[CANCER RESEARCH 54, 2514-2520, May 1, 1994J

ABSTRACT

Cell surface melanoma-associated antigens can mediate cell-cell orcell-substrate adhesion, signal transduction, proteolysis, or immune recognition and play a key role in determining invasive and metastaticcompetence of the tumor cells. The melanoma-associated antigen, A32,was defined by a murine monoclonal antibody and was immunoprecipitated as a single 113 kDa integral membrane glycoprotein containing sialicacid and HNK-1 carbohydrate moieties. Immunohistochemistry revealedthe presence of A32 antigen on most melanomas and nevi but not onnormal epidermal melanocytes. Of the normal tissues tested, only endothelium, smooth muscle, cerebellum, and hair follicles expressed the A32antigen. Tryptic peptides of the A32 antigen obtained after immunoafUnity chromatography showed sequence identity to MUC18 antigen, a member of the immunoglobulin supergene family. Melanoma cells adhered toaffinity-purified A32 antigen immobilized to a solid phase, and the adhesion was blocked by either soluble A32 antigen or monoclonal antibodyagainst the HNK-1 carbohydrate moiety. These findings, together with theobservation that A32 antigen is concentrated In cell-cell contact borden,suggest that this antigen is an adhesion molecule with a possible role intumor invasion and metastasis.

INTRODUCTION

Distinct biological features characterizing each stage of tumordevelopment and progression toward a more aggressive phenotypehave been defined in cultured human melanocytic cells (1). Theantigenic profile of melanocytic cells from different stages of tumorprogression has been studied using MAbs3 (2, 3). Melanoma-associated antigens most highly linked to tumor progression include adhesion receptors, which can be divided into cell-substrate and cell-cellinteractive molecules (4, 5). Of the cell-substrate adhesion receptors,the @33subunit of the vitronectin receptor has been demonstrated todistinguish between radial growth phase (early) and vertical growthphase tumorigenic (advanced) primary melanoma (6), and the expression of this subunit is associated with the metastatic potential of cells(7). Correlative studies have also demonstrated an increase in a2f31and a4f31 integrins with melanoma progression toward an increasinglyinvasive phenotype (8). Of the cell-cell adhesion receptors, ICAM-1,which is a member of the immunoglobulin supergene family, isassociated with melanoma invasiveness, and its expression is a negative prognostic indicator for survival of patients (9, 10). Expressionof ICAM-1 can increase adhesion of lymphocytes expressing theligand lymphocyte function-associated antigen-i (i 1), but the mechanisms of ICAM-1 interactions leading to increased metastatic abilityare not known. Besides ICAM-i, melanoma cells express three additional members of the immunoglobulin supergene family, i.e., vascu

Received 9/24/93; accepted 3/1/94.Thecostsof publicationof thisarticleweredefrayedin partby thepaymentof page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

I This investigation was supported by Grants CA-25874 and CA-10815 from the NIH.

2 To whom requests for reprints should be addressed, at The Wistar Institute of

Anatomy and Biology, 3601 Spruce Street, Philadelphia, PA 19104.3 The abbreviations used are: MAb, monoclonal antibody; F@, fetal calf serum; SDS,

sodium dodecyl sulfate; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; cDNA,complementary DNA; PBS, phosphate-buffered saline; PCR, polymerase chain reaction;PVDF, polyvinyl difluoride; BSA, bovine serum albumin; CAM, cell adhesion molecule;ICAM,intercellularadhesionmolecule;NCAM,neuralCAM;PECAM,platelet-endothelial CAM; PAP, peroxidase anti-peroxidase.

lar CAM (i2, 13), NCAM (14), and MUC18 antigen (15). Of these,only the MUC18 antigen is expressed on most melanoma lesions, andits expression correlates significantly with metastatic potential (16).However, the biological functions of MUC18, NCAM, or vascularCAM expressed by melanoma cells i.e., their potential involvement inadhesion, are not known.

In the present study, we describe the identification of a melanomaassociated antigen, A32, by amino acid sequence analysis of anaffinity-purified Mr 113,000 protein. We show that the A32 antigenhas sequence identity with MUC18 antigen and that the A32/MUC18antigen is a cell surface molecule involved in adhesion of melanomacells.

