Identification and Characterization of a Mr 50,000 Adrenal ...Identification and Characterization of a Mr 50,000 Adrenal Protein in Human Hepatocellular Carcinoma1 Mehmet Ozturk,2

(CANCER RESEARCH 49. 6764-677.1. December I. 1989]

Identification and Characterization of a Mr 50,000 Adrenal Protein in HumanHepatocellular Carcinoma1

Mehmet Ozturk,2 Philippe Motte,3 Hiroshi Takahashi, Mark FrÃ¶hlich,Byron Wilson, Linda Hill, Brigitte Bressac,and Jack R. Wands4

Molecular l/epatolony I.ahoratory and Cancer Center, Massachusetts General Hospital and Department of Medicine. Harvard Medical School f.\t. O.. P. M., H. T.,L. H., K. B..J. R. H'./; and Ilanurd-MIT Division of Health Science Â¡M.F., B. HJ. Boston. Massachusetts 02114

ABSTRACT

A M, 50,000 cell surface protein antigen (p50) was identified on ahuman hepatocellular carcinoma derived cell line (FOCUS) by twomonoclonal antibodies (SI- 31 and SI- 90). This antigen was subsequently

shown to be expressed in vivo in human hepatocellular carcinoma. All18 tumors tested by Western immunoblotting demonstrated high levelsof p50 with undetectable amounts observed in the adjacent normal livercounterparts. Further characterization revealed that p50 is a monomericpoly peptide with a neutral pi (6.5-7.2) and appears not to be glycosylated.

The cellular localization was determined by direct antibody binding tointact cells, immunoprecipitation of '"I-labeled cell surface proteins, and

Western immunoblotting of subcellular fractions. p50 was found on thecell surface as well as in the cytoplasm. In vitro monoclonal antibodybinding studies indicate that the protein is expressed in all humanmalignant cells (n = 34) tested thus far regardless of the embryonictissue of origin and the degree of differentiation. p50 was present at verylow levels in normal tissues with the notable exception of high expressionin adrenal glands. The protein is conserved in mammalian evolution sincea similar protein was also found in bovine adrenals. The molecularcharacteristics and the pattern of expression of p50 indicate that thisnormal adrenal protein is associated with the transformed phenotype.

INTRODUCTIONHCC' is one of the most frequent tumors in the world today

and is responsible for approximately 1,000,000 deaths annually(1). It is known that substantial changes have occurred in thecellular and biochemical composition of the liver during hepa-tocarcinogenesis (2). However, very little information is available regarding the factors that distinguish the malignant fromnormal phenotype. In general, the malignant phenotype ischaracterized by an augmented rate of cell proliferation andcapability of invasion (3). The cell surface may play a centralrole in the uncontrolled behavior of malignant cells. For example, it has been suggested that the plasma membrane maydetermine several essential properties of the malignant phenotype such as increased growth rate, prolonged survival, decreased adhesion, loss of contact inhibition, increased invasive-ness and motility, expression of repressed antigens, and escapefrom immune surveillance (4). It is noteworthy that severaloncogenes code for plasma membrane-associated proteins andsome have recently been shown to be cell surface receptors forgrowth factors (5).

Received 4/13/89; revised 7/13/89; accepted 8/24/89.The costs of publication of this article were defrayed in pan hy (he payment

of page charges. This article musi therefore be hereby marked advertisemeni inaccordance wi(h 18 U.S.C'. Section 1734 solely to indicate this fact.

1This work was supported in part hy (Â¡rantsCA-35711, AA-02666. and III)-20469 from Nili and a urani from Association pour la Recherche sur le Cancer.

2To whom requests for reprints should he addressed, at Molecular HepalologyLaboratory and Cancer Center. Massachusetts General Hospital. 149 13th Street.7tli Floor. Charlestown. MA 02129.

' Recipient of a fellowship from Association pour la Recherche sur le Cancer,Villejuif (France) and from the NCI-INSERM Bilateral Program.

4 Recipient of a Research Career Scientist Development Award AA-OO048.'The abbreviations used are: I ICC. hepatocellular carcinoma: KMKM, Karle's

modified Faglc's medium; PHS. phosphate buffered saline; C'S. calf scrum; SAM,'"l-laheled sheep F(ab')j anti-mouse immunoglobulins: SDS-PAGE, sodiumdodecyl sulfate-polyacrylamide gel electrophorcsis; TBS. Iris-buffered saline.

Identification of tumor specific cell surface molecules byantibodies raised against tumor cells has been an attractive wayto study the properties of the transformed phenotype (3, 6, 7).Despite the large number of antibodies described for specificrecognition of certain tumor cells, information about the characteristics of these antigens has been limited. However, severalinvestigators have described monoclonal antibodies that identify cell-surface protein antigens directly implicated in the transformed phenotype (8-10) or associated with an activated cellular oncogene (11). Investigation of cell surface moleculesinvolved in malignant hepatocytes has only recently begun andvery few antigens have been characterized in detail (12-18).Two protein antigens (Mr 90,000 and 115,000, respectively)have been identified on several hepatoma cell lines and HCCtumors (12, 13). The M, 115,000 antigen was not detectable innormal liver. However, there is only limited published information on such antigens with respect to their molecular characteristics and normal tissue distribution. We have previouslydescribed several hcpatoma-associated antigens (18, 19). In aneffort to better understand some of the cellular changes associated with transformation of hepatocytes to the malignantphenotype, we have produced several libraries of monoclonalantibodies against a HCC cell line (FOCUS) (20). In this reportwe describe a M, 50,000 transformation-associated cell surfaceprotein (p50) present at high levels on hepatoma cells. Thisprotein is also abundant in adrenal cells.

