Oncogenes in developmentOncogenes in development EILEEN D. ADAMSON La Jolla Cancer Research Foundation, 10901 North Torrey Road, La Jolla, Pines CA 92037, USA Synopsis Section no

Development 99, 449-471 (1987)Printed in Great Britain © The Company of Biologists Limited 1987

Review Article 449

Oncogenes in development

EILEEN D. ADAMSON

La Jolla Cancer Research Foundation, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA

Synopsis

Section no.

(1) Introduction(2) EGF receptor/c-erb-B(3) CSF-1 receptor/c-/nw(4) c-src(5) c-abl(6) Other oncogene kinases(7) The ras family of genes(8) c-fos(9) c-myb

page449449452452453454454455457

(10) The myc gene family(11) c-sis(12) Other proto-oncogenes(13) Proto-oncogene expression in terato-

carcinoma cells(14) Conclusions and summary(15) References

Kev words: oncoeene.

457459460

460461462

(1) Introduction

Realization that the transforming oncogenes (v-onc)of the acutely oncogenic retroviruses are homologousto cellular genes (and were probably derived fromthem) brought several areas of research together withexciting prospects for advances in virology, carcino-genesis, evolution and development. The proto-onco-genes (c-onc) are likely to be crucially involved ingrowth regulation and/or differentiation because oftheir conservation throughout evolution and becauseof the well-known growth deregulation effects pro-duced by the \-oncs. It was therefore reasoned thatthe normal counterpart of these genes should beactive during embryonic development and that identi-fication of a specific tissue or stage where c-oncs areexpressed should help to identify their roles in all cellsand provide a source of material to study the mechan-isms of action. The preliminary results suggest growthmodulatory roles for most oncogenes, and develop-mental studies have provided clues to c-onc roles thatwould not have been forthcoming from studies on celllines.

Several c-onc products have now been identifiedwith specific cellular proteins, and these have con-firmed their importance to growth regulation. Theyare growth factor or hormone receptors (such asc-erb-B, or epidermal growth factor [EGF] receptor;c-fms, or colony stimulating factor-1 [CSF-1] receptor

and c-erb-A, or thyroid hormone receptor) or growthfactors (such as c-sis, or B chain of the platelet-derived growth factor [PDGF]). Table 1 lists theproto-oncogenes that have been studied in develop-ing or differentiating systems. For general reviews,see Miiller, 1983; Hunter, 1984; Weinberg, 1984;Varmus, 1984; Heldin & Westermark, 1984; Sin-kovics, 1984; Klein & Klein, 1985; Bishop, 1985; andMuller, 1986.

(2) EGF receptor/c-erf>-B

The best known of the tyrosine kinase familyof c-oncs is the EGF receptor, which is a largerhomologue of the v-erb-B protein that causes avianerythroblastosis in chickens infected with avian eryth-roblastosis virus (AEV) (Downward etal. 1984). TheEGF receptor has been recognized as a cell-surfaceglycoprotein whose activity is triggered after bindingEGF in the cellular environment. After receptorclustering and endocytosis, lysosomal compartmentsdegrade both receptor and ligand. After this eventthere is very little known about how the cell receivesthe signal to commence DNA synthesis and to entermitosis. Many steps of the process may be necessary,but after the discovery that the receptor also has atyrosine phosphokinase enzyme activity (Ushiro &Cohen, 1985) that leads to the phosphorylation ofitself and of other cellular substrates, the clearest clue

450 E. D. Adamson

Table 1. Some proto-oncogenes and their protein products

c-oncProtein identity,homology or size

I. Tyrosine kinase and related c-onc

c-erb-B EGF receptor170 K

c-neu

c-fms

c-src

c-mos

c-abl

c-fesc-fps

II. GTPasesc-Ki-raj-2]c-H-nu-1 \

185 KHomology to EGF-R

CSF-1 receptor140 K

60K

37K

150K

92 KJ98 KJ

21K

III Nuclear productsc-fos 55 K

c-myc ^

N-myc 1L-mycJ

c-myb

IV. O^ersc-erfovl

c-iis

62K66K

75 K

T3 receptor,homology toglucocorticoidreceptor

PDGF B chain14 K

Cell location anddistribution

Plasma membrane ofmesodermal, ectodermal& endodermal cells

Plasma membrane

Plasma membrane ofmacrophages & extra-embryonic cells

Cytoplasmic face ofmembranes.Adhesion plaques

Cytoplasmic.Embryotestis & ovary

Plasma membrane

Cytoplasm, plasmamembrane

Membrane cytoplasmicface in a widevariety of cells

Nucleus of extra-embryonic tissues,haematopoietic cells& macrophages Allother cells at lowerlevel.