MATERIALS AND METHODS

Antibodies. Hybridoma cells producing MAb A32 (IgGi,K) were obtainedafter fusion of the murine myeloma cell line P3X63Ag8.653 with spleen cellsderived from BALB/c mice which were immunized by i.p. injection of 1.5 Xi0@human primaryWM9O2Bmelanoma cells and boosted with the sametreatment procedures 2 and 7 weeks later. Fusion of mouse spleen cells,selection, cloning, and growth of hybridoma cells were done following standard protocols (17). MAb A32 was selected by its immunoreactivity withcultured melanoma cells but not with melanocytes as detected by phasecontrast fluorescence microscopy. Secondary antibodies including isothiocyanate- and enzyme-labeled goat anti-mouse IgO were purchased from JacksonImmuno Research Laboratories (West Grove, PA). Hybridoma cells producingMAb HNK-1 which specifically reacted with HNK-i carbohydrate moiety (18)

were obtained from the American Type Culture Collection (Rockville, MD).MAbs MUC18, MUC18BA2, and MUC18BA4 defining the MUC18 antigenhave been described (19). MAb 4G6 against human PECAM-1 was kindlyprovided by Dr. S. Albelda, Philadelphia, PA.

Cell Culture and Cell Extracts. WM1366melanomacells froma primarycutaneous melanoma were cultured in W489 medium supplemented with 2%FCS and 5 1.Lg/mlinsulin (Sigma, St. Louis, MO). Subconfluent monolayerswere harvested from Coming roller bottles (Corning, NY), and cell extractswere prepared by lysing pellets of 3—6X i0@cells in 0.5 ml of lysis buffercontaining 20 mM Tris-HC1 (pH 83), 150 m@i NaG, 2 m@s phenylmethylsul

fonyl fluoride, 5 mMEDTA, 1% deoxycholic acid, 1% Triton X-i00, and 0.1%SDS. Cells were resuspended, and cell fragments were pelleted at 4°Cfor 50mm at 16,000 x g.

Isolation and culture conditions for melanocytes and nevus cells have beendescribed (20). Endothelial cells from human umbilical vein were grown inM199 medium (Sigma) supplemented with 10% FCS, heparin (50 @Wml),pituitary extract, and 2% glutamate. Human monocytic leukemia cells, U937,

were maintained in RPM! 1640 supplemented with 10% FCS. Human carcinoma cell lines (SW620, SW707, BXCP, and KATO-ifi) were maintained inW489 medium with 2% FCS (21).

Antigen Purification and Peptide Microsequencing. A32 antigen waspurified by affinity chromatography with affinity-purified MAb A32. Approximately 300 ml of cell lysate were cycled over a column containing 8 mlpacked volume of purified MAb A32 coupled to CNBr-activated Sepharose (5mg of antibody/ml CNBr Sepharose 4B; Pharmacia LKB, Uppsala, Sweden).The antigen was eluted with 100 m@itriethylamine (pH 11.5), and the peakprotein fraction was immediately neutralized, dialyzed against PBS at 4°C,andconcentrated using Centriprep concentrators (Amicon, Beverly, MA). Part of

the purified A32 antigen was analyzed by SDS-PAGE (22) followed byWestern blotting (23), and the remaining preparation was frozen at —70°C.

Affmity-purified A32 antigen (50—60 @g)was subjected to SDS-PAGEunder reducing conditions with 5—8 @gloaded protein per lane. The separatedbands were blotted onto PVDF membranes (Bio-Rad, Hercules, CA) and then

2514

Isolation and Functional Characterization of the A32 Melanoma-associated Antigen'

le-Ming Shih, David E. Elder, David Speicher, Judith P. Johnson, and Meenhard Herlyn2The Wistar Institute, PhiIadelphia@ Pennsylvania [f.M. S., D. S., M. H.]; The Department of Pathology at the University of Pennsy1vania@ PhiIade1phia@ Pennsylvania 19104fD. E. E.J; and The University ofMunich, Munich, Germany fJ. P. J.J

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A32/MUC18 ANTIGEN ON MELANOMA

stained with Amido Black for NH2-terminalsequencing and internal sequencing. Protein bands blotted to the PVDF membranes were excised and digestedin situ with trypsin. Separation and sequence analysis of peptides have beendescribed (24).