MATERIALS AND METHODS

Tissue Specimens. Specimens of HCC with adjacent nontumorousliver were obtained at autopsy or from surgical specimens (kindly-provided by Dr. Michael Kew, University of Witwatcrsrand, Johannas-bourg. South Africa). Normal adult and fetal human tissues wereobtained at autopsy. Tissues from bovines were freshly collected andall tissues stored at -70Â°Cbefore analysis.

Cells. Peripheral blood lymphocytes were obtained from normallaboratory personnel and isolated from whole blood by ficoll-hypaqucgradient centrifugaron. The human hepatocellular carcinoma cell line(FOCUS) was established in our laboratory (20). Most of the cell lineslisted in Table 2 were obtained from the American Type CultureCollection, Rockville, MD, with the following exceptions. PC' 12 cells

were kindly supplied by Dr. Lee Kaplun. Drug-sensitive cpidermoidcell line KB-3-1 and its drug-resistant derivative KB-VI were kindlysupplied by Dr. Ira Pastan from Nili. All cell lines except KB-V1 weremaintained in EMEM (MA Bioproducts, Walkersville, MD) supplemented with 10% fetal calf serum inactivated at 56Â°C,10 ^M noncssen-

tial amino acids. 1,000 iÂ¿/m\penicillin, and 100 Â¿jg/mlstreptomycin.KB-VI cell line was grown in the above media in the presence of 1 ^8/ml vinblastinc (Sigma, St. Louis, MO).

Monoclonal Antibodies. An early passage of the F'OCUS cell line

(20) was used to immuni/.e BALB/c mice. Mice first received an i.p.injection (200 Â¿il)of 4 x IO6 viable cells in 50% Freund's complete

adjuvant. Secondary immuni/ations were performed 6-10 weeks laterby i.v. inoculation of the same number of living cells suspended in abuffer consisting of 0.01 M sodium phosphate (pll 7.2) and 0.14 Msodium chloride (PBS). Three days later splcnocytcs obtained fromimmunized mice were fused with the parent myeloma cell line SP2O.

6764

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ADRENAL PROTEIN IN TUMOR CELLS

Selection of hybrids in hypoxanthine-, aminopterin-, and thymidine-containing medium and cloning by limiting dilution were performed asdescribed (21). The mouse hybridoma cell line (Pab 122-clone 122)producing a monoclonal antibody reactive with p53 antigen was obtained from the American Type Culture Collection (22). Ascitcs fluidwas prepared by injection of hybridoma cells i.p. into 2,6,10,14-tetra-

methylpentadecane (Pristane, Aldrich) primed BALB/c mice. Purification and iodination of monoclonal antibodies were performed aspreviously described (21).

Screening of Hybridomas. Hybridomas were initially screened foranti-FOCUS antibodies with an indirect binding assay using SAM(specific activity, 8-12 ^Ci/ng proteins. New England Nuclear, Boston.MA). In brief, binding assays were carried out in 96-well filter-bottomedplates (V&P Scientific Inc., San Diego, CA). After blocking of nonspecific protein binding sites on the plate with CS. IO5target cells in 100

Â¡AEMEM containing 20% CS were incubated with 100 ^1 hybridomaculture supernatant for l h at 20Â°Cwith gentle agitation. Cells were

drawn onto filters in the wells by suction and washed three times withPBS containing 20% CS (PBS-CS). Subsequently, 10' of SAM dilutedin PBS-CS was added and allowed to incubate for I h. The cells werewashed three times and dried filters were counted by a gamma wellcounter.

Cell Binding Assay. In order to measure the binding of '"I-labeled

monoclonal antibodies to the intact cells, FOCUS cells were grownovernight in 12-well plates as monolayers. Before analysis cells werekept on ice for 15 min and the medium was discarded. After additionof fresh culture medium (1 ml/well) containing '"I-labeled monoclonalantibodies (5 x IO5cpm/ml). cells were incubated for an additional Ih at 4Â°C.Unbound material was discarded by gentle washing of intact

cells with fresh media and cell-bound radioactivity was counted aftersuspension of cells in EDTA/versene buffer (18).

Solid Phase Radioimmunoassay. This assay was carried out in 96-well filter-bottomed plates. Total homogenates were prepared fromcells or tissue samples in 5 volumes of PBS containing 0.1% sodiumazide using a polytron. Homogenates containing increasing concentration of proteins (50 ^1) were applied directly to the filter papers. Afterabsorption of proteins onto filters by an incubation of l h at roomtemperature, excess of binding sites were saturated by incubation withCS (100 Ml).One hundred Mlof 125I-labeledantibody solution (~ 100,000

cpm in 20% CS and 1% nonspecific mouse IgG in PBS) was added andsamples incubated for l h at room temperature. Filters were thenwashed three times with PBS-CS and counted by a gamma well counter.

Cell-Surface Labeling. Proteins on the surface of FOCUS cells wereiodinated with '"I by the lactoperoxidase method as described (23). Inbrief, 2-5 x 10" cells were suspended in 0.5 ml PBS at 20Â°C.One mCiof Na '"I (Amersham), and 40 mg lactoperoxidase (Sigma) were addedand followed by four successive I5-/Â¿1pulses of 0.04% H2O2 at 5-minintervals. To stop the reaction, 0.02 M potassium iodine was added andcells were washed three times in PBS.

Metabolic Labeling of Cells. Subconfluent FOCUS cells were grownovernight under normal culture conditions in methionine-frec mediumcontaining 10% dialyzed fetal calf serum. Cells were then labeled for 1h in the same medium with 20 ÃŸC'\/m\["Sjmethionine (Amersham

specific activity, >1000 Ci/mmol) and chased by incubating in theculture medium with 1 mM L-methionine (Sigma) for 15, 30, and 60min. Cells were then harvested and washed three times in PBS. For['Hjglucosamine labeling studies, subconfluent cells were grown in theculture medium containing 500 Â¿iCi/mlof ['Hjglucosamine (Amer

sham, specific activity, 30 Ci/mmol) for 18 h and harvested and washedas described above.

