Inability of Anti-epidermal Growth Factor Receptor Monoclonal Antibody to Block “Autocrine” Growth Stimulation in Transforming Growth Factor-secreting Melanoma Cells*

Mouse monoclonal antibodies to the human epider- mal growth factor (EGF) receptor were raised by im-munizing with plasma membrane vesicles prepared from A431 cells. This paper describes the characteri- zation of one of the IgG anti-receptor monoclonal antibodies generated and its use to probe the role of transforming growth factor (TGF) in the autonomous growth of a melanoma cell line in culture. This anti- body blocks: 1) the binding of 12’I-EGF to the A431 EGF receptor; 2) the EGF stimulation of the EGF- dependent protein kinase in vitro; and 3) human fibroblast DNA synthesis and proliferation in culture. It can precipitate the EGF receptor from metabolically labeled A431 cells and human fibroblasts and these re- ceptors have indistinguishable peptide maps. No EGF receptor could be detected by immunoprecipitation after fibroblasts were treated with EGF or conditioned medium from the melanoma cells which secrete EGF- like TGF (aTGF). The antibody itself did not down-regulate the receptor but could block down-regulation caused by EGF and aTGF. Despite its ability to block EGF-stimulated growth and down-regulation in fibro- blasts, the antibody was unable to block the growth and soft agar colony formation of aTGF-secreting melanoma cells, nor could the antibody detect EGF recep- tor in these cells under the conditions developed to prevent down-regulation

Mouse monoclonal antibodies to the human epidermal growth factor (EGF) receptor were raised by immunizing with plasma membrane vesicles prepared from A431 cells. This paper describes the characterization of one of the IgG anti-receptor monoclonal antibodies generated and its use to probe the role of transforming growth factor (TGF) in the autonomous growth of a melanoma cell line in culture. This antibody blocks: 1) the binding of 12'I-EGF to the A431 EGF receptor; 2) the EGF stimulation of the EGFdependent protein kinase in vitro; and 3) human fibroblast DNA synthesis and proliferation in culture. It can precipitate the EGF receptor from metabolically labeled A431 cells and human fibroblasts and these receptors have indistinguishable peptide maps. No EGF receptor could be detected by immunoprecipitation after fibroblasts were treated with EGF or conditioned medium from the melanoma cells which secrete EGFlike TGF (aTGF). The antibody itself did not downregulate the receptor but could block down-regulation caused by EGF and aTGF. Despite its ability to block EGF-stimulated growth and down-regulation in fibroblasts, the antibody was unable to block the growth and soft agar colony formation of aTGF-secreting melanoma cells, nor could the antibody detect EGF receptor in these cells under the conditions developed to prevent down-regulation and lysosomal degradation of the EGF receptor. These studies suggest that these melanoma cells do not have the intact EGF receptor and that the secretion of aTGF by these cells plays no role in their growth in culture. The absence of receptor cannot be explained by down-regulation by secreted aTGF.
A variety of transformed cell lines have been shown to secrete growth factors (1). A subset of these growth factors are capable of allowing nontransformed target cells to assume anchorage-independent growth. Such mitogens have been operationally defined as TGF' (for review, see Ref. 2). One class * This work was supported by grants from the National Cancer Institute (Canada) and Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$Medical Scholar of the Canadian Life and Health Insurance Association. To whom reprint requests should be addressed.
of TGFs are capable of competing with EGF for binding to the EGF receptor (3-8) and these have been termed aTGFs (2). Blocking of the EGF receptor by anti-EGF receptor antibodies can block the stimulation of anchorage-independent growth of target cells by exogenously added aTGF (9). Therefore, the EGF receptor plays an integral role in the mechanism of action of aTGF. The mechanism by which the EGF receptor contributes to the TGF-induced transformed phenotype is not known. However, the EGF receptor shares properties with the products of a variety of oncogenes in that it contains a domain with intrinsic protein kinase activity (10, 11) capable of phosphorylating its substrates at tyrosine residues (12). The activity of the kinase is stimulated similarly by EGF and aTGF (13,14). The ability of this tyrosine kinase to phosphorylate antibodies to ~60"'" (15, 16) and sequence data revealing homology with the v-erb-B oncogene protein (17) imply a structural relationship between the gene for the EGF receptor and the src-related subset of oncogenes. Therefore, the hypothesis has arisen that a cell capable of synthesizing an aTGF and the EGF receptor would, by an "autocrine" route, stimulate the activity of the EGF-dependent protein kinase and initiate the cascade of events that would ultimately lead to autonomous growth independent of exogenous signals (2,7).
