[CANCER RESEARCH 46, 4726-4731, September 1986]

Expression of Epidermal Growth Factor Receptor in Human Cultured Cells andTissues: Relationship to Cell Lineage and Stage of Differentiation1

Francisco X. Real,2 Wolfgang J. Rettig,3 Pilar Garin Chesa,4 Myron R. Melamed, Lloyd J. Old, and

John MendelsohnLaboratory of Human Cancer Immunology [F. X. K., W. J. K., L. J. O.J, Department of Pathology [P. G. C., M. R. M.], and Laboratory of Receptor Biology [J. MJ,Memorial Sloan-Kettering Cancer Center, New York, New York 10021

ABSTRACT

Mouse monoclonal antibodies have been used to study the distributionof the epidermal growth factor receptor in human cultured cells andtissues. As expected, most epithelial cells expressed epidermal growthfactor receptor (EGFr), whereas cells of hematopoietic origin wereEGFr". However, EGFr was found to be differentially expressed on

cultured cells of neuroectodermal origin. Normal melanocytes and aproportion of melanomas are EGFr~, whereas a distinct subset of othermelanomas, astrocytomas, and neuroblastomas is EGFr*. Expression ofthe EGFr in melanomas was closely related to the expression of pheno-typic traits of differentiation. Less differentiated melanomas have anepithelioid morphology and are nonpigmented, la*, and EGFr*; in con

trast, more differentiated melanomas have a dendritic morphology andare pigmented, Ia~, and EGFr". In the melanoma cell panel used, expres

sion of EGFr did not correlate with rate of proliferation. Expression ofEGFr in tissues also showed a lack of correlation with the proliferativestate of cells. Our findings indicate that expression of EGFr is related tocell lineage and specific stages of cellular differentiation, rather than onlyto cell proliferation. The data suggest that receptor content may beelevated in a large number of tumor cell lines.

INTRODUCTION

EGF5 is a polypeptide which has growth-promoting activity

for a number of cell types in culture and also in vivo (1). EGFexerts its effects on cells through a cell surface receptor (EGFr)identified as a glycoprotein with a molecular weight of 170,000(2). Upon EGF binding, the receptor undergoes autophospho-rylation through the kinase activity of its cytoplasmic domain(3,4). This domain has been shown to have amino acid sequencehomology with the protein encoded by the \-erb B oncogene(5). EGF has been shown to have amino acid sequence homology with the tumor-derived type-a TGF (6), and type-a TGFscan interact with the receptor for EGF (7, 8). The relationshipof EGF and EGFr to type-«TGFs and to the protein encodedby a known oncogene, respectively, has drawn considerableattention to the possible involvement of the EGF/EGFr systemin the malignant transformation of cells.

In this paper, we have examined the reactivity of two mAbdetecting the human EGFr molecule with a large panel of celllines and fresh normal and tumor tissues. Our studies showthat EGFr is expressed on many epithelial, mesenchymal, andneuroectodermal cell types, but it is not expressed on hematopoietic cells. In a detailed analysis of melanocytic cells, follow-

Received 3/5/86; revised 5/21/86; accepted 5/29/86.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported in part by Grants CA-08748, CA-34079, and CA-42060 from the NIH and by the Oliver S. and Jennie R. Donaldson CharitableTrust, Inc.

2To whom requests for reprints should be addressed, at 1275 York Avenue,New York, NY 10021.

3 Recipient of a Fellowship from the Deutsche Forschungsgemeinschaft.' Recipient of a Fulbright Fellowship from the Spanish Ministry of Education.5The abbreviations used are: EGF, epidermal growth factor; EGFr, receptor

for epidermal growth factor; TGF, transforming growth factor; mAb, monoclonalantibody: PBS, phosphate-buffered saline; PAA, pigmentation-associated antigen.

ing the approach proposed by Houghton et al. (9), EGFrexpression was detected on epithelial, nonpigmented ("early")melanoma cultures, whereas dendritic, pigmented ("late") mel

anoma cultures and normal fetal and adult melanocytes lackedEGFr expression. These findings confirm and extend previousstudies suggesting that EGFr expression may serve as a markerfor specific stages of cellular differentiation and that it is notrestricted to actively proliferating cells.

