Role of Epidermal Growth Factor in Carcinogenesis

(CANCER RESEARCH 46, 1030-1037, March 1986]

Christa M. Stoscheck1 and Lloyd E. King, Jr.2

Veterans Administration Medical Center Research Service, Nashville 37203, and Department of Medicine, Division of Dermatology, Vanderbilt University Medical Center, Nashville, Tennessee 37232

Abstract rylates certain proteins on their tyrosine residues. Another mech anism may involve receptor clustering or a combination of clus For cell growth and division to occur, a large variety of meta tering and kinase activity which, in turn, produces an intracellular bolic processes must be carefully coordinated in the cell. Through signal, (b) Modulation of EGF-stimulated receptor activity (auto- evolutionary pressures, specific hormones and growth factors have acquired the ability to trigger a complex coordinated "pleio- regulation) occurs by at least three modes. First, binding and tropic growth response" in their target cells. This complex re kinase activities may be inhibited by phosphorylation of certain regulatory sites of the receptor. Second, receptor activity may sponse is mediated by specific cellular receptors and intracellular be reduced by receptor degradation following its interaction with messengers. Teleologically then, it makes sense that in onco- EGF. Third, sequestration of the EGF receptor in intracellular genesis this growth regulating network is utilized by the produc compartments may prevent EGF activation of this pool of recep tion of proteins which mimic growth factors, the activated form tors. of their receptors or, the messengers themselves. Several lines Several lines of evidence indicate that the EGF-stimulated cell of evidence indicate that the epidermal growth factor-stimulated regulatory system may play a role in carcinogenesis. This will be growth regulatory system is involved in cellular proliferation, both discussed in detail below. In brief, the first line of evidence normal and neoplastia Some of the effects of epidermal growth indicates that EGF directly elicits transformation-associated phe- factor in carcinogenesis are separable from its direct, growth notypes in certain target cells. Many of these effects are revers stimulatory effects. Thus, the role of epidermal growth factor in ible upon the removal of EGF, but in a small but highly significant carcinogenesis is more complex than is its role in stimulating number of already partially transformed cells, they are not re growth. versible; e.g., EGF has been observed to potentiate chemical transformation in vivo and viral transformation in vitro. The Introduction second line of evidence that EGF plays a role in carcinogenesis is that many cancer cells produce a peptide, termed Â«-TGF, EGF3 is a M, 6045 polypeptide which can stimulate or inhibit which is structurally related to EGF and activates the EGF proliferation or differentiation of a wide variety of cells (1-3). receptor. However, to date, it is not definitively determined While EGF is thought to play a role in almost every tissue in the whether a-TGF is essential for the transformed properties of its body both during development and in the adult, the exact nature parent cell. Another, albeit indirect, line of evidence is that EGF of this role is not clear. The inability to experimentally lower EGF depresses the immune system whose function is to destroy levels in mice by removing the salivary glands (4), the major site newly arising tumors (immune surveillance). The EGF-stimulated of EGF synthesis in the mouse (5), has hampered clarification of cell regulatory system could conceivably also be activated by a the role of EGF. This inability to alter EGF levels surgically is mutation of the receptor eliminating the requirement for elevated probably due to EGF being synthesized at several anatomical EGF (or TGF levels). This may be the mechanism by which sites in the body (6-9), where EGF functions more in a paracrine erythroblastosis or lymphoid leukosis viruses cause a variety of rather than an endocrine manner. Additionally, the use of anti cancers including erythroblastosis, sarcomas, and carcinomas. bodies against EGF are unlikely to lower EGF levels because of Also discussed are data which contradict the hypothesis that the the very fast turnover rate (half-life, 1.5 min) of EGF in vivo (10). EGF-stimulated cell regulatory system plays a role in carcinogen Since a specific receptor located at the cell surface is required esis. in order for EGF to interact with the cell, the availability of antisera against the EGF receptor which blocks EGF binding (11) offers a new approach in which to deplete cells of an EGF signal. EGFElicitsTransformation-associatedPhenotypesinNormal The EGF receptor is an integral membrane protein with many Cells complex functions which have recently been reviewed in detail elsewhere (12). Briefly, these functions fall into two categories; The addition of EGF to normal cells elicits certain responses signal transmission and autoregulation. (a) Signal transmission which are associated with neoplastic transformation. For exam may involve an intrinsic EGF-stimulated kinase which phospho- ple, EGF induces a partial loss of density dependent inhibition of growth and the dependency on serum for growth (2,13,14), an Received 5/28/85; revised 9/16/85; accepted 11/6/85. 1Recipient of a grant from the Veterans Administration Merit Review Board, increase in the level of phosphotyrosine in proteins (15-18), and American Cancer Society Institutional Research Grant IN-25X, and NIH Biomedicai an increase in cellular metabolism including sugar and amino Research Support Grant RR-05424. To whom requests for reprints should be acid transport, ATP turnover, and ornithine decarboxylase activ addressed. 2 Recipient of grants from the NIH (USPHS Grant AM 26518 and Biomedicai ity (19-20, 52, 53). EGF induces the expression the mRNA of c- Research Support Grant RR-05424), and funded by the Veterans Administration. fos and c-myc genes, cellular proto-oncogenes (22). Cellular 'The abbreviations used are: EGF, epidermal growth factor; TGF, transforming protooncogenes are the normal cellular homologs of viral onco- growth factor; PDGF, platelet-derived growth factor; TPA, 12-O-tetradecanoylphorbol-13-acetate; pp60, M, 60,000 phosphoprotein; p28, M, 28,000 protein; p21, genes. Oncogenic viruses are thought to have acquired the M, 21,000 phosphoprotein. oncogene from normal cellular RNA. Through uncontrolled

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1986 American Association for Cancer Research. EGF AND CARCINOGENESIS expression (plus, in some cases, mutation as well) they induce transformed by the Kirsten murine sarcoma virus as is their EGF uncontrolled growth in the host cell (23). EGF also elicits certain receptor-positive parental cell line (70), indicating that a-TGF responses which are associated with cancer such as the loss of production may be a circumstantial consequence of cellular fibronectin, and an augmentation of the secretion of plasminogen transformation. activator (24-27). Growth of cells in soft agar, considered by The production of autostimulatory growth factors by cancer many (28-30) to be one of the best assays for showing the cells appears to be a general phenomenon. For example, simian tumorigenicity of a cell, is potentiated by EGF or EGF-like factors sarcoma virus-transformed cells, glioma, rhabdomyosarcoma, (31-33). This potentiation by EGF is even greater for partially and human osteosarcoma cells produce PDGF or PDGF-like transformed cells (34) and tumor cells (35, 36). Thus, EGF or proteins (71-73). Although PGDF alone does not cause cellular EGF-like proteins contribute toward the malignant phenotype of transformation, its homologue, the v-s/s-transforming protein, transformed cells in vivo. As discussed below, certain trans does (74-76). Other examples of autostimulatory growth factors formed cells produce EGF-like proteins which may feed back to are the production of nerve growth factor or an insulin-like growth the cell and elicit certain transformation-associated phenotypes. factor by melanoma cells, fibrosarcoma, and mammary tumor cells (77-81). High levels of tumor-produced growth factors can

