- Email: [email protected]
EPIDERMAL GROWTH FACTOR AND RELATED MOLECULES
1243
induced ulcers in dogs. Although EGF remains the single most effective experimental treatment for this condition, difficulties with factor delivery and its possible role in cancer have limited its introduction as a therapeutic agent. Study of EGF has also suggested other potential applications--eg, when perfused into sheep, EGF slows hair follicle development,4 making it easy to remove the fleece but leaving a redundant shearer with a naked and potentially sunburnt animal. The importance of EGF as a potential therapeutic agent has been extended by the realisation that EGF-like activities may be involved in wound healing and could alter cell growth in cancer. The search for cancer-specific factors that could induce a transformed phenotype on normal test cells in tissue culture led to the discovery of transforminggrowth-factor-a (TGF-&agr;, also known as sarcoma growth factor or SGF) in the conditioned media of cancer cells.5,6 TGF-&agr; was subsequently shown to be a normal gene product closely related to EGF that is produced in abnormal amounts by some tumour cells. The EGF and TGF genes have been mapped to chromosomes 4 and 2, respectively. A third gene is encountered through vaccination, since the poxvirus genome encodes a factor homologous in structure and function to EGF called vaccinia virus growth factor (WGF); this factor, although not essential for viral function, may contribute to the epidermal eruption seen at the vaccination site. Our understanding of EGF-initiated signals and the modulation of cancer cell growth has been extended by studies of the specific cell surface receptor shared by EGF and TGF-a. The receptor is a large glycoprotein (MW 175 000) that was originally detected with radiolabelled EGF and subsequently isolated with antireceptor monoclonal antibodies. Further analysis was greatly accelerated by the introduction of genetic engineering techniques in the 1980s. The full primary structure of the receptor has been established, so aiding interpretation of biochemical findings about the ligand and receptor interaction.7,8 From these studies came the dramatic realisation that the receptor bears a striking resemblance to the transforming protein of a cancer virus. Thus, avian
experimentally
Peptide Regulatory Factors EPIDERMAL GROWTH FACTOR AND RELATED MOLECULES
M. D. WATERFIELD
Ludwig Institute for Cancer Research, University College and Middlesex School of Medicine, Courtauld Building, 91 Ridinghouse Street, London W1P 8BT THE discovery of epidermal growth factor (EGF) and the subsequent search for its physiological role and mechanism of action have all the hallmarks of a marvellous but unfinished detective story. The tale begins in the early 1960s in the laboratory of Victor Hamburger, where Rita LeviMontalcini and Stanley Cohen were looking for factors that supported nerve cell development and growth.! As part of this study, tumour cell extracts were treated with snake venom containing phosphodiesterases to rule out the participation of nucleic acids-surprisingly, the venom itself was found to have a nerve-growth-factor-like activity. Lateral thinking led these workers to examine mouse salivary glands for similar factors. Remarkably, they found that male mice stored large amounts of a factor in these glands, for reasons that remain unclear. The activity, awarded the name EGF, was first detected when Stanley Cohen injected salivary gland extracts into newborn mice and found that the treated animals opened their eyes earlier than did control mice. The ability to induce this effect was used as a bioassay to purify EGF in sufficient amounts to establish its aminoacid sequence and to provide a pure characterised factor for study of its biochemistry and physiology.2 The discovery that EGF was involved in the control of gastric acid secretion came with the purification of urogastrone from urine of pregnant women (who have a low frequency of ulcers) by H. Gregory, who showed that this substance was human EGFand that it could cure
DA, Butler S, Riemersma RA, Thomson M, Oliver MF. Adipose tissue and platelet fatty adds and coronary heart disease in Scottish men. Lancet 1984; ii:
14. Wood
117-21.
15. Miettinen TA, Naukkarinen V, Huttunen JK, Mattila S, Kumlin T. Fatty add composition of serum lipids predicts myocardial infarction. Br Med J 1982; 285: 993-96 16. Wood DA, Riemersma RA, Butler S, et al. Linoleic and eicosapentaenoic acids in adipose tissue and platelets and nsk of coronary heart disease. Lancet 1987; i: 177-83. 17. Thomson M, Logan RL, Sharman M, Lockerbie L, Riemersma RA, Oliver MF. Dietary survey in 40-year old Edinburgh men. Hum Nutr Appl Nutr 1982; 36A: 272-80. 18. Thomson M, Fulton M, Elton RA, Brown S, Wood DA, Oliver MF. Alcohol consumption and nutrient intake in middle-aged Scottish men. AmJ Clin Nutr 1988; 47: 139-45. 19. Fehily AM, Phillips KM, Yarnell JWG. Diet, smoking, social class and body mass index in the Caerphilly Heart Disease Study. Am J Clin Nutr 1984; 40: 827-33. 20. Homstra G, Lewis B, Chait A, Turpeinen O, Karvonen MJ, Vergroesen AJ. Influence of dietary fat on platelet function in men. Lancet 1973; i: 1155-57. 21. Ahrens EH Jr, Hirsch J, Insull W Jr, Tsaltas TT, Blomstrand R, Peterson ML. The influence of dietary fats on serum lipid levels in man. Lancet 1957; i: 943-53. 22. Weiner BH, Ockene IS, Levine PH, et al. Inhibition of atherosclerosis by cod-liver oil in a hyperlipidemic swine model. N Engl J. Med 1986, 315: 841-46. 23. Dehmer GJ, Popma JJ, van den Berg EK, et al. Reduction in the rate of early restenosis after coronary angioplasty by a diet supplemented with n-3 fatty adds. N Engl J Med 1988; 319: 733-40. 24. Demke DM, Peters GR, Linet OI, Metzler CM, Klott KA. Effects of a fish oil concentrate in patients with hypercholesterolemia. Atherosclerosis 1988; 70: 73-80.
25. McLennan PL, Abeywardena MY, Charnock JS. Influence of dietary lipids on arrhythmias and infarction after coronary ligation in rats. Can J Physiol Pharmacol 1985; 63: 1411-17.