MATERIALS AND METHODS

Cultured Cells. Tumor cell lines were established and maintained aspreviously described (10, 11). Most human cell lines and cultures ofnormal cells were obtained from the Human Cancer ImmunologyLaboratory cell bank. Neuroblastoma cell lines were kindly providedby Dr. J. L. Biedler (Sloan-Kettering Institute, New York, NY) and Dr.J. T. Casper (Milwaukee Children's Hospital, Milwaukee, WI). Mela-

nocyte cultures were kindly provided by Dr. A. N. Houghton (Sloan-Kettering Institute) and were maintained in culture as described (12).

Monoclonal Antibodies. The production and characterization ofmAbs 225 (IgGl) and 528 (IgG2a) have been reported (13, 14). Hybri-domas were obtained from fusion of splenocytes from mice immunizedwith partially purified EGFr from A431 cells (13, 14). mAb 225 waspurified on DEAE-Sepharose (Pharmacia, Piscataway, NJ) and ProteinA-agarose (Calbiochem-Behring, La Jolla, CA) as described (14).

Serological Methods. Reactivity of antibody with cell surface antigensof cultured cells was assayed using a rabbit anti-mouse immunoglobulinhemadsorption assay. mAb 225 was used at a concentration of 1 mg/ml, and mAb 528 was used as hybridoma culture supernatant. Briefly,serial dilutions of antibody were incubated for l h at 20°Cwith target

cells growing on Falcon 3040 Microtest II plates (Falcon Labware,Oxnard, CA). Nonadherent target cells were tested on Microtest platesusing the concanavalin A method (15). Target cells were washed with3% 7-globulin-free fetal bovine serum in PBS and incubated for l hwith a 0.2% suspension of type O RBC coated with rabbit anti-mouseimmunoglobulin by the chromium chloride method (16). Target cellswere washed and evaluated for rosette formation. Titration end pointsrefer to the highest antibody dilution showing 10% of the target cellsforming rosettes.

Biochemical Analysis. Cells were metabolically labeled with |'II|-

glucosamine and extracted with Tris/Nonidet P-40 buffer as described(17). Alternatively, cell membranes were isolated from cultured cellsand labeled with 125Iusing the chloramine T method (18). Immunopre-cipitation tests and sodium dodecyl sulfate-polyacrylamide gel electro-phoresis were performed following published procedures ( 17, 18).

Immunohistochemical Techniques. Tissues were quick frozen in iso-penthane precooled in liquid nitrogen. Sections of 5-^m thickness weremounted on gelatin-coated slides, air dried for 30 min at 20°C,and

fixed in cold acetone for 10 min. The avidin-biotin complex methodwas used as described (19). Briefly, endogenous peroxidase was blocked,and sections were incubated overnight with antibody. mAb 225 wasused at a concentration of 201<n/inl, and mAb 528 was used as undilutedhybridoma culture supernatant. Sections were washed with PBS, incubated with biotinylated horse anti-mouse immunoglobulin for 30 min,washed, and incubated with an avidin-biotinylated horseradish peroxidase complex. The final reaction product was visualized with diaminobenzidine-H2O2. Sections were counterstained with hematoxylin.Specificity controls included unrelated mouse mAb and PBS.

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DISTRIBUTION OF THE RECEPTOR FOR EGF

RESULTS

Cultured cells and tissues were typed using two monoclonalantibodies, mAb 225 and mAb 528, detecting human EGFr.Unlike several other mAbs reactive with the EGFr described sofar (20, 21), mAb 225 and mAb 528 do not react with bloodgroup-related epitopes which are part of the carbohydratemoiety of the EGFr molecule (21). Both antibodies block thebinding of EGF to the receptor (14). The results obtained withmAb 225 and mAb 528 in this study were essentially identical.