Nuclear matrix ofmost cells In sometumours & embryonictissues.

Nucleus of haemato-poietic cells

Cytoplasmic &nuclear

Secreted protein

Activities

EGF bindingTGFa bindingTyr. kinase

Tyr. kinase

CSF-1 bindingTry. kinase

Tyr. kinasephosphorylatesmany cellularproteins, e.g.,vinculin, vimentin,filamin, p36.

Ser/thr kinase

Tyr. kinasephosphorylatesvincuhn

Tyr. kinase

GTP + GDPbinding.GTPase.

DNA bindingwith an accessoryprotein

DNA binding

DNA binding

Bindsthyroxine

Homo- orheterodimerbinds toPDGF-R

Possible roles

Signal transductionformitogenesis and differ".Stim° of tooth eruption,eye-opening, lung devel.

Receptor for anunknown ligand?

Signal transduction formitogenesis and differ"

Neurone &muscle development9

B-cell differ1?

Macrophage devel.9

Adenylate cyclaseregulation?

GO to Gl transition.Differentiation

Proliferation.Regulates DNAsynthesis.

Differentiation ofhaematopoietic cells9

Metabolic regulator?

MitogenesisWound healingEarly embryonicgrowth factor?

Chromosomallocationhuman/mouse

7/7

17/?

5/?

20/9

8/?

9/7

15/9

10/711/71/7

14/?

8/15

6/?

17/7

22/7

References

1

2.

3.

4

5.

6.

7

8.

9.

10.

11.

12.

13

K, 10"3iMr

References1. Hayman et al. 1983; Downward el al. 1984; Carpenter 1984. 2. Schechter a al. 1984, 1985; Hung el al. 1986. 3. Rettenmier et al. 1985; Coussens et al.

1986. 4. Purchio et al. 1978; Takeya & Hanafusa, 1983; Sejersenflo/. 1986. 5. Van Beveren et al. 1981; Papkoff«oA 1982, 1983; Baldwin, 1985. 6. Witte «al 1978, 1979, 1980; Reddy a al. 1983. 7. Baibacid et at. 1980; Hampe et al. 1982; Caimier & Samarut, 1986; Ferrari et al. 1985. 8. Tsuchida a al. 1982;Dhar et al. 1982; Hall et al. 1983; MOUer et al. 1982; Cooper a al. 1984. 9. Van Beveren et at. 1983; Deschamps et al. 1985. 10 Mellon et al. 1978; Alitalo etal 1983; Kaczmarek et al. 1985: Eisenman a al. 1985; Wan et al. 1985; Jakobovitz et al. 1985; Zimmerman et al. 1986; Nau et al. 1985. U .Gondana / .1985; Sheiness & Gardinier, 1984; Janssen a al. 1986. 12 HoUenberg et al. 1983; Weinberger a al. 1985, 1986. 13. Devare et al. 1983; Doolittle et al. 1983.

appeared to have been identified. This is because theproportion of phosphotyrosine, compared with phos-phoserine and phosphothreonine in a normal cell, isvery small, about 0-03 % of the total. The mechanism

of oncogenesis via tyrosine phosphorylation has beenpursued vigorously, but the outcome so far has notgiven a clear picture of the roles of tyrosine phos-phokinases in growth regulation. For reviews that

Oncogenes in development 451

cover the enzymic and metabolic aspects of the EGFreceptor, see Thompson & Gill, 1985; Herschman,1985; Kris, Libermann, Avivi & Schlessinger,1985; Carpenter & Zendoqui, 1986; Soderquist &Carpenter, 1986.

(2.1) The EGF-receptor geneNormal tissues and cells appear to have a single genefor EGF receptor but differential splicing leads to theproduction of at least two mRNAs of about 6 and10 kb (Ullrich et al. 1984; Lin et al. 1984; Xu et al.1984). Both of these appear to code for full-lengthprotein of 170 x 103 Afr (170 K) (Simmen et al. 1984).The gene is amplified in human A431 cells and inseveral types of tumours such as gliomas (Libermannet al. 1984), squamous cell carcinoma (Cowley et al.1984) and retinoblastomas where the protein is alsooverexpressed. Presumably the expression of highlevels of receptor provided a growth advantage to thetumour cells but it is not known if this was the originallesion.