Lepran I, Nemecz G, Koltai M, Szekeres L. Effects of a linoleic acid-rich diet on the acute phase of coronary occlusion in conscious rats: influence of indomethacin and aspirin. J Cardiovasc Pharmacol 1981; 3: 847-53. 27. Riemersma RA, Sargent CA, Saman S, Rebergen SA, Abraham R. Dietary fatty acids and ischaemic arrhythmias. Lancet 1988; i: 285-86 28. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witzum JL Beyond cholesterol. N Engl J Med 1989; 320: 915-24 29. Homig DH, Glatthaar BE. Vitamin C and smoking: increased requirements of smokers. In: Homig DH, Hanck AB, eds. Vitamins: nutrients and therapeutic agents Bern: Hans Huber, 1985. 139-55. 30. Riemersma RA, Wood DA, Macintyre C, Elton R, Gey F, Oliver MF. Plasma vitamin E and the risk of angina in Scottish men. Vitamin E. biochemistry and health implications. New York: New York Acad Sci. 1988: Abstract 25. 31. Marmot MG. Interpretation of trends on coronary heart disease mortality. Acta Med 26.
Scand (Suppl) 1985; 701: 58-65. 32. Berry EM, Hirsch J, Most J, McNamara DJ, Thomton J. The relationship of dietary fat to plasma lipid levels as studied by factor analysis of adipose tissue fatty acid composition in a free-living population of middle-aged American men AmJ Clin Nutr 1986; 44: 220-31 33. Jacobsen
BJ, Trygg K, Hjermann I, Thomassen MS, Real C, Norum KR. Acyl pattern of adipose tissue triglycerides, plasma free fatty acids, and diet of a group of men participating in a primary coronary prevention program (The Oslo Study). Am J Clin Nutr 1983; 38: 906-13.
1244
erythroblastosis virus transforms cells by introducing
a
truncated EGF receptor, active in the absence ofligand, into cells which then lose control of their proliferative capacity. This observation shows that the effects of a growth factor such as EGF or TGF-a can be mimicked by the generation of an altered receptor. Since many growth factors and receptors have now been characterised, the potential role of such receptor subversion in cancer and other diseases must be examined carefully. What role do EGF and TGF-a normally fulfil and how are these factors involved in abnormal cell regulation? Whilst answers to these questions remain incomplete, there have been many interesting insights into the possible function of the two molecules.
PHYSIOLOGY AND PATHOPHYSIOLOGY OF EGF AND TGF-OC
Although the sites of synthesis of EGF and TGF-K have been difficult to establish because of limitations in the techniques available for detection of the peptides, measurement ofmRNA synthesis has been helpful. TGF-cx mRNA6but not EGF mRNA 10 is expressed in some tissues of the embryo and in the placenta. TG F-a, is made in normal keratinocytes" and activated macrophages12 and its mRNA has been found in the brain13 and pituitary.’4 The only readily demonstrable site of EGF synthesis in the adult mouse, apart from the salivary gland, is the kidney, but the observation that EGF can be found in milk and urine suggests that other sites may be important.*" Many cells can respond to EGF and TGF-oc: these peptides can modulate development of epidermis, breast, and gut; act as angiogenic factors; and may mediate hypercalcaemia.6,10 It is therefore possible that such processes could be manipulated by antagonists for the factors once the structure and function of the ligands and their receptor have been established.
Altered Production
Fig I-The biosynthetic precursors of EGF, TGF-o,andWGFand projected 3-D structure of mouse EGF. Insertion of precursors into the cell membrane is shown. The 3-1 of mouse EGF was obtained by 2-D magnetic resonance (deduce by Montelione et al9 and redrawn from Burgess et al’O). structure
with homologous but distinct aminoacid sequences isolate( from different animals and from cells infected with th! poxviruses, and of artificial mutant molecules generated b) recombinant DNA (rDNA) techniques, should lead t( defmition of the structural requirements for recepto binding. Such studies may also encourage the developmen of specific EGF antagonists. EGF-like sequences occur ir several other proteins, where they probably serve a! building blocks rather than as growth factor precursors Thus evolution has used an important structural motif foi two different purposes.
of TGF-ot
Although transformed cells have not yet been shown to produce EGF, many produce TGF-a. Such expression in cells with EGF receptors would be expected to contribute to the generation of neoplastic growth by means of an autocrine circuit, but the relevance of such a mechanism to the genesis of human tumours is unclear.
STRUCTURE OF EGF AND TGF-a,
EGF and TGF-a are synthesised as precursors of 121716 and 16017 aminoacids, respectively; the precursor molecules have aminoterminal signal sequences and transmembrane sequences near their c-termini that direct their insertion into membranes as transmembrane proteins. Subsequent exposure of the precursor aminoterminal domains to specific proteases releases the active factors of about 50 aminoacids. The biological function of the precursor in membranes is unclear, but it might provide an immobilised growth factor capable of stimulating adjacent cells. Large quantities of recombinant EGF and TGF-a are now available and two-dimensional magnetic resonance techniques have been used by several groups to derive three-dimensional structural models (fig 1).9,10 EGF has also been crystallised, 17 so more detail of the structure will eventually become available via X-ray crystallographic analysis. The availability of many natural EGF molecules
EGF RECEPTOR AND RELATED MOLECULES
availability of large amounts of pure EGF from salivary glands led to the measurement of receptor numbers and their affinity for ligand; it was also possible to show that certain cells from all three germ layers could The
mouse
express receptors. The vulval carcinoma cell line A431 which over-expresses the receptor (having 2 000 000 rather than 50 000-100 000 receptors), has provided an essential tool for study of receptor biochemistry and an early indication of abnormal receptor expression in tumours. The receptor was identified as a 175 000 MW glycoprotein; the first clue to the mechanism used by the receptor to transduce a signal across the cell membrane was found by Cohen when he showed that EGF could stimulate an intrinsic tyrosine kinase activity that autophosphorylates the receptor.18 The aminoacid sequence of the receptor was deduced both from protein structure analysis of receptor purified with monoclonal antibodies and from the isolation of a receptor cDNA sequences This information made it possible to interpret a mass of biochemical data and derive a model for the first growth factor receptor. The model in fig 2 shows a receptor with an external ligand binding domain separated from an internal tyrosine kinase domain by a hydrophobic transmembrane region. This organisation has been found in ten other receptors or putative receptors whose sequences have now been deduced.18,19 The plateletderived growth factor (PDGF) and insulin receptors define
1245
subgroups that are distinguished because they are homodimers or have an insert in the tyrosine kinase domain, respectively (fig 2). To solve the structural basis of ligand-receptor binding through biophysical studies, the external domains of the EGF and insulin receptors have been expressed as soluble proteins by means of recombinant DNA techniques. two other
The tyrosine kinase domain of the EGF receptor contains
several aminoacids that are conserved in all kinases, some of known to be important for ATP binding.2O Mutation of ATP binding residues such as lysine 761 inactivates EGF signal transduction, thereby confirming the importance of the kinase activity. Remarkably little is known about the signalling pathway downstream from this or any other tyrosine kinase; intensive searches for potential substrates, other than the kinases themselves, have not been rewarding. The EGF receptor is autophosphorylated at three sites and appears neither to have any effect on kinase activity nor to influence transduction of a mitogenic signal by the receptor. Mutagenesis experiments involving inactivation of the kinase or truncation of the c-terminal domain that contains the autophosphorylation sites suggest that the kinase could mediate intemalisation, recycling, and down-regulation of the receptor.21 Since EGF must be present for about 8 hours to generate a mitogenic signal in cells in tissue culture, the fate of the receptor, as mediated by its kinase activity, could be crucial. Receptor function can be modulated by components of other signalling pathways such as protein kinase C. Thus PDGF or bombesin-mitogens that activate protein kinase C-reduce EGF binding affinity and inhibit EGF receptor tyrosine kinase activity. The effect is partly mediated by phosphorylation of a threonine residue in a basic region of the receptor immediately below the transmembrane region (T654) ZZ How is it possible for the EGF binding signal to be transferred through the hydrophobic transmembrane domain to turn on the kinase, and how does phosphorylation of T654 just below the membrane reduce EGF affinity for the external domain? The most popular, but by no means proven, notion is that a conformational change induced by
which
are
I
I
between EGF/TGF-a
I
Fig 2-Relation receptor and the putative neu, insulin, and PDGF receptors. PDGF=platelet-derived growth factor.