To test whether we could intervene in this process of autostimulation, we made use of a monoclonal antibody we recently raised to the EGF receptor. In this paper, we show that this antibody precipitates the EGF receptor and behaves as a competitive inhibitor of EGF action. It competes with EGF for binding to the human EGF receptor, blocks EGF stimulation of the EGF-dependent protein kinase, blocks EGF stimulation of human fibroblast growth, and blocks EGFstimulated down-regulation of its receptor. We determined the effect of this antibody on the growth of a human melanoma cell line that secretes aTGF (8, 18) and on the downregulation of their EGF receptor. We show that the EGF receptor in these aTGF-secreting cells is not detectable by our antibody by biologic and biochemical criteria. The antibody cannot block the autocrine growth stimulation and the autocrine down-regulation of the receptor, nor can it detect the receptor in these cells under conditions where EGFstimulated lysosomal degradation of the receptor is blocked. These findings are compatible with the interpretation that the growth of this melanoma cell line in culture does not depend on the interaction of secreted aTGF with the EGF receptor.

EXPERIMENTAL PROCEDURES
Materials-Culture media and serum were from Gibco. Mouse EGF was purified from male Swiss Webster mouse (Simonsen) submaxil-lary glands (19 Preparation of Monoclonal Antibodies to the EGF Receptor-The monoclonal antibody to the human EGF receptor on A431 plasma membranes was prepared essentially as described by Galfr6 and Milstein (21) with slight modification. Purified A431 plasma membrane vesicles were used as immunogen on BALB/c mice and the immunization schedule was according to that reported for high frequency of antigen-specific hybridoma production (22). Briefly, 100 pg of membrane protein in Freund's complete adjuvant was injected subcutaneously into the recipient mouse on day 0 as the primary dose, followed by 20 pg of membrane proteins in Freund's incomplete adjuvant injected subcutaneously and intramuscularly on days 14 and 28. From days 42-48, smaller amounts of membrane protein in PBS were injected intraperitoneally and intravenously by the schedule of Stihli et al. (22). On day 49, spleen cells from the immunized mouse were fused with the myeloma cell line Sp2/0 (kindly supplied by Dr. J. Falk, University of Toronto). The fused cells were seeded into five 96-well tissue culture plates containing an unimmunized spleen cell feeder layer. The culture medium was further supplemented with 10 pg/ml insulin and 2 mM glutamine. Positively growing hybridoma cells were seen after 10 days in greater than 90% of the wells. The culture medium was screened for antibody capable of interfering with "'I-EGF binding to formaldehyde-fixed A431 cells as described below. Fifteen hybridoma cultures were identified and these were cloned by limiting dilution at least twice prior to freezing. Three clones of these hybridomas were selected and used in this study. Antibodies were purified (23, 24) from culture media conditioned by these clones or from the ascitic fluid produced in syngeneic mice injected with the hybridoma cells.
EGF Radioreceptor Assay and Hybridoma Screening-The standard assay was essentially as described previously (5,25). Briefly, 40,000 A431 cells were formaldehyde-fixed in 24-well plates (Costar). Antibody or EGF, dissolved in binding buffer consisting of PBS containing 0.1% BSA and 0.1 M Hepes, pH 7.4, was added into the wells with 9 -E G F (20,000 cpm; specific activity, 160 pCi/pg) in a total volume of 200 pl. Incubations were for 2 h at 22 'C and were followed by removal of unbound counts with binding buffer. The bound counts were removed with 0.1 M NaOH containing 1% SDS and the radioactivity was determined by a y counter with 45% counting efficiency. For screening of hybridoma culture supernatant for antibodies that interfered with '=I-EGF binding, 20,000 A431 cells per well were plated in 96-multiwell plates (Costar). Formaldehyde fixation and the binding reactions were the same as with the larger well plates except that washing of unbound counts was facilitated using a cell harvester (Titertek, Flow Laboratories). the wash was for 20 5.
EGF-stimulated Protein Kinase Activity-Protein kinase activity in intact A431 plasma membranes was assayed as previously described (11). Briefly, the membrane pellet was suspended in a 50-rl volume pH 7.2, 1 mM MnC12, 0.01% BSA with or without 0.3 p~ EGF, and of reaction buffer containing a final concentration of 20 mM Hepes, various concentrations of monoclonal antibody. After an initial 30min incubation in an ice bath, the reaction was initiated by the addition of [y-3ZP]ATP (2 pCi, 0.5 p~) and terminated 10 min later by the addition of 6 X Laemmli (26) sample buffer and boiling for 5 min. The extent of phosphorylation was determined by running the reaction mixture on a 7.5% SDS-polyacrylamide gel (26) followed by autoradiography. Molecular weight markers, run in a parallel lane, were myosin, @-galactosidase, phosphorylase B, BSA, and ovalbumin.