Interaction of Transforming Growth Factors with the EGF cause complications in tumor patients such as hypoglycemia and hypercalcemia (82-84). Receptor Several different "a-TGFs" were detected in human urine (29, Some, although not all, transformed cells produce EGF-like 30, 85). Two of these were present at significantly higher levels proteins, termed a-TGFs, which interact with the EGF receptor. in the urine of cancer patients than in normal humans. These These TGFs mimic most, if not all, of the actions of EGF. For patients had disseminated cancers such as small cell lung, renal, example, a-TGFs bind with high affinity to the EGF receptor, colon, and breast carcinomas, as well as melanomas and sar stimulate EGF receptor autophosphorylation, cause down regu comas. In vitro incubation in acid of the larger molecular weight, lation of the EGF receptor, stimulate cell growth, and promote cancer-associated TGF produced the smaller cancer associated precocious tooth eruption and eyelid opening (31, 32, 34, 37- TGF, indicating that both forms are derived from the same 42). Structural similarities have been shown between human and molecule. One of the other molecules exhibiting a-TGF activity rat a-TGF with human and mouse EGF (40 and 44% sequence was identified to be urogastrone, and the identity of the other homology, respectively (43-46). The DNA sequence encoding a- factors are not known but may be precursor forms of urogas TGF was observed in normal cells (46), indicating that it is an trone (86). These latter factors did not exhibit higher levels in the unmodified normal protein which is expressed in cancer cells in urine of cancer patients. Since it is currently not known how to abnormal quantities and/or at the wrong stage of the cells' differentiate TGF from EGF activities, immunological or sequenc development. a-TGF is not an EGF-like peptide from precursor ing data are necessary to identify these factors as EGF or TGF EGF (47, 48). The production of these EGF-like TGFs, by virus, related molecules. The implication of these clinical studies is that chemical, and spontaneously transformed cells may be respon (a) human cancer cells may produce their own growth factors in sible for the loss of EGF binding after transformation (34,38, 41, vivo; and (b) measurement of these factors in urine may serve 51,67-69,137,138,146). as a diagnostic tool. a-TGFs promote the growth of cells in soft agar. For some cell lines, a second transforming growth factor termed 0-TGF, EGF Promotion of Viral Carcinogenesis which is structurally and functionally unrelated to EGF, is addi tionally required, although for other cell types it may be inhibitory EGF enhances viral transformation of cells (87). Rat embryo (56). The combination of the two TGFs is referred to in the older cells infected by adenovirus formed almost 5 times as many literature as sarcoma growth factor. 0-TGF production is induced colonies in soft agar when EGF was present. Additionally, the by viral transformation in cells but is not a viral product itself colonies appeared sooner, grew faster, and had a more diffuse and, in fact, is found in certain nontransformed normal cells such morphology than did non-EGF treated cells. These effects were as platelets, submaxillary gland, kidney, liver, muscle, heart, very similar to those produced by 12-O-tetradecanoylphorbol- brain, and placenta (57-62). a-TGFs are also not viral products 13-acetate, a classic tumor promotor. Classic tumor promotors and have been detected in normal adult urine (29, 30), as well have no or very weak tumorigenicity by themselves but can as in embryos and placenta (63-66). In fact, a-TGF may be a markedly enhance the carcinogenicity of low doses of chemical fetal form of EGF with its expression derepressed in transformed carcinogens. The mechanism by which tumor promotors and cells in manner similar to that of a-fetoprotein. Nevertheless, EGF may enhance carcinogenesis may in part be due to stimu since autoproduction of TGFs should confer a growth advantage lation of cell growth. to the cancer cells which produce it, it might be expected that EGF plays even a more active role in the transformation of TGFs should be found in rapidly growing cancers. cultured granulosa cells by the Kirsten murine sarcoma virus Several lines of evidence indicate that TGFs may play a role (88). In the absence of EGF, infected cells formed few transfor in the cellular transformation process. Abelson sarcoma virus- mation foci, whereas in its presence, the cells formed many foci transformed cells release TGFs whereas cells infected with trans of rounded cells. It should be noted that EGF does not produce formation defective variants do not (55). Cells infected with these effects in noninfected cells. Removal of EGF resulted in temperature sensitive mutants of Kirsten and Moloney murine the reversion of the majority of the infected granulosa cells to a sarcoma virus produce TGFs only at temperatures permissive normal phenotype, indicating that the presence of EGF is essen for cellular transformation (54, 67). On the other hand, cells tial for the transformed phenotype. The presence of the remain which do not have functional EGF receptors are as readily ing persistent transformed foci was dependent on their prior

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1986 American Association for Cancer Research. EGF AND CARCINOGENESIS exposure to EGF. Thus EGF has the capacity to elicit a trans properties of the kinase site or clustering, which in turn may formed phenotype in partially transformed cells. This phenotype have important roles in growth regulation. Alternatively, these may be "locked" into place allowing cells to become EGF inde alterations of EGF receptor activity by chemical carcinogens may pendent. simply be circumstantial. In this light it should be noted that EGF did not appear to be acting as a classic tumor promotor phorbol esters have the capacity to stimulate cell growth in cells in the granulosa cells since its transforming activity was sepa that do not respond to EGF and, depending on cell type, may rable from its growth promoting activity. This was shown by the either inhibit or be synergistic with EGF-induced mitogenesis following results: (a) EGF did not stimulate the growth of either (104-106). normal or virally infected granulosa cells, and (b) the time required for formation of transformation foci was not accelerated. Transformation of normal cells, in contrast to certain estab Immunosuppressive Activity of EGF lished cell lines, requires the presence of at least two comple mentary oncogenes (89-92), thus in this case, EGF is apparently Although immunosuppression may not directly produce can substituting for the second oncogene. Since the Kirsten sarcoma cers, it may prevent the elimination of spontaneously arising virus expresses the ras oncogene (which creates the traits of tumors by the immune system. Thus, Â¡mmunosuppression usu focus formation and tumorigenesis) is complemented by onco ally is considered to permit cancer growth rather than to actually genes responsible for immortalization (92), EGF may partially cause it. An example of the role of an impaired immune system substitute for oncogenes in this second group. Alternatively, in neoplastic disease is the increased incidence of cancer ob EGF may be an enhancer of ras function. EGF enhances the served after the destruction of T-cells by the human T-cell guanine nucleotide binding activity of ras transforming proteins leukemia virus type III which causes the acquired immune defi as well as enhancing the phosphorylation state of at least one ciency syndrome (107). of them (28). Injection of EGF 1 day before the injection of an antigen (sheep RBC) suppressed the production of antibodies to this antigen. EGF Promotion of Chemical Carcinogenesis Since the IgG response was affected more greatly than the IgM response, and since the IgG response is more T-cell dependent One of the first observations that indicated that EGF may play than is the IgM response, helper T-cells were suggested to be a role in carcinogenesis is that EGF enhances the carcinogenic the target of EGF induced immunosuppression. Recently, it has potential of methylcholanthrene in skin (93, 94). A specific inter been shown that the antigen-presenting function of lymphocytes action of chemical tumor promoters with the EGF receptor was affecting the growth of T-cells is potentiated by EGF In vitro shown in cultured cells where TPA and benzopyrene inhibited the binding of EGF but not insulin, multiplication stimulating (108). It should be noted that EGF does not have a general toxic activity, concanavalin A, nerve growth factor, low density lipo- effect on lymphocytes. This was shown by the observation that protein, or murine type C ectopie viral glycoprotein. Decreased EGF was somewhat stimulatory for Â¡mmunoglobulin production EGF binding was found to be caused by lowered receptor affinity if injected 3 days after the injection of the antigen, and that it did rather than decreased receptor number (95-101). not prevent the development of immunological memory (109, This effect of these chemical tumor promoters on EGF binding 110). Suppression of T-cell function by EGF was also implicated is thought to be mediated by intracellular processes since they by studies showing that EGF depressed the delayed type hyper- have no effect on the binding of EGF to broken cell preparations sensitivity response to 2,4-dinitro-1-fluorobenzene (111). Sali (100). Protein kinase C fulfills many of the requirements of such vary gland extract (containing high levels of EGF) delays the an intracellular mediator: it is activated by a variety of chemical rejection of skin allografts (112), a process also predominantly tumor promoters and decreases EGF receptor affinity for EGF mediated by T-cells. when added to EGF receptor preparations in vitro (102). The In addition to these relatively short term immunosuppressive mechanism by which protein kinase C inhibits EGF receptor effects, EGF may also have a long term effect. The possibility activity may be via direct phosphorylation of a threonine residue that EGF may cause long term effects on the immune system which is near the membrane spanning region of the EGF receptor was first shown by Takeda and Grollman (113), who injected (103). It should be emphasized that this is not an autophospho- crude salivary gland extract into mice, causing the thymus and rylation site. EGF stimulates the phosphorylation of its own lymph nodes to atrophy. In a subsequent study, purified EGF receptor on tyrosine residues near the cytoplasmic end of the was observed to cause a decrease in DNA synthesis in the molecule. thymus, spleen, and bone marrow, although an increase in DNA In addition to decreasing EGF receptor affinity, chemical tumor synthesis was observed in other tissues (114). promoters also decrease tyrosyl kinase activity of the EGF receptor (49,95,102). Since an increase in tyrosyl kinase activity, Although EGF stimulates cortisol secretion by the adrenal rather than a decrease, is usually associated with transformation, glands (99), it appears unlikely that EGF induction of cortisol it is unclear how these effects promote carcinogenesis. In fact, synthesis would mediate the immunosuppressive activity of EGF. EGF receptor activities recover within hours in the continued Thymic and lymphoid atrophy induced by injections of crude presence of TPA (96, 98). Thus, since TPA promotion of carci submandib.ular extracts was not prevented by prior extirpation nogenesis requires several days, it is not likely that phorbol ester of the adrenal glands (113). The ability of thymus and spleen action is mediated through decreasing EGF receptor binding and membranes to bind EGF (115) is another indication that EGF kinase activities. Perhaps chemical carcinogens promote other may have a direct effect on the immune system, since growth functions of the receptor such as protein recognition and binding and development of lymphocytes occurs in these tissues.