to the external domain causes receptor dimerisation and consequently brings into apposition the two receptor kinase domains. These domains then phosphorylate each other and somehow activate a mechanism for signal transmission that involves interaction with other proteins 21 The identity of the proteins in the cascade that conveys the signal to the nucleus and alters gene regulation remains unknown.
ligand binding
Receptor Encoded by c-neu The c-neu gene (also
called c-erb-B-2 and HER-2) encodes a second receptor with close structural similarity to the EGF receptor (fig 2) that was discovered by two different routes. The first involved isolation of the neu oncogene from neuroglioblastomas induced in newborn rats by administration of N-nitroso urea to their pregnant mothers.23 The rat oncogene was found because it transformed NIH3T3 cells (as does the ras oncogene) and it has subsequently been shown to encode a transmembrane protein of 180 000 MW. The second approach, used by three different research groups, identified the human c-neu gene, cDNA, and protein with probes based on knowledge of the EGF receptor and its DNA sequence. The putative ligand for this receptor is unknown. SUBVERSION OF EGF RECEPTOR FUNCTION
As part of the analysis of the sequence of peptides derived from the EGF receptor, Julian Downward in my laboratory searched our sequence database for proteins of similar sequence.’ This search showed that Toyoshima’s group in Japan had recently obtained the sequence of the transforming gene v-erb-B of avian erythroblastosis virus (AEV)24 and that the sequence closely resembled that of the human EGF receptor. Thus the chicken virus oncogene is probably derived from the chicken EGF receptor gene. Realisation that v-erb-B encodes a truncated receptor lacking both the external ligand binding domain and a smaller segment of the c-terminus that includes part of the autophosphorylation site domain is especially remarkable. These data suggested to us that expression of a truncated receptor was a mechanism that allowed a ligandindependent transforming signal to be introduced by a virus or generated by mutation in a cell. Detailed analysis of the EGF receptor alterations that are necessary to create a transforming protein for avian cells25 showed that deletion of the ligand binding domain is sufficient to induce constitutive self renewal of erythroblasts; this effect is enhanced by further c-terminal truncation but the resultant molecules are less potent than the v-erb-B protein itself. Thus it is possible that other mutations in the truncated receptor remain to be defined. Rearrangements in the EGF receptor gene in human tumours have not yet been found, except in some brain tumours. Subsequent studies have shown that similar mechanisms have been used to create other oncogenes. Thus the fms transforming protein is generated from the macrophage colony stimulating factor receptor by a single aminoacid change in the ligand binding domain and removal of a negative regulatory site at its c-tem-linus .16 Of special interest is the conversion of the normal c-neu-encoded receptor to a transforming protein by a mutation that leads to substitution of a glutamic acid residue for a valine in the transmembrane domain.23 A similar change engineered in the EGF receptor does not generate a transforming protein and the mechanism of activation of c-neu remains a mystery.
1246
Analysis of the transforming potential of normal EGF receptor expression has shown that over-expression in the presence of ligand can partly transform test cells ;27 this is exactly the alteration seen in the A431 cell line mentioned above. Such over-expression, accompanied by gene amplification (as seen in A431) in some cases, has been seen in squamous tumour cell lines and less frequently in primary lung and bladder tumours. Over-expression of the c-neuencoded receptor, for which we do not know the ligand, has been documented in some breast adenocarcinomas and ovarian cancers28 and may be of prognostic value.28,29 Levi-Montalcini and Cohen were awarded their Nobel prize in 1986 for their work in unravelling the role of EGF and other growth factors in normal and abnormal growth and development. The detective story that they initiated is unfinished, but their innovative discoveries proved to be a stimulus for many scientists and the insights gained are rapidly aproaching clinical application. More than forty companies are trying to commercialise EGF. Some applications are diagnostic; others involve clinical trials. EGF is being evaluated as an aid to repair of skin burns and of damaged tympanic membrane of the ear, and in the treatment of glaucoma. It is sobering to think that the supplementary EGF in cows’ milk given to babies to achieve the levels in human milk would require about 600 tons of EGF a year. CONCLUSIONS
EGF and TGF-a are synthesised as transmembrane protein precursors that are cleaved by proteases to generate active factors of approximately 50 aminoacids. Both factors bind to the same receptor to stimulate an intrinsic tyrosine kinase that autophosphorylates the receptor. This activity is essential for signal transduction, a process that also involves receptor dimerisation. Moreover, the kinase may mediate receptor intemalisation and down-regulation, although the subsequent path of mitogenic signal processing is not understood. Overproduction of TGF-a or of its receptor and the expression of altered truncated receptors may play a role in cancer. The c-neu protein, which is closely related to the EGF receptor in structure and can also be converted to oncogene, is over-expressed in certain cancers. An understanding of the structure and function of EGF and TGF-a, together with their receptor and related molecules, may lead to the design of antagonists with therapeutic an
potential. REFERENCES 1.