Transfections and Flow Cytometric Analyses. cDNA clone drop 4.7from human melanoma cell line Mel-JuSo encoding MUC18 was cloned intothe expression vector, pCB6+, carrying the cytomegalovirus promoter andneomycin resistance gene. U937 cells (2 X 10@)were washed with PBS andresuspended in 0.4 ml PBS. PCB6+/MUC18 vector (10 @g)was added to the

cell suspension which was transferred to a plastic electroporation cuvette(Bio-Rad, Richmond, CA). The cell suspension was subjected to electroporation (960 @LFDand 450 V) using a Gene-pulser electroporation unit (Bio-Rad).After pulsing, cells were transferred into Petri dishes containing neomycin

selection medium (RPM! with 10% FCS and 400 @.&g/mlG418 sulfate).Transfected cells were cloned by serial dilution, and each clone was evaluated

by flow cytometry. For this assay, cultured cells were stained for 40 mm withMAb A32 (10 @.tWml)and then incubated at 4°Cfor 40 mm with fluorescein

isothiocyanate-conjugatedgoat IgG anti-mouse IgO. Cells were analyzed usingan Ortho Cytofluorograf 50H connected to a 2150 Data Handling System(Ortho Diagnostics, Inc., Westwood, MA). As a negative control, mouse serumwas used instead of MAb A32.

Nucleotide Sequencing. Polyadenylated mRNA was isolated from 5 X i0@WM1366 melanoma cells using the FastTrack mRNA isolation system (In

vitrogen Corporation, San Diego, CA). cDNA was prepared from polyadenylated RNA according to the manufacturer's protocol using Moloney murineleukemia virus RNase H-RT (Gibco-BRL). Amplification by PCR was per

formed as described (20). Oligonucleotide primers were synthesized on aBiosearch 8700 (Milligen Biosearch, MA) DNA synthesizer. Primer sequenceswere sense 5'-GGGGCIACTCTGGCCCIXIACF and antisense 5'-GAGGAATGACAAGTGTCCCTA.PCRproductswerecloneddirectlyintotheTAvector pCRII (Invitrogen). Plasmid DNA was isolated using a kit purchasedfrom Qiagen (Tchapsworth, CA). DNA was sequenced with a Sequenase 2.0DNA sequencing kit (United States Biochemical, Cleveland, OH) using aqueous 5' [a-35SJdATP at 600 mCi/mmol (Amersham Corporation, ArlingtonHeights, IL).

Immunohistochemistry and Immunofluorescence. Normal human neonatal foreskin, normal adult skin from breast areas, and lesions obtained from

biopsies performed for diagnostic or therapeutic purposes including nevi andprimary and metastatic melanoma samples were from the Hospital of theUniversity of Pennsylvania (Philadelphia, PA). Normal tissues includingesophagus, trachea, thymus, lymph node, spleen, pancreas, liver, kidney,skeletal muscle, cerebrum, and cerebellum were obtained from autopsy casesand from the National Disease Research Interchange (Philadelphia, PA).Cryosections from these specimens were fixed with 2.7% paraformaldehyde in

PBS for 10 mm. After incubation with blocking reagent (PBS containing 3%BSA) for 1 h at room temperature,sections were treated with 50 ,.il ofaffinity-purified MAb A32 (20 @.tg/mlin PBS containing 1% BSA) for 1 h at

room temperature, stained by the PAP method with the HistoGen PAP immu

nostaining kit (Biogenex, San Ramon, CA), and counterstained with 0.1%hematoxylin (Sigma). Immunofluorescence staining has been described (24)

Immunoprecipitation and Lectin Analyses. Subconfluent WM1366 melanoma and SW707 rectal carcinoma cells were detached and labeled with 100,.Lg/mlNHS-Biotin (Pierce Chemicals) for 1 h at room temperature. Cells werelysed in lysis buffer, and the lysate was precleared with protein G-Sepharose(Pharmacia) at 4°Cfor 2 h and incubated with affmity-purified MAb A32 (3g.Lg/ml)or irrelevant mouse IgG-conjugated protein G-Sepharose at 4°Cfor 12h. Immunoprecipitates were washed 5 times with lysis buffer and boiled inLaemmli buffer for 10 mm. Following electrophoresis on a 10% SDS-polyacrylamide gel, samples were electrotransferred onto PVDF membranesblocked with 3% BSA and 0.5% Tween 20 in PBS and incubated with a 1:1000dilution of alkaline phosphatase-conjugated streptavidin for 1 h at roomtemperature. The labeled protein band was visualized by incubating membranes with 5-bromo,4-chloro,3-indolyl phosphate and nitroblue tetrazolium