Metabolic Labeling of Cells-Monolayer cultures were prepared by seeding 5 X lo6 melanoma cells, 5 X 10' foreskin fibroblasts, or 1.5 X IO4 A431 cells in complete growth medium onto 10-cm, 6-cm, and 3.5-cm tissue culture plates, respectively. After an overnight incubation, the plates were washed 3~ with PBS and labeled for 16 h with [%]methionine (specific activity, 1220 Ci/mmol) in methionine-free F12 medium containing 1% fetal bovine serum. Melanoma cells were labeled with 500 pCi/ml of [%]methionine, foreskin fibroblasts with 100 pCi/ml, and A431 cells with 50 pCi/ml. When indicated, EGF, anti-EGF receptor monoclonal antibody, methylamine (10 mM), or tunicamycin (0.5 pg/ml) was added to the labeling medium.
Melanoma Cell-conditioned Medium-A2058 melanoma cells were grown to confluence (5 X 10" cells/plate) on 1O-cm plastic plates in F12 with 10% fetal bovine serum. The medium was aspirated and the cells were washed 3X with PBS and incubated in serum-free F12. Conditioned medium was collected every 4 days and replaced with fresh F12. The first collection was discarded. The medium was concentrated by lyophilization followed by reconstitution with and dialysis against 1 M acetic acid. The acetic acid was removed by lyophilization and the residue was reconstituted with F12 at %o the original volume. After dialysis against F12, this medium was used as described.
Down-regulation of the EGF Receptor-Human foreskin fibroblasts (5 X 106) or melanoma cells (5 X lo6) were labeled with ["Slmethionine as described above in the presence or absence of BlD8 antibody (100 pg/ml), EGF (1 X lo4 M), or both, as indicated. In some experiments, the fibroblasts were first labeled with [8SS]methionine for 16 h as above, then washed 3X with PBS containing 0.1% BSA. The labeled cells were then incubated with serum-free F12 medium previously conditioned by melanoma cells or with unconditioned F12 medium with or without B1D8 antibody.
Immunoprecipitation of EGF Receptor-After labeling the cells with [=S]methionine using the protocols described above, the monolayer was washed 3X with PBS containing 2 mM EDTA. The cells were then solubilized in RIPA buffer containing 5 mM EDTA (11). After removal of nonsolubilized material by centrifugation at 100,000 X g for 30 min, the EGF receptor was immunoprecipitated quantitatively as described previously (11). Briefly, monoclonal antibody was added at a final concentration of 10" M and incubated for 30 min. Pansorbin, as a 10% suspension, was added to the tubes and incubated for 30 min. The bacterial pellet was washed by centrifugation through a 1 M sucrose cushion, then sequentially with RIPA buffer, 0.5 M NaCl, and HZ0 prior to electrophoresis of the immunoprecipitated labeled proteins on a 7.5% SDS-polyacrylamide gel (26). The gel was fixed, stained (26), and treated with ENHANCE (New England Nuclear) prior to drying. Autoradiographic exposure was carried out at -75 "C.
Two-dimensional Tryptic Peptide Analysis-Tryptic peptide mapping of [36S]methionine-labeled EGF receptor from A431 cells and human foreskin fibroblasts was performed on receptor protein eluted from heat and vacuum-dried unfixed and unstained 7.5% polyacrylamide gels (16). The eluted protein was concentrated and digested with trypsin as described by Beemon and Hunter (27) and 15,000 cpm of the "S-tryptic peptides were resolved by electrophoresis followed by ascending thin-layer chromatography in the second dimension as described by Hunter and Sefton (28). After drying, the thin-layer plate was chromatographed at 37 "C in isopropyl alcohol containing 200 mg/ml 2,5-diphenyloxazole. This step did not alter the position of the peptides. The peptides were located by fluorography by exposure of the TLC plates to x-ray film at -75 "C.