Levi-Montalcini R, Cohen S. Effects of the extracts of the mouse submaxillary salivary glands on the sympathetic system of mammals. Ann NY Acad Sci 1960; 85: 324-41.
Carpenter G, Zendegui JG. Epidermal growth factor, its receptor, and related proteins. Exp Cell Res 1986; 164: 1-10. 3. Gregory H. Isolation and structure of urogastrone and its relationship to epidermal 2.
growth factor. Nature 1975; 257: 325-27. 4. Thornboum GD, Waters MJ, Dolling M, Young IR Fetal maturation and epidermal growth factor. Proc Aust Soc Pharmacol Sci 1981; 12: 11-15. 5. De Larco JE, Todaro GJ. Growth factors from murine sarcoma virus-transformed cells. Proc Natl Acad Sci USA 1978; 75: 4001-05. 6. Derynck R. Transforming growth factor &agr;. Cell 1988; 54: 593-95. 7. Downward J, Yarden Y, Mayes E, et al. Close similarity of epidermal growth factor receptor and v-erbB oncogene protein sequences. Nature 1984. 307: 521-27. 8. Ullrich A, Coussens L, Hayflick JS, et al. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 cells. Nature 1984; 309: 418-25 9. Montelione GT, Wuthnch K, Nice EC, Burgess AW, Scheraga HA. Identification of two anti-parallel beta sheet conformations in the solution structure of murine epidermal growth factor by proton magnetic resonance. Proc Natl Acad Sci USA 1986; 83: 8594-98. 10. Burgess AW, Lloyd CJ, Smith S, et al. Munne epidermal growth factor: structure and function. Biochemistry 1988; 27: 4977-85.
Therapeutics TRIAL OF PREDNISOLONE FOR PREVENTION OF MELARSOPROL-INDUCED
ENCEPHALOPATHY IN GAMBIENSE SLEEPING SICKNESS
JACQUES PEPIN1 CLAUDE GUERN2,3 LUCIEN ETHIER2,3
FRANCOIS MILORD2,3 BOKELO MPIA3 DIABAKANA MANSINSA3
University of Manitoba, Winnipeg;1 University of Sherbrooke, Sherbrooke, Canada;2 and Zone de Sante Rurale, Nioki, Zaire3
prospective randomised trial, 620 patients who had Trypanosoma brucei gambiense trypanosomiasis with central nervous system involvement were treated either with prednisolone plus melarsoprol or with melarsoprol only. 598 patients were evaluable: morbidity and death associated with melarsoprolinduced encephalopathy was reduced in patients who were given prednisolone. The two groups did not differ either in the incidence of other complications of melarsoprol therapy or in relapse rate after melarsoprol therapy. The cost of prednisolone would be outweighed by savings on the treatment of encephalopathies in such patients. Summary
In
a
Coffey RJ, Derynek P, Wilcox JN, Bringham TS, Goustin AS, Moses HL, Pittelkow MR. Production and auto-induction of transforming growth factor &agr; in human keratinocytes. Nature 1987; 328: 817-20. 12. Madtes DK, Raines EW, Sakarissen KS, et al. Induction of transforming growth factor-&agr; in activated human alveolar macrophages. Cell 1988; 53: 285-93. 13. Wilcox JN, Derynck L. Localization of cells synthesising transforming growth factor-alpha mRNA in the mouse brain. Cell 1988; 8: 1901-04. 14. Kobrin MS, Samsoondar J, Kudlow JE. A transforming growth factor secreted by untransformed bovine anterior pituitary cells in culture. J Biol Chem 1986; 261: 11.
14414-19.
Gray A, Dull TJ, Ullnch A. A nucleotide sequence of epidermal growth factor cDNA predicts a 128 000 molecular weight protein precursor. Nature 1983; 303: 722-25. 16. Derynck R, Roberts AB, Winkler ME, Chen EY, Goeddel DV. Human transforming growth factor-&agr;: precursor structure and expression in Ecoli. Cell 1984; 38: 287-97. 17. Higuchi Y, Marimoto Y, Harinata A, Yasuoka N. Crystalisation and preliminary X-ray studies of human epidermal growth factor.J Biochem 1988; 103: 905-06 18. Carpenter G. Receptors for epidermal growth factor and other polypeptide mitogens. 15.
Ann Rev Biochem 1987; 56: 881-915. 19. Yarden J, Ullrich A. Growth factor receptor tyrosine kinases. Am Rev Biochem 1988; 57: 443-79. 20. Hanks SK, Quinn AM, Hunter T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 1988; 241: 42-52. 21. Schlessinger J. Signal transduction by receptor oligomerisation. Trends Biochem Sci 1988; 13: 443-47. 22. Cochet C, Gill GN, Meisenhelder J, Cooper JA, Hunter T. C-kinase phosphotylates the epidermal growth factor receptor and reduces its epidermal growth factor stimulated-tyrosine kinase activity. J Biol Chem 1984; 259: 2553-58. 23. Bargmann CI, Hung M-C, Weinberg RA. Multiple independent activations of the c-neu oncogene by a point mutation in the transmembrane domain. Cell 1986; 45: 649-57. 24. Yamamoto T, Nishida T, Miyajima N, Kawai S, Doi T, Toyoshima K. The erbB gene of avian erythroblastosis virus is a member of the scr gene family. Cell 1986; 45: 649-57. 25. Khazaie K, Dull TJ, Graf T, Schlessinger J, Ullrich A, Beug H, Vennstrom B Truncation of the human EGF receptor leads to differential transformating potentials in primary avian fibroblasts and erythroblasts. EMBO J 1988; 7: 3061-71. 26. Roussel MF, Downing JR, Rettenmier CW, Sherr CJ. A point mutation in the extracellular domain of the human CSF-1 receptor (c-fms proto-oncogene) activates its transforming potential. Cell 1988; 55: 965-77. 27. Di Fiore PP, Pierce JH, Fleming TP, et al. Overexpression of the human EGF receptor confers an EGF-dependent phenotype to NIH3T3 cells. Cell 1987; 51: 1063-70. 28. Slamon DJ, Godolphin W, Jones LA, et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 1989; 244: 707-12. 29. Nicholson S, Sainsbury JRC, Halcrow P, Chambers P, Farndon JR, Harris AL Expression of epidermal growth factor receptors associated with lack of response to endocrine therapy in recurrent breast cancer. Lancet 1989; i. 182-85.