(Sigma) as chromagen and substrate. For lectin analysis, affinity-purified A32antigen on PVDF membranes was probed with the following peroxidase- oralkaline phosphatase-labeled lectins: concanavalin A, Lens culinaris, Maackiaamurensis, Triticum vulgaris, Arrachis hypogaea, Phytolacca americana, Teragonolobus purpureas, and Wisteria floribunda. Maackia amurensis was pur

kD

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Fig. 1. Immunoprecipitation and immunoblot analysis of A32 antigen expressed onWM1366 melanoma cells. The 1 13-kDa A32 antigen was immunoprecipitated from

biotinylated membrane fractions of melanoma cells (Lane 2) but not from membranefractions of SW7O7 colon carcinoma cells (negative control; Lane 1). Lane 3, theaffinity-purified A32 antigen after SDS-PAGE (10%) and Coomassie blue staining. Thecorresponding gels were transferred to a PVDF membrane and probed with MAb A32under nonreducing (Lane 4) and reducing conditions (Lane 5). A32 antigen purified byaffinity chromatography from WM1366 melanoma cells was probed with MAb HNK-1(Lane 6). Western blot analysis of cell lysates from nontransfected (Lane 7) and transfected (lane 8) U937 cells probed with MAb A32 demonstrated a single 113-kDa proteinband.

2515

I 2@ 4 5678

chased from Boehringer-Mannheim Corporation (Indianapolis, IN); all otherswere from Sigma.

Adhesion Assay. Nitrocellulose-coated plastic Petri dishes were preparedby a modification of the method of Lemmon et al. (25). A 9-cm2 strip ofnitrocellulose (Schleicher & Schuell, Keene, NH) was dissolved in 15 ml

methanol, and 1 ml of the solution was spread over the surface of a 35-mmpolystyrene Petni dish (VWR, Philadelphia, PA) and allowed to dry in a

laminar flow hood. Five @lof purified A32 antigen (200 @Wml)and othercontrol samples including mouse serum or BSA (200 p@g/ml)were applied tothe nitrocellulose-coated dishes which were then incubated at 37°Cfor 1 h,followed by washes and blocking with W489 culture medium containing 20%FCS at 37°Cfor 1 h. Two-ml cell suspensions (1 X 106cells/ml) of melanomacells, carcinoma cells, and MUC18-transfected or nontransfected U937 cells inserum-free culture medium were added to the dishes and incubated for 20 mmat 37°C.After three washes with PBS, cells were fixed with 2.7% paraformaldehyde, and cell numbers were determined under a light microscope. Resultsare expressed as the total number of adherent cells from five randomly chosen

fields at X150.

RESULTS

Biochemical and Structural Characterization of the A32 Antigen. The A32 antigen was identified by immunoprecipitation of biotinylated membrane extracts from WM1366 melanoma cells. Asshown in Fig. 1, the apparent molecular mass of A32 antigen was 113kDa under nonreducing conditions (Fig. 1, Lane 2). After reductionwith 2-mercaptoethanol, the molecular mass shifted to 130 kDa (Fig.1, Lane 5), indicating disulfide bridge-stabilizing structures withinthis monomeric membrane-bound molecule. The A32 antigen purifiedfrom lysates of WM1366 melanoma cells on an affinity column ofMAb A32 coupled to CNBr-activated Sepharose 4B eluted as a singleprotein peak with 100 mi@striethylamine. SDS-PAGE analysis of thisfraction under nonreducing conditions revealed an intense proteinband migrating at 113 kDa on Coomassie blue staining (Fig. 1, Lane3) andWesternblotting (Fig. 1,Lane 4), respectively,when probedwith MAb A32.

MAb HNK-1 reacted with the affinity-purified A32 antigen, mdicating the presence of the HNK-1 carbohydrate moiety on the protein(Fig. 1, Lane 6). Glycosylation of the A32 antigen was further studied

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A32/MUC18 ANTIGEN ON MELANOMA

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1 : MGLPRLVCAFLLAACCCCPRVAGVPGEAEQPAPELVEVEV GSTALLKCGLVPGEAEQ PAPELVEVEV GSTA

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121: LGPRSTASSS AS L NI QVNPLGIPVN SKEPEEVATCE NI QVNPLGIPVN 5K

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301 : NGVLVLEPARKEHSGRYECQAWNLDTMISLLSEPQELLVNYVSDVRVSPAAPERQEGSSL

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GPV LQLHDLKRGGGYRCVASV PSIPGLNRTQ