Immunoprecipitation of Proteins. Cell pellets obtained after cell surface or metabolic labeling were extracted in 0.1 M phosphate buffer(pH 7.2) containing 150 mM NaCl, 10 mM EDTA, 20 mM EGTA, 10mM NaF, 0.1% deoxycholate. and 1% Triton X-IOO. For immunopre-cipitation studies SF 90 was covalently bound to protein A-Sepharose.The SF 90 ascitic fluid was dialyzed against 0. l M sodium borate buffer(pH 8.2) and one volume of the dialyzed fluid was incubated with onevolume of protein A-Sepharose beads (Pharmacia) overnight. The beadswere then washed with 0.2 M triethanolaminc buffer (pH 8.2) andincubated in the same buffer containing 30 mM diethylpimilimidate

(Sigma) for 45 min. The beads were finally washed in 30 mM ethanol-amine in 0.2 M tricthanolamine (pH 9.2) and stored in PBS. All theexperimental steps were carried out at 4Â°Cunless noted otherwise. Cell

lysates were first incubated with formalin-fixed Staphylococcus aiireus

cells (Boehringer) for l h and then with a nonspecific monoclonalantibody linked-beads for 2 h. The specific immunoprecipitations werecarried out overnight using the SF 90 monoclonal antibody linkedbeads. After extensive washing, beads were resuspended directly inSDS-PAGE sample buffer (24), heated at 95Â°Cfor 5 min and subjected

to SDS-PAGE using 10% polyacrylamide slab gels. After electropho-

retic protein separation, gels were processed for fluorography usingAmplify solution (Amersham) according to manufacturer instructions.Autoradiographs were carried out at â€”80Â°Cusing X-ray films (Kodak

X-Omat AR, Eastman Kodak Co., Rochester. NY).Western Immunoblotting. Detergent solubilized cell and tissue ex

tracts were used for these studies. Detergent-solubilized cell lysateswere prepared from confluent cells harvested from culture flasks usingEDTA/versene buffer, washed twice in PBS. and suspended in detergentbuffer (100 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.1% aprotinin, 1mM phenylmethylsulfonyl fluoride, and 0.5% NP-40). After 15-minincubation on ice with occasional vortexing. lysates were centrifugedfor 15 min at 10,000 rpm at 4Â°C.Supernatants were harvested and

adjusted to a protein concentration of I mg/ml and stored frozen at-80Â°C until use. Detergent solubilized tissue extracts were prepared

similarly by homogenizing tissue samples in 10 volumes of buffer usinga polytron homogenizer with detergent being added after homogeniza-tion.

For western immunoblotting studies cell and tissue extracts containing the same amount of protein (50-100 ng) were diluted in SDS-sample buffer with or without 2-mercaptoethanol (5% v/v final concentration). Samples containing 2-mercaptoethanol were heated at 95Â°C

for 3 min. For two-dimensional gel electrophoresis. samples (0.5-3 mgprotein/ml) were diluted with an equal volume of a mixture composedof 9 M urea, 4% NP-40, 5% glycerol, 10 mM dithiothreitol. and 2%ampholytes (ampholines, pH 3.4-10, LKB, Orsay, France) and incubated for 2 h at 25Â°C.Next, sample (15 p\) was applied to gels

prefocused by application of 200 V for 0.5 h. Isoelectric focusing wasperformed for 14,000 V x h on 1-mm diameter cylindrical gels containing 4% acrylamide, 2% NP-40. 9 M urea, and 2% of a mixture ofampholytes pH 3.5-10 (LKB), pH 3-10 (Serva, France), and pH 3-10(Pharmacia) at a ratio of 2:2:1, respectively. Focused gels, equilibratedin the migration buffer for 5 min at 25Â°Cwere laid on top of the I-

mm-thick second-dimension-resolving gel containing 13.5% acrylamide. Separation was performed according to Laemmli (24) at 25Â°C.

After electrophoresis in SDS-PAGE, the separated proteins were transferred electrophotoretically to nitrocellulose sheets (BA-85; 0.45-^mpore size: Schleicher & Schuell, Keene, NH) using a transblot cell(Biorad) at 50 V over 16 h. Prestained molecular weight markers(Biorad) were used to follow electrophoretic separation and transfer ofproteins to nitrocellulose filters. To block nonspecific binding sites, thesheet containing the transferred proteins was incubated with blockingbuffer [0.02 M Tris/HCl, and 0.5 M NaCl, pH 8.0 (TBS) containing 1%nonfat milk] at room temperature for 1 h. The nitrocellulose sheet wasthen incubated with the monoclonal antibody solution.

For indirect western immunoblotting investigations, SF 90 ascitesfluid diluted in blocking buffer (1:500) was incubated with the nitrocellulose paper for 2 h. The nitrocellulose paper was then washed threetimes in washing buffer (TBS containing 0.05% Tween 20) for 15 min.Nitrocellulose filters were incubated for 2 h with SAM (1 x IO6cpm/

ml) diluted in the probe buffer (blocking buffer added with 0.05%Tween 20). After a final washing step under the same conditions,nitrocellulose filters were dried and exposed for autoradiography.