induced ulcers in dogs. Although EGF remains the single most effective experimental treatment for this condition, difficulties with factor delivery and its possible role in cancer have limited its introduction as a therapeutic agent. Study of EGF has also suggested other potential applications--eg, when perfused into sheep, EGF slows hair follicle development,4 making it easy to remove the fleece but leaving a redundant shearer with a naked and potentially sunburnt animal. The importance of EGF as a potential therapeutic agent has been extended by the realisation that EGF-like activities may be involved in wound healing and could alter cell growth in cancer. The search for cancer-specific factors that could induce a transformed phenotype on normal test cells in tissue culture led to the discovery of transforminggrowth-factor-a (TGF-&agr;, also known as sarcoma growth factor or SGF) in the conditioned media of cancer cells.5,6 TGF-&agr; was subsequently shown to be a normal gene product closely related to EGF that is produced in abnormal amounts by some tumour cells. The EGF and TGF genes have been mapped to chromosomes 4 and 2, respectively. A third gene is encountered through vaccination, since the poxvirus genome encodes a factor homologous in structure and function to EGF called vaccinia virus growth factor (WGF); this factor, although not essential for viral function, may contribute to the epidermal eruption seen at the vaccination site. Our understanding of EGF-initiated signals and the modulation of cancer cell growth has been extended by studies of the specific cell surface receptor shared by EGF and TGF-a. The receptor is a large glycoprotein (MW 175 000) that was originally detected with radiolabelled EGF and subsequently isolated with antireceptor monoclonal antibodies. Further analysis was greatly accelerated by the introduction of genetic engineering techniques in the 1980s. The full primary structure of the receptor has been established, so aiding interpretation of biochemical findings about the ligand and receptor interaction.7,8 From these studies came the dramatic realisation that the receptor bears a striking resemblance to the transforming protein of a cancer virus. Thus, avian
experimentally
Peptide Regulatory Factors EPIDERMAL GROWTH FACTOR AND RELATED MOLECULES
M. D. WATERFIELD
Ludwig Institute for Cancer Research, University College and Middlesex School of Medicine, Courtauld Building, 91 Ridinghouse Street, London W1P 8BT THE discovery of epidermal growth factor (EGF) and the subsequent search for its physiological role and mechanism of action have all the hallmarks of a marvellous but unfinished detective story. The tale begins in the early 1960s in the laboratory of Victor Hamburger, where Rita LeviMontalcini and Stanley Cohen were looking for factors that supported nerve cell development and growth.! As part of this study, tumour cell extracts were treated with snake venom containing phosphodiesterases to rule out the participation of nucleic acids-surprisingly, the venom itself was found to have a nerve-growth-factor-like activity. Lateral thinking led these workers to examine mouse salivary glands for similar factors. Remarkably, they found that male mice stored large amounts of a factor in these glands, for reasons that remain unclear. The activity, awarded the name EGF, was first detected when Stanley Cohen injected salivary gland extracts into newborn mice and found that the treated animals opened their eyes earlier than did control mice. The ability to induce this effect was used as a bioassay to purify EGF in sufficient amounts to establish its aminoacid sequence and to provide a pure characterised factor for study of its biochemistry and physiology.2 The discovery that EGF was involved in the control of gastric acid secretion came with the purification of urogastrone from urine of pregnant women (who have a low frequency of ulcers) by H. Gregory, who showed that this substance was human EGFand that it could cure
DA, Butler S, Riemersma RA, Thomson M, Oliver MF. Adipose tissue and platelet fatty adds and coronary heart disease in Scottish men. Lancet 1984; ii:
14. Wood
117-21.
15. Miettinen TA, Naukkarinen V, Huttunen JK, Mattila S, Kumlin T. Fatty add composition of serum lipids predicts myocardial infarction. Br Med J 1982; 285: 993-96 16. Wood DA, Riemersma RA, Butler S, et al. Linoleic and eicosapentaenoic acids in adipose tissue and platelets and nsk of coronary heart disease. Lancet 1987; i: 177-83. 17. Thomson M, Logan RL, Sharman M, Lockerbie L, Riemersma RA, Oliver MF. Dietary survey in 40-year old Edinburgh men. Hum Nutr Appl Nutr 1982; 36A: 272-80. 18. Thomson M, Fulton M, Elton RA, Brown S, Wood DA, Oliver MF. Alcohol consumption and nutrient intake in middle-aged Scottish men. AmJ Clin Nutr 1988; 47: 139-45. 19. Fehily AM, Phillips KM, Yarnell JWG. Diet, smoking, social class and body mass index in the Caerphilly Heart Disease Study. Am J Clin Nutr 1984; 40: 827-33. 20. Homstra G, Lewis B, Chait A, Turpeinen O, Karvonen MJ, Vergroesen AJ. Influence of dietary fat on platelet function in men. Lancet 1973; i: 1155-57. 21. Ahrens EH Jr, Hirsch J, Insull W Jr, Tsaltas TT, Blomstrand R, Peterson ML. The influence of dietary fats on serum lipid levels in man. Lancet 1957; i: 943-53. 22. Weiner BH, Ockene IS, Levine PH, et al. Inhibition of atherosclerosis by cod-liver oil in a hyperlipidemic swine model. N Engl J. Med 1986, 315: 841-46. 23. Dehmer GJ, Popma JJ, van den Berg EK, et al. Reduction in the rate of early restenosis after coronary angioplasty by a diet supplemented with n-3 fatty adds. N Engl J Med 1988; 319: 733-40. 24. Demke DM, Peters GR, Linet OI, Metzler CM, Klott KA. Effects of a fish oil concentrate in patients with hypercholesterolemia. Atherosclerosis 1988; 70: 73-80.
25. McLennan PL, Abeywardena MY, Charnock JS. Influence of dietary lipids on arrhythmias and infarction after coronary ligation in rats. Can J Physiol Pharmacol 1985; 63: 1411-17.