42 1 : LVKLAIFGPP WMAFKERKVW VKENMVLNLS CEASGHPRPT ISWNVNGTAS EQDQDPQRVL

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Fig. 2. A, comparison of amino acid sequences between reported MUC18 and purified A32 antigen. Six fragments of trypsin-digested A32 peptides were selected and subjected tointernal and NH2-terminalsequencing, and the results are shown in the lower part of the sequence. The amino acid sequence of the MUCI8 protein was deduced from the nucleotidesequence of the MUC18 eDNA. Square, discrepancies of amino acid sequence. Underline, deduced transmembrane domain. B, the original published sequence starting at residue 115is compared to the corrected sequence and the peptide sequence. Dash, a missed codon in the previous report. The altered amino acid residues are underlined in the corrected sequence.

using a variety of enzyme-labeled lectin probes. A32 antigen contamed specific carbohydrate moieties including sialic acids, t-mannose, N-acetyl-2,3-D-galactose, and (N-acetylglucosamine)2 as mdicated by reactivity of A32 antigen reacted with concanavalin A, Lensculinaris, Maackia amurensis, and Triticum vulgaris (not shown). Asdetermined by neuraminidase treatment to remove sialic acid fromA32 antigen, approximately 20% of the oligosaccharide on the A32antigen is composed of terminal sialic acid residues.

@2t@'@@almicrosequencing analysis and tryptic mapping of theprotein were then performed. A homology search of protein databasesshowed that the A32 antigen was almost identical to the membraneanchored MUC18 antigen (15). As shown in Fig. 2A, sequences ofpeptides completely matched the deduced amino acid positions 24—44, 98—112,239—259,378—388,and 459—477of the MUC18 mole

cule with the exception of the peptide corresponding to amino acids134—152,which differed in the first four residues of the NH2-terminalA32 antigen and the deduced MUC18 amino acid sequence reportedpreviously (15). Nucleotide sequence analysis of this region by reverse transcriptase-polymerase chain reaction of WM1366 mRNAand direct sequencing of the MUC18 cDNA clone drop 4.7 showedthat they were identical. The sequence between nucleotides 360 and412 is 5'-CCG CGG TCC CAG GAG TAC CGC ATC CAG CrCCGC GTC TAC AAA GCF CCG GAG GAG. A comparison of thecorrected peptide sequence with the original published sequence isshown in Fig. 2B.

To further examine the relationship between the A32 and MUC18antigens, human U937 cells transfected with MUC18 cDNA wereexamined for immunoreactivity with MAb A32. Western blot analysis

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A32/MUCI8 ANTIGEN ON MELANOMA

tivities of MAb A32 and a MAb specific for human PECAM-1, whichrecognizes endothelial cells but not vascular smooth muscle (notshown). Except for blood vessels, no reactivity of MAb A32 wasdetected in normal lymph nodes, thymus, spleen, trachea, esophagus,colon, placenta, liver, kidney, pancreas, skeletal muscle, or peripheralblood lymphocytes and granulocytes (not shown).

Cellular Distribution of A32 Antigen. MAn A32 bound to all 27melanoma cell lines and 1 small cell lung carcinoma cell line (TKB-2)tested but not to 25 cell lines derived from colon carcinoma, pancreatic carcinoma, cervical carcinoma, or lymphocytic and monocyticleukemias (not shown). Antibody staining reactivity was diffuse onthe surface of singly growing melanoma cells but concentrated atcell-cell contact sites as compared with free borders in cells clusteredtogether in small colonies (Fig. 6, A and B). Cultured endothelial cellsshowed a similar immunostaining pattern (Fig. 6C).

Functional Characterization of A32 Antigen. Cell adhesion tosolid phase-bound A32 molecules was evaluated using nitrocellulosecoated Petri dishes (Fig. 7A). After 20 mm of incubation at 37°C,cellsfrom three different melanoma cell lines (WM1366, WM9O2B, andWM39P) adhered to the nitrocellulose area coated with A32 antigenbut not to the control area coated with irrelevant protein. In contrast,four different carcinoma cell lines (SW620, SW707, BXCP, andKATO-III) did not adhere under the same experimental conditions.U937 cells, either transfected or nontransfected with MUC18 cDNA,did not bind to A32 antigen (Fig. 7B). The adhesion of WM1366melanoma cells to immobilized A32 antigen was blocked in cellspreincubated with soluble A32 antigen (20 @Wml)for 20 mm at 37°C(Fig. 8). However, MAb A32 did not block the adhesion even atconcentrations as high as 300 p@g/ml.Since A32 antigen expresses theHNK-1 oligosaccharide, which mediates homophilic binding of neuraladhesion molecule P0 (26), we studied the contribution of this glycanto the adhesion mechanism of the A32 antigen. As shown in Fig. 8,preincubation of WM1366 melanoma cells with MAb HNK-1 (10