For direct western immunoblots, nitrocellulose filters after priorblocking of excess protein binding sites, were incubated directly for 2h with 1 x IO6cpm/ml of '25I-labeled monoclonal antibody. The blots

were washed, dried, and exposed as described above.Cell Fractionation. The fractionation of FOCUS cells into the Triton

X-100 soluble, cytoskeletal and nuclear fraction was performed according to the protocol described by Rotter et al. (25). Briefly. FOCUS cellsgrown overnight in 100-mm culture dishes were washed with cold PBS

6765



ADRENAL PROTEIN IN Tl'MOR CELLS

and once with extraction buffer (50 m\i NaCI, 10 niM 4-(2-hydroxy-ethyl)-l-piperazineethanesulfonic acid. pH 7.4, 2.5 niM MgCl2, 300mM sucrose. 1 HIMphenylmethylsulfonyl fluoride. 10Â¿<g/mlof aproti-nin, pepstatin A, and leupcptin). Extraction was achieved by adding theabove buffer with \''< Triton X-100 to the washed cells, for 5 min on

ice. The Triton X-100 soluble fraction was removed. The cytoskeletonswere solubili/ed by scraping them into the extraction buffer containing0.5% deoxycholate and lrÂ¿Tween 40. followed by homogenization bypipetting. The nuclei were pelleted at 2000 rpm for 2 min at 4Â°Cand

the solubilized cytoskeletons removed. The nuclear pellet was dissolvedin 10 mM NaH2PO4, 100 HIM NaCI, 1% Triton X-100, 5% sodiumdeoxycholate, 0.1 TÃSDS containing the same concentration of proteaseinhibitors described above. Proteins solubilized in each fraction wereanalyzed by Western immunoblotting using I25I-SF 90 as the tracer

antibody.Immunohistochemistry. Both immunofluorescence and Â¡mmunoper-

oxidase assays were used for the cellular and tissue localization of p50.Indirect immunofluorescence studies were performed as described previously (14). Cryostat sections (4 ^m) of snap-frozen or paraffin-embedded tissue specimens were used for indirect immunoperoxidasestaining with monoclonal antibodies. Snap-frozen tissues were embedded in OTC compound (Miles Scientific, Naperville, IL), sectioned,and dried onto glass slides. For paraffin-embedded preparations, tissuesections were first fixed in PBS containing 2% paraformaldehyde(Sigma) for 2 h and directly processed for paraffin-embedding. Slideswere deparaffinized with xylene and rehydrated by passage throughgraded alcohols to a final wash in PBS. Indirect immunoperoxidasestudies were carried out using monoclonal antibodies and the VectastainABC kit to mouse immunoglobulins (Vector Laboratories, Burlingame,CA). Diluted normal horse serum was added to the slides and incubatedat room temperature for 30 min. The horse serum was removed byblotting and ascitic fluids diluted in PBS (1:100 to 1:1000) were addedand the incubation was carried out for 16 h at 4Â°C.Slides were washed

in PBS for 10 min and biotinylated horse anti-mouse IgG was addedfor 60 min at room temperature. After washing, the sections wereincubated in 0.3% H2O2 followed by methanol for 20 min in order toblock endogenous peroxidases; the slides were subsequently washed for20 min. Avidin/biotin complex was added, incubated for 60 min andthe slides washed as described before. The slides were then incubatedwith 0.5 mg/ml 3,3'-diaminobenzidine in 0.05 M tris/phosphate buffer

(pH 7.5) containing 0.0059o hydrogen peroxide for 15 min. The reactionwas stopped by washing of slides with distilled water and tissues werecounterstained with methyl green or hematoxylin, rehydrated, andmounted. A nonrelevant murine monoclonal antibody to tetanus toxoid(B2TT) was used as a negative control.

RESULTS

Monoclonal Antibody Production. In order to produce monoclonal antibodies to hepatoma cell-surface-associated antigenicdeterminants we used a strategy based on; (a) immunization ofmice with single cell suspensions of viable FOCUS cells and(b) screening of hybridomas by a radioimmunoassay using livecells as the antigen source. From the initial fusions 140 monoclonal antibodies were selected on the basis of their high bindingactivity to immunizing FOCUS cells. Hybridomas producingmonoclonal antibodies SF 31 and SF 90 described here wereobtained from a cell fusion experiment between hyperimmu-nized BALB/c mouse splenocytes and the SP2O mouse myeloma cell line (18). Similarly AF5 antibody was obtained fromanother fusion experiment.

Characterization of Monoclonal Antibodies. Ascitic fluids containing monoclonal antibodies SF 31 and SF 90 were firsttested for binding activity to FOCUS, other human hepatomacell lines, and peripheral blood lymphocytes. Both antibodiesreacted with all human hepatoma cell lines tested but not withnormal lymphocytes (Table 1). Monoclonal antibodies SF 31and SF 90 were of the IgG, isotype as determined by double

Table I Reactivity ofSF Vi)anil SF il monoclonal antibodies with humanhepatoma cell lines

S/NÂ°

Hepatoma cell line SF90 SF3I

FOCUSMahlavuPLC/PRF5HEP

3BHEPG2SK-HEP

IWhiteblood cells12

31229

114112210

2<211

391102133102I0Â±

2<2

a S/N, signal (S) to noise (N) ratio where S and N are cpm bound (mean Â±SD: n = 3) with SF 90 and nonspecific (B;TT) monoclonal antibodies, respectively. While blood cells have been used as a negative control.

COMPETITIVE INHIBITION

I

I

â€¢Q

i

SF90

Â«SF31OSF90oAF5

SF31

05

o irr'2 â€¢io'10io'8

UNLABELED MAb (M)

Fig. l. Competitive inhibition experiments show ing that monoclonal antibodies SF 90 and SF 31 are directed to the same or closely related epitope(s). Thebinding of '"I-SF 90 is inhibited by SF 90 and SF 31 but not with AF5 (top).similar results are obtained with '"I-SF 31 (bottom).

antibody radioimmunoassay. Fig. 1 shows that both antibodieswere directed against the same or very closely related epitope(s).I25I-SF 90 antibody demonstrated high binding to FOCUS cells

(5,114 Â±347 cpm) in the absence of unlabeled antibody (Fig.1, top). The binding of I25I-SF 90 to FOCUS cells was progres

sively inhibited in the presence of increasing concentrations ofunlabeled SF 90 (538 Â±76 cpm in the presence of IO"9 M cold

SF 90 representing 90% inhibition). The same experiment wascarried out in the presence of AF5, a monoclonal antibodyknown to be directed against a distinct and separate antigenpresent on FOCUS;6 no inhibition of binding was observed. In

contrast, SF 31 inhibited the binding of labeled SF 90 in amanner very similar to unlabeled SF 90 (Fig. 1, top). Indeed80% inhibition was achieved in the presence of 10~9 M SF 31

antibody. Fig. 1, bottom, shows the results of a similar experiment performed with I25l-labeled SF 31. The binding of i:5I-SF

31 to FOCUS cells was inhibited with unlabeled SF 31 and SF90 antibodies and not with AF5 antibody.