Lepran I, Nemecz G, Koltai M, Szekeres L. Effects of a linoleic acid-rich diet on the acute phase of coronary occlusion in conscious rats: influence of indomethacin and aspirin. J Cardiovasc Pharmacol 1981; 3: 847-53. 27. Riemersma RA, Sargent CA, Saman S, Rebergen SA, Abraham R. Dietary fatty acids and ischaemic arrhythmias. Lancet 1988; i: 285-86 28. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witzum JL Beyond cholesterol. N Engl J Med 1989; 320: 915-24 29. Homig DH, Glatthaar BE. Vitamin C and smoking: increased requirements of smokers. In: Homig DH, Hanck AB, eds. Vitamins: nutrients and therapeutic agents Bern: Hans Huber, 1985. 139-55. 30. Riemersma RA, Wood DA, Macintyre C, Elton R, Gey F, Oliver MF. Plasma vitamin E and the risk of angina in Scottish men. Vitamin E. biochemistry and health implications. New York: New York Acad Sci. 1988: Abstract 25. 31. Marmot MG. Interpretation of trends on coronary heart disease mortality. Acta Med 26.
Scand (Suppl) 1985; 701: 58-65. 32. Berry EM, Hirsch J, Most J, McNamara DJ, Thomton J. The relationship of dietary fat to plasma lipid levels as studied by factor analysis of adipose tissue fatty acid composition in a free-living population of middle-aged American men AmJ Clin Nutr 1986; 44: 220-31 33. Jacobsen
BJ, Trygg K, Hjermann I, Thomassen MS, Real C, Norum KR. Acyl pattern of adipose tissue triglycerides, plasma free fatty acids, and diet of a group of men participating in a primary coronary prevention program (The Oslo Study). Am J Clin Nutr 1983; 38: 906-13.
1244
erythroblastosis virus transforms cells by introducing
a
truncated EGF receptor, active in the absence ofligand, into cells which then lose control of their proliferative capacity. This observation shows that the effects of a growth factor such as EGF or TGF-a can be mimicked by the generation of an altered receptor. Since many growth factors and receptors have now been characterised, the potential role of such receptor subversion in cancer and other diseases must be examined carefully. What role do EGF and TGF-a normally fulfil and how are these factors involved in abnormal cell regulation? Whilst answers to these questions remain incomplete, there have been many interesting insights into the possible function of the two molecules.
PHYSIOLOGY AND PATHOPHYSIOLOGY OF EGF AND TGF-OC
Although the sites of synthesis of EGF and TGF-K have been difficult to establish because of limitations in the techniques available for detection of the peptides, measurement ofmRNA synthesis has been helpful. TGF-cx mRNA6but not EGF mRNA 10 is expressed in some tissues of the embryo and in the placenta. TG F-a, is made in normal keratinocytes" and activated macrophages12 and its mRNA has been found in the brain13 and pituitary.’4 The only readily demonstrable site of EGF synthesis in the adult mouse, apart from the salivary gland, is the kidney, but the observation that EGF can be found in milk and urine suggests that other sites may be important.*" Many cells can respond to EGF and TGF-oc: these peptides can modulate development of epidermis, breast, and gut; act as angiogenic factors; and may mediate hypercalcaemia.6,10 It is therefore possible that such processes could be manipulated by antagonists for the factors once the structure and function of the ligands and their receptor have been established.
Altered Production
Fig I-The biosynthetic precursors of EGF, TGF-o,andWGFand projected 3-D structure of mouse EGF. Insertion of precursors into the cell membrane is shown. The 3-1 of mouse EGF was obtained by 2-D magnetic resonance (deduce by Montelione et al9 and redrawn from Burgess et al’O). structure
with homologous but distinct aminoacid sequences isolate( from different animals and from cells infected with th! poxviruses, and of artificial mutant molecules generated b) recombinant DNA (rDNA) techniques, should lead t( defmition of the structural requirements for recepto binding. Such studies may also encourage the developmen of specific EGF antagonists. EGF-like sequences occur ir several other proteins, where they probably serve a! building blocks rather than as growth factor precursors Thus evolution has used an important structural motif foi two different purposes.
of TGF-ot
Although transformed cells have not yet been shown to produce EGF, many produce TGF-a. Such expression in cells with EGF receptors would be expected to contribute to the generation of neoplastic growth by means of an autocrine circuit, but the relevance of such a mechanism to the genesis of human tumours is unclear.
STRUCTURE OF EGF AND TGF-a,
EGF and TGF-a are synthesised as precursors of 121716 and 16017 aminoacids, respectively; the precursor molecules have aminoterminal signal sequences and transmembrane sequences near their c-termini that direct their insertion into membranes as transmembrane proteins. Subsequent exposure of the precursor aminoterminal domains to specific proteases releases the active factors of about 50 aminoacids. The biological function of the precursor in membranes is unclear, but it might provide an immobilised growth factor capable of stimulating adjacent cells. Large quantities of recombinant EGF and TGF-a are now available and two-dimensional magnetic resonance techniques have been used by several groups to derive three-dimensional structural models (fig 1).9,10 EGF has also been crystallised, 17 so more detail of the structure will eventually become available via X-ray crystallographic analysis. The availability of many natural EGF molecules
EGF RECEPTOR AND RELATED MOLECULES
availability of large amounts of pure EGF from salivary glands led to the measurement of receptor numbers and their affinity for ligand; it was also possible to show that certain cells from all three germ layers could The
mouse
express receptors. The vulval carcinoma cell line A431 which over-expresses the receptor (having 2 000 000 rather than 50 000-100 000 receptors), has provided an essential tool for study of receptor biochemistry and an early indication of abnormal receptor expression in tumours. The receptor was identified as a 175 000 MW glycoprotein; the first clue to the mechanism used by the receptor to transduce a signal across the cell membrane was found by Cohen when he showed that EGF could stimulate an intrinsic tyrosine kinase activity that autophosphorylates the receptor.18 The aminoacid sequence of the receptor was deduced both from protein structure analysis of receptor purified with monoclonal antibodies and from the isolation of a receptor cDNA sequences This information made it possible to interpret a mass of biochemical data and derive a model for the first growth factor receptor. The model in fig 2 shows a receptor with an external ligand binding domain separated from an internal tyrosine kinase domain by a hydrophobic transmembrane region. This organisation has been found in ten other receptors or putative receptors whose sequences have now been deduced.18,19 The plateletderived growth factor (PDGF) and insulin receptors define
1245
subgroups that are distinguished because they are homodimers or have an insert in the tyrosine kinase domain, respectively (fig 2). To solve the structural basis of ligand-receptor binding through biophysical studies, the external domains of the EGF and insulin receptors have been expressed as soluble proteins by means of recombinant DNA techniques. two other
The tyrosine kinase domain of the EGF receptor contains
several aminoacids that are conserved in all kinases, some of known to be important for ATP binding.2O Mutation of ATP binding residues such as lysine 761 inactivates EGF signal transduction, thereby confirming the importance of the kinase activity. Remarkably little is known about the signalling pathway downstream from this or any other tyrosine kinase; intensive searches for potential substrates, other than the kinases themselves, have not been rewarding. The EGF receptor is autophosphorylated at three sites and appears neither to have any effect on kinase activity nor to influence transduction of a mitogenic signal by the receptor. Mutagenesis experiments involving inactivation of the kinase or truncation of the c-terminal domain that contains the autophosphorylation sites suggest that the kinase could mediate intemalisation, recycling, and down-regulation of the receptor.21 Since EGF must be present for about 8 hours to generate a mitogenic signal in cells in tissue culture, the fate of the receptor, as mediated by its kinase activity, could be crucial. Receptor function can be modulated by components of other signalling pathways such as protein kinase C. Thus PDGF or bombesin-mitogens that activate protein kinase C-reduce EGF binding affinity and inhibit EGF receptor tyrosine kinase activity. The effect is partly mediated by phosphorylation of a threonine residue in a basic region of the receptor immediately below the transmembrane region (T654) ZZ How is it possible for the EGF binding signal to be transferred through the hydrophobic transmembrane domain to turn on the kinase, and how does phosphorylation of T654 just below the membrane reduce EGF affinity for the external domain? The most popular, but by no means proven, notion is that a conformational change induced by
which
are
I
I
between EGF/TGF-a
I
Fig 2-Relation receptor and the putative neu, insulin, and PDGF receptors. PDGF=platelet-derived growth factor.