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Relationship of the EGF Receptor to Transforming Proteins transforming gene yet still induces tumors albeit with a latency period of weeks. The mechanism by which the avian leukosis The EGF receptor itself is related to certain transforming virus induces tumors seems to be through promotor insertion proteins both structurally and functionally. Transforming proteins into the host genome. If the promotor is placed in front of a are produced by an oncogene in a viral genome. Deletion of this cellular gene whose unrestricted transcription can cause uncon gene renders the virus incapable of inducing carcinogenesis. trolled growth, the host organism will develop cancer. In a Functionally, both the EGF receptor and several transforming chicken with erythroblastosis, the viral promotor was mapped in proteins, which are also protein kinases, have the unusual prop front of the c-erbB gene which is thought to be the gene for the erty of being able to phosphorylate proteins on tyrosine residues. EGF receptor. Additionally, increased levels of RNA which hy Of all transforming proteins known, 30% have tyrosyl kinase bridize to probes against v-erbA and v-erbB, were detected in activity. These include src, erbB, abl, yes, fgr, ros, fes(fps), and the erythroblasts. Thus the inserted promotor appears to suc fms (116, 117). Phosphotyrosine represents usually less than cessfully promote the transcription of c-ero proteins, most likely 0.03% of the total phosphoaminoacids in nontransformed cells the EGF receptor. It is not clear in this study whether the whole and increases approximately 30-fold in transformed cells (118). or truncated form (as in v-erbB) of the receptor is produced in This property is also shared with other receptors for growth the transformed cells and whether the receptor is appropriately promoting factors such as PDGF, insulin, and somatomedin C membrane bound. (116). Thus it is thought that tyrosyl kinase activity may play a It is not entirely clear whether increased kinase activity nec role in growth control. The pathways which tyrosyl kinases essarily leads to cell proliferation. For example, infection of cells control have not been identified at this time. by the avian erythroblastosis virus does not appreciably increase Protein substrate specificities may be similar between the phosphotyrosine levels in vivo (118, 126, 129). EGF, which various tyrosyl kinases. Both the EGF receptor and ppoO^0, the presumably increases tyrosine kinase activity in target cells, protein encoded by the transforming gene of the Rous sarcoma inhibits the growth of several lines of mammary, liver, and virus, phosphorylate a M, 36,000 normal cell protein at the same pituitary cancer cells, as well as several other tumor cell lines site (119). These kinases also phosphorylate synthetic peptide (35,51,130-132). EGF inhibits A-431 cell proliferation (16,133), substrates related to the tyrosine autophosphorylation site with although it increases intracellular phosphotyrosyl levels 3-fold similar specificities (120). pp60srcphosphorylates certain antibod (17). In fact, monoclonal antibodies acting as EGF agonists (but ies directed against it; these antibodies also serve as substrates also those which have no effect on receptors) inhibit the growth for the EGF receptor, although they do not have the capacity to of A-431 tumors in nude mice (134). Stimulation of EGF receptor precipitate it (121,122). Thus, the EGF receptor and pp60srcmay kinase activity without concurrent stimulation of growth can also both stimulate cell growth through the same pathway, i.e., by occur when cells are exposed to cyanogen bromide-treated EGF phosphorylation of the same regulatory proteins due to similar (135). Also, cells containing 10 times the normal concentration substrate specificities. of the tyrosyl-kinase c-src, the cellular homologue of the trans Structurally, the cytoplasmic portion of the receptor has an forming protein v-src do not express the transformed phenotype average of 25% homology to the kinase domain of several (136). These examples show that increased tyrosyl kinase activ transforming proteins. Even greater homology (95%) has been ity is not necessarily correlated with increased cell proliferation. observed with the eroÃŸ-transforming protein of the avian ery- Thus, qualitative as well as quantitative changes may contribute throblastosis virus (123,124). This extent of homology suggests toward the transforming capacity of a tyrosyl kinase. Such that the erythroblastosis virus incorporated the genome for the qualitative rather than quantitative changes have previously been cytoplasmic portion of the EGF receptor from its host. Since the observed for proteins encoded by ras genes. These examples erythroblastosis virus infects chickens, whereas the EGF recep also indicate that, in certain cases, EGF may be antagonistic tor sequence used for comparison is from humans, the homology toward cancer cell growth. Since EGF is relatively nontoxic the of v-erbB to the chicken EGF receptor sequence may be even use of EGF as well as monoclonal antibodies to the EGF receptor higher. Another point which should be made is that this evidence (134) should be considered in cancer therapy. indicates that the cytoplasmic portion of the EGF receptor may be remarkably conserved between birds and humans, two highly Measurement of EGF Receptor Levels in Cancer Cells divergent species. Overexpression of the erbB gene in avian erythroblastosis virus infected cells, possbily by causing high These is no simple correlation of EGF receptor levels to cancer. tyrosyl kinase activity, may lead to cellular transformation. erbB In many transformed cells, including those transformed by vi protein kinase activity has not, until recently, been detectable ruses, the level of EGF receptors decreases (34, 54, 55, 68, 69, (125-127). The recent evidence for erbB activity may be due to 137,146). This decline is thought to be caused by the production the use of noninhibitory antibodies to purify the protein. Further of a-TGFs, which can down regulate the receptor (32), by the work is necessary to ascertain whether v-erbB actually does transformed cells. Some preliminary evidence indicates that this have intrinsic kinase activity. It is also essential to compare its relationship may not necessarily hold true. No correlation was kinetic parameters with those of the EGF receptor to determine observed in variants of a mammary carcinoma cell line and in whether the protein is in an activated state or not. various lung cancer cells between EGF-receptor levels and the The EGF receptor itself can become an oncogene if a viral capacity of the conditioned medium to stimulate cells to grow in promotor or activator is inserted into the cellular gene. This soft agar (137, 146). On the other hand, this assay is relatively appears to be one mechanism by which the avian leukosis virus, nonspecific because /3-TGF and PDGF, which are also produced a slowly oncogenic retrovirus, causes erythroblastosis (128). A by many transformed cells, also potentiate cellular growth in soft slowly oncogenic retrovirus is a retrovirus that does not carry a agar (31,37,139). Thus altered production of these latter factors