Expression of EGFr on Cultured Cells. The results of typinga panel of 154 tumor cell lines by anti-mouse immunoglobulinhemadsorption assays are shown in Fig. 1. Among tumors ofneuroectodermal derivation, 19 of 36 melanomas, 19 of 19astrocytomas, and 8 of 9 neuroblastomas were reactive withmAb 225. Two retinoblastomas, one medulloblastoma, and oneprimitive neuroectodermal tumor were unreactive. In tests withsome melanoma cell lines only a proportion of the target cellsformed immune rosettes even at the highest antibody concentrations tested, suggesting the existence of two subpopulationsof cells with respect to EGFr expression, one being EGFr* andone being EGFr". Titration end points of mAb 225 on EGFr-expressing melanoma cell lines ranged from 10~4 to 10~8 (Fig.

2). Expression of EGFr clearly correlated with the differentiation phenotype of melanoma cultures. Most melanoma celllines with an epithelioid morphology and no or little pigmentwere EGFr+. In contrast, all melanoma cell lines with dendriticmorphology and strong pigmentation were EGFr". Among

melanoma cell lines with spindle morphology, some wereEGFr+, and some were EGFr". EGFr* and EGFr" melanomas

could also be distinguished by their expression of other differ-

TUMOR TYPE

MELANOMA

ASTROCYTOMA

NEUROBLASTOMA

COLONCARCINOMA

RENAL CARCINOMA

BLADDERCARCINOMA

BREAST CARCINOMA

LUNG CARCINOMA

OVARIAN CARCINOMA

OTHERCARCINOMAS

SARCOMA

LEUKEMIA/LYMPHOMA

INDIVIDUAL CELL LINES

•0©©©©»0©©OOOOOOOO©©©©»•©©•ooooooooo•©••©•©••©••••••••A••ooooooo

©©©©©•©©•©©@©©o©©•©o©©o®o©©•©©©©©©©©•©00oooooooooooooo

I'i/5R 100

50

02x10-3 1.6x10-5 1.2x10-8 2x10-3 1.6x10-5 1.2x10-»

Fig. 2. Expression of EGFr on cultured normal and malignant cells. A: •,SK-MEL-187: •SK-MEL-178; O, SK-MEL-I9; D, SK-MEL-23; A, normal adultskin melanocytes. B: •,SK-RC-1; •.SK-RC-7; A, normal kidney epithelial cells.C: O, HL-60; A, normal peripheral blood mononuclear cells. I>. •,A3095; A,normal adult skin fibroblasts. Serological assay by anti-mouse immunoglobulinhemadsorption assay using mAb 225 (starting concentration, I mg/ml).

