growth factors in biology and medicine...growth factors in biology and medicine.-(ciba foundation...

Growth factors in biology and medicine

Ciba Foundation Symposium 116

1985

Pitman London

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Ciba Foundation Symposium 116

1985

Pitman London

0 Ciba Foundation 1985

ISBN 0 272 79818 5

Published in October 1985 by Pitman Publishing Ltd., 128 Long Acre, London WC2E 9AN, UK. Distributed in North America by CIBA Pharmaceutical Company (Medical Education Divi- sion), P.O. Box 18060, Newark, NJ 07101, USA

Suggested series entry for library catalogues: Ciba Foundation symposia

Ciba Foundation Symposium I16 x + 283 pages, 42 figures, 14 tables

British Library Cataloguing in Publication Data Growth factors in biology and medicine.-(Ciba

Foundation symposium; 116) 1. Somatotropin 2. Human growth-Endocrine aspects I. Evered, David 11. Nugent, Jonathan 111. Whelan, Julie IV. Series 612’.6 QP572.S6

Printed in Great Britain at The Bath Press, Avon

Contents

Symposium on Growth Factors in Biology and Medicine, held at the Ciba

The topic of this symposium was proposed by Professor Russell Ross Foundation, London, 22-24 January I985

Editors: David Evered, Jonathan Nugent (Organizers) and Julie Whelan

Sir Michael Stoker Introduction 1

J. K. Heath and A. R. Rees Growth factors in mammalian embryogenesis 3 Discussion 15

Y. Yarden and J. Schlessinger The EGF receptor kinase: evidence for allos- teric activation and intramolecular self-phosphorylation 23 Discussion 40

J. S. Huang and S. S. Huang Role of growth factors in oncogenesis: growth factor-proto-oncogene pathways of mitogenesis 46 Discussion 59

E. Rozengurt, A. Rodriguez-Pena and J. Sinnett-Smith Signalling mitogenesis in 3T3 cells: role of Ca*+-sensitive, phospholipid-dependent protein kinase 66 Discussion 82

M. Callahan, B. H. Cochran and C. D. Stiles The PDGF-inducible ‘compe- tence genes’: intracellular mediators of the mitogenic response 87 Discussion 92

R. Ross, D. F. Bowen-Pope and E. W. Raines Platelet-derived growth factor: its potential roles in wound-healing, atherosclerosis, neoplasia, and growth and development 98 Discussion 106

H. Thoenen, S. Korsching, R. Heumann and A. Acheson Nerve growth factor 113 Discussion 123

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vi CONTENTS

T. M. Dexter, C. M. Heyworth and A. D. Whetton The role of haemopoietic cell growth factor (interleukin 3) in the development of haemopoietic cells 129 Discussion 144

A. W. Burgess Haemopoietic growth factors: structure and receptor interactions 148 Discussion 158

J. Zapf, E. Schoenle and E. R. Froesch In vivo effects of the insulin-like growth factors (IGFs) in the hypophysectomized rat: comparison with human growth hormone and the possible role of the specific IGF carrier proteins 169 Discussion 180

T. Hunter, C. B. Alexander and J. A. Cooper Protein phosphorylation and growth control 188 Discussion 198

P. M. Blumberg, K. L. Leach, B. Konig, A. Y. JengandN. A. Sharkey Rec- eptors for the phorbol ester tumour promoters 205 Discussion 216

J. E. De Larco and D. A. Pigott Ectopic peptides released by a human mela- noma cell line that modulate the transformed phenotype 224 Discussion 235

R. W. Holley, J. H. Baldwin, S. Greenfield and R. Armour A growth regulatory factor that can both inhibit and stimulate growth 241 Discussion 246

Final general discussion Receptor modulation and signalling systems 253 Biological functions of growth factors 259 Medical aspects of growth factors 261

Index of contributors 271

Subject index 273

Participants

P. M. Blumberg Molecular Mechanisms of Tumor Promotion, Laboratory of Cellular Carcinogenesis & Tumor Promotion, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205, USA

K. D. Brown Department of Physiology, AFRC Institute of Animal Physio- logy, Babraham, Cambridge, CB2 4AT, UK

A. W. Burgess Melbourne Tumour Biology Unit, Ludwig Institute for Cancer Research, Post Office, Royal Melbourne Hospital, Melbourne, Vic- toria 3050, Australia

R. R. Burk Department of Biotechnology, K-681.5.44, CIBA-GEIGY Limited, CH-4002 Basle, Switzerland

M. Clemens Mammalian Protein Synthesis and Interferon Research Group, Department of Biochemistry, St George’s Hospital Medical School, Cranmer Terrace, London SW17 ORE, UK

M. P. Czech Department of Biochemistry, University of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, Massachusetts 01605, USA

J. E. De Larco Laboratory of Chemoprevention, Division of Cancer Etio- logy, National Cancer Institute (Bldg 41, Rm CSOS), National Institutes of Health, Bethesda, Maryland 20205, USA

T. M. Dexter Department of Experimental Haematology , Paterson Labora- tories, Christie Hospital & Holt Radium Institute, Wilmslow Road, With- ington, Manchester M20 9BX, UK

H. Gregory Biosciences Dept I , ICI Pharmaceuticals Division, Mereside, Alderley Park, Macclesfield, Cheshire SKlO 4TG, UK

A. J. R. Habenicht Medizinische Klinik, Ruprecht-Karls-Universitat Heidel- berg, Bergheimstrasse 58,6900-Heidelberg-l , Federal Republic of Germany