(2.2) Distribution of EGF-binding activity indeveloping tissuesEGF receptors are present on a wide range of celltypes including cells of ectodermal, mesodermal andendodermal origin (reviewed by Adamson & Rees,1981). They are also present on embryonic/fetaltissues and extraembryonic tissues during murinedevelopment (reviewed by Adamson, 1983). But inspite of this, their role in development is not yet clear.EGF receptors increase in number during the ges-tation period so that the new-born's liver expressesmore than any other tissue at any stage (Adamson &Meek, 1984). Crude membrane preparations from allfetal tissues (with the exception of the parietal yolksac) contain EGF-binding activity, and the affinity ofthese receptors for 125I-EGF decreases somewhatduring development. The earliest time of detection ison giant trophoblast cells of a 5-day blastocyst grownfor 2-3 days in culture (Adamson & Meek, 1984).Fetal receptors are functional in that they bind EGFand they can be down-regulated by excess EGFinjected into the placenta or amniotic cavity (Adam-son & Warshaw, 1981). When fetal organs areexplanted and cultured with EGF, incorporation of[3H]thymidine into DNA is stimulated (Adamson,Deller & Warshaw, 1981). Autophosphorylating ac-tivity of EGF receptors is first detected in 10-daymouse embryos at the time of onset of organogenesis(Hortsch, Schlessinger, Gootwine & Webb, 1983).The changing characteristics of EGF receptorssuggests that roles may be different at different stagesof development, although changing cell populationscould also explain changing receptor characteristics in

a tissue such as fetal liver which has high levels ofhaemopoietic cells in the early stages.

Specific tissues have been targets of research aimedto find a role for EGF in their development. EGFstimulates skin differentiation in the neonatal mouseand accelerates eye opening and tooth eruption(Cohen, 1962). Palatal epithelium expresses EGFreceptors that may play a role in palate closure (Tyler& Pratt, 1980; Turley, Hollenberg & Pratt, 1985).Lung development and maturation are affected byEGF (Catterton, Escobedo & Sexson, 1979; Gross etal. 1986; Goldin & Opperman, 1980). Growth ofnewborn rat liver, kidney and craniofacial structuresis retarded by EGF (Hoath, 1986). Chick neural crestcells (Erickson & Turley, 1987), rat oocyte duringmaturation (Dekel & Sherizly, 1985), and postnataland fetal development of rat gastric mucosa (Dem-binski & Johnson, 1985; O'Loughlin et al. 1985;Conteas, DeMorrow & Majumdar, 1986) areinfluenced by EGF.

(2.3) Occurrence of EGF receptors in teratocarcinomacellsThe stem cell lines (embryonal carcinoma, EC) ofmurine teratocarcinomas appear to have very few(F9) or no EGF-binding sites (OC15, PC13, P19).When differentiation occurs, either in aggregate cul-tures (Adamson & Hogan, 1984) or in monolayers(Rees, Adamson & Graham, 1979; Mummery et al.1985), receptors appear which respond to EGF bypromoting cellular proliferation. Teratocarcinoma-derived differentiated cell line PSA5E (visceral endo-derm-like) also has EGF receptors, while PYS andF9AC cells (parietal endoderm-like) do not. Thisdistribution is in agreement with the tissue distri-bution determined from embryo studies. Surpris-ingly, OC15 EC cells have considerable EGF-recep-tor-kinase activity (equivalent to about 3-daydifferentiated cells), but it appears to be largelyintracellular or possibly masked (Weller, Meek &Adamson, 1987). Therefore, we have to modify ouroriginal hypothesis that early embryonic ectodermcells would be negative for this protein based onfindings in EC cells. Indeed, a human EC cell-line(PA1) has been shown to express a low level of EGFreceptors on the cell surface (Carlin & Andrews,1985).

(2.4) Activation of the EGF-receptor oncogeneprotein during developmentIf the EGF receptor is to function it must receivesignals by ligand binding to the extracellular bindingdomain. Two known growth factors bind to the EGFreceptor, EGF/urogastrone and transforming growthfactor a (TGFa). See reviews by Adamson (1983,1986) and Roberts & Sporn (1985).