SK-MEL-188

SK-MEL-37

SK-MEL-31

SK-MEL-133

SK-MEL-131

SK-MEL-33

SK-MEL-90

SK-MEL-93-m

SK-MEL-13

SK-MEL-29

SK-MEL-119

SK-MEL-28

SK-MEL-93-H

SK-MEL-93-ET

SK-MEL-73

SK-MEL-113

MeWoSK-MEL-30

SK-MEL-64

SK-MEL-19

SK-MEL-23

Io

O

O

oo

EGFr PAA

ooo

ooo

oo

ooooooooo

o•ooooooooo

Fig. I. Each symbol represents typing results with an individual cell line. Fig. 3. Coordinate expression of EGFr, la antigens, and PAA on 21 melanomaMelanoma: first row. SK-MEL-11, -13, -28, -29, -31, -33, -37, -81, -90, -131, -19, ce" lines- Expression (or nonexpression) of EGFr is accompanied by expression-23, -30, -43, -64, -72, -73, -93-II; second row: SK-MEL-133, -139, -144, -147, (or nonexpression) of la antigens in 15 of 21 cell lines. On the other hand,-169, -173, -178, -186, -187, -93,-IH, -93-IV, -113, -119, -120, -174, -176, M14, expression of PAA follows a pattern reciprocal to that of EGFr in 12 of 14 cellMeWo. Astrocytoma: SK-MG-1, -2, -3, -4, -7, -8, -9, -11, -12, -13, -15, -16, -17, lines tested- ••positive reaction; O, negative reaction.-21; U251MG; U343MG; SK-GS-1; T98; MS. Neuroblastoma: SK-N-BE(l),-BE(2); SMS-KAN; CHP-234; LA-N-1; MC-NB-1; SK-N-SH; IMR-32; SK-N-MC. Colon: SK-CO-1, -10, -12, -13, -15; SVV48; SW403; SW480; SW837;SW1083; SW1116; SW1222; SW1417; HT-29; CaCo-2; SK-CO-11; SW620.Renal: SK-RC-1, -2, -4, -7, -8, -10, -15, -17, -26, -29, -35, -38, -41, -42, -44, -46,-48, -51; Caki-I. Bladder. SW1410; SW780; SW800; T24; RT4; 253J. Breast.SK-BR-3, -5, -7; MCF-7; BT-20; AlAb; MDA-MB-136; MDA-MB-231; CAMA.Lung: SK-LC-8, -9, -13, -LL, -6. Ovary: SK-OV-3, -4, -6; OV-2774; AIO. Othercarcinomas: CAPAN-1, CAPAN-2 (pancreas); A431 (vulva); MKN-45 (stomach);GCC-SV (choriocarcinoma); AA (uterus); SK-HEP-1 (hepatoma); ME-180 (cervix); PC-3 (prostate). Sarcomas: RD-2; V-201; A3095; A2394; TE-85; SAOS-2.Leukemia/lymphoma: MOLT-4, T-45, P-12, RPMI-8402, CCRF-CEM, CCRF-HSB2 (T-cell); Daudi, SK-DHL-2, BALL-1 (B-cell); NALM-1, NALL-1, NALM-16 (null cell); K562 (erythroleukemia); HL-60 (promyelocytic leukemia). Assayby rabbit anti-mouse immunoglobulin hemadsorption. •,positive reaction, titer10~'°to 10'"; ©,positive reaction, titer 10~7 to 10"'; 0, positive reaction, titer10"4 to 10"3;O, no reaction. Detailed results of the serological assays are available

upon request to the authors.

entiation antigens. When the expression of EGFr and la antigen, a marker for "early" melanoma cultures (9, 22), was

compared, cotyping was observed for 15 of 21 cell lines examined (Fig. 3). In contrast, expression of PAA, a marker for"late" melanoma cultures (23, 24), followed a pattern reciprocal

to that of EGFr in 13 of 14 cell lines tested (Fig. 3). Thus,expression of EGFr seems to be restricted to specific stages ofdifferentiation of cultured melanomas and closely follows thepattern of expression predicted by Houghton et al. for an earlydifferentiation marker. Cultured melanocytes from normal fetaland adult skin were EGFr~ (Fig. 2).

Seventy cell lines derived from epithelial cancers were tested4727

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DISTRIBUTION OF THE RECEPTOR FOR EGF

with mAb 225. All 19 renal carcinoma cell lines tested were225+. Fifteen of 17 colon carcinomas, 8 of 9 breast carcinomas,

6 of 6 bladder carcinomas, 4 of 5 lung carcinomas, and 4 of 5ovarian carcinomas were strongly reactive with mAb 225. Anumber of cell lines derived from other epithelial cancers (pancreas, uterus, stomach, vulva, liver) and choriocarcinomas werealso EGFr+. A431 cells, which are known to express 2-3 x 10"

EGFr molecules/cell on the cell surface (25, 26), were alsostrongly reactive with mAb 225. Titration end points of mAb225 ranged from 10~5to 10~'°for EGFr+ carcinoma cell lines.

Short-term cultures of normal kidney epithelial cells derivedfrom three individuals and short-term cultures of keratinocytesobtained from normal skin were also reactive with mAb 225,although the titration end points were generally 10- to 100-foldlower than with most carcinomas (Fig. 2).

In contrast to our findings with carcinomas, EGFr could notbe detected on any of 14 cell lines of hematopoietic origintested, including leukemias and lymphomas with T-, B- andnull cell phenotype, as well as one erythroleukemia and onepromyelocytic leukemia. Four cell lines obtained by transformation of peripheral blood lymphocytes with Epstein-Barr virusand peripheral blood mononuclear cells were also EGFr" (Fig.