J. K. Heath Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS, UK

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viii PARTICIPANTS

C.-H. Heldin Institute of Medical & Physiological Chemistry, Biomedical Centre, University of Uppsala, Biomedicum, Box 575, S-751 23 Uppsala, Sweden

R. W. Holley" The Salk Institute, PO Box 85800, San Diego, California 92138-9216, USA

J. S. Huang Edward A. Doisy Department of Biochemistry, St Louis Univer- sity Medical Center, 1402 S. Grand Boulevard, St Louis, Missouri 63104, USA

T. Hunter Molecular Biology & Virology Laboratory, The Salk Institute, PO Box 85800, San Diego, California 92138-9216, USA

G. L. King The J o s h Diabetes Center, 1 Joslin Place, Boston, Massachu- setts 02215, USA

R. H. Michell Department of Biochemistry, University of Birmingham, PO Box 363, Birmingham, BT15 2TT, UK

B. Ozanne Department of Microbiology, University of Texas, Health Science Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235, USA

E. Reich Department of Biotechnology, Pharmaceutical Division, CIBA- GEIGY Limited, CH-4002 Basle, Switzerland

R. Ross Department of Pathology, C514, Health Sciences Building, SM-30, University of Washington, School of Medicine, Seattle, Washington 98195, USA

E. Rozengurt Membrane & Growth Control Laboratory, Imperial Cancer Research Fund, PO Box 123,44 Lincoln's Inn Fields, London WC2A 3PX, UK

J. Schlessinger Department of Chemical Immunology, The Weizmann Insti- tute of Science, 76100 Rehovot, Israel

C. D. Stiles Department of Microbiology & Molecular Genetics, Harvard Medical School and The Dana Farber Cancer Institute, 44 Binney St, Bos- ton, Massachusetts 02159, USA

* Professor Holley was unable to attend the symposium. His paper was presented by Professor Tony Hunter.

PARTICIPANTS ix

Sir Michael Stoker (Chairman) Clare Hall, University of Cambridge, Cam- bridge, CB3 9AL, UK

S. Tag (Ciba Foundation Bursar) The Scientific and Technical Research Council of Turkey, Research Institute for Basic Sciences, PO Box 74 Gebze, Kocaeli, Turkey

H. Thoenen Department of Neurochemistry, Max Planck Institute for Psy- chiatry, Am Klopferspitz MA, 8033 Planegg-Martinsried, Federal Republic of Germany

N. A. Wright Department of Histopathology, Royal Postgraduate Medical School, Hammersmith Hospital, Ducane Road, London W12 OHS, UK

J. Zapf Department of Medicine, University Hospital, Ramistrasse 100, CH- 8091 Zurich, Switzerland

Introduction

SIR MICHAEL STOKER

Clare Hall, University of Cambridge, Cambridge CB3 9AL, UK

I985 Growth factors in biology and medicine. Pitman, London (Ciba Foundation Symposium 116) p 1-2

Most of those taking part in this symposium will have been to at least one other meeting on growth factors in the recent past. But Ciba Foundation symposia are rather special, and I suspect that this may turn out to be a particularly rewarding meeting. There is a drawback to the choice of an out- sider like myself as chairman. Usually the chairman is the person who has been most closely involved in organizing the meeting, but I have had absolutely nothing to do with this side of things. The responsibility for planning this symposium lies with the Foundation and with others here (the original propo- sal, of course, came from Russell Ross). I should also explain at the outset that I suffer from a disease which is not well known in medical circles but I suspect is quite common; this is dysacronyrnia. Those who work on growth factors are among the worst culprits in generating acronyms. There are a number of these already, but what are you going to do when the number of known factors gets up to 40 or 50? I personally wish that people would use words for new factors. However, I suspect that you will continue to think of more acronyms as further factors are discovered.

It has often puzzled me why people didn’t call these factors ‘hormones’. In fact, they have been called mitogenic hormones. But although they may all turn out to be hormones in one sense, in that they are ways of communicating between different cell types, there is an important distinction, in that the classical hormones were nearly all discovered because of diseases in which the source of the hormone had been ablated, naturally or sometimes surgically. Consequently, with the classical hormones, a lot is known about their actual physiological effects. The situation of growth factors is different, in that most of them have been discovered as products of modern techniques of cell biology. We are now learning a lot about their molecular mechanisms, their pathways, and to some extent their regulation, but we know extremely little about what they are there for. That problem will be at the back of our minds in this

1

2 INTRODUCTION

symposium and will be discussed by some of you during it. I suppose that those working on the haemopoietic growth factors are nearest to the goal of finding out what the factors actually do physiologically, and there is even one example of ablation, namely pernicious anaemia. But on the whole, at least until fairly recently, we have not known where these factors come from, and what reg- ulates their production.

Apart from that, much of the discussion will be about their mechanism of action and its regulation, especially at the molecular level. After all, factors and hormones are a way of communicating between a gene in one cell via a product which then affects the expression of genes (usually more than one) in another cell. This pathway between the genes in the two cells is what concerns us. The actual growth factor itself is only a part of that pathway. There are many other links in the chain, from the specific receptors to the protein kinases, and the various other molecules and systems, before the expression of the genes in the target cell. The common or dissimilar nature of these pathways is a point of interest which will come up, and the possibility of synergistic interaction between pathways.

If one thinks of the scientific fathers of this whole area, for me, Stanley Cohen is the chief among them, but Gordon Sat0 also deserves much credit for identifying the multifactorial needs of cells-the idea that cells have an address which involves more than one symbol, or more than one number in the telephone system, so to speak. The interaction between these factors will no doubt arise during our discussions.