Identification and Cellular Localization of p50. Monoclonalantibodies SF 31 and SF 90 were used to identify a p50 proteinand subsequently characterize its molecular properties and cel-

' Unpublished observations.

6766



ADRENAL PROTEIN IN Tl'MOR CELLS

lular localization. Since the same cell binding and Westernimmunoblotting results were obtained with both antibodies, wepresent here studies on SF 90. As shown in Fig. 2A, I25I-SF 90

demonstrated high binding to intact FOCUS cells. The specificity of binding was demonstrated by lack of significant bindingof a nonrelevant antibody (125I-HT13) and inhibition of the

binding of labeled SF 90 in the presence of 100 fig/mi tinlabeledSF 90 (Fig. 2A). In order to identify the cell surface antigenrecognized by SF 90 we performed a selective cell surfacelabeling with I25I followed by an immunoprecipitation withantibody. SDS-PAGE analysis demonstrates that SF 90 im-munoprecipitates a M, 50,000 polypeptide (p50; Fig. 2B). Asshown in Fig. 1C, indirect immunofluorescence studies conducted with SF 90 showed a reticular cytoplasmic stainingpattern in acetone-fixed FOCUS cells. Immunofluorescencestudies performed with a nonrelevant antibody (B2TT) showedno staining under the same experimental conditions (resultsnot shown). These studies indicate that p50 initially found onthe cell surface of FOCUS cells was also highly abundant withinthe cytoplasm. To explore the intracellular distribution of p50further, we first prepared cytosolic (250,000 x g supernatant)and noncytosolic (pellet) fractions of a FOCUS cell homogenatein extraction buffer. p50 was localized in the noncytosolicfraction (data not shown). FOCUS cells were subsequentlyfractionated into three components. We employed a schemethat separates cells into nuclear, cytoskeletal, and Triton-soluble fractions by using extraction buffers with defined detergentcompositions (25). Equivalent amounts of these subcellularfractions were then analyzed for the presence of p50 by Westernimmunoblotting. As shown in Fig. 3, p50 was localized to theTriton-soluble fraction. However, low but detectable amountswere also found in association with the cytoskeletal framework.There was no detectable p50 in the nuclear fraction.

Characterization of p50. FOCUS cells were labeled with [35S]-methionine or with |'H]glucosamine under various conditions

and cell extracts were immunoprecipitated with SF 90. Fig. 4shows that after a [15S]methionine pulse for 1 h, a M, 50,000

polypeptide could be immunoprecipitated. No apparent shift ofmolecular weight was observed when cells were chased withunlabeled methionine for 1 h. A single p50 protein was obtainedfrom cell extracts and relative migration of p50 on SDS-PAGEwas not altered under nonreducing and reducing [5% (v/v) 2-

mercaptoethanol] conditions. We were unable to Â¡mmunopre-cipitate any antigen after metabolic labeling of FOCUS cells inthe presence of ['Hjglucosamine (results not shown). A deter-

gent-solubilized extract from FOCUS cells was analyzed bytwo-dimensional cell electrophoresis followed by Western immunoblotting with 125I-SF90. Fig. 5 demonstrates some micro-

heterogeneity of p50. In addition to two M, 50,000 proteinswith slightly different pi values (6.5 and 7.2), a M, 40,000protein (p40) with a basic pi (8.2) was also observed. This p40molecular weight form was occasionally seen in Western blotsin variable amounts from tissue specimens. In some instances,where the same cell pellet or tissue specimens were analyzed byhomogenization of aliquots at various intervals, we observedthe appearance of the smaller p40 form suggesting that it maybe a breakdown product of p50.

Ubiquitous Expression of p50 Â¡nHuman Tumor Cell Lines.Tumor cell lines other than hepatocellular carcinoma derived(Table 1) were tested for the expression of p50 antigen by directand indirect cell binding, immunofluorescence, immunoprecipitation, and Western immunoblotting assays. These studiesincluded a variety of human tumor cell lines representing carcinomas and adenocarcinomas but also other types such assarcoma, melanoma, neuroblastoma, and teratocarcinomas.p50 was detected in all human tumor cells tested (Table 2). Amonkey kidney cell line (COS-1) obtained by transformation ofCV-1 cells with origin defective SV40 DNA (26) also expressedhigh levels of a M, 50.000 protein recognized by SF 90 antibody.In contrast, SF 90 failed to bind to rat tumor cell lines PC-12(adrenal pheochromocytoma) and Mca-RH777 (hepatoma)(data not shown).

In Vivo Expression of p50 on Human Hepatocellular Carcinoma. After the initial identification of p50 on cultured tumorcell lines, we studied its display on human hepatocellular carcinoma tissues and compared our results to the adjacent normalliver counterparts. In the first place, immunoperoxidase studiesshowed consistent and homogenous staining of hepatoma cellsin 18 different tumors. As shown in Fig. 6, there was strongimmunostaining of a hepatoma tumor (Fig. 6B) with no detectable staining in the normal adjacent liver counterpart (Fig.6A). These results were then confirmed by Western immunoblotting. Fig. 6C shows in vivo results derived from eight pairedhepatoma and adjacent normal liver specimens when probed

Fig. 2. Localization of p50 antigen Â¡nFOCUS cells. A, there is high binding of '"I-

labelcd SF 90 to monolayers of FOCUS cellsgrown overnight before analysis (left column]compared to the insignificant binding of a non-relevant antibody (anti-hCG monoclonal antibody '"I-HT13. center column). Note thatbinding of I2SI-SF 90 was inhibited with an

excess of unlabeled SF 90 (right column), lÃ¬.imniunoprecipitation of p50 with SF 90 from'"l-surface labeled FOCUS cells. C indirectimmunofluorescence staining of acetone-fixed/permeabilized FOCUS cells with SF 90shows intense intracellular staining particularly in the pcrinuclear area.