@g/ml)for 20 mm decreased cell adhesion to A32 antigen by 60%.The adhesion of melanoma cells to A32 antigen was temperaturesensitive since lower incubation temperature during the assay greatlydecreased cell binding activity to 10 and 38% at 4 and 24°C,respectively. Cell aggregation was not observed in MUC18-transfectedU937 cells or in the cell mixture of transfectant and nontransfectantcells (data not shown).

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I 2 3 4 5 6 7 8 9Fig. 4. Expression of A32 antigen in melanocytic cell lines as determined by Western

blot. Membrane extracts of equal cell numbers from the following melanocytic cultureswere subjected to SDS-PAGE (10%) followed by immunoblotting with MAb A32. LanesI and 2, two different normal melanocyte primary cultures; Lanes 3 and 4, two benignnevus cell cultures; Lanes 5 and 6, two primary melanoma cell lines (WM9O2B andWM983A, respectively); Lanes 7 and 8, two metastatic melanoma cell lines (WM164 andWM266—4, respectively). Lane 9, A32 antigen-negative SW707 carcinoma cell line as thenegative control.

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Fig. 3. In situ expression of A32 antigen in different stages of tumor progression in thehumanmelanocyticsystem.Frozensectionsfrom surgicalspecimenswere fixedandincubated with MAb A32 (10 @Wml)and visualized by the PAP method. Each dot in thefigure represents a different sample, and the percentage of positive cells within eachsampleis shownon they-axis.Bloodvesselswithineachsectionwereusedas internalpositive controls.

of cell lysates from transfected U937 cells probed with MAb A32demonstrated a single 113-kDa protein band (Fig. 1, Lane 8), andflow cytometry showed that only cells transfected with MUC18cDNA were positive. Western blot analysis of purified A32 antigenprobed with three MAbs (MUC18, BA4, and BA2) against differentepitopes on the MUC18 antigen revealed the same reactivity patternas MAb A32. These results confirmed that the A32 antigen isolated byus was identical to the MUC18 antigen.

In Situ and in Vitro Expression of A32 Antigen on HumanMelanocytes at Different Stages of Tumor Progression. Fig. 3shows the reactivity of MAb A32 to normal skin and melanocyticlesions representing various stages of tumor progression. No reactivitywas detected in melanocytes either from newborn foreskin, normaladult skin, or skin adjacent to melanocytic lesions. However, themajority of dermal nevus cells and primary and metastatic melanomacells (including amelanotic melanoma cells) expressed A32 antigen.Expression in each sample of melanocytic lesion was heterogeneous,and A32 antigen expression levels in nevus cells were lower than inmelanoma cells. Acquired nevi expressed A32 antigen at higher levelsthan did congenital nevi, and the expression pattern in nevi wasindependent of the maturation and differentiation stages of nevi.

Western blot analysis under nonreducing conditions of eight melanocytic cell lines of equal cell numbers representing different stagesof tumor progression (two normal melanocytes, two nevi, two primary, and two metastatic melanomas) and probed with 10 @g/mlof

MAb A32 revealed a single 113-kDa band in all cases, with increasingexpression of A32 antigen from normal melanocytes to malignantmelanoma cells (Fig. 4). Phorbol ester (O-i2-tetradecanoyl phorbol13-acetate) in melanocyte growth medium did not affect A32 antigenexpression (data not shown).

In addition to its expression on melanoma cells, hair follicles, andsmooth muscle of blood vessels, A32 antigen was detected in humannevus cells, endothelium of blood vessels, arrector pili muscles, andcerebellar cortex (Fig. 5). A32 antigen expression in endothelial cellsin situ was further confirmed by the colocalization of immunoreac

2517

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A32/MUC18 ANTIGEN ON MELANOMA

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Fig. 5. In situ expression of A32 antigen on human acquirednevi and normal tissues as detected by MAb A32 using PAPimmunohistochemistry. A, mature dermal nevus; B, dermalnevus with neural differentiation; C, endothelium of bloodvessel (from chorionic villus); D and E, gross view of cerebellum (D, negative control; E, stained with MAb A32); F and C,microscopic view of cerebellum (F, negative control; C, stainedwith MAb A32). Molecular and follicular layers of cerebellumare shown.