2).Among cell lines of mesenchymal origin 3 of 4 osteosarco-

mas, one pleomorphic rhabdomyosarcoma, and one synovialcell sarcoma were reactive with mAb 225. Six cultures of fetaland adult fibroblasts were tested and found to be EGFr* (Fig.

2).Biochemical Characterization. Immunoprecipitates obtained

from ['H]glucosamine-labeled cell extracts of a number of celllines (SK-MEL-178, SK-MEL-187, SK-MG-4, U251-MG, LA-N-l, SK-RC-7, T24, TE671, and A431) with mAB 225 were

analyzed by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis, and a single A/r 170,000 component or a doublet witha molecular weight of 170,000 and 150,000 was detected. A M,170,000 component was also immunoprecipitated from the '"I-labeled glycoprotein fraction of SK-RC-41 cells. No EGFrmolecules were detected in immunoprecipitation experimentswith mAb 225 using [3H]glucosamine-labeled extracts of 4different melanoma cell lines shown to be EGFr+ using theanti-mouse immunoglobulin rosetting assay. This discrepancyis probably related to a lower number of EGFr molecules/cellin the melanomas, which is reflected in lower antibody titersand was confirmed by the results of binding assays with 125I-

EGF (data not shown).Expression of EGFr in Fresh Normal and Tumor Tissues.

Frozen sections of a number of normal and tumor tissues weretyped for EGFr expression using the avidin-biotin immunoper-oxidase method, and the results are shown in Table 1. Amongnormal tissues, reactivity was found with both epithelial andnonepithelial cells. All types of epithelia tested expressed EGFr,including simple epithelium (stomach, small bowel, colon, en-docervix, endometrium, kidney tubules, pneumocytes), pseu-dostratified epithelium of the bronchus, transitional epitheliumof the bladder, and stratified epithelium (esophagus, exocervix,and skin) (Fig. 4). In squamous epithelia, reactivity was observed with cells in all layers (Fig. 4). mAb 225 was consistentlyreactive with epithelial cells in glandular structures: sebaceousand sweat glands in the skin; acini and ducts of the pancreas;and glands located in the bronchus, esophagus, colon, andprostate. However, reactivity was not restricted to epithelialcells: vascular endothelial cells were strongly reactive in alltypes of blood vessels in all tissues; in addition, smooth musclecells in the gastrointestinal tract, in blood vessel walls, and in

Table 1 Reactivity of mAb 225 and mAb 528 with normal and malignant human tissues

NormaltissuesSkinEpidermis

+°Sweat

glands+SebaceousglandstEsophagusEpithelium

+Glands+StomachEpithelium

+Glands+Small

bowelEpithelium+Glands

+ColonEpithelium

+Glands+PancreasAcini

+Ducts+IsletsLiverHepatocytes

+Bileducts+LungBronchus

+Alveoli+UterusEndometrium

+Myometrium+CervixExocervix

+Endocervix+KidneyGlomeruli

—Proximaltubule+Distal

tubule+Collectingtubule+BladderEpithelium

+ProstateGlands

+Smooth

muscle+Skeletal

muscle—Lymph

node—Endothelial

cellstBrainNeurons

—GlialcellsSympathetic

gangliaGanglioncells-Satellite

cells+Nervefibers +Malignant

tissuesPrimarymelanoma (5)*—Metastatic

melanoma (7)—Neuroblastoma

(2)—Primitive

neuroectodermal tumor (2)—Colon

carcinoma (4)+Kidney

carcinoma (6)+Lung

carcinoma (2)+Bladder

carcinoma (2)+Hepatocarcinoma

(1)+Prostate

carcinoma (1)+Adrenal

carcinoma (1)—Sarcoma

(2)+Chordoma

(2)

" +, positive reaction; —,no reaction.* Numbers in parentheses, number of specimens of each tumor type tested.

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DISTRIBUTION OF THE RECEPTOR FOR EGF

Fig. 4. Immunohistochemical analysis ofEGFr expression in normal and malignant tissues detected with mAb 225 (avidin-biotincomplex method), a, squamous epithelium ofnormal exocervix; b, squamous epithelium ofnormal skin; c, squamous carcinoma of thelung: (I. normal sweat glands. Sections werecounterstained with hematoxylin. Originalmagnification, x 400.