?J10~s>iâ€¢5Ã§.0.<OIÂ§

5^^^^Â»^QAâ€”-â€”'.-:'

:::::.::::::_.Ã„

^SSF90*HU 3* SF90*+SF90

B

-p50

6767




m O in o u">

KDo

13075

-50

-39

-27

Fig. 3. Distribution of p50 in the subcellular fractions of FOCUS cells. Cellswere fractionated into Triton \-100 soluble, cytoskeletal. and nuclear fractions.Proteins present in each fraction were analyzed by Western immunoblotling using'"I-SF 90. A M, 50.000 protein is detected in Triton-soluble and cytoskeletalfractions, but not in the nuclear fraction.

Minutes; 0 i5 30 60

KDo130-

75-

50-

39-

27-

17-Fig. 4. SDS-PAGE analysis of FOCI'S cell proteins immunoprecipitated with

SF 90 after metabolic labeling with ["S]niiMhionine before (0 minute) and after

chasing up to 60 min with excess of unlabeled mcthionine.

pi

kDo130-

75-

50-

39-

27-

I I ill

QadI

PO

odI

Fig. 5. Western immunoblotting of a 2D-gel of separated FOCUS cell extractwith '"I-SF 90 showing three major species. p40 (pi K.3) and p50 (plo.S and pi

7.0).

with SF 90. All of the eight tumors exhibited p40 and p50antigens whereas no expression was detected on adjacent normal liver. We obtained identical results with 10 additionalhepatoma/normal liver pairs (data not shown). Thus, all hepa-tomas studied to date expressed detectable levels of this protein,suggesting a constant expression in the malignant phenotype.

Antigenic Expression in Normal Adult and Fetal Tissues. Theexpression of the p50 antigen in normal human tissues wasfirst investigated by Western immunoblotting: the followingadult tissues were found to be negative for this antigen; liver,kidney, pancreas, skin, muscle, heart, lung, lymphocytes, smalland large intestine, spleen, stomach, and esophagus. In contrast,tissue extracts obtained from normal adrenal gland showed ahighly immunoreactive band at M, 50,000 (data not shown).Fig. 7, depicts the pattern of expression in fetal tissues. Similarto our observation in adult adrenal glands, fetal adrenal glandsalso express a highly immunoreactive antigen comigrating withp50. In contrast, no expression was detected in fetal liver,kidney, lung, muscle, and skin. In order to test if the p50antigen was absent or present at low levels in normal tissues,we also used a more sensitive solid-phase radioimmunoassay.Homogenates prepared from FOCUS cells and from variousnormal tissues were probed with radiolabeled SF-90. As shown

in Fig. 8/1, there was a linear relationship between the proteinconcentration and 125I-SF 90 binding. We then investigated

several adult tissue homogenates under the same conditions.Fig. IB shows that all tissues tested have detectable levels ofp50. The expression was low in liver, kidney, lung, heart, andspleen but very high in the adrenals.

p50 Expression in Other Mammalian Species. Because of thehigh expression of p50 in human adrenal tissue, we studiedbovine adrenals as well. Fig. 9 illustrates immunoperoxidasestaining of human and bovine adrenal tissues. Both tissuesshowed intense and homogeneous staining. Staining of paraffin-embedded bovine adrenal allowed us to localize the immuno-reactivity to adrenal medullary cells and to a subpopulation ofcells in the zona glomerulosa of the cortex. No staining wasobserved with a nonrelevant B^TT monoclonal control antibodytested in the same conditions. Western immunoblotting studieson detergent-soluble extracts from adrenals revealed that the

6768




Table 2 p50 antigen expression in human tumor cell lines

CelllineEpitheliumFOCUSSK-HEP

1MahlavuPLC/PRF5HEP

3BHEPG2\\1DRSW403CACO-2LS-180SK-CO1COLO

320DMCALU6CALU3SK-LU-1SK-MES

1A427A498HELAC33AAN3CAÃ‡A

OV3BT20SW-13KB-3-1KB-V

1Embryonic

tissueJEG-3JARNeural

tissueSK-MEL-5IMR-32Connective

tissueSK-UT-1Gonadal

tissueTERA-2CATES

IBLvmphoid

tissue'RAJIDennitionHepatomaAdenocarcinomaHepatomaHepatomaHepatomaHepatoblastomaAdenocarcinomaAdenocarcinomaAdenocarcinomaAdenocarcinomaAdenocarcinomaAdenocarcinomaAnaplastic

carcinomaAdenocarcinomaAdenocarcinomaSquamous

carcinomaCarcinomaCarcinomaCarcinomaCarcinomaAdenocarcinomaAdenocarcinomaCarcinomaAdenocarcinomaEpidermoid

carcinomaEpidermoidcarcinomaChoriocarcinoma

trophoblastC'horiocarcinomatrophoblastMalignant

melanomaNeuroblastomaLeiomyosarcomaEmbryonal

carcinomaEmbryonalcarcinomaHin

kin's lymphoma maxillaTissue

oforiginImmunoreactivity"Liver

++Liver++Liver++Liver++Liver++Liver++Colon++Colon++Colon++Colon++Colon++Colon+Lung++Lung++Lung++Lung++Lung++Kidney++Cervix++Cervix+Endometrium+Ovary++Breast+Adenal

cortex+Oral++Oral++++++Melanocytes

++Adrenalmedulla++Uterus

++Testis

++Testis++++

" +, a S/N >2.5 and ++. a S/N >5 in a direct binding assay using 1!!I-SF 90 as the probe. S/N is the ratio of cpm bound to human tumor cell line divided by cpm

bound to a mouse cell line (NIH 3T3). Most cell lines were also tested by Western immunoblotting and demonstrated a M, 50,000 protein on detergent solubilizcdcell extracts.