to the external domain causes receptor dimerisation and consequently brings into apposition the two receptor kinase domains. These domains then phosphorylate each other and somehow activate a mechanism for signal transmission that involves interaction with other proteins 21 The identity of the proteins in the cascade that conveys the signal to the nucleus and alters gene regulation remains unknown.
ligand binding
Receptor Encoded by c-neu The c-neu gene (also
called c-erb-B-2 and HER-2) encodes a second receptor with close structural similarity to the EGF receptor (fig 2) that was discovered by two different routes. The first involved isolation of the neu oncogene from neuroglioblastomas induced in newborn rats by administration of N-nitroso urea to their pregnant mothers.23 The rat oncogene was found because it transformed NIH3T3 cells (as does the ras oncogene) and it has subsequently been shown to encode a transmembrane protein of 180 000 MW. The second approach, used by three different research groups, identified the human c-neu gene, cDNA, and protein with probes based on knowledge of the EGF receptor and its DNA sequence. The putative ligand for this receptor is unknown. SUBVERSION OF EGF RECEPTOR FUNCTION
As part of the analysis of the sequence of peptides derived from the EGF receptor, Julian Downward in my laboratory searched our sequence database for proteins of similar sequence.’ This search showed that Toyoshima’s group in Japan had recently obtained the sequence of the transforming gene v-erb-B of avian erythroblastosis virus (AEV)24 and that the sequence closely resembled that of the human EGF receptor. Thus the chicken virus oncogene is probably derived from the chicken EGF receptor gene. Realisation that v-erb-B encodes a truncated receptor lacking both the external ligand binding domain and a smaller segment of the c-terminus that includes part of the autophosphorylation site domain is especially remarkable. These data suggested to us that expression of a truncated receptor was a mechanism that allowed a ligandindependent transforming signal to be introduced by a virus or generated by mutation in a cell. Detailed analysis of the EGF receptor alterations that are necessary to create a transforming protein for avian cells25 showed that deletion of the ligand binding domain is sufficient to induce constitutive self renewal of erythroblasts; this effect is enhanced by further c-terminal truncation but the resultant molecules are less potent than the v-erb-B protein itself. Thus it is possible that other mutations in the truncated receptor remain to be defined. Rearrangements in the EGF receptor gene in human tumours have not yet been found, except in some brain tumours. Subsequent studies have shown that similar mechanisms have been used to create other oncogenes. Thus the fms transforming protein is generated from the macrophage colony stimulating factor receptor by a single aminoacid change in the ligand binding domain and removal of a negative regulatory site at its c-tem-linus .16 Of special interest is the conversion of the normal c-neu-encoded receptor to a transforming protein by a mutation that leads to substitution of a glutamic acid residue for a valine in the transmembrane domain.23 A similar change engineered in the EGF receptor does not generate a transforming protein and the mechanism of activation of c-neu remains a mystery.
1246
Analysis of the transforming potential of normal EGF receptor expression has shown that over-expression in the presence of ligand can partly transform test cells ;27 this is exactly the alteration seen in the A431 cell line mentioned above. Such over-expression, accompanied by gene amplification (as seen in A431) in some cases, has been seen in squamous tumour cell lines and less frequently in primary lung and bladder tumours. Over-expression of the c-neuencoded receptor, for which we do not know the ligand, has been documented in some breast adenocarcinomas and ovarian cancers28 and may be of prognostic value.28,29 Levi-Montalcini and Cohen were awarded their Nobel prize in 1986 for their work in unravelling the role of EGF and other growth factors in normal and abnormal growth and development. The detective story that they initiated is unfinished, but their innovative discoveries proved to be a stimulus for many scientists and the insights gained are rapidly aproaching clinical application. More than forty companies are trying to commercialise EGF. Some applications are diagnostic; others involve clinical trials. EGF is being evaluated as an aid to repair of skin burns and of damaged tympanic membrane of the ear, and in the treatment of glaucoma. It is sobering to think that the supplementary EGF in cows’ milk given to babies to achieve the levels in human milk would require about 600 tons of EGF a year. CONCLUSIONS
EGF and TGF-a are synthesised as transmembrane protein precursors that are cleaved by proteases to generate active factors of approximately 50 aminoacids. Both factors bind to the same receptor to stimulate an intrinsic tyrosine kinase that autophosphorylates the receptor. This activity is essential for signal transduction, a process that also involves receptor dimerisation. Moreover, the kinase may mediate receptor intemalisation and down-regulation, although the subsequent path of mitogenic signal processing is not understood. Overproduction of TGF-a or of its receptor and the expression of altered truncated receptors may play a role in cancer. The c-neu protein, which is closely related to the EGF receptor in structure and can also be converted to oncogene, is over-expressed in certain cancers. An understanding of the structure and function of EGF and TGF-a, together with their receptor and related molecules, may lead to the design of antagonists with therapeutic an
potential. REFERENCES 1.