Growth factors in mammalian embryogenesis

JOHN K. HEATH and ANTHONY R. REES*

Department of Zoology and *Laboratory of Molecular Biophysics, University of Oxford, South Parks Road, Oxford, OX1 3PS, UK

Abstract. Malignant murine embryonal carcinoma (EC) cells have been studied as a means of characterizing the identity and action of growth regulatory molecules in the early postimplantation mouse embryo. The differentiation of EC cells in vitro is accompanied by significant changes in the control of cell proliferation, including the acquisition of dependence on specific exogenous growth factors for cell multiplication. This is at least partly controlled by the developmentally regulated expression of specific growth factor receptors and their intracellular response systems. The development of defined media has allowed the identification of the principal factors required for EC cell proliferation in vitro. These factors are synthesized in vivo by the extra-embryonic tissues of the developing embryo and by the differentiated progeny of EC cells in vitro.

EC cells secrete a potent growth factor (embryonal carcinoma-derived growth factor, ECDGF) which has been purified and partly characterized. ECDGF induces proliferation of the differentiated progeny of EC cells and specific normal embryonic cell types in vitro, suggesting that ECDGF may act as an embryonic growth factor in viva

Together these findings suggest that proliferation in the embryo may be controlled by reciprocal interaction between primitive ectoderm cells and their differentiated derivatives, mediated by the developmentally regulated expression of specific soluble growth factors.

1985 Growth factors in biology and medicine. Pitman, London (Ciba Foundation Sympo- sium 116) p 3-22

The mechanisms underlying the coordinated growth of the mammalian embryo are of fundamental interest, both from the viewpoint of understanding cellular processes in development and from the practical consequences that may accrue from the ability to manipulate embryonic growth and development. The early postimplantation stages of development offer an attractively simple situation in which to analyse the role of growth factors and their response systems in the control of embryonic growth; the tissue types are few in number, and

3

4 HEATH & REES

relatively easy to obtain in pure form. This issue has taken on added interest with the finding that expression of a variety of cellular oncogenes is developmentally regulated in the early postimplantation embryo (Muller et a1 1982), suggesting that embryonic growth processes may, in certain respects, resemble those occurring in more widely studied fibroblastic cell types.

The control of cell proliferation during cleavage is poorly understood, but since complete preimplantation development can occur in relatively simple basal nutrient media, it does not appear to depend upon exogenous growth factors or other macromolecular substances. It is at the stage immediately after implantation, when embryonic growth rapidly accelerates, the requirements for whole embryo growth in vitro become more sophisticated (reviewed New 1978) and interactions between cell types in the control of cellular multiplication becomes evident. Of key importance in postimplantation development are a group of stem cells, the primitive ectoderm, from which all the cell types in the fetus and extra-embryonic membranes are ultimately derived.

The earliest differentiation events in postimplantation development are the formation of the extra-embryonic tissues which surround the developing fetus in vivo and are specialized structures required for survival in utero. The first cell type to be formed from the primitive ectoderm is the primitive endoderm, which gives rise to the visceral endoderm and the parietal endoderm. The next event is the formation of extra-embryonic mesoderm, which forms the amnion and, together with the visceral endoderm, the yolk sac. This is the organ that is responsible for embryonic nutrition in early development, and forms a physical barrier between the fetus and the uterine environment. Com- ponents of the mesoderm also give rise to the extra-embryonic blood vessels and the embryonic haemopoietic system, located in the blood islands of the yolk sac.

Teratocarcinomas

A major asset in the analysis of growth regulatory mechanisms operating in these early phases of development is the existence of teratocarcinomas, which are malignant tumours experimentally derived from the primitive ectoderm. Teratocarcinomas comprise two components: a malignant stem cell, the embryonal carcinoma (EC) cell, and its differentiated derivatives. Homo- geneous EC cell populations can be maintained in culture and induced to differentiate either by manipulation of the culture conditions, or in some cases by the addition of drugs such as retinoic acid (RA). EC cells closely resemble their normal counterparts of the primitive ectoderm. Perhaps the most striking demonstration of this feature is that certain EC cell lines, when incorporated into the preimplantation embryo, will participate in normal deve-

GROWTH FACTORS IN EMBRYOGENESIS 5

lopment and give rise to fully functional differentiated adult tissues (Papaioan- nou et al 1975). It was therefore anticipated that the analysis of teratocarcinoma proliferation in vitro would both reveal the identity of specific factors which may regulate embryonic cell proliferation and yield insights into possible embryonic growth control pathways.

The control of EC cell proliferation

We have principally studied one EC cell line, PC13, which can be maintained as a continuous homogeneous cell line in culture. PC13 EC cells undergo a restricted repertoire of differentiation in vitro in response to RA, forming an apparently homogeneous cell type, END, which appears to resemble in phenotype, morphology and behaviour the extra-embryonic mesoderm of the postimplantation embryo. Critical to the analysis of mechanisms of cell proliferation in this system is the fact that controlled differentiation in culture is extremely efficient: less than l / l O s cells retain the EC phenotype after five days exposure to RA. It is therefore possible to compare homogeneous populations related by a single differentiation step. The action of RA is inductive rather than selective (Rayner & Graham 1982), and RA is not required for the maintenance of the END cell phenotype. Studies of clonal EC cell differentiation in response to RA have suggested that exposure to RA for at least one full cell cycle is necessary to induce differentiation (Rayner & Graham 1982, Rayner & Pulsford 1984).