452 E. D. Adamson

(2.4.1) EGF

This 53 amino-acid polypeptide (6K) is synthesizedand stored in very large amounts in male adult mouse(but not rat or human) submandibular salivary gland.It is detected in saliva, amniotic fluid, milk, urine andmost tissues of several species. Although EGF hasbeen detected in fetal mice (Nex0, Hollenberg,Figueroa & Pratt, 1980) and fetal rats (Matrisian,Pathak & Magun, 1982), it seems unlikely to bederived from the fetus itself, and Popliker et al. (1986)have presented evidence that the EGF found in fetalmice is derived from the mother. The EGF geneoccurs as a large gene encoding a precursor thatcontains several EGF-like peptides as well as EGF,together with a membrane-spanning domain (Scott etal. 1983). Using a cloned probe for prepro EGFmRNA, Rail et al. (1983) have shown high-level genetranscription in the mouse salivary gland and inkidney. Female kidney expresses about 2- to 4-foldhigher levels than male, and this is a reversal of thesalivary gland levels (Popliker et al. 1986; Gubits etal.1986; Salido et al. 1986). However, only the salivarygland precursor appears to be processed to the active6K form. The kidney 130 K precursor form is notknown to be an active mitogen. The importance ofthese reports is the realization that EGF is notsynthesized until at least 2 weeks postpartum in thekidneys and even later (after weaning) in the malesalivary gland. Then why does the fetus express EGFreceptors?

(2.4.2) TGFa

Rat TGFar (5K) has been purified, sequenced, syn-thesized, molecularly cloned and shown to have35-40 % homology with EGF (Marquardt, Hunkapil-lar, Hood & Todaro, 1983; Tarn et al. 1984; Lee,Rose, Webb & Todaro, 19856). TGFa binds to EGFreceptors and produces exactly the same effects,including mitogenesis and skin maturation. It can alsosynergize with TGF/3 to stimulate anchorage-inde-pendent growth of normal fibroblasts. TGFa alsooccurs as a larger precursor form (Derynck et al. 1984)as a transmembrane protein. TGFa is found innormal rat tissues (Lee et al. 19856; Stromberg,Pigott, Ranchalis & Twardzik, 1982; Proper, Bjorn-son & Moses, 1982; Twardzik, 1985; Massagu6, 1985.In contrast to EGF, TGFa'has been detected as earlyas day 7 in mouse embryos using a specific radioim-munoassay procedure. The levels fall and then rise toa second peak in day-13 fetuses (Twardzik, 1985), inparallel with the levels of mRNA (Lee, Rochford,Todaro & Villareal, 1985a). Later in development,the placenta could be a major source of TGFQ-(Stromberg etal. 1982). These studies imply that earlyembryonic development could be affected by TGFa-binding to EGF receptors, but the details of which

embryonic tissues synthesize the growth factor andwhere TGFa'finds its target are still to be discovered.

(3) CSF-1 receptor/c-frns

The x-fms product is the transforming protein ofthe McDonough strain of the feline sarcoma virus(SM-FeSV). Its proto-oncogene homologue is amembrane-inserted phosphorylated glycoprotein ofapproximately 165 K in mouse and 170 K in cat. Theidentity of the c-fms gene was first suggested by Sherret al. (1985) who showed that it is very similar if notidentical to the monocyte/macrophage cell surfacereceptor for CSF-1. The c-fms gene product is atyrosine kinase, and the gene is therefore a memberof the src family of related oncogenes (Rettenmier,Chen, Roussel & Sherr, 1985; Coussens et al. 1986).

The tissue expression of c-fms appears to be limitedto the monocyte/macrophage lineage (spleen, bonemarrow and fetal liver) and to developing extraem-bryonic tissues in mouse and human where it isexpressed in a stage- and tissue-specific manner(Muller et al. 1983a). Highest levels of accumulatedtranscripts are present in 17th-19th day placenta withlower amounts in amnion, visceral yolk sac andchorion. The question arises whether this expressionin extraembryonic tissues is due to high levels ofmacrophages found there. This will only be answeredwhen in situ localizing methods are performed. How-ever, evidence from a human teratocarcinoma cellline indicates that c-fms may be specific to placentalcells. HT-H cells differentiate spontaneously inmonolayer cultures to trophoblast-like cells that se-crete hCG a sub-units and that express c-fms mRNA(Izhar et al. 1986). This phenotype is cell-type specificsince undifferentiated stem cells and other types ofcells that differentiate from HT-H cells in aggregatesdo not express c-fms and do not secrete hCG. Be Wohuman choriocarcinoma cells also express c-fms,further supporting localization in placental cells(Muller et al. 1983a,6).