Levi-Montalcini R, Cohen S. Effects of the extracts of the mouse submaxillary salivary glands on the sympathetic system of mammals. Ann NY Acad Sci 1960; 85: 324-41.
Carpenter G, Zendegui JG. Epidermal growth factor, its receptor, and related proteins. Exp Cell Res 1986; 164: 1-10. 3. Gregory H. Isolation and structure of urogastrone and its relationship to epidermal 2.
growth factor. Nature 1975; 257: 325-27. 4. Thornboum GD, Waters MJ, Dolling M, Young IR Fetal maturation and epidermal growth factor. Proc Aust Soc Pharmacol Sci 1981; 12: 11-15. 5. De Larco JE, Todaro GJ. Growth factors from murine sarcoma virus-transformed cells. Proc Natl Acad Sci USA 1978; 75: 4001-05. 6. Derynck R. Transforming growth factor &agr;. Cell 1988; 54: 593-95. 7. Downward J, Yarden Y, Mayes E, et al. Close similarity of epidermal growth factor receptor and v-erbB oncogene protein sequences. Nature 1984. 307: 521-27. 8. Ullrich A, Coussens L, Hayflick JS, et al. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 cells. Nature 1984; 309: 418-25 9. Montelione GT, Wuthnch K, Nice EC, Burgess AW, Scheraga HA. Identification of two anti-parallel beta sheet conformations in the solution structure of murine epidermal growth factor by proton magnetic resonance. Proc Natl Acad Sci USA 1986; 83: 8594-98. 10. Burgess AW, Lloyd CJ, Smith S, et al. Munne epidermal growth factor: structure and function. Biochemistry 1988; 27: 4977-85.
Therapeutics TRIAL OF PREDNISOLONE FOR PREVENTION OF MELARSOPROL-INDUCED
ENCEPHALOPATHY IN GAMBIENSE SLEEPING SICKNESS
JACQUES PEPIN1 CLAUDE GUERN2,3 LUCIEN ETHIER2,3
FRANCOIS MILORD2,3 BOKELO MPIA3 DIABAKANA MANSINSA3
University of Manitoba, Winnipeg;1 University of Sherbrooke, Sherbrooke, Canada;2 and Zone de Sante Rurale, Nioki, Zaire3
prospective randomised trial, 620 patients who had Trypanosoma brucei gambiense trypanosomiasis with central nervous system involvement were treated either with prednisolone plus melarsoprol or with melarsoprol only. 598 patients were evaluable: morbidity and death associated with melarsoprolinduced encephalopathy was reduced in patients who were given prednisolone. The two groups did not differ either in the incidence of other complications of melarsoprol therapy or in relapse rate after melarsoprol therapy. The cost of prednisolone would be outweighed by savings on the treatment of encephalopathies in such patients. Summary
In
a
Coffey RJ, Derynek P, Wilcox JN, Bringham TS, Goustin AS, Moses HL, Pittelkow MR. Production and auto-induction of transforming growth factor &agr; in human keratinocytes. Nature 1987; 328: 817-20. 12. Madtes DK, Raines EW, Sakarissen KS, et al. Induction of transforming growth factor-&agr; in activated human alveolar macrophages. Cell 1988; 53: 285-93. 13. Wilcox JN, Derynck L. Localization of cells synthesising transforming growth factor-alpha mRNA in the mouse brain. Cell 1988; 8: 1901-04. 14. Kobrin MS, Samsoondar J, Kudlow JE. A transforming growth factor secreted by untransformed bovine anterior pituitary cells in culture. J Biol Chem 1986; 261: 11.
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Gray A, Dull TJ, Ullnch A. A nucleotide sequence of epidermal growth factor cDNA predicts a 128 000 molecular weight protein precursor. Nature 1983; 303: 722-25. 16. Derynck R, Roberts AB, Winkler ME, Chen EY, Goeddel DV. Human transforming growth factor-&agr;: precursor structure and expression in Ecoli. Cell 1984; 38: 287-97. 17. Higuchi Y, Marimoto Y, Harinata A, Yasuoka N. Crystalisation and preliminary X-ray studies of human epidermal growth factor.J Biochem 1988; 103: 905-06 18. Carpenter G. Receptors for epidermal growth factor and other polypeptide mitogens. 15.
Ann Rev Biochem 1987; 56: 881-915. 19. Yarden J, Ullrich A. Growth factor receptor tyrosine kinases. Am Rev Biochem 1988; 57: 443-79. 20. Hanks SK, Quinn AM, Hunter T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 1988; 241: 42-52. 21. Schlessinger J. Signal transduction by receptor oligomerisation. Trends Biochem Sci 1988; 13: 443-47. 22. Cochet C, Gill GN, Meisenhelder J, Cooper JA, Hunter T. C-kinase phosphotylates the epidermal growth factor receptor and reduces its epidermal growth factor stimulated-tyrosine kinase activity. J Biol Chem 1984; 259: 2553-58. 23. Bargmann CI, Hung M-C, Weinberg RA. Multiple independent activations of the c-neu oncogene by a point mutation in the transmembrane domain. Cell 1986; 45: 649-57. 24. Yamamoto T, Nishida T, Miyajima N, Kawai S, Doi T, Toyoshima K. The erbB gene of avian erythroblastosis virus is a member of the scr gene family. Cell 1986; 45: 649-57. 25. Khazaie K, Dull TJ, Graf T, Schlessinger J, Ullrich A, Beug H, Vennstrom B Truncation of the human EGF receptor leads to differential transformating potentials in primary avian fibroblasts and erythroblasts. EMBO J 1988; 7: 3061-71. 26. Roussel MF, Downing JR, Rettenmier CW, Sherr CJ. A point mutation in the extracellular domain of the human CSF-1 receptor (c-fms proto-oncogene) activates its transforming potential. Cell 1988; 55: 965-77. 27. Di Fiore PP, Pierce JH, Fleming TP, et al. Overexpression of the human EGF receptor confers an EGF-dependent phenotype to NIH3T3 cells. Cell 1987; 51: 1063-70. 28. Slamon DJ, Godolphin W, Jones LA, et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 1989; 244: 707-12. 29. Nicholson S, Sainsbury JRC, Halcrow P, Chambers P, Farndon JR, Harris AL Expression of epidermal growth factor receptors associated with lack of response to endocrine therapy in recurrent breast cancer. Lancet 1989; i. 182-85.