Fig. 6. The presence of p50 on human hep-atocellular carcinoma is shown. Positive andhomogenous staining of a human hepatocel-lular carcinoma (/f ]. negative staining in theadjacent normal liver counterpart (A ). Westernimmunoblots of paried hepatoma i / i and normal liver (N) samples probed with SF 90 followed by SAM (C). Note the presence of M,40.000 and 50.000 immunoreactive proteinsin all hepatoma samples as compared to theadjacent uninvolved liver.

1 23456783NT NTNT NTNTNTNTN T Â£

bovine analogue of p50 had an approximate molecular weightof 53,000 (data not shown).

Comparative Studies with Anti-p50 and Anti-p53 MonoclonalAntibodies. Since the molecular weight of p50 and anothertumor antigen p53 are similar, we performed studies to determine if the two proteins were related. It has previously been

determined that p53 was undetectable in HeLa cells (27). Thus,we prepared cell homogenates and determined the presence ofp50 compared to p53 by Western immunoblotting. It is noteworthy that we found no detectable p53 in HeLa cell extractswhereas COS-1 cells were highly positive (data not shown). Incontrast, we observed that p50 antigen was present in both

6769




-p50

Fig. 7. Expression of p50 antigens on human fetal tissues as determined byWestern immunoblotting with '"I-SF 90. Compared to a FOCUS cell lysate (lane/). p50 was undctectable in fetal liver (lane 2). kidney (lane 3). lung (lane 4).muscle (lane 5), and fetal skin (lane 6), but highly expressed in adrenal gland(lane 7).

5 10 (5 20

PROTEINS (Â¿Â¿g/well)

5 S

_B---1iJoÃ›Ã»Ã›Ã».

Fig. 8. Measurement of p50 levels by solid-phase radioimmunoassay. In A,binding of '"I-SF 90 to FOCUS cell lysate is proportional to the amount of totalprotein tested. In B, results obtained on normal tissue homogenates. As comparedto FOCUS, the adrenal glands show high levels of binding activity whereas p50expression is low in other tissues including liver.

HeLa as well as COS-1 cells (Tables 2 and 3). Finally, wecompared the direct binding of 125I-SF 90 and 125I-Pabl22 toCV-1 and COS-1 cells by a solid-phase radioimmunoassay; 125I-SF 90 binding to both cell lines was similar. In contrast, 125I-Pabl22 antibody binding was low to CV-1 cells but very highto COS-1 cells. Based on these immunochemical observations,we are led to believe that p50 and p53 are unrelated proteins.

DISCUSSION

Two murine monoclonal antibodies produced by immunization with intact single cell suspensions of FOCUS cells allowed

us to identify a novel M, 50,000 protein antigen highly expressed in HCC-derived cell lines. Several other hepatoma-associated protein antigens have been previously described byus (14, 15, 18, 19) and by others (12, 13, 16, 17). p50 appearsto be different from those other described hepatoma-associatedantigens due to its constant expression in hepatoma cells bothin vitro and in vivo and lack of expression in normal hepatocytes(18). A protein of similar molecular weight has been previouslyidentified by a monoclonal antibody to PLC/PRF/5 hepatomacells (14). However, the two proteins are unrelated since p50reported here is expressed not only in hepatoma but also inother human tumor cells.

The concept of retrodifferentiation implies that the patternof gene expression of dedifferentiated cells resembles that ofthe corresponding embryonic cells (28). It has been assumedthat hepatocarcinogenesis involves retrodifferentiation and Â«-fetoprotein is a typical oncodevelopmental protein productbecause its levels are high in fetal liver and liver tumors but lowin the adult liver (28, 29). Similarly, oncogenes such as c-mycand c-ras have been found to be highly expressed in hepatomaand fetal liver but low in adult liver (29-31). We found p50expression was high in hepatoma but undetectable in both adultand fetal liver. This pattern of expression suggests that unlikea-fetoprotein, p50 is not an oncodevelopmental protein andappears to be unrelated to protooncogenes (c-myc and c-ras)that are highly expressed in malignant hepatocytes (29-31).

p50 was found to be present in all human tumor cell linestested thus far (Table 2). Although our panel of cell linesconsisted mainly of tumors derived from epithelial tissues (carcinomas and adenocarcinomas), it also contained cell linesderived from nonepithelial origins (leiomyosarcoma, neuroblastoma, and malignant melanoma). Thus, the expression ofp50 appears to be unrelated to embryonic tissue of origin. It isalso noteworthy that p50 was equally present in tumor cell lineswith variable degrees of differentiation. For example, p50 waspresent in all of six colon tumor cell lines (Table 2). Most ofthese cell lines are well differentiated such as SK-CO-1, WiDr,CaCo-2, SW403, and LS 180 (32, 33) whereas one of them(Colo 320 DM) was derived from a moderately undifferentiatedadenocarcinoma of the sigmoid colon (34). Colo 320 DM cellline has been shown to be undifferentiated and does not expresscarcinoembryonic antigen (34). We found relatively constantlevels of p50, as measured by direct binding assays, in all ofthese colon tumor cell lines (data not shown). Similarly, wetested five lung tumor cell lines (Calu-6, Calu-3, SK-LU-1, SK-MES-1, and A 427). Calu-3 is a well-differentiated adenocarcinoma (35, 36), whereas Calu-6, A 427, and SK-LU-1 arepoorly differentiated. SK-MES-1 is an epidermoid carcinomacell line (36). p50 was highly expressed on the cell surface ofall five lung carcinoma cell lines. Our data suggests that p50expression in tumor cells is not related to embryonic tissue oforigin, in vitro morphology, or degree of cellular differentiation.