The survival and multiplication of PC13 EC cells in culture is dependent on high (>lo%) concentrations of fetal calf serum (FCS) in the medium. As the concentration of FCS is lowered, EC cell survival is rapidly impaired, and the cells die before any change in the rate of cell proliferation becomes evident. The essential macromolecular factors required for EC cell growth were defined by the development of serum-free media which will support the growth of EC cells in vitro (Rizzino & Crowley 1980, Heath & Deller 1983). These requirements are relatively simple, namely a source of lipid (high density plasma lipoproteins, HDL, and low density plasma lipoproteins, LDL) and iron (transferrin). Of interest is that limited substitution for lipoprotein supplementation can be achieved by physiological concentrations of insulin-like growth factor I1 (IGF 11) or high concentrations of insulin. It is important to note that these factors are necessary for cell viability and do not affect the rate of transit through the cell cycle. There is therefore no requirement for exogenous growth factors to control EC cell multiplication in vitro.

EC cells also express high affinity receptors for LDL (Goldstein et al1979), transferrin (Karin & Mintz 1981) and IGF I1 (Nagarajan et at 1982) but not

HEATH & REES 6

insulin (Heath et al 1981). This suggests that these factors reflect physiological requirements for primitive ectoderm proliferation in vivo. Are these factors expressed in the postimplantation embryo? Studies employing antibodies directed against transferrin and individual apolipoproteins, and specific recombinant DNA probes, have established that these factors are specifically expressed and secreted by the visceral endoderm in vivo (Shi & Heath 1984, Meehan et a1 1984). Insulin and proinsulin expression has also been detected in the fetal rat yolk sac using both recombinant DNA probes and radioimmunoassay, although the tissue localization of insulin expression within the yolk sac is at present unknown (Muglia & Locker 1984).

An insight into the location of IGF I1 expression in the embryo was obtained from the finding that media conditioned by PC13 END cells contain activities which compete with the binding of 12sI-labelled IGF I1 to its cell surface receptor. Fractionation of PC13 END cell-conditioned media by gel filtration reveals three peaks of inhibitory activity (Heath & Isacke 1983). These include two high relative molecular mass fractions (approximate M , of 150000 and 40 000) which may represent forms of a soluble IGF 11-binding protein, since fractions containing these activities have the ability to specifically bind IGF I1 directly (unpublished observations). The lower molecular weight material (approximate M, of 12 000) contains IGF-like biological activity (unpublished observations) and exhibits similar chromatographic behaviour to authentic IGF-like molecules secreted by other cultured cell lines. To investigate this further, we prepared specific antibodies directed against authentic rat IGF I1 and examined 3sS-labelled EC and END cell culture supernatants for the expression of IGF 11-related molecules by immunoprecipitation (Fig. la). Radiola- belled protein species (M, values of 35 000,16 000 and 14 000) were identified. A similar pattern of expression, including an M , = 18 000 species, was observed in medium conditioned by extra-embryonic mesoderm and amnion, but not by parietal or visceral endoderm (Fig. 1b,c). Rat IGF I1 secreted by the BRL cell line is known to be initially synthesized as a M, = 20000 precursor, which is then processed through a series of intermediate prohormone forms to the final M , = 7000 IGF I1 molecule (Acquaviva et a1 1982). Sequence analysis of the rat IGF I1 gene (Dull et a1 1984) has identified possible process- ing sites in the prohormone which could yield the putative intermediate IGF I1 species corresponding to the M , = 18 000,16 000 and 14 000 forms observed here, and similarly sized immunoreactive IGF I1 prohormone forms have been identified in BRL-conditioned media (Moses et a1 1980). The identity of the M, = 35 000 species is unclear. Peptide mapping does not reveal any relation to the other species (unpublished observations). It is significant, however, that the growth hormone-independent IGF 11-binding protein of rat plasma has an apparent M , = 35 000, based on cross-linking studies (D’Ercole & Wilkins 1984), and a form of IGF 11-binding protein of approximate M,


35 000 has been detected in human term amniotic fluid (Chochinov et a1 1976). It is possible therefore that immunization with IGF I1 also results in the production of antibodies directed against the binding protein by some unknown mechanism. We conclude that IGF 11-like molecules and their cognate soluble binding proteins are specifically expressed by extra-embryonic mesoderm derivatives in the embryo, and that IGF I1 expression is switched on as PC13 EC cells differentiate in vitro.

The macromolecular factors required for the proliferation of PC13 EC cells in culture are therefore present in the early postimplantation mouse embryo, and furthermore are synthesized by the early direct differentiated derivatives of the primitive ectoderm. This suggests that the survival and proliferation of the primitive ectoderm is controlled by interaction with its differentiated progeny. Indeed, primitive ectoderm survival in culture has been empirically found to depend on the presence of either an overlying layer of primitive endoderm cells (G. Shia, personal communication) or fibroblast feeders (which might be anticipated to secrete IGF 11-like molecules: Adams et a1 1983). Isacke & Deller (1983) provided a direct demonstration that PC13 EC cells can depend on their differentiated progeny for survival and multiplication by observing that clonal growth of PC13 EC cells in low concentrations of FCS without further supplementation could occur in the presence of PC13 END cell feeders.

