region of the gene or quantitativeand/or qualitative changes in the expression of positive or negativetranscriptional regulators. Thus, it will be ofinterest to determine whetherthere are any changes in the regulation of tumor-type allb gene.

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EXPRESSION OF aIIb@3 IN HUMAN PROSTATE CARCINOMA

27. Tang, D. G., Onoda, J. M., Steinert, B. W., Grossi, I. M., Nelson, K. K., Umbarger,L., Diglio, C. A., Taylor, J. D., and Honn, K. V. Phenotypic properties of culturedtumor cells: integrin allb(33 expression, tumor-cell-induced platelet aggregation, andtumor-cell adhesion to endothelium as important parameters of experimental metastasis. lot. J. Cancer, 54: 338—347, 1993.

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ACKNOWLEDGMENTS

We thank David Kagawa and Homan Kian for technical assistance, Drs.Xiang Gao and Yong Chen for assisting with PCR, and Dr. Michael Hagen(Department of Molecular Medicine and Genetics, Wayne State University,Detroit, MI) for sequencing the PCR products. We are grateful to Dr. DeanTang for critical discussions throughout the period of this study.

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1996;56:5071-5078. Cancer Res   Mohit Trikha, Jozsef Timar, Steven K. Lundy, et al.   Integrin

3βIIbαHuman Prostate Carcinoma Cells Express Functional

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