Antigens shared by a variety of tumor cells have been previously described (10, 37-41). It is of interest that p50 and apreviously identified p53 transformation-related protein havesimilar physical properties such as relative molecular mass andpi (10, 37). p53 was first identified in SV40-transformed cellsas a cellular-encoded tumor antigen (42, 43). Other studies haveshown that p53 is a transformation-related antigen detected notonly in viral transformed cells but also in most mouse andhuman tumor cells both in vitro and in vivo (37, 44). Despitesome physical similarities, p50 and p53 appear to be differentmolecules. First, p53 was shown to be a nuclear protein intransformed cells (45). We have localized p50 to the cell surface

6770




Fig. 9. Immunoperoxidiase stainings withSF 90 performed on a fresh frozen sections ofnormal human adrenal (A) and on a paraffin-embedded section of bo\ine adrenal tissues(B). Note intense staining in adrenal medulla(am) and in a cortex cell population of thezona glomerulosa of the cortex (:g) of bovineadrenal tissue.

yi

am

Table 3 Binding of'"l-SF 90 (anli-pSO) and '"l-Pabl22 (anli-p53) antibodieslo parental CV-I and SWO-transformed COS-I cells

Antibody bound cpm/50 //g protein

Cell line '"I-SF 90 125l-Pabl22

CV-ICOS-1

12.537 Â±11112.399 + 351

824 Â±62,839 Â±101

and to the cytoplasm but not in the nucleus of FOCUS cells(Fig. 4). The cellular localization of p50 was found to be similarin other cells tested such as choriocarcinoma (JEG-3 and JAR)and adrenal cortical adenocarcinoma (SW-13) cells (data notshown). In addition, p53 was shown to be undetectable in sometransformed cell lines such as HeLa (46) and highly present inothers such as COS-1. We confirmed these observations byWestern immunoblotting using Pabl22 monoclonal antibodyagainst p53 antigen (22). Moreover, as demonstrated in Table3, we present evidence that p50 was highly and equally expressed in CV-1 parental and SV40 transformed COS-1 celllines. In contrast, p53 levels were low in parental CV-1 but veryhigh in COS-1 cells. p53 was first identified as a cellular antigenin SV40-transformed cells and under these conditions, its levelsare increased substantially (42, 43). We are led to believe fromthese observations that the p50 and p53 proteins are unrelated.Since p53 has been shown to be directly involved in cellulartransformation (45-47), studies that examine the possible functions of p50 in the transformation process will also be ofinterest.

Although p50 appears to be transformation associated, it isnot a tumor-specific antigen since high expression was foundin a normal tissue such as adrenal gland. The tissue localizationwas primarily confined to the medulla, a tissue of neuroecto-dermal embryonic origin (48). When we compare our findingsto those major adrenal proteins described by others (49), p50appears to be a novel protein. Creutz et ai. (50) identifiedseveral chromaffin granule binding proteins that have molecularweight and pi values similar to that of p50. It will be interestingto investigate pSO's potential for binding to chromaffin gran

ules. Other transformation-related proteins were also found to

be highly expressed in adrenal glands. For example, bovineadrenal medullae express high levels of p60"" comparable tothat of p60'~'"' in chicken embryo fibroblasts infected by Rous

sarcoma virus (51). Another protein has also been found to behighly expressed both in normal adrenal tissues and primaryliver tumors with correspondingly low levels in normal liver,namely P-glycoprotein, a cell surface protein encoded by amulti-drug resistance gene (52). Multi-drug resistance gene wasfound to be highly expressed in these tissues both at the mRNAand protein levels (53-55). It is unlikely that both proteins arerelated since P-glycoprotein has a molecular weight of 170,000and unlike p50, its expression in transformed cells was limitedto the drug-resistant phenotype (55). In addition, we tested suchcell lines for the presence of p50 by direct antibody binding andWestern blotting assays. One of these cell lines (KB-3-1) is drugsensitive and does not express P-glycoprotein. The other cellline (KB-V1) was derived from the same cell line by selectionfor the drug-resistant phenotype (56). This derivative containsamplified drug-resistance genes and expresses high levels of P-glycoprotein (53). We found high expression of p50 in both celllines (Table 2) in similar amounts (data not shown).

There has been renewed interest on the relationship of adrenal anabolic steroids and HCC. It has long been recognizedthat HCC is more common in men (1) and may be induced bychronic administration of such agents. HCC tumors have regressed following withdrawal of the anabolic agent (57). Morerecently, during experimental hepatocellular tumor inductionin rats, Ostrowsky et al. (58) found a 20-fold increase in the

hepatic androgen receptor and suggested increased androgensensitivity in this tumor model was analogous to the observations of increased androgen receptor expression in HCC. Although p50 was detected mainly in the adrenal medulla, it wasalso found in a subpopulation of cortical cells. It will be ofinterest to pursue the identification of p50 by molecular cloningas well as to study its cellular function(s) as a presumed normaladrenal protein whose expression is strikingly increased intransformed hepatocytes.

6771




ACKNOWLEDGMENTS

We thank Rolf Carlson for his excellent technical assistance andDrs. Lee Kaplan, Ira Pastan, Shiv Pillai, and Michael Kew for providingsome of the cell lines and specimens investigated in this report.

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1989;49:6764-6773. Cancer Res Mehmet Ozturk, Philippe Motté, Hiroshi Takahashi, et al. Protein in Human Hepatocellular Carcinoma

50,000 AdrenalrMIdentification and Characterization of a

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Identification and Characterization of a Mr 50,000 Adrenal ...Identification and Characterization of a Mr 50,000 Adrenal Protein in Human Hepatocellular Carcinoma1 Mehmet Ozturk,2