Differentiation and cell proliferation

The differentiation in culture of PC13 EC cells in response to retinoic acid results in a developmentally regulated change in the underlying mechanisms that control cell multiplication. The population doubling rate slows, the cells lose the capacity to give rise to progressively growing tumours in syngeneic hosts (Rayner & Graham 1982), and they acquire a finite proliferative lifespan in vitro (Rayner & Pulsford 1984).

Part of this developmental control of the cell cycle is manifested as a change in the requirements for cell proliferation in culture. PC13 END cell multiplication appears to be regulated by at least two factors. The first is cell density, since the labelling index of PC13 END cells in vitro rises progressively with cell density in both high and low concentrations of serum (in marked contrast to the behaviour of established fibroblast cell lines, which demonstrate the opposite effect of density on cell proliferation) (Fig. 2). The underlying basis of this effect of cell density on PC13 END cell multiplication is at present unclear. It may be the result of autostimulation of cell multiplication by factors released by PC13 END cells into the culture media (IGF 11-like molecules?) or may result from some direct cell contact-mediated phenomenon.


The second factor controlling the growth of PC13 END cells is the availabi- lity of serum-derived mitogens, since the labelling index of PC13 END cells also depends on the concentration of FCS in the culture medium (Fig. 2). The possibility of manipulating PC13 END cell multiplication by withdrawing serum-derived mitogens allows us to identify the specific growth factors required for cell multiplication. These studies reveal that END cell multiplication in vitro can be induced by a number of defined growth factors, such as epidermal growth factor (EGF) and insulin. The cell's ability to respond to these exogenous growth factors is, at least in part, due to the appearance of the corresponding specific functional cell surface receptors for EGF (Rees et al 1979) and insulin (Heath et al 1981) as EC cells differentiate in vitro. Teratocarcinoma cells therefore exhibit developmentally regulated expression of both specific growth factors and their cellular receptor systems.

It is clear, however, that there must also be a concomitant, developmentally regulated change in intracellular growth control mechanisms, since PC13 EC cells proliferate in the absence of exogenous mitogens, and apparently lack part of the functional apparatus required for response to exogenous growth factors. Differentiation in vitro is therefore accompanied not only by the expression of specific growth factors receptors, but by the coupling of growth factor receptor-derived signals to intracellular growth control mechanisms. A second aspect pointing to the existence of some developmentally regulated intracellular control mechanism is that whereas PC13 EC cells have a high probability of self-renewal, the response of PC13 END cells to exogenous

FIG. la. Immunoprecipitation of [35S]methionine-labelled PC13 EC and PC13 END cell culture supernatant. 2 X 106 PC13 EC and PC13 END cells were labelled for 16 h in 2ml of ECM medium (Heath & Deller 1983) (containing 1/100th the normal concentration of methionine) supplemented with 200 pCi "5SJmethionine. Immunoprecipitation of culture supernatant was performed as described (Shi & Heath 1984). Truck A: molecular weight markers. Trucks B-D: EC cell culture supernatant. Trucks E-G: PC 13 END cell culture supernatant. Trucks B and E: total 35S-labelled culture supernatant. Trucks C and F: immunoprecipitation with rabbit anti-rat IGF I1 IgG. Trucks D and G: immunoprecipitation with control rabbit (anti-keyhole limpet haemocya- nin) IgG. Arrows indicate the position of precipitated radiolabelled species.

FIG. 1 b. Immunoprecipitation of [3sS]methionine-labelled culture supernatant from parietal endoderm (tracks A-C) and amnion (trucks D-F). Tissue dissection, culture and immunoprecipitation as described by Shi & Heath (1984). Trucks A and D: total labelled culture supernatant. Trucks B and E: immunoprecipitation by control rabbit IgG. Trucks C and F: immunoprecipitation by rabbit anti-rat IGF I1 IgG.

FIG. lc. Immunoprecipitation of ['sS]methionine-labelled culture supernatant from yolk sac visceral endoderm (trucks A-C) and yolk sac mesoderm (trucks D-F). Tissue dissection, culture and immunoprecipitation as described by Shi & Heath (1984). Trucks B and E: immunmoprecipi- tation of culture supernatant by control rabbit IgG. Trucks C und F: immunoprecipitation by rabbit anti-rat IGF I1 IgG.

10 HEATH & REES

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FIG. 2. Effect of cell density and fetal calf serum (FCS) concentrations on labelling index of PC13 END and 1OT4 fibroblasts. Cells were plated in 2ml of Dulbecco's modified Eagle basal nutrient medium containing 10% (vol:vol) FCS into 35 mm diameter gelatin-coated tissue culture dishes at the indicated density. The following day the medium was changed in half the dishes to medium containing 1% (vol:vol) FCS. PHlThymidine was added to a final concentration of 1 pCi/ml (final thymidine concentration, 10-6M) 48 h later. The cells were incubated for a further 18 h and then fixed in methanokacetic acid (3:1, vol:vol) and processed for autoradiography. V, 1OT4: 10% FCS; V , 1OT4: 1% FCS; 0, PC13 END: 10% FCS; 0, PC13 END: 1% FCS.

mitogens is heterogeneous. Only a fraction of the cell population can respond to exogenous signals by entering DNA synthesis (see Fig. 2), and PC13 END cells cannot be maintained in cycle indefinitely, even in the continuous presence of exogenous growth factors, and despite the fact that nearly all the cells express detectable growth factor receptors (Rayner & Pulsford 1984).

Reciprocity and cell proliferation

Although PC13 END cell differentiation is accompanied by the appearance of the ability to respond to exogenous growth factors, it is as yet unclear what the normal source of these growth regulatory molecules might be. It was significant therefore to discover that co-culture of PC13 EC cells with their differentiated progeny, or with fibroblast target cells, results not only in the enhanced survival of EC cells but in the induction of DNA synthesis and cell proliferation in these heterologous target cells (Isacke & Deller 1983).