(4) c-src

The src proteins were the first tyrosine kinases to bediscovered and form the archetype for this group ofoncogene products, \-src encodes the transformingprotein of the avian Rous sarcoma virus and c-srcencodes a similar 60K protein. The demonstrationthat protein kinases are involved in oncogene-mediated transformation stems from the finding thatpp60"lc is tightly associated with a protein kinaseactivity (Collett & Erikson, 1978; see review by


Wyke, 1983). The Rous SV product also phosphory-lates phosphatidylinositol and diacylglycerol (Sugi-moto, Whitman, Cantley & Erikson, 1984), andso its function is linked with other oncogeneproducts which also affect phospholipid metabolism.A pp60VJnc related tyiosine kinase has been purifiedfrom bovine brain cerebral cortex (Neer & Lok, 1985)as a 61K protein that can be phosphorylated on serineas well as tyrosine. It exhibits an autophosphorylatingactivity and also phosphorylates precipitating anti-body (Resh & Erikson, 1985). Its location is on thecytoplasmic face of the cell as well as perinuclear. Theamino terminus of pp6ffrc is myristylated, and this isneeded both for localization to the inner face of theplasma membrane and for cellular transformation(Kamps, Buss & Sefton, 1985).

When the pp60v~jrc gene is transfected into NIH3T3 cells, gap junction communication between ad-jacent cells is inhibited and the activity of proteinkinase C is enhanced (Chang et al. 1985). Theseactivities suggest that if c-src has a similar function, itcould be important to development. It was foundseveral years ago that chick embryonic neural tissuesexpress high levels of c-src (Cotton & Brugge, 1983).Immunoassays show that most tissues have eight- toten-fold lower levels of c-src than brain, retina andspinal ganglia. Immunocytochemical studies showhighest levels in neural tube, brain and heart of stage-32 chicks, with lower levels in eye, limb bud and liver.A similar distribution is found in human fetuses withhighest levels in cerebral cortex, spinal cord and heart(Levy, Sorge, Meymandi & Maness, 1984; Gessler &Barnekow, 1984). Thus the expression of c-src corre-lates strongly with the differentiation of electrogenictissues when proliferation has ceased and expressionpersists in terminally differentiated neurones.

Recent immunocytochemical staining results ofManess, Sorge & Fults (1986) show that developingneural tissues in chick exhibit pp6O*rc expression attwo different stages. On or before stage 4, transientlocalization is seen in the neural ectoderm. Thisdeclines by stage 12 (45-49 h) and later rises again interminally differentiated neurones at about stage 21(day 3-5) in the neural retina (Sorge, Levy & Maness,1984) and stage 17 (day 2-5) in the cerebellum (Fults,Towle, Lauder & Maness, 1985). Therefore it ap-pears that c-src may play a role at proliferative stagesalso. An analogous pattern of c-src expression inDrosophila has been shown by in situ hybridization(Simon, Drees, Kornberg & Bishop, 1985). c-srcmRNA is abundant in early embryos during gastru-lation, low in larvae, and high in neural tissue andsmooth muscle and pupae at later stages.

In vitro culture systems have been used to elucidatethe location, effects and roles of c-src. Immunostain-ing shows that c-src is distributed all over cell bodies,

processes and growth cones of chick dorsal rootganglion cultures (Maness, 1986). Primary cultures ofneurones or astrocytes from rat brain contain 15- to20-fold more c-src protein than fibroblasts, and thekinase specific-activity of c-src in neurones is 6- to 12-fold higher than in astrocytes (Brugge et al. 1985).Intriguingly, neuronal but not astrocytic c-src differsin size by a post-translational modification in theamino-terminal half. The murine teratocarcinomacell line, PCC7, can be induced to differentiate intoneuronal structures and a parallel elevation (3- to 5-fold) in c-src mRNA can be detected (Sejersen,Bjorklund, Sumegi & Ringertz, 1986). An 8- to 20-fold increase in src protein levels is observed after 5days of induction of P1951801A1 murine EC cellswith retinoic acid, corresponding to the appearanceof neuritic processes and other neuronal markers.The induced src protein is the slower mobility formassociated with neurones, a finding that suggests theusefulness of teratocarcinoma cell model systems(Lynch, Brugge & Levine, 1986).