<+.•'.

the myometrium were EGFr+. In the central nervous system

neuronal and glial cells were unreactive. In sympathetic ganglia,satellite cells and nerve fibers were stained, whereas neuronswere EGFr". Nerve fibers were also stained elsewhere.

Tissues from a range of tumor types were tested for reactivitywith anti-EGFr mAbs. Most epithelial cancers were EGFr+,

including carcinomas of the kidney (6 of 6), colon (4 of 4), lung(2 of 2), bladder (2 of 2), prostate, and hepatocarcinoma. Twosarcomas were tested, and both expressed EGFr. Five primarymelanomas and 7 metastatic melanomas were tested and foundto be unreactive with mAb 225.

DISCUSSION

Expression of EGFr has previously been demonstrated forcultured human astrocytes, fibroblasts, vascular endothelialcells, astrocytomas, and carcinomas (reviewed in Ref. I), and'"I-EGF binding assays have shown considerable variation inthe number of EGFr molecules/cell (25-28) for these differentcell types. These studies have suggested that quantitative differences in the expression of EGFr may affect the biological effectsinduced by the growth factor on cells (13, 29).

The molecular structure and the role of EGFr in cell proliferation have been studied in considerable detail in A431 carcinoma cells ( 13, 29, 30) and in a small number of other culturedcell types (1, 31, 32). However, little is known about thedistribution of EGFr in normal and tumor tissues in vivo (33)and the role of the EGF/EGFr system in the control of growthand differentiation of normal cells. The development of mAbsdetecting growth factor receptors has provided probes for thestudy of their distribution and biological function.

In our study, EGFr expression was detected on a wide rangeof normal and tumor cell types using a rabbit anti-mouseimmunoglobulin resetting assay. In a small panel of cell linestested, reactivity with mAb 225 correlated well with the numberof EGFr molecules/cell detected in binding assays with 125I-

EGF (data not shown). Based on these results, and those ofother investigators (34), as few as 1000-3000 receptors/cell canbe detected using the rabbit anti-mouse immunoglobulin he-madsorption assay. Most normal and malignant epithelial cellswere EGFr* in culture and in tissue sections, as were most

mesenchymal cell types. On the other hand, none of the cells

of hematopoietic origin studied and only a proportion of neu-roectoderm-derived cells were EGFr* in culture or in tissue

sections. Similar findings in fresh tissues have been reported byGusterson et al. (33) in an immunohistochemical analysis ofthe reactivity of another mAb which detects human EGFr,EGFR1. The interpretation of the results of Gusterson et al. iscomplicated by the fact that antibody EGFR1 reacts with acarbohydrate determinant shared by Blood Group A structures(35). Gullick et al. (36) have recently reported the expressionof EGFr on squamous cell carcinomas, and Gusterson et al.(37) have found increased levels of EGFr in soft tissue sarcomasusing mAb EGFR1. The wide (although not universal) expression of the EGFr on cultured cells may indicate a role in cellproliferation. However, this is not substantiated by the immu-nohistochemical findings. Many cell types which are not actively proliferating were found to be EGFr+, including vascular

endothelial cells, glandular epithelial cells, smooth muscle cells,and peripheral nerve fibers. Therefore, the expression of EGFrmay not only be related to the state of cell proliferation, butalso to their cell-lineage derivation (as suggested by the difference between hematopoietic and nonhematopoietic cells) or thestage of differentiation within a given cell lineage.

The best evidence for a differentiation stage-restricted expression of EGFr comes from our analysis of normal and malignantcells of the melanocyte lineage. Expression of EGFr on melanoma cell lines was generally found to be associated withconstitutive expression of la antigens, an epithelioid or spindlecell morphology, and no or light pigmentation. In contrast,lack of expression of EGFr on cultured melanomas was generally associated with dendritic cell morphology, strong pigmentation, and expression of PAA, an antigen of mature melano-somes. Based on the studies of Houghton et al. a differentiationpathway for cells of the melanocyte lineage has been proposed(9). When melanomas were compared to melanocytes, well-differentiated melanomas shared phenotypic traits with normaladult skin melanocytes, while melanomas with an intermediatedegree of differentiation were more closely related to normalfetal or newborn skin melanocytes. Finally, Houghton et al.proposed that less-differentiated melanomas are phenotypicallyrelated to a melanocyte precursor cell, the melanoblast, whichhas so far remained unidentified.