This suggested that at least one source of PC13 END cell growth factors was their undifferentiated parent EC cells. The agent responsible for the induction of proliferation of PC13 END cells by their EC cell progenitors, termed embryonal carcinoma-derived growth factor (ECDGF), has been purified from serum-free culture media conditioned by PC13 EC cells (Heath & Isacke 1984). ECDGF is a single-chain molecule of apparent M , of 17500 and is a potent growth factor for a variety of cell types, including PC13 END cells and fibroblast cell lines. It differs in both its structure and its range of suscept- ible target cell types from other well-defined growth factors such as EGF, platelet-derived growth factor, fibroblast growth factor or endothelial cell growth factor.

These findings suggest that there can exist a reciprocal interdependence of PC13 EC cells and their differentiated progeny for survival and proliferation, mediated through the developmentally regulated expression of soluble growth factors, such as IGF I1 and ECDGF, and their cellular response systems. This type of interaction may occur during normal postimplantation development in vivo. ECDGF is furthermore a strong candidate for a novel growth factor species which acts to influence the proliferation of specific cell types in postimplantation development. That this may indeed be the case is sup- ported by the finding that ECDGF induces DNA synthesis in primary cultures of extra-embryonic yolk sac mesoderm (thought to be the normal tissue equivalent of PC13 END cells), cultured under serum-free conditions in vitro (Fig. 3). ECDGF may furthermore be relatively tissue-specific in its action in the mouse embryo, since it does not appear to affect the multiplication or differentiation of either visceral endoderm or parietal endoderm cultured in vitro under equivalent conditions (unpublished observations, J. K. Heath, E. Myst- kowska & A. Wills).

We have argued that the switch from one type of cell cycle control mechanism to another is regulated by differentiation, and that EC cells and END cells can influence each other’s behaviour in culture. There is evidence to suggest, however, that growth factors may additionally play a part, at least indirectly, in the differentiation process itself. This comes from the examina- tion of EC cell lines which undergo ‘spontaneous’ differentiation in vitro. OC15S1 EC cells differentiate in vitro when plated at clonal density in medium containing a high concentrations of FCS, forming a cell type similar to that produced by the RA-induced differentiation of PC13 EC cells (Rees et a1 1979). In the presence of exogenous ECDGF, low density OC15S1 EC cell differentiation is inhibited, giving rise to a proportion of colonies containing rapidly growing EC cells (Fig. 4). A similar, although quantitatively less efficient, effect is seen in the presence of exogenous IGF 11, but not with other growth factors such as EGF or PDGF (Fig. 4). It is not yet clear whether ECDGF and IGF I1 act directly on OC15S1 cells themselves, or indirectly

12 HEATH & REES

through the differentiated progeny, which in turn act to inhibit EC cell differentiation. It is interesting, however, that the effect of ECDGF on OC15S1 EC cell differentiation is overridden by RA. This suggests that the differentiation-inducing properties of RA may be due to an ability to block endogenous or exogenous growth factor action in EC cells, which is consistent with the inhibitory effects of RA on the action of growth factors in other systems (e.g. Todaro et al 1978).

FIG. 3. Effect of exogenous growth factors on the labelling index of yolk sac mesoderm cells cultured in vitro. Yolk sacs were dissected from 9.5 days post coitum mouse embryos and germ layer separation was effected by incubation for 2 h in 0.05% pancreatin, 0.01% trypsin dissolved in Earle’s balanced salt solution containing 25 mM-HEPES, pH7.2, at 4°C. The enzyme-treated yolk sacs were then cultured for 2h at 37°C in Dulbecco’s modified Eagle medium (DME) containing 10% FCS (by volume). The endoderm and mesoderm were separated with fine forceps and the mesoderm was dissociated by exposure for 15 min at 4°C to0.125% trypsin, 10 mM-EDTA in Ca*+, Mg2+-free phosphate-buffered saline. Dissociated cells were plated into 10 mm diameter gelatin-coated tissue culture wells in DME:F-12 medium (5050, v01:vol) supplemented .with 10% (by volume) FCS. The following day the medium was changed to ECM serum-free medium, and supplemented 24 h later as indicated. After a further 6 h of culture the cells were exposed to [3H]thymidine and processed for autoradiography as described in the legend to Fig. 2. C , ECM medium alone; S, 10% FCS; egf, 50ng/ml EGF; igf, 50ngYml rat IGF 11; ins, 20ng/ml porcine insulin; pd, 50ng/ml PDGF; ec, 10ng/ml ECDGF; cm, ECM conditioned by overnight exposure to PC13 EC cells at a density of 105cells/ml.


I 0.7

FIG. 4. Inhibition of OC15S1 EC cell differentiation by exogenous growth factors. lo) OC15S1 EC cells were plated in lOml DME:F-12 medium (50:50, v01:vol) containing 10% FCS into gelatin-coated 25 cm2 tissue culture flasks and the media were supplemented as indicated. The cells were cultured for 10 days at 37°C and then fixed with methano1:acetic acid (3:1, vol:vol) and stained with 0.5% crystal violet in water. The proportion of colonies containing dark-staining EC cells was scored. ec, 10ng/ml ECDGF; ra, 5 X lO-’M-all trans-retinoic acid (added from 10-*M stock dissolved in dimethyl sulphoxide at 10-*M): egf, SOng/ml EGF; igf, 50ng/ml IGF 11; Pd, 50ng/ml PDGF; c, no additions.