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1986 American Association for Cancer Research. EGF AND CARCINOGENESIS and possibly others by the cells can independently modulate the oncogene product as EGF must in granulosa cells (88). capacity of conditioned medium to support cell growth in soft The complexity of hormonal interactions with cells makes the agar. Another confounding aspect of measuring Â«-TGFlevels is precise elucidation of their role in carcinogenesis difficult. For that simple competitive binding assays on live cells may not example, the same hormone may stimulate, inhibit, or have no produce valid results. Several substances, including PDGF, rap effect on proliferation, depending on the cell type and its state idly lower the affinity of the EGF receptor (96, 97, 140-143), of differentiation. Thus, the choice of target cells can highly which could make it appear as if the substance was competing influence the results of an experiment where a hormone may with EGF. More accurate assessment of Â«-TGFlevels should have no effect, enhance, or inhibit carcinogenesis. involve high performance liquid chromatography to partially char acterize the TGF and a binding assay utilizing cellular membranes rather than live cells (115). The absence of Â«-TGF from the Acknowledgments medium may not necessarily mean that the cell is not producing We thank R. Gates and M. Chinkersfor a critical readingof the manuscript,and it; interaction of TGF with its receptor may occur intracellularly, S. Bennett for preparation of the manuscript. i.e., within the endoplasmic reticulum, Golgi or exocytotic vesi cles (144). Thus, intracellular levels of specific messenger RNA for TGFs may more accurately quantitate TGF production. An References other reason for the lack of a correlation of TGF production and 1. Carpenter, G. Epidermal growth factor. Handb. Exp. Pharmacol., 57: 90- receptor levels is that different cell types may have varying 126,1981. 2. Carpenter, G., and Cohen, S. Human epidermal growth factor and the capacities to recycle EGF receptors rather than necessarily proliferationof humanfibroblasts. J. Cell Physiol.,88: 227-237, 1976. degrading them in response to ligand binding (145). 3. King, L. E., Jr., and Carpenter,G. F. Biochemistryand physiologyof the skin. In some cases EGF receptor levels are higher in malignant as In: L. Goldsmith (ed.), Epidermal Growth Factor, Ed. 1, pp. 269-281. New York: Oxford UniversityPress, 1983. compared to normal tissues. In several different squamous cell 4. Byyny, R. L., Orth, D. N., Cohen, S., and Doyne, E. S. Epidermal growth lung carcinomas EGF receptor levels were highly elevated as factor: effects of androgens and adrenergicagents. Endocrinology,95: 776- compared to the normal lung. In contrast, other lung carcinomas 782,1974. 5. Turkington, R. W., Males, J. L., and Cohen, S. Synthesis and storage of (in particular, small cell carcinoma) had undetectable EGF recep epithelial-epidermalgrowth factor in submaxillary gland. Cancer Res., 37: tor levels (146-148). Increased levels of EGF receptors were 252-256,1971. also observed in some breast cancer cells as compared to normal 6. Elder, J. B., Williams, G., Lacey, E., and Gregory, H. Cellular localizationof human urogastrone/epidermalgrowth factor. Nature (Lond.),277: 464-467, cells, but in others the levels of EGF receptors were lower (149). 1978. Another instance in which increased levels of EGF receptors 7. FallÃ³n,J.H., Loughlin,S. E., Morrison, R. S., and Bradshaw, R. A. Epidermal growth factor immunoreactivematerialin the central nervoussystem:location were detected are in brain tumors (150). This finding may at and development. Science(Wash. DC),224:1107-1109,1984. least partially be explained by the presence of different ratios of 8. Oka, Y., and Orth, D. N. Human plasma epidermal growth factor/beta- nonneuronal versus neuronal cells in the tumors, since neurons urogastrone is associated with blood platelets. J. Clin. Invest., 72: 249-259, 1983. have a particularly low EGF binding capacity as compared to 9. Shikata, H., Utsumi,N., Hiramatsu,M., Minami, N., Nemoto, N., and Shidata, other cells. In some tumors the number of EGF receptor gene T. Immunohistochemicallocalization of nerve growth factor and epidermal copies per cell was increased, which could also explain high EGF growth factor in guinea pig prostate gland. Histochemistry, 80: 411-413, 1984. receptor expression (150). An increased number of EGF receptor 10. Gregory, H., Walsh, S., and Hopkins, C. R. The identificationof urogastrone gene copies has also been reported for A-431 epidermoid car in serum, saliva and gastric juice. Gastroenterotogy,77: 313-318,1979. 11. Stoscheck, C. M., and Carpenter, G. Characteristics of antibodies to the cinoma cells (124,151, 152). epidermalgrowth factor receptor-kinase.Arch. Biochem.Biophys.,227:457- In conclusion, EGF receptor levels may be decreased, in 468,1983. creased, or not changed in cancer cells as compared to their 12. Stoscheck, C. M., and King, L. E., Jr. Characteristicsof EGFand its receptor and their relationship to transforming proteins. J. Cell. Biochem., in press, normal counterpart. In many cases a decrease in EGF receptor 1986. levels is associated with the production of a-TGF. In some cases 13. Kirkland, W. L., Yang, N. S., Jorgensen, T., Langley, C., and Furmanski, P. Growth of normal and malignant human mammary epithelialcells in culture. an increase in EGF receptor levels is associated with gene J. Nati. Cancer Inst., 63: 29-39, 1979. amplification. 14. Westermark, B. Densitydependentproliferationof humanglia cellsstimulated by epidermal growth factor. Biochem. Biophys. Res. Commun., 69: 304- 309,1976. Overall Perspective 15. Cooper, J. A., and Hunter, T. Similaritiesand differencesbetween the effects of epidermal growth factor and Rous sarcoma virus. J. Cell Bid., 97: 878- It has been clear for quite some time that hormonal systems 883,1981. 16. Gill,G. N., and Lazar, C. S. Increasedphosphotyrosinecontent and inhibition are involved in cancer growth. The identification of oncogenes of proliferation in EGF-treated A431 cells. Nature (Lond.), 293: 305-307 and their products is shedding new light on the involvement of 1981. 17. Hunter, T., and Cooper, J. A. Epidermalgrowth factor induces rapid tyrosine hormones in cancer. In addition to the previously discussed phosphorylationof proteins in A-431 human tumor cells. Cell, 24: 741-752 homology of the EGF receptor to v-erbÃŸ,homology of PDGF to 1981. the putative transforming protein p28em"sisand homology of G- 18. Nakamura, K. D., Martinez, R., and Weber, M. J. Tyrosine phosphorylation of specific proteins after mitogen stimulation of chicken embryo fibroblasts. proteins (GTP-binding proteins which transduce a hormone bind Mol. Cell Biol., 3: 380-390,1983. ing signal to adenyl cyclase) to p21rashave been observed (153- 19. Barnes, D., and Colowick, S. P. Stimulation of sugar uptake in cultured flbroblast by EGF and EGF-bindingarginine esterase. J. Cell Physiol., 89: 155). Functional similarities, such as tyrosyl kinase activity, be 633-640,1976. tween these oncogene products and their homologous cellular 20. Diamond, I., Legg, A., Schneider, J. A., and Rozengurt, E. Glycolysis in products have also been observed (12,116,125-127,156). If a quiescent cultures of 3T3 cells. J. Biol. Chem., 253: 866-871,1978. 21. DiPasquale,A., White, D., and McGuire,J. Epidermalgrowth factor stimulates simple substitution by an oncogene product for its cellular hom putrescinetransport and omithine decarboxylaseactivity in cultivated human ologue occurs, a hormone stimulus should substitute for the fibroblasts. Exp. Cell Res., 76: 317-323, 1978.