While a proportion of cultured melanomas and neuroblasto-

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DISTRIBUTION OF THE RECEPTOR FOR EGF

mas were EGFr+, no reactivity was observed with any of the

melanomas or neuroblastomas tested by immunohistochemicalmethods. For immunohistochemical staining mAb 225 had tobe used at much higher concentrations (20 fig/ml) than forresetting assays (positive reactions at end-point concentrationsranging from 0.1 pg/ml to 100 ng/ml). This suggests that ourinability to detect EGFr expression on fresh melanoma tissuesis related to the lower sensitivity of the technique used.

The expression of EGFr by "early" melanomas could have at

least two different explanations. EGFr may indeed be a differentiation antigen of pigmented cells, which is only expressedby melanocyte precursors and early melanomas but lost at laterstages of melanocyte differentiation. Alternatively, expressionof the EGFr may be related to the transformation event givingrise to melanomas of early phenotype. Melanomas with otherphenotypes would arise through a different mechanism, notinvolving EGFr expression. EGFr expression in the EGFr+

melanomas could result from increased and/or abnormalexpression of the gene coding for EGFr. The gene coding forEGFr has been mapped to human chromosome 7 (20), andpolysemy (38, 39) and translocations (40) involving this chromosome, as described in several melanoma cell lines (38-40),could lead to increased gene copy number. Another mechanismthat might lead to increased gene copy number is gene amplification. The gene coding for EGFr has undergone amplification in A431 and in other cell lines derived from squamouscarcinomas (41-43), and this is accompanied by expression ofa high number of EGFr molecules in the cell surface. In A431cells the EGFr gene is also rearranged, resulting in the secretionof a truncated EGFr-related M, 95,000 protein (41, 44). Lib-ermann et al. (45) have shown amplification and increasedexpression of the gene coding for EGFr in fresh tumor tissuefrom a proportion of astrocytomas which express high levels ofEGFr, while Xu et al. (46) have not observed gene amplification,increased expression, or gene rearrangements in 5 EGFr+ car

cinoma cell lines. Our studies with normal and malignant cellsshowed that many tumor cells had EGF receptors which weredetectable with higher antibody dilutions, by one or two ordersof magnitude. This suggests that many malignant cells bearmarkedly increased numbers of EGFr. Studies aimed at determining whether gene amplification, increased expression, and/or gene rearrangements account for the differential expressionof EGFr in our melanoma cell lines are currently under way inour laboratory.

The distribution of EGFr in tissues and cell lines as definedin this study raises fundamental questions about some of thecurrent hypotheses concerning the biological function of theEGF/EGFr system. Our studies indicate that expression of theEGFr does not simply correlate with proliferative activity in allcell types. Instead, the biological effects of EGF upon interaction with its receptor seem to be cell-type specific. This view issupported by the inhibitory effect of EGF on acid secretion inthe stomach (47), and by the EGF-mediated induction of bothproliferation and contraction of rat aortic smooth muscle cells(48). For cells of the melanocyte lineage the effects of EGFmay be distinct at different stages of differentiation. The studypresented here with cells of this lineage provides a new experimental model to investigate the interplay of cell proliferation,transformation, and cellular differentiation.

ACKNOWLEDGMENTS

The authors wish to acknowledge Dr. A. N. Houghton for valuablecontributions. Dr. N. Dracopoli for valuable discussions, and Dr. H. F.Oettgen for continued support.

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1986;46:4726-4731. Cancer Res   Francisco X. Real, Wolfgang J. Rettig, Pilar Garin Chesa, et al.   Stage of DifferentiationCultured Cells and Tissues: Relationship to Cell Lineage and Expression of Epidermal Growth Factor Receptor in Human

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