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procedures (by heating the slides in a microwave oven for 5 mmfollowed by proteolytic treatment) suggest that MAb A32 may beuseful in the differential diagnosis of amelanotic melanomas. Withthis MAb, we also found that benign nevi express MUC18 antigen.This is in contrast to the previously reported observations with MAbMUC18 which reacted only with melanomas. These differences mayreflect different affinities ofthe antibodies since the immunoreactivityof MAb MUC18 on melanocytic tumors is much lower than that ofMAb A32 under the same experimental conditions. However, nevuscells generally express lower amounts of MUC18 antigen than melanoma cells as determined by in situ immunostaining and immunoblotting analyses. The induction of MUC18 antigen expression onnormal melanocytes grown in vitro but not in situ may reflect the factthat MUC18 antigen expression is associated with a proliferating stateof cultured melanocytes or may be due to the absence of keratinocytes, which can suppress expression of melanoma-associated antigens on normal melanocytes (26).

In addition to MAb A32 reactivity with vascular smooth musclesand hair follicles (19), we found that endothelial cells of blood vesselsfrom various tissue origins (except umbilical vein) and cerebellarcortex were MAb A32-positive. Endothelial cells also express threeother immunoglobulin superfamily adhesion molecules, ICAM-i, PECAM-i, and vascular cell adhesion molecule-i, which are thought to

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DISCUSSION

In this report, we describe the identification and characterization ofa surface antigen expressed by most malignant melanomas but not bynormal melanocytes. The A32 antigen is immunoprecipitated as asingle 113 kDa glycoprotein and is identical to the MUC18 antigenbased on analysis of NH2-terminal and tryptic peptide microsequencesand on MAb A32 immunoreactivity of human U937 cells transfectedwith MUC18 cDNA. Evidence presented here demonstrates that theA32 antigen is an adhesion molecule.

MUC18, like ICAM-1, NCAM, Li, fascilin II, and carcinoembryonic antigen, belongs to the immunoglobulin supergene family characterized by varying numbers of immunoglobulin-like domains intheir extracellular domains (15). In this family, MUC18 and ICAM-1are the most specific melanoma-associated antigens, and their expression in melanoma correlates with poor prognosis. Using MAb A32 asthe immunoprobe, we have confirmed that most human melanomasexpress A32 antigen abundantly, whereas normal epidermal melanocytes are negative for this antigen. The rate of detection of metastaticmelanoma by MAb A32 is above 95%, which is higher than for otherknown MAbs against MUC18 antigen. MUC18 antigen expression asdetected by MAb A32 is relatively specific for melanoma becauseother human tumors tested do not appear to express the A32 antigen.The high sensitivity and specificity of MAb A32 and its detectabilityin formalin-fixed and paraffin-embedded tissue after antigen retrieval

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A32/MUCS8 ANTIGEN ON MELANOMA

U937 and L cells. MAb A32 did not block adhesion, suggesting that

the epitope recognized by MAb A32 is distant from the active siteinvolved in adhesion. However, cell adhesion to A32IMUC18 antigenwas blocked by MAb HNK-i, suggesting that the HNK-1 carbohydrate epitope is important in the adhesion mechanism.

In addition to its role in cell-cell adhesion, A32/MUC18 antigenmight be involved in the interactions between cells and extracellularmatrix since the second immunoglobulin loop of A32/MUC18 antigencontains an amino acid stretch (LKEEKN) that is highly similar to theglycosaminoglycan recognition sequence (LKREKN) located at thesame site in NCAM and PECAM-i (35). If the LKEEKN sequence inA32/MUC18 is functional in binding glycosaminoglycan, which is

abundantly secreted by melanocytic cells, it is possible that a proteoglycan might modify the cell-cell adhesion mediated by A32IMUC18antigen. The temperature dependence of adhesion is reminiscent ofthat exhibited by cadherins and integrins (36) but different from thetemperature-independent nature of adhesion mediated by selectins(37).