CANCER RESEARCH VOL. 46 MARCH 1986 1034

22. Muller, R., Bravo, R., Burckhardt, J., and Curran, T. Induction of c-/os and RNA encoding epidermal growth factor and seven related proteins. Science protein by growth factors precedes activation of c-myc. Nature (Lond.),312: (Wash. DC),226: 236-240, 1983. 716-720,1984. 49. Decker, S. J. Effects of epidermal growth factor and 12-0-tetradecanoyl- 23. Bishop, J. M. Cellularoncogenes and retroviruses. Annu. Rev. Biochem.,52: phorbol-13-acetateon metabolismof the epidermalgrowth factor receptor in 301-354,1983. normal human fibroblasts. Mol. Cell. Biol., 4: 1718-1724,1984. 24. Chen, L. B., Gudor, R. C., Sur, T. T., Chen, A. Bâ€žandMosesson, M. W. 50. Fisher, P. B., Boersig, M. R., Grahan, G. M., and Weinstein, I. B. Production Control of a cell surface major glycoprotein by epidermal growth factor. of growth factors by type 5 adenovirus transformed rat embryo cells. J. Cell. Science(Wash. DC), 797: 776-778, 1977. Physiol., 774: 365-370,1983. 25. Eaton, D. L., and Baker, J. B. Phorbol ester and mitogens stimulated human 51. Imai,Y., Leung, C. K. H., Friesen,H. G., and Shiu, R. P. C. Epidermalgrowth fibroblast secretion of plasmin-activatable plasminogen activator and pro factor receptors and effect of epidermal growth factor on growth of human tease nexin, an antiactivator/antiplasmin. J. Cell Biol., 97: 323-328,1983. breast cancer cells in long-term tissue culture. Cancer Res., 42: 4394-4398, 26. Hashimoto, K., Singer, K. H., Ã¼de,W.B., Shafran, Â«.,Weber,P., Morioka, 1982. S., and Lazarus, G. S. Plasminogenactivator in cultured human epidermal 52. Hollenberg,M. D., and Cuatrecasas, P. Insulin and epidermalgrowth factor. cells. J. Invest. Dermatol., 81: 424-429,1983. Human-fibroblastreceptors related to deoxyribonucleic acid synthesis and 27. Lee, L. S., and Weinstein, I. B. Epidermalgrowth factor, like phorbol esters, amino acid uptake. J. Biol. Chem., 250: 3845-3853,1975. induces plasminogen activator in HeLa cells. Nature (Lond.),274: 696-697, 53. Talha, S., and Harel, L. Early stimulation of ATP turnover induced by growth 1978. factors. Synergistic effect of EGF and insulin and correlation with DNA 28. Kamata,T., and Feramisco,J. R. Epidermalgrowth factor stimulatesguanine synthesis. Exp. Cell Res., 758: 311-320,1985. nucleotide binding activity and phosphorylation of ras oncogene proteins. 54. Ozanne, B., Fulton, R. J., and Kaplan, P. L. Kirsten murine sarcoma virus Nature (Lond.),370: 147-150, 1984. transformed cell lines and a spontaneous transformed rat cell line produce 29. Sherwin, S. A., Twardzik, D. R., Bonn, W. H., Cockley, K. D., and Todaro, transforming factors. J. Cell Physiol., 705: 163-180,1980. G. J. High-molecular-weighttransforming growth factor activity in the urine 55. Twardzik, D. R., Todaro, G. J., Marquardt, H., Reynolds, F. H., and Stephen- of patients with disseminated cancer. Cancer Res., 43: 403-407,1983. son, J. R. Transformation by Abelsonmurine leukemiavirus involvesproduc 30. Twardzik, D. R., Sherwin,S. A., Ranchalis,J., and Todaro, G. J. Transforming tion of a polypeptide growth factor. Science (Wash. DC), 276: 894-897, growth factors in the urine of normal, pregnant, and tumor-bearinghumans. 1982. J. Nati. Cancer Inst., 69: 793-798, 1982. 56. Roberts, A. B., Anzano, M. A., Wakefield, L. M., Roche, N. S., Stem, D. F., 31. Anzano, M. A., Roberts, A. B., Smith, J. M., Sporn, M. B., and DeLarco, J. and Sporn, M. B. Type ÃŸtransforminggrowth factor: a bifunctionalregulator E. Sarcoma growth factor from conditioned medium of virally transformed of cellulargrowth. Proc. Nati. Acad. Sci. USA,82: 119-123, 1985. cells is composed of both type a and type /3transforming growth factors. 57. Assoian, R. K., Komoriya, A., Meyer, C. A., Miller, D. M., and Sporn, M. B. Proc. Nati. Acad. Sci. USA,80: 6264-6268,1983. Transforminggrowth factor-0 in human platelets. J. Biol. Chem., 258: 7155- 32. Carpenter, G., Stoscheck, C. M., Preston, Y. A., and De Larco, J. E. 7160,1983. Antibodies to the epidermal growth factor receptor block the biological 58. Childs, C. B., Proper,J. A., Tucker, R. F., and Moses, H. L. Serum contains activities of sarcoma growth factor. Proc. Nati. Acad. Sci. USA, 80: 5627- a platelet-derivedtransforminggrowth factor. Proc. Nati. Acad. Sci. USA,79: 5630,1983. 5312-5316,1982. 33. Roberts, A. B., Anzano, M. A., Lamb, L. C., Smith, L. M., Frolik, C. A., 59. Frolik, C. A., Dart, L. L., Meyers, C. A., Smith, D. A., and Sporn, M. B. Marquardt, H., Todaro, G. J., and Sporn, M. B. Isolationfrom murine sarcoma Purification and initial characterization of a type beta transforming growth cells of noveltransforming growth factors potentiated by EGF.Nature(Lond.), factor from human placenta. Proc. Nati. Acad. Sci. USA, 80: 3676-3680, 295:417-419, 1982. 1983. 34. Todaro, G. J., Fryling, C., and DeLarco, J. E. Transforming growth factors 60. Proper, J. A., Bjomson, C. L., and Moses, H. L. Mouse embryos contain produced by certain human tumor cells: polypeptides that interact with polypeptide growth factor(s) capable of inducing a reversible neoplasmic epidermal growth factor receptors. Proc. Nati. Acad. Sci. USA, 77: 5258- phenotypein non-transformedcells in culture. J. Cell Physiol., 770:169-174, 5262,1980. 1982. 35. Hamburger,A. W., White, C. P., and Brown, R. W. Effect of epidermalgrowth 61. Roberts, A. B., Anzano, M. A., Lamb, L. C., Smith, J. M., and Sporn, M. B. factor on proliferation of human tumor cells in soft agar. J. Nati. Cancer Inst., New class of transforming growth factors potentiated by epidermal growth 67:825-830,1981. factor: isolation from nonneoplastic tissues. Proc. Nati. Acad. Sci. USA, 78: 36. Pathak, M. A., Matrisian, L. M., Magun, B. E., and Salmon, S. E. Effect of 5339-5343,1981. epidemial growth factor on clonogenicgrowth of primary human tumor cells. 62. Roberts, A. B., Frolik, C. A., Anzano, M. A., and Sporn, M. B. Transforming Int. J. Cancer, 30: 745-750, 1982. growth factors from neoplastic and nonneoplastic tissues. Fed. Proc., 42: 37. Anzano, M. A., Roberts, A. B., Meyers, C. A., Komoriya, A., Lamb, L. C., 2621-2625,1983. Smith, J. M., and Sporn, M. B. Synergistic interaction of two classes of 63. Stromberg, K., Pigott, D. A., Ranchalis,J. E., and Twardzik, D. R. Human transforming growth factors from murine sarcoma cells. Cancer Res., 42: term placentacontains transforming growth factors. Biochem. Biophys. Res. 4776-4778,1982. Commun., 706: 354-361,1982. 38. Massague, J. Epidermal growth factor-like transforming growth factor. I. 64. Matrisian, L. M., Pathak, M., and Magun, B. E. Identificationof an epidermal Isolation, chemical characterization and potentiation by other transforming growth factor-related transforming growth factor from rat fetuses. Biochem. factors from feline sarcoma virus-transformed rat cells. J. Biol. Chem., 258: Biophys. Res. Commun., 707: 761-769, 1982. 13606-13613,1983. 65. Nexo, E., Hollenberg,M. D., Figueroa,A., and Pratt, R. M. Detectionof EGF- 39. Massague, J. Epidermal growth factor-like transforming growth factor. II. urogastrone and its receptor during fetal mouse development. Proc. Nati. Interaction with epidermal growth factor receptors in human placenta mem Acad. Sci. USA, 77: 2782-2785,1980. branes and A431 cells. J. Biol. Chem., 258:13614-13620,1983. 66. Twardzik, D. R., Ranchalis, J. E., and Todaro, G. J. Mouse embryonic 40. Reynolds, F. H., Jr., Todaro, G. J., Fryling,C., and Stephenson,J. R. Human transforminggrowth factors relatedto those isolatedfrom tumor cells.Cancer transforming growth factors induce tyrosine phosphorylation of EGF recep Res., 42: 590-593,1982. tors. Nature (Lond.),292: 259-262, 1981. 67. DeLarco, J. E., Preston, Y. A., and Todaro, G. J. Properties of a sarcoma- 41. Twardzik, D. R., Todaro, G. J., Reynolds,F. H., and Stephenson,J. R. Similar growth-factor-like peptide from cells transformed by a temperature-sensitive transforming growth factors (TGFs)produced by cells transformed by differ sarcoma virus. J. Cell Physiol., 709:143-152,1981. ent isolates of feline sarcoma virus. Virology, 724: 201-207,1983. 68. Moses, H. L., Branum, E. L., Proper,J. A., and Robinson,R. A. Transforming 42. Tarn,J. P. Physiologicaleffects of transforming growth factor in the newborn growth factor production by chemicallytransformed cells. Cancer Res., 47: mouse. Science (Wash. DC),229: 673-675, 1985. 2842-2848,1981. 43. Gregory, H. Isolation and structure of urogastrone and its relationship to 69. Twardzik, D. R., Todaro, G. J., Reynolds,F. H., and Stephenson,J. R. Similar epidermalgrowth factor. Nature (Lond.),257: 325-327,1975. transforming growth factors (TGFs)produced by cells transformed by differ 44. Savage, C. R., Jr., Inagami, T., and Cohen, S. The primary structure of ent isolates of feline sarcoma virus. Virology, 724: 201-207, 1983. epidermalgrowth factor. J. Bid. Chem., 247: 7612-7621,1972. 70. Pruss, R. M., Herschman, H. R., and Klement, V. 3T3 variants lacking 45. Marquardt, H., Hunkapiller, M. W., Hood, L. E., and Todaro, G. J. Rat receptors for epidermal growth factor are susceptible to transformation by transforming growth factor type 1: structure and relationship to epidermal Kirsten sarcoma virus. Nature (Lond.),274: 272-274,1978. growth factor. Science(Wash. DC), 223: 1079-1082,1984. 71. Betsholtz,C., Heldin,C. H., Nister, M., Ek, B., Wasteson,A., and Westermark, 46. Derynck, R., Roberts, A. B., Winkler, M. E., Chen, E. Y., and Goeddel, D. V. B. Synthesis of a PDGF-like growth factor in human glioma and sarcoma Humantransforming growth factor-alpha: precursorstructure and expression cells suggests the expression of the cellular homologue of the transforming in E. coli. Cell, 38: 287-297,1984. protein of simian sarcoma virus. Biochem. Biophys. Res. Commun., 777: 47. Gray, A., Dull, T. J., and Ullrich,A. Nucleotide sequenceof epidermalgrowth 176-182,1983. factor c-DNA predicts a 128,000-molecularweight protein precursor. Nature 72. Heldin, C. H., Westermark, B., and Wasteson, A. Chemical and biological (Lond.),303: 722-725, 1983. properties of a growth factor from human cultured osteosarcoma cells: 48. Scott, J., Urdea, M., Quiroga, M., Sanchez-Prescador,R., Fong, N., Selby, resemblancewith platelet-derivedgrowth factor. J. Cell. Physiol., 705: 235- M., Rutter, W. J., and Bell,G. I.Structure of a mousesubmaxillarymessenger 246,1980.