Cell Growth Assays-Human foreskin fibroblasts from confluent plates were distributed into 96-well plates (Costar) at a density of 5000 cells/well in F12 with 10% fetal bovine serum. After 18 h, the medium was replaced with 180 pl of F12 containing 0.2% fetal bovine serum. Forty-eight h later, various concentrations of EGF and monoclonal antibody were added to each well (six replicates) in 20 p1 of F12 containing 0.1% BSA. After 18 h, 2.5 pCi of [3H]thymidine (78.2 Ci/mmol) was added to each well. Six h later, the medium was replaced with 50 pl of a 0.25% trypsinization solution and incubated for 15 min at 22 "C. The cells were collected onto glass fiber filters using a cell harvester with a water wash. Radioactivity on the filters was determined by scintillation counting using an Omnifluor (New England Nuclear) in toluene scintillation fluid.
The experiments to determine the effects of antibody on cell proliferation were initiated by seeding 20,000 fibroblasts or A2058 cells into triplicate 35-mm plates in F12 with 10% fetal bovine serum. After 18 h, the medium was replaced with F12 containing various concentrations of monoclonal antibody and EGF. NO serum was added to the melanoma cell medium; 2% fetal bovine serum was added to the fibroblast medium. Cell numbers were determined 7-10 days later using a Coulter Counter (Hialeah, FL), model ZP after trypsinization of the monolayer.
Colony-forming Assay-EGF receptor antibody was dissolved at final concentrations of 0, 1, 10, 100, or 200 &g/ml in F12 culture medium containing 10% fetal bovine serum, 0.3% Agar Noble, and 2 X 10' A2058 cells and added to 35-mm culture plates on top of a 1ml base layer of 0.5% agar in F12 containing 10% fetal bovine serum. The plates were incubated at 37 "C in a humidified atmosphere containing 95% air and 5% CO,. The plates were refed after 7 and 14 days with 1 ml of 0.3% agar in the same medium. After 21 days, the colonies were counted or photographed unfixed and unstained at lowpower magnification. Colonies greater than 20 cells were counted as positive.

RESULTS
Three hybridomas producing monoclonal antibody raised against A431 plasma membrane vesicles and selected for their ability to interfere with '251-EGF binding to EGF receptors were stabilized and characterized. One of the hybridomas produced IgG, and two IgG,. In this communication, we describe in some detail the characterization of an anti-EGF receptor I&, secreted by hybridoma BlD8.
The ability of the B1D8 antibody to compete with Iz5I-EGF for binding to formaldehyde-fixed A431 cells is shown in Fig.  1. Half-maximal inhibition of binding occurred at an antibody concentration of 2 X IO-* M (3 pglml). A molar concentration of 30-fold more antibody than cold EGF was required for equivalent displacement of '9-EGF.
The antibody was capable of precipitating the M, = 170,000 receptor from A431 cells labeled with [35S]methionine and from normal human foreskin fibroblasts (Fig. 2). However, no EGF receptor could be precipitated from human melanoma cells even when conditions were arranged to obtain 300-fold more label in protein than was used for A431 cells and 50fold more than for the fibroblasts. To determine whether the fibroblast and A431 EGF receptors are structurally similar, peptide maps were made of the M, = 170,000 proteins isolated by immunoprecipitation and SDS-polyacrylamide gel electrophoresis (Fig. 3). The two-dimensional peptide maps of the A431 and fibroblast receptors were virtually indistinguishable. The antibody could precipitate a nonglycosylated M , = 130,000 protein from A431 cells labeled in the presence of tunicamycin indicating that the antibody recognizes the protein backbone of the receptor (data not shown).
Because B1D8 was capable of competing with EGF for binding to the A431 EGF receptor, we determined the effect of the antibody on the EGF-dependent protein kinase (Fig.  4). A431 plasma membrane vesicles were incubated with various doses of antibody with or without EGF and the ability of  the EGF-dependent protein kinase to phosphorylate the EGF receptor in uitro was assessed. At a concentration of 100 pg/ ml (0.7 p~) , the antibody could completely block the stimulation of the kinase activity by EGF.
EGF has been shown to cause the down-regulation of its own receptor (29). After binding of EGF to the receptor, the EGF-receptor complex is internalized with ultimate degradation of the ligand receptor in lysosomes (30)(31)(32)(33). To determine the effect of the antibody on this process, normal human fibroblasts were incubated with or without antibody and EGF Autocrine Growth Overnight incubation with antibody at 100 pg/ml had no effect on the receptor. However, when cells were incubated with EGF and antibody, receptor down-regulation was completely blocked (Fig. 5).