Studies with RSV infections or \-src introducedinto cultured cells have also given results that suggestboth proliferation and differentiation can be inducedby the gene. For instance, marrow cultures areinduced to greater selfrenewal of haematopoieticprogenitor cells (Boettiger, Anderson & Dexter,1984), while retrovirus carrying \-src introduced intoPC12 rat phaeochromocytoma cells induces somefeatures characteristic of differentiation (neurite ex-tension) (Alema, Casalbore, Agostini & Tato, 1985).v-src-containing viruses suppress differentiation inchondroblasts as measured by the synthesis of chon-drocyte-specific products (Alema, Tato & Boettiger,1985). However, \-src has different properties to c-srcand may also be regulated differently.

(5) c-abl

The Abelson leukemia virus (Ab-MLV) induces Band T cell lymphomas in mice by means of theexpression of v-abl, which encodes a tyrosine kinaseof 160 K. Cooperation with EGF receptors appears tobe necessary for v-abl to transform murine fibroblastsinto tumorigenic cells (Gebhardt, Bell & Foulkes,1986). An homologous c-abl of 150 K is found innormal mouse cells (Goff, Gilboa, Witte & Balti-more, 1980). c-abl is actively transcribed duringembryo and fetal development with high levels oftranscripts in extraembryonic tissues as well as in thefetus proper (Miiller et al. 1982). Its expression ishighest on day 10 when organogenesis is progressingrapidly. Thereafter, mRNA levels fall during ges-tation but are detectable throughout. Low levels havebeen detected in several teratocarcinoma cell lines(Sejersen, Sumegi & Ringertz, 1985).

454 E. D. Adamson

(6) Other oncogene kinases

Very little is known about the expression of the othertyrosine kinases in development. The proto-onco-gene c-ros has features in common with the EGFreceptor family and displays tissue-specific and de-velopmentally regulated expression (Neckameyer,Shibuya, Hsu & Wang, 1986). The oncogene \-kit,which is the transforming gene of HZ4 feline retrovi-rus, has homologous regions to PDGF receptor andc-fms, but is presumably a truncated version with notransmembrane domain (Besmer et al. 1986).

Distantly related to the tyrosine kinase encodingoncogenes is v-raf, which has ser/thr kinase activity.The c-raf-1 gene has been cloned and utilized toevaluate expression in mouse embryos. mRNA is notdetected, while a related but distinct gene, A-raf, istranscribed in 14-day embryos with less in 18-dayembryos and placenta. Moderate levels were found incertain adult tissues (Huleihel et al. 1986).

The cellular homologue of the transforming geneproduct, p2>lc'mos, of the Moloney murine sarcomavirus (Papkoff, Nigg & Hunter, 1983) was thought tobe unexpressed in mouse tissues. By sensitive SInuclease and Northern assays, expression has beendetected in whole embryos at moderate levels, inplacenta, kidney and brain at very low levels, and athigh levels in adult testes and ovary. Transcript sizesdiffer in different tissues and it was suggested thattissue-specific regulation of the size of mos transcriptscould be transactivated, for example, by hormones,and could give rise to functionally different proteinproducts (Propst & Vande Woude, 1985).

(7) The ras family of genes

The ras genes were first identified as the viral onco-genes of the Harvey and Kirsten rat sarcoma viruses(Ellis, DeFeo, Furth & Scolnick, 1982). Cellular rasgenes have been identified in most species, and theyconstitute a family of three human genes whichencode a remarkably well-conserved protein, desig-nated p21 (Defeo et al. 1981). The p21 protein islocated on the cytoplasmic face of the plasma mem-brane in both normal and transformed cells. In vitro,p21 binds GTP (Finkel, Der & Cooper, 1984), but thenormal gene product hydrolyses GTP at a rate 8- to10-fold higher than that of the transforming protein(Sweet et al. 1984). ras proteins are structurally andfunctionally analogous to the G proteins which areinvolved in adenylate cyclase regulation (Gilman,1984; Hurley et al. 1984). The potential relationshipbetween adenylate cyclase and p21 may be part of thecontrol of cell division in Xenopus laevis oocytes.Progesterone-induced meiotic activation of germinalvesicle breakdown (GVBD) is mediated, at least in