CANCER RESEARCH VOL. 46 MARCH 1986 1035

73. Nister, M., Heldin, C. H., Wasteson, A., and Westermark, B. A glioma-derived 100. Feam. J. C., and King, A. C. EGF receptor affinity is regulated by intracellular analog to platelet-derived growth factor: demonstration of receptor competing calcium and protein kinase C. Cell, 40: 991-1000,1985. activity and immunological cross-reactivity. Proc. Nati. Acad. Sci. USA, 87: 101. Shoyab, M., DeLarco, J. E., and Todaro, G. J. Biologically active phorbol 926-930,1984. esters specifically alter affinity of epidermal growth factor membrane recep 74. Gelman, E. P., Wong-Staal, F., Kramer, R. A., and Gallo, R. C. Molecular tors. Nature (Lond.), 279: 387-391, 1979. cloning and comparative analysis of the genomes of simian sarcoma virus 102. Cochet, C., Gill, G. N., Meisenhelder, J., Cooper, J. A., and Hunter, T. C- and its associated helper virus. Proc. Nati. Acad. Sci. USA, 78: 3373-3377, kinase phosphorylates the epidermal growth factor receptor and reduces its 1981. epidermal growth factor-stimulated tyrosine protein kinase activity. J. Biol. 75. Robbins, K. C., Devare, S. G., and Aaronson, S. A. Molecular cloning of Chem., 259: 2553-2556,1984. integrated simian sarcoma virus: genome organization of infectious DNA 103. Hunter, T., Ling, N., and Cooper, J. A. Protein kinase C phosphorylation of clones. Proc. Nati. Acad. Sci. USA, 78. 2918-2922.1981. the EGF receptor at a threonine residue close to the cytoplasmic face of the 76. Stiles, C. D. The molecular biology of platelet-derived growth factor. Cell, 33: plasma membrane. Nature (Lond.), 377: 480-483, 1984. 653-655, 1983. 104. Dicker, P., and Rozengurt, E. Synergistic stimulation of early events and DNA 77. DeLarco, J. E., and Todaro, G. J. A human fibrosarcoma cell line producing synthesis by phorbol esters, polypeptide growth factors, and retinoids in multiplication-stimulating activity (MSA)-related peptides. Nature (Lond.), 272: cultured fibroblasts. J. Supramol Struct., 77: 79-93, 1979. 356-358,1978. 105. Frantz, C. N., Stiles, C. D., and Scher, C. D. The tumor promoter 12-0- 78. Bajzer, S.. Pavelio, K., and Vuk-Pavlovic, S. Growth self-incitement in murine tetradecanoyl-phorbol-13-acetate enhances the proliferative response of melanoma B16: a phenomenological model. Science (Wash. DC), 225: 930- BALB/3T3 cells to hormone growth factors. J. Cell Physiol., 700: 413-424, 932, 1984. 1979. 79. Knaver, D. J., Iyer, A. P., Banerjee, M. R., and Smith, G. T. Identification of 106. Pruss, R. M., and Herschman, H. R. Variants of 3T3 cells lacking mitogenic somatomedin-like polypeptides produced by mammary tumors of BALB/c response to epidermal growth factor. Proc. Nati. Acad. Sci. USA, 74: 3918- mice. Cancer Res., 40: 4368-4372, 1980. 3921,1977. 80. Pavelic, K., Bolanca, M., Vecek, N., Pavelic, J.. Marotti, T., and Vuk-Pavlovic, 107. Shaw, G. M., Hahn, B. H., Arya, S. K., Groopman, J. E., Gallo, R. C., and S. Carcinomas of the cervix and corpus uteri in humans: stage-dependent Wong-Staal, F. Molecular characterization of human T-cell leukemia (lympho- blood levels of substance)s) immunologically cross-reactive with insulin. J. tropic) virus type III in the acquired immune deficiency syndrome. Science Nati. Cancer Inst., 68. 891-894, 1982. (Wash. DC). 226: 1165-1172,1984. 81 Sherwin, S. A., Sliski, A. H., and Todaro, G. J. Human melanoma cells have 108. Acres, R. B., Lamb, J. R., and Feldman, M. Effects of platelet-derived growth both nerve growth factor and nerve growth factor-specific receptors on their factor and epidermal growth factor on antigen-induced proliferation of human cell surfaces. Proc. Nati. Acad. Sci. USA, 76: 1288-1292,1979. T-cell lines. Immunology, 54: 9-16,1985. 82. Gorden. P., Hendricks, C. M., Kahn, C. R., Megyesi, K., and Roth, J. 109. Koch, J. H., Fifis, T., Bender, V. J., and Moss, B. A. Molecular species of Hypoglycemia associated with non-islet-cell tumor and insulin-like growth epidermal growth factor carrying immunosuppressive activity. J. Cell. factors. N. Engl. J. Med., 305: 1452-1455, 1981. Biochem., 25: 45-59, 1984. 83. Ibbotson, K. J., D'Souza, S. M., Ng. K. W., Osbome, C. K., Niall, M., Martin, 110. Koch, J. H., and Rowe, J. Immunosuppressive activity of submaxillary gland T. J., and Mundy, G. R. Tumor-derived growth factor increases bone rÃ©sorp extracts of the mouse. I. Effect on antibody formation in response to sheep tion in a tumor associated with humoral hypercalcemia of malignancy. Science red blood cells. Eur. J. Immunol., 6: 583-588,1976. (Wash. DC), 227. 1292-1294, 1983. 111. Roberts, M. L., Freston, J. A., and Reade, P. C. Suppression of immune 84. Ibbotson, K. J., D'Souza, S. M., Smith, D. D., Carpenter, G., and Mundy, G. responsiveness by a submandibular salivary gland factor. Immunology, 30: R. EGF receptor antiserum inhibits bone resorbing activity produced by a rat 811-814,1976. Leydig cell tumor associated with the humoral hypercalcemia of malignancy. 