Using '''I-EGF binding as a means of detecting EGF receptors, it has been observed that cells that secrete cuTGFs do not bind EGF (34). It has been suggested that the inability to observe receptors in cuTGF-secreting cells results from "autocrine" down-regulation of the receptors (25). Since B1D8 antibody is capable of blocking EGF-stimulated down-regulation in fibroblasts, we determined whether we could block the down-regulation of the fibroblast receptor by melanomaconditioned medium. The serum-free conditioned medium of the melanoma cells collected after 4 days of incubation of confluent cells contains EGF-displacing activity, as determined in a radioreceptor assay, equivalent to 10"' M EGF (data not shown). This melanoma-conditioned medium causes only partial down-regulation of fibroblast EGF receptors (Fig.  5). When the melanoma-conditioned medium was concentrated 10-fold, down-regulation of fibroblast EGF-receptor was nearly complete and B1D8 could block this process (Fig.  5). While the antibody could block down-regulation of fibroblast receptors in the presence of lo-' M EGF and in the presence of aTGF (concentrated melanoma cell-conditioned medium), incubation of antibody with melanoma cells did not result in the appearance of immunoprecipitable EGF receptor (Fig. 5).
The mechanism by which EGF causes the disappearance of EGF receptor involves lysosomal degradation of the internalized protein. Methylamine has been shown to completely block this proteolysis (33). We therefore attempted to observe melanoma EGF receptor in cells treated with methylamine.
No M , = 170,000 protein could be specifically precipitated by B1D8 under these conditions (Fig. 5). The inability to detect receptor in melanoma cells with B1D8 antibody raised the possibility that the melanoma receptor is immunologically distinct from the A431 and fibroblast receptors. We used our two other monoclonal antibodies in a similar attempt to detect  fibroblasts (lanes 1-9) and melanoma cells (lanes 10-13) were metabolically labeled as described in Fig. 2 with certain additions. The EGF receptors were precipitated using B1D8 antibody, run on 7.5% polyacrylamide gels, and detected by autoradiography. EGF receptor in these cells without success (data not shown).
The effect of B1D8 on EGF-stimulated DNA synthesis in human fibroblasts was assessed. Fibroblast DNA synthesis was stimulated in a dose-dependent manner by EGF (Fig. 6). Co-incubation of these cells with varying doses of antibody and EGF resulted in a shift in the dose-response curves such that higher doses of EGF were required in the presence of antibody to give DNA synthesis comparable to cells without antibody.
The antibody could also block EGF-stimulated fibroblast proliferation. Fibroblasts in 2% fetal bovine serum stimulated with 10"' M EGF achieved a higher cell density than cells not exposed to EGF. Antibody blocked this EGF-stimulated proliferation in a dose-dependent manner with 200 pglml(l.3 p~) blocking the EGF effect by greater than 70% (Fig. 7). In contrast, the same doses of antibody were unable to block the 10-fold increment in melanoma cell density achieved after 10 days of incubation in serum-free medium (Fig. 7).
TGFs can cause appropriate target cells to grow in an anchorage-independent manner. The melanoma cells, like many tumor cell lines, are also capable of colony growth in soft agar. Because these cells secrete aTGF, it has been assumed that aTGF imparts this ability to these cells. Since anti-EGF receptor antibodies have been shown to block cuTGF-stimulated anchorage independence when TGF is added exogenously to the cultures (91, we tested whether our anti-receptor antibody could block the autocrine effect of cuTGF. Various doses of antibody were added to the melanoma cells suspended in soft agar and the effect on colony number and size was noted. Up to a dose of 200 pg/ml (1.3 p~) antibody, no effect on colony formation was observed. The effect of 200 pg/ml antibody is represented in Fig. 8.