Conclusions

The analysis of teratocarcinoma growth regulation has revealed an intimate relationship between the control of cell proliferation and differentiation, and has also revealed the identity of some candidate embryonic growth regulatory molecules, such as IGF I1 and ECDGF. At least some of these particular growth regulatory molecules are expressed in a lineage-specific manner in the postimplantation embryo. As EC cells undergo differentiation, the extra- cellular requirements for cell multiplication change. This is due to a developmentally regulated switch in the expression of specific growth factors such as IGF I1 and ECDGF and the appropriate receptor and post-receptor response systems. Furthermore, there is indirect evidence that in certain cases

14 HEATH & REES

specific growth factors can inhibit the process of differentiation itself, suggesting that cell multiplication and differentiation are linked together. Of more general importance in considering the control of cell proliferation in the embryo is that the specific pattern of growth factor and receptor expression in this case fits together in a complementary fashion, whereby the survival and proliferation of one cell type is dependent upon specific products expressed by the other. This points to the existence of embryo growth control by reciprocal interactions between stem cells and their differentiated progeny, a situation that may be common in other systems. It is, however, necessary to emphasize that this type of in vitro analysis reveals the repertoire of possible growth regulatory mechanisms that can occur and does not, of itself, indicate which factors actually play a predominant role in vivo.

Acknowledgements

We are grateful to the Cancer Research Campaign and the Medical Research Council for financial support. We also thank Clare Isacke, Wai-kang Shi and Alan Wills for productive collaboration, and Chris Graham and Mike Rayner for discussion and comment.

REFERENCES

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Adam SO, Nissley SP, Handwerger S, Rechler MM 1983 Developmental patterns of insulin-like growth factor-I and -11 synthesis and regulation in rat fibroblasts. Nature (Lond) 302:150-153

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Goldstein J , Brown M, Kreiger M, Anderson R, Mintz B 1979 Demonstration of low density lipoprotein receptors in mouse teratocarcinoma stem cells and description of a method for producing receptor deficient mutant mice. Proc Natl Acad Sci USA 76:2843-2847

Heath JK, Deller MJ 1983 Serum-free culture of PC13 murine embryonal carcinoma cells. J Cell Physiol 115:225-230

Heath JK, Isacke CM 1983 Reciprocal control of teratocarcinoma proliferation. Cell Biol Int Rep 7:561-562

Heath JK, Isacke CM 1984 PC13 Embryonal carcinoma derived growth factor. EMBO (Eur Mol Biol Organ) J 3:2957-2962

Heath J, Bell S, Rees AR 1981 Appearance of functional insulin receptors during the differentiation of embryonal carcinoma cells. J Cell Biol91:293-297


Isacke CM, Deller MJ 1983 Teratocarcinoma cells exhibit growth cooperativity in virro. J Cell Physiol117:407-414

Karin M, Mintz B 1981 Receptor mediated endocytosis of transferrin in developmentally totipo- tent mouse teratocarcinoma stem cells. J Biol Chem 256:3245-3252

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DISCUSSION

Stoker: Does the fact that there is density-dependent, low serum growth of the differentiated derivatives mean that there must be some sort of autocrine system at work?

Heath: Our presumption is that an autocrine mechanism is operating. When conditioned medium from high density cultures of END cells is put on low density cultures, there is stimulation of growth; but the stimulation isn’t as great as the stimulation seen at high density. We conclude that if there is an autocrine component, it may not be the full explanation. There may also be, for example, cell contact-related processes.

Stoker: If you plate the cells on a layer of high density irradiated feeder cells, do you get the same result?

16 DISCUSSION

Heath: We haven’t done that. Ross: In any given situation, where you find that a particular growth factor is

active or is not active, and you say that there are, or are not, enough receptors, how can you be sure that that is so? If the growth factor is being made by a cell, then the receptor is being down-regulated by that growth factor, so with the usual approaches one would not find receptors for the growth factor, nor any specific evidence for that growth factor. How have you made that determina- tion, therefore?

Heath: Our data relate entirely to the identification of unoccupied receptors by radioligand binding assay. You are right in that this method does not enable us to identify the receptors which might be blocked by the endogenous production of growth factors.

Ross: You said that embryonal carcinoma cells are serum-requiring, but serum contains at least PDGF, EGF, and transforming growth factor beta (TGF-P). So how can you go on to say that specific factors are not required and there are no receptors for these factors?

Heath: If you look at the conditioned medium of embryonal carcinoma (EC) cells, and ask whether the absence of, say, EGF receptors is due to the endogenous expression of EGF-like molecules, you might anticipate finding EGF-competing activity in the conditioned medium, but we don’t find it. The same is true for PDGF and insulin. In the case of END cells, in which IGFII receptors appear to be undetectable, or present in very low numbers, we do find IGFII-competing activity in the conditioned medium. This might argue for secretion of IGFII-like molecules blocking cell surface receptors.

Ross: How are you going to deal with the problem of totally endogenous growth factor formation and ligand-receptor binding that may not be a cell surface phenomenon, in particular cases?

Heath: The only way to tackle that is to use cDNA probes, or specific antibodies, to look for intracellular expression of the factors or their receptors. King: One possible way of detecting the down-regulation of receptors is by

using antibody to phosphotyrosine. If you postulate that the cells are producing a growth factor, such as a PDGF-like growth factor, and secreting it, and then it binds to the receptor on the cell itself, to down-regulate that receptor, there should be growth factor-receptor complexes which are autophosphorylated. Using antibody to phosphotyrosine, you should still see a band where the receptor should be, even in the down-regulated state.