112. Kongshaun, P. A. L., and Bliss, J. Q. Effect of mouse submandibular gland Endocrinology, 776: 469-471, 1985. extracts on survival of H-2 incompatible skin allografts. Immunol., 79: 363- 85. Kimball, E. S., Bohn, W. H., Cockley, K. D., Warren, T. C., and Sherwin, S. 367,1970. A. Distinct high-performance liquid chromatography pattern of transforming 113. Takeda, T., and Grollman, A. Inhibitory action of submaxillary gland on thymus growth factor activity in urine of cancer patients as compared with that of and lymphoid tissues of the mouse. Am. J. Physiol., 275:1337-1342,1968. normal individuals. Cancer Res., 44: 3613-3619, 1984. 114. Yeh, V. C., Scheving, L. A., Tsai, T. H., and Scheving, L. E. Circadian stage 86. Hirata, Y., and Orth, D. N. Epidermal growth factor (urogastrone) in human dependent effects of epidermal growth factor on deoxyribonucleic acid syn fluids: size heterogeneity. J. Clin. Endocrinol. Metab., 48: 673-679,1979. thesis in ten different organs of the adult mouse. Endocrinology, 709: 644- 87. Fisher, P. B., Bozzone, J. H., and Weinstein, I. B. Tumor promoters and 651,1984. epidermal growth factor stimulate anchorage-independent growth of adeno- 115. O'Keefe, E., Hollenberg, M. D., and Cuatrecasas, P. Epidermal growth factor virus-transformed rat embryo cells. Cell, 78. 695-705, 1979. characteristics of specific binding in membranes from liver, placenta and other 88. Harrison, J., and Auersperg, N. Epidermal growth factor enhances viral target tissues. Arch. Biochem. Biophys., 764: 518-526, 1974. transformation of granulosa cells. Science (Wash. DC), 273: 218-219, 1981. 116. Heldin, C. H., and Westermark, B. Growth factors: mechanism of action and 89. Land, H., Parada, L. F., and Weinberg, R. A. Cellular oncogenes and multistep relation to oncogenes. Cell, 37: 9-20, 1984. carcinogenesis. Science (Wash. DC), 222: 771-778,1983. 117. Roussel, M. F., Rettenmier, C. W., Look, A. T., and Sheer, C. J. Cell surface 90. Land, H., Parada, L. F., and Weinberg, R. A. Tumorigenic conversion of expression of v-fms-coded glycoproteins is required for transformation. Mol. primary embryo fibroblasts requires at least two cooperating oncogenes. Cell Biol., 4:1999-2009, 1984. Nature (Lond.), 304: 596-602, 1983. 118. Sefton, B. M., Hunter, T., Beemon, K., and Eckhart, W. Evidence that the 91. Newbold, R. F., and Overell, R. W. Fibroblast immortality is a prerequisite for phosphorylation of tyrosine is essential for transformation by Rous sarcoma transformation by EJ c-Ha-ras oncogene. Nature (Lond.), 304: 648-651, virus. Cell, 20:807-816, 1980. 1983. 119. Erikson, E., Shealy, D. J., and Erikson, R. L. Evidence that viral transforming 92. Ruley, H. E. Adenovirus early region 1A enables viral and cellular transforming gene products and epidermal growth factor stimulate phosphorylation with genes to transform primary cells in culture. Nature (Lond.), 304: 602-606, similar specificity. J. Biol. Chem., 256: 11381-11384, 1981. 1983. 120. Pike, L. J., Gallis, B., Casnelfe, J. E., Bomstein, P., and Krebs, E. G. Epidermal 93. Reynolds, V. H., Boehm, F. H., and Cohen, S. Enhancement of chemical growth factor stimulates the phosphorylation of synthetic tyrosine containing carcinogenesis by an epidermal growth factor. Surg. Forum, 76: 108-109, peptides by A431 cell membrane. Proc. Nati. Acad. Sci. USA, 79: 1443- 1965. 1447,1982. 94. Rose, S. P., Stahn, R., Passovoy, D. S., and Herschman, H. Epidermal 121. Chinkers, M., and Cohen, S. Purified EGF receptor-kinase interacts specifi growth factor enhancement of skin tumor induction in mice. Experientia cally with antibodies to Rous sarcoma virus transforming protein. Nature (Basel), 32:913-915, 1976. (Lond.), 290: 516-519,1981. 95. Friedmann, B., Frackelton, A. R., Jr., Ross, A. H., Connors, J. M., Fujiki, H., 122. Kudlow, J. E., Buss, J. E., and Gill, G. N. Anti-pp60 src antibodies are Sugimura, T.. and Rosner. M. R. Tumor promoters block tyrosine-specific substrates for EGF-stimulated protein kinase. Nature (Lond.), 290: 519-521, phosphorylation on the epidermal growth factor receptor. Proc. Nati. Acad. 1981. Sci. USA, 87: 3034-3038. 1984. 123. Downward, J., Yarden, Y., Mayes, E., Scrace, J. G., Totty, N., Stockwell, P., 96. King, A. C., and Cuatrecasas, P. Resolution of high and low affinity epidermal Ullrich, A., Schlessinger, J., and Waterfield, M. D. Close similarity of epidermal growth factor receptors. J. Biol. Chem., 257: 3053-3060, 1982. growth factor receptor and v-erb-B oncogene protein sequences. Nature 97. Lee, L. S., and Weinstein, I. B. Tumor-promoting phorbol esters inhibit binding (Lond.), 307: 521-527, 1984. of epidermal growth factor to cellular receptors. Science (Wash. DC), 202: 124. Ullrich, A., Coussens, L., Hayflick, J. S., Dull, T. J., Gray, A., Tarn, A. W., 313-315,1978. Lee, J., Yarden, Y., Libermann, T. A., Schlessinger, J., Downward, J., Mayes, 98. Lee, L. S., and Weinstein, I. B. Mechanism of tumor promoter inhibition of E. L. V., Whittle, Nâ€žWaterfield, M. D., and Seeburg, P. H. Human epidermal cellular binding of epidermal growth factor. Proc. Nati. Acad. Sci. USA, 76: growth factor receptor cDNA sequence and aberrant expression on the 5168-5172,1979. amplified gene in A431 epidermoid carcinoma cells. Nature (Lond.), 309:418- 99. Singh-Asa, P., and Waters, M. J. Stimulation of adrenal cortisol biosynthesis 425,1984. by epidermal growth factor. Mol. Cell. Endocr., 30: 189-199, 1983. 125. Decker, S. J. Phosphorylation of the erbB gene product from an avian