DISCUSSION
In this communication we present evidence that the monoclonal antibody B1D8 is directed a t a determinant in or near the binding site of EGF on the human EGF receptor. Thus, this antibody can interfere with the binding of EGF to the receptor, preventing a variety of effects of EGF on its target cells. In uitro, the antibody can block the activation of EGFdependent tyrosine-specific protein kinase by EGF and in normal human fibroblasts can block EGF-stimulated DNA synthesis, proliferation, and EGF receptor down-regulation. This antibody differs from those described by Schreiber et al. (35) which mimic EGF action. The antibody can also be used to precipitate the EGF receptor from A431 human epidermoid carcinoma cells and normal human fibroblasts. Peptide maps of the receptor from the cancer cells and normal fibroblasts are indistinguishable. Others have shown that the EGF receptors from A431 cells and human placenta have similar peptide maps (17). Thus, the human EGF receptor appears to be similar in normal and cancer cells and does not appear to vary with the embryologic origin of the tissue. Despite this apparent conservation of the receptor, no receptor could be immunoprecipitated from human melanoma cells. It had been hypothesized that these melanoma cells lack "'I-EGF cell surface binding sites because the receptors were occupied by aTGF which resulted in autocrine growth stimulation and autocrine down-regulation of the receptor (34). However, conditioned medium from these melanoma cells, which contains aTGF, could cause only a barely detectable loss of EGF receptors in normal fibroblasts. When the aTGF was concentrated, it could cause complete down-regulation of the fibroblast receptor. Thus, the concentrations of EGF-displacing activity in the unconcentrated conditioned medium of the melanoma cells, although a t a level adequate to give nearmaximal fibroblast growth, was insufficient to cause disappearance of fibroblast receptors. Furthermore, B1D8 antibody could block down-regulation of fibroblast receptor by lo-' M EGF and the aTGF secreted by the melanoma cells. If the absence of EGF receptor in melanoma cells was a result of interaction of secreted aTGF with cell surface EGF receptor, then it could be predicted that antibody could block this down-regulation. Nevertheless, incubation of melanoma cells with the antibody did not result in the emergence of the EGF receptor. Since newly synthesized receptor and aTGF are processed as secretory proteins, it is possible that the receptor and aTGF interact within the secretosomes, initiating the process of down-regulation and mitogenic stimulation prior to emergence on the cell surface. However, the subsequent degradation of the receptor would be expected to proceed as if the growth factor-receptor interaction had occurred on the cell surface, that is via the lysosomes. Methylamine, through its action on lysosomes, has been shown to prevent the disappearance of immunoprecipitable EGF receptor in fibroblasts incubated with EGF (33). However, incubation of melanoma cells with methylamine also failed to allow the detection of the receptor, indicating that these cells do not express sufficient levels of receptor to be detected by immunoprecipitation. Recently, a wide variety of human tumor cells probed for the expression of several oncogenes was found not to express the erb-B gene (36). Since this gene codes for a portion of the EGF receptor (17), it can be concluded that these cancer cells were not expressing the erb-B domain of the EGF receptor even though their parental cells probably did. It would be important to confirm, using hybridization techniques, whether this line of melanoma cells has also discontinued expressing the gene for the erb-B and EGF-binding domains of the EGF receptor.
Our inability to detect EGF receptor synthesis in these melanoma cells by immunoprecipitation may have resulted from a level of receptor below our detection limits (about 500 receptors per cell). However, if these cells were synthesizing cell surface receptor which was necessary for their autonomous growth, then blocking of the EGF-binding site of the receptor, regardless of number, with antibody should have impaired the growth of these cells in serum-free medium or as soft agar colonies. The failure of the antibody to block the growth of these aTGF-secreting cells suggests that there were no EGF receptors on these cells accessible to the monoclonal antibody or that, if present, blocking of EGF receptors by antibody had no effect on growth. The possibility that aTGF acts through a mechanism distinct from the EGF receptor has been suggested. Affinity labeling of aTGF membrane binding proteins labeled both the EGF receptor and a M, = 60,000 membrane protein, the latter not compatible with EGF (37). This finding suggested the existence of an aTGF receptor distinct from the EGF receptor. However, subsequent experiments using more highly purified aTGF have failed to reveal the M, = 60,000 protein (38) and antibodies to the EGF receptor are able to block colony formation by target cells stimulated by the addition of purified aTGF to the medium.
The data now suggest that the effects of aTGF are mediated entirely through the same receptor as for EGF. We interpret the failure to detect EGF receptor in aTGFsecreting melanoma cells by immunoprecipitation or biologic methods to imply that these cells do not synthesize the receptor or at least the domain of the receptor that binds EGF. Alternatively, the receptor synthesized in these cells is immunologically distinct from the receptor in A431 cells and fibroblasts. However, the similarity of receptor from different human sources and the failure of three distinct monoclonal antibodies to detect the receptor makes this possibility somewhat less likely. If indeed this cell line does not synthesize EGF receptor, the autonomous production of the EGF-like growth factor may not be necessary for the autonomous growth of these cells in culture. However, these cells probably produce other growth factors which do not act through the EGF receptor (flGF) and these may play an autocrine role in the autonomous growth. In the host, however, the production of aTGF may play an important paracrine role for the tumor by recruiting the normal surrounding cells to provide support for tumor cell growth such as the induction of angiogenesis.