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Fig. 6. Immunolocalization ofA32 antigen in CUltUredmelanoma and endothelial cells.WM1366 melanoma cells and endothelial cells (from umbilical vein endothelium) growing on coverslips were incubated with MAb A32 (20 @sg/ml)followed by fluoresceinconjugated goat IgO anti-mouse secondary antibody. Staining was diffusely distributed onthe cell membrane in individual melanoma cells (A) but preferentially concentrated tointercellular borders on both melanoma cells (B) and endothelial cells (C) when cellsclustered into small colonies and cell-cell contact was achieved.

integrity (27, 28). In the nervous system, several members of theimmunoglobulin superfamily are expressed: NCAM (14), neuron-glialcell adhesion molecule (29), Thy-i (30), Li (31), MAG (32), SC-i(33), and P0 (34). These molecules are suggested to be involved incell-cell recognition, adhesion, and axonal growth. The close structural similarity between A32IMUC18 antigen and other immunoglobulin superfamily members points to a similar function. Indeed, wefound that A32/MUC18 antigen is involved in cell adhesion. Melanoma cells adhered to purified A32/MUC18 antigen immobilized tosolid phase in an interaction which was competitively blocked by A32protein. This finding, together with the observation that A32IMUCi8antigen preferentially localizes to the cell-cell contact sites in culturedendothelial and melanoma cells, suggests that A32/MUC18 antigen isan adhesion molecule. The inability of MUC18 cDNA-transfectedU937 cells to aggregate and adhere to immobilized A32/MUC18protein suggests that A32/MUC18 antigen binds to an as yet unidentified molecule on the surface of melanoma cells through heterophilicbinding. However, the possibility of homophilic adhesion for A32/MUC18 antigen cannot be ruled out entirely, since A32IMUC18-mediated homophilic binding in U937 cells may be too weak to bedemonstrated experimentally in our aggregation and solid phase adhesion assays and the active site(s) for homophiic binding on A32/MUC18 molecules might not be properly expressed by transfected

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Fig. 7. A, attachment of melanoma cell to affinity-purified A32/MUC18 antigen.Chromatographically purified A32/MUC18 antigen was immobilized on a nitrocellulosecoated polystyrene dish. The dish was blocked with 20% FCS, and WM1366 melanomacells were allowed to attach in serum-free medium at 37°Cfor 20 mm; they were thenwashed and observed under phase microscopy. The area coated with A32/MUC18 isdemarcatedly outlined by attached cells. B, cell adhesion assay for different cell lines toimmobilized A32/MUC18 antigen. Melanoma cells from three different cell lines(WM1366, WM9O2B, and WM39P) adhered to A32 antigen, whereas cells from fourdifferent carcinoma cell lines (KATO-Ill, BXCP, SW620, and SW707) and U937 humanleukemia cells, either nontransfected or transfected (U937T) with MUC18 cDNA, did notsignificantly bind to A32 antigen. For negative control, WM1366 melanoma cells wereallowed to adhere to a BSA (200 @WmI)-coateddish (control). Data are the average oftriplicate determinations.

2519

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A32/MUCI8 ANTIGEN ON MELANOMA

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Fig. 8. Effects of treatments on cell adhesion of WM1366 melanoma cells to immobiized A32 antigen. Adhesion was greatly reduced in WM1366 melanoma cells preincubated with MAb to HNK-1 or soluble A32/MUC18 protein to solid-phase bound A32antigen. MAb A32 (300 @tg/ml)did not block cell adhesion in WM1366 melanoma cells.

The results presented here demonstrate that the A32/MUCi8 antigen is an adhesion molecule and a tumor marker for human melanoma, although it is also expressed in some normal tissues such asendothelial cells, smooth muscle cells, and cerebellum. This studysuggests that melanoma cells and normal cells use the same moleculeand strategy for adhesion. Possibly, the A32/MUC18 antigen contributes to metastasis by increasing adhesion between melanoma cells andendothelium/vascular smooth muscle. Detailed functional studies ofthe A32/MUC18 antigen in animal models will clarify the active roleof this antigen in tumor invasion and progression.

ACKNOWLEDGMENTS

We acknowledge the expert technical assistance of Michelle Myrga andPatricia Van Belle.

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1994;54:2514-2520. Cancer Res   Ie-Ming Shih, David E. Elder, David Speicher, et al.   Melanoma-associated AntigenIsolation and Functional Characterization of the A32

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