CANCER RESEARCH VOL. 46 MARCH 1986

1036

erythroblastosis virus-transformed chick fibroblast cell line. J. Biol. Chem., surface receptors by tumor promoters. Biochem. Biophys. Res. Commun., 260:2003-2006, 1985. 86:1037-1043, 1979. 126. Gilmore,T., DeClue,J. E., and Martin, S. Protein phosphorylationat tyrosine 142. Pessin,J. E., Gitomer, W., Oka, Y., Oppenheimer,C. L., and Czech, M. P. 0- is induced by the v-erbB gene product in vivo and in vitro. Cell, 40:609-618, Adrenergic regulationof insulin and epidermalgrowth factor receptors in rat 1985. adipocytes. J. Biol. Chem., 258: 7386-7394, 1983. 127. Kris, R. M., Lax, I., Gullick, W., Waterfield, M. D., Ullrich,A., Fridkin, M., and 143. Rozengurt, E., Brown, K. D., and Pettican, P. Vasopressin inhibition of Schlessinger. J. Antibodies against a synthetic peptide as a probe for the epidermalgrowth factor binding to cultured mouse cells. J. Biol. Chem., 256: kinase activity of the avian EGF receptor and v-erbB protein. Cell, 40: 619- 716-722,1981. 625,1985. 144. Huang, J. S., Huang, S. S., and Deuel, T. F. Transforming protein of simian 128. Fung, K. T., Lewis, W. G., Crittendon, L. B., and Kung, H. Activation of the sarcomavirus stimulates autocrine growth of SSV-transformedcells through cellular oncogene c-erbB by LTR insertion: molecular basis for induction of PDGFcell-surface receptors. Cell, 39: 79-87, 1984. erythroblastosis by avian leukosis virus. Cell, 33: 357-368, 1983. 145. Gresik, E. W., Van Der Noen, H., and Barka, T. Transport of 125l-epidermal 129. Radke, K., and Martin, G. S. Transformation by Rous sarcoma virus: effects growth factor into milk and effect of sialoadenectomyon milk EGF in mice. of src gene expression on the synthesis and phosphorylation of cellular Am. J. Physiol.,247: E349-E354,1984. polypeptides. Proc. Nati. Acad. Sci. USA, 76: 5212-5216, 1979. 146. Todaro, G. J., DeLarco, J. E., and Cohen, S. Transformation by murine and 130. Johnson, L. K., Baxter. J. D., Vlodavsdy, I., and Gospodarowicz, D. EGFand feline sarcomaviruses specificallyblocks bindingsof epidermalgrowth factor expression of specific genes: effects on cultured rat pituitary cells are to cells. Nature (Lond.),264: 26-31, 1976. dissociable from the mitogenic response. Proc. Nati. Acad. Sci. USA, 77: 147. Hendler,F.J., and Ozanne,B. W. Humansquamouscell lung cancersexpress 394-398,1980. increased epidermal growth factor receptors. J. Clin. Invest., 74: 647-651, 131. Knowles,A. F., Salas-Prato,M., and Villela,J. Epidermalgrowth factor inhibits 1984. growth while increasing the expression of an ecto-calcium-ATPase of a 148. Hirata, Y., Uchihashi,M., Fujuta, T., Matsukura, S., Motoyama, T., Kaku, M., human hepatoma cell line. Biochem. Biophys. Res. Commun., 726: 8-14, and Koshimizu, K. Characteristics of specific binding of epidermal growth 1985. factor (EGF)on human tumor cell lines. Endocrino!.Jpn., 30: 601-607,1983. 132. Schonbrunn, A., Krasnoff, M., Westendorf, J. M., and Tashjian, A. H., Jr. 149. Fitzpatrick, S. L., LaChance, M. P., and Schulz, G. S. Characterization of EGFand thyrotropin-releasinghormone act similarlyon a clonal pituitary cell epidermalgrowth factor receptor and action on human breast cancer cells in strain. J. Cell Biol., 85: 786-797,1980. culture. Cancer Res., 44: 3442-3447, 1984. 133. Barnes, D. W. Epidermal growth factor inhibits growth of A431 human 150. Libermann,T. A., Bartal, A. D., Yarden, Y., Schlessinger,J.. and Soreq. H. epidermoid carcinoma in serum-freeculture. J. Cell Biol., 93:1-4,1982. Expression of epidermal growth factor receptors in human brain tumors. Cancer Res., 44: 753-760, 1984. 134. Masui, H., Kawamoto, T., Sato, J. D., Wolf, B., Sato, G., and Mendelsohn,J. Growth inhibition of human tumor cells in athymic mice by anti-epidermal 151. Lin, C. R., Chen, W. S., Kruiger, W., Stolarsky, L. S., Weber, W., Evans, R. growth factor receptor monoclonalantibodies. Cancer Res., 44:1002-1007, M., Verma, I. M., Gill, G. N., and Rosenfeld, M. G. Expression cloning of human EGF receptor complimentary DNA: gene amplification and three 1984. related messenger RNA products in A431 cells. Science (Wash. DC), 224: 135. Schreiber, A. B., Yarden, Y., and Schlessinger,J. A nonmitogenic analog of 843-848,1984. epidermalgrowth factor enhancesthe phosphorylationof endogenous mem brane proteins. Biochem. Biophys. Res. Commun., 707: 517-523,1981. 152. Merlino, G. T., Xu, Y., Ishii, S., Clark, A. J. L., Semba, K., Toyoshima, K., 136. Parker, R. C., Varmus, H. E., and Bishop, J. M. Expression of v-src and Yamamoto, T., and Pastan, I. Amplification and enhancedexpression of the epidermal growth factor receptor gene in A431 human carcinoma cells. chicken c-src in rat cells demonstrates qualitative differences between pp60 Science(Wash. DC),224: 417-419,1984. v-src and pp60 c-src. Cell, 37:131-134,1984. 153. Hurley, J. B., Simon, M. I., and Teplow, D. B. Homologies between signal 137. Salomon, D. S., Zwiebel, J. A., Bano, M., Losonczy, I., Fennel, P., and transducing G proteins and ras gene products. Science (Wash. DC), 226: Kidwell, W. R. Presence of transforming growth factors in human breast 860-862,1984. cancer cells. Cancer Res., 44: 4069-4077,1984. 154. Waterfield, M. D., Scrace, G. T., Whittle, N., Stroobant, P., Johnsson, A., 138. Sherwin, S. A., Minna, J. D., Gazdar, A. F., and Todaro, G. J. Expression of Wasteson, A., Westermark, B., Heldin, C. H., Huang, S., and Deuel, T. F. epidermal and nerve growth factor receptors and soft agar growth factor Platelet-derivedgrowth factor is structurally relatedto the putative transform production by human lung cancer. Cancer Res., 41: 3538-3542,1981. ing protein p28 sis of simian sarcoma virus. Nature (Lond.), 304: 35-39, 139. McClure, D. B. Anchorage-independent colony formation of SV-40 trans 1983. formed BALB/C-3T3 cells in serum-free medium: role of cell- and serum- 155. Toda, T., Uno,I., Ishikawa, T., Powers, S., Kataoka,T., Broek. D., Cameron, derived factors. Cell, 32: 999-1006, 1983. S., Broach, J., Matsumoto, K., and Wigier. M. In yeast, ras proteins are 140. Bowen-Pope, D. F., Dicorteto, P. E., and Ross, R. Interactions between the controlling elementsof adenylatecyclase. Cell, 40: 27-36,1985. receptors for platelet-derivedgrowth factor and epidermal growth factor. J. 156. Kataoka, T., Powers, S., Cameron,S., Fasano,O., Goldfarb, M., Broach, F., Cell Biol., 96:679-683, 1983. and Wigier, M. Functionalhomologyof mammalianand yeast ras genes. Cell, 141 Brown, K. D., Dicker, P., and Rozengurt, E. Inhibition of EGF binding to 40:19-26, 1985.

CANCER RESEARCH VOL. 46 MARCH 1986 1037

Christa M. Stoscheck and Lloyd E. King, Jr.

Cancer Res 1986;46:1030-1037.

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