Secondly, the antibodies to phosphotyrosine could be helpful in detecting receptors for growth factors, where we don’t know the receptors that the factors are working through. In other words, if a growth factor has to be released and then re-binds to a receptor to mediate its action, the occupation of the specific receptor can be detected using antibodies to phosphotyrosine, if it goes through a tyrosine kinase. This method can also be used in differentiating


the multiplication-stimulating activity (MSA) receptor and the IGFI receptor, since they are quite different in molecular weight.

Heath: An obvious experiment would be to add antibodies to the IGFI or IGFII receptors and see if they block or lower the labelling index.

Ross: If the receptor is down-regulated, I don’t know how you would find the tyrosine kinase, if the receptor has disappeared from the cell surface by internalization.

Hefdin: The osteosarcoma cell line which produces a PDGF-like growth factor shows no specific binding of ‘251-labelled PDGF by the conventional techniques. One can detect a PDGF-stimulatable phosphorylation of the receptor, however, but the signal is less strong than when using membranes from normal human fibroblasts (C. Betshultz et al, unpublished results). So it seems that the phosphorylation assay is more sensitive than the binding assay.

Ross: Are these from membrane preparations or from whole cell lysates? Heldin: Membrane preparations or metabolically labelled intact cells, im-

munoprecipitated with an antiserum against phosphotyrosine. Hunter: The EGF receptor is not autophosphorylated in A431 cells, which

are transformed by Harvey murine sarcoma virus and express a factor that causes permanent down-regulation. You don’t detect an autophosphorylated form of the receptor; you just see fewer receptors. So even if tyrosine phosphorylation is stimulated by the factor, the receptor is presumably internalized and lost quickly.

King: The cytoplasmic insulin receptor appears to be similar to the plasma membrane receptor in its ability to autophosphorylate.

Hunter: That may be the case, but looking at an autocrine system, as we were, we don’t see an autophosphorylated form of the receptor.

Heath: It has to be said that even if that experiment worked, as suggested by Dr King, it would not be evidence that it was working through an autocrine mechanism.

Schfessinger: Perhaps you don’t see the phosphorylation of the internal portion of the receptor, Dr Hunter, because you are using an antibody against the external portion of the receptor. The internal portion of the receptor would not face degradative enzymes, so if you produced an antibody against the internal portion, you might see cell phosphorylation. You don’t precipitate the v-erb-B internal portion with an antibody to the external part of the receptor.

Hunter: Yes, it’s true that we only looked at intact receptor molecules. Schfessinger: In down-regulation there is degradation mainly of the external

portion, because the cytoplasmic portion is not exposed to enzymes which degrade the receptor.

Hunter: Drs M. Willingham and I. Pastan (unpublished results) say that the cytoplasmic portion is also rapidly degraded in EGF-treated cells.

Dexter: The growth factor effect that Dr Heath sees could presumably be

18 DISCUSSION

restricted to tumour cells. Do you have evidence that the same growth factors have an effect on normal cells?

Heath: These growth factors do in fact work on normal mesodermal cells from the mouse embryo. We don’t as yet have direct evidence that ECDGF is either expressed by or works on embryonic ectoderm. That is because we lack the necessary probes and antibodies to identify expression in such a small amount of tissue.

Dexter: It’s nice that you can demonstrate an effect upon normal cells. Leukaemic cells, for example, can be induced to differentiate in vitro in response to many stimuli, while the corresponding normal cells do not show the same response.

Stiles: The other point to remember is that these embryonal carcinoma cells, or lines very much like them, will, if mixed with disaggregated cells from a normal mouse embryo, take part in forming a perfectly normal mouse; therefore they are not so far removed from normal embryonic cells.

Zapf: What are the effects of IGFI on differentiation? Heath: If someone would give us some IGFI, we would be glad to look at

this! Czech: I was interested in your studies with IGFII, because insulin mimicked

IGFII at high concentrations. The fact that IGFII receptors do not bind insulin suggests that the IGFI receptor, rather than the IGFII receptor, is mediating these effects. It would therefore be interesting to compare dose-response curves for IGFI and IGFII.

Heath: I agree. The direct binding of insulin to EC cells is not detectable, and the inhibition of binding of IGFII to these cells by insulin is very poor. We can’t therefore rule out the expression of type1 receptors, but if they are expressed, they are probably present in relatively low numbers. We haven’t done cross- linking studies, which might also clarify the issue.

Czech: Many differentiated cells, such as liver cells, have large amounts of IGFII receptor and much lower amounts of. IGFI receptors or insulin receptors, yet the biological responses (either the short-term transport and other enzymic effects or long-term proliferation effects) seem to be mediated by the IGFI or the insulin type1 receptor rather than the type11 receptor (Mottola & Czech 1984). This would argue that in these cells the IGFII receptor is not related to the long-term biological responses you are looking at, or even the short-term effects.

Heath: Are you saying that the type1 receptor mediates the growth responses, as well as the metabolic responses? The effect of IGFII on the END cells is on cell proliferation; we haven’t measured glucose transport. Whereas in EC cells, IGFII seems to principally affect viability, or survival, which may well be a metabolic phenomenon. However, the other effect is the inhibition of differentiation in the spontaneously differentiating EC cells, which may be a growth factor effect.

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