part, by inhibition of the oocyte adenylate cyclase(Finidori-Lepicard et al. 1981), but it appears not toinvolve the inhibitory guanine-nucleotide bindingsubunit G,. Sadler, Schechter, Tabin & Moller (1986)showed that monoclonal antibodies to p21-ras in-hibited adenylate cyclase activity and gave acceler-ated maturation of Xenopus laevis oocytes in a dose-dependent manner. It is possible either that p21protein interacts with the pathway of normal celldivision regulated by progesterone or that antibodiescross react with the oocyte G proteins. Ras proteinsappear to interact with phospholipase C in a Gprotein-like manner (Fleischman, Chahwala & Cant-ley, 1986), and c-Ki-ras-2a has some homology withlipocortin and related proteins (Kretsinger & Creutz,1986).

The ras gene products are clearly involved in animportant aspect of cell proliferation since evennormal p21c""" when expressed at elevated levels canresult in the immortalization and transformation ofmouse cells (but not human cells). c-Ha-ras and c-Ki-ras expression is detected at all stages of mouseembryonic development at almost unvarying levels(Miiller et al. 1982; Muller, 1983; Slamon & Cline,1984). This would suggest a general metabolic role indevelopment, ras proteins are remarkably conservedevolutionarily, with homologous proteins occurringin yeast which have similar GTP-binding and hydro-lytic properties (Temeles et al. 1985) and inDrosophila (Mozer, Marlor, Parkhurst & Corces,1985). Of the three v-Ha-ras-related cellular genes inD. melanogaster, each is transcribed into two sizes ofmRNAs. The larger is expressed at similar abundanceduring the life cycle stages, while the shorter tran-script is more abundant in embryonic stages (Lev,Kimchi, Hessel & Segev, 1985). In situ hybridizationwas used to locate and identify active tissues. Distri-bution is uniform in embryos but is restricted todividing cells in larvae. However, in the adult, bothdividing and nondividing tissues contain high levels ofras transcripts, including ovaries, cortex of brain andganglia, thus suggesting roles in growth and in differ-entiation (Segal & Shilo, 1986). An homologous p23protein in Dictyostelium discoideum is expressed athighest levels in growing organisms, and this declineswith the onset of differentiation (Pawson et al. 1985).Similarly, F9 murine teratocarcinoma cells expresshigh levels of c-Ha-ras mRNA and this is moderatelydiminished during differentiation (Campisi et al.1984). However, c-Ki-ras expression increases after48 h of stimulation of differentiation of mouse eryth-roleukemic cells with DMSO. Other oncogenes arealso induced, including fos, myb, and myc, while tenothers remain unchanged (Todokoro & Ikawa, 1986).

The differentiation inducing properties of c-Ha-raswere tested by introducing sarcoma viruses carrying


ras oncogenes into PC12 cells (Noda etal. 1985) or bymicroinjecting purified normal or activated Ha-rasproteins (Bar-Sagi & Feramisco, 1985). There is noeffect of normal c-ras on PC12 differentiation, butactivated Ha-ras products induce differentiation inboth cases. Antibody to p21-ras protein microinjectedinto PC12 cells inhibits nerve-growth-factor-induceddifferentiation (Hagag, Halegoua & Viola, 1986),and this indicates that c-ras does indeed play a role indifferentiation.

The expression of c-Ki-ras appears to be cell cycledependent in a chemically transformed mouse fibro-blast cell line with highest expression in mid to lateGo/Gi (Campisi etal. 1984). In addition, antibody toras microinjected into quiescent NIH 3T3 cells priorto serum stimulation blocks a large population of cellsfrom entering S phase later while control antibodiesdo not. A time course shows that c-ras activity isneeded just before S phase or about 8 h after additionof serum (Mulcahy, Smith & Stacey, 1985). A similartime of c-Ha-ras gene activation is found after partialhepatectomy in rats, and this returns to normal after 3days suggesting that proto-oncogene regulation is anormal regulated process in non-neoplastic growthprocesses (Goyette, Petropoulos, Shank & Fausto,1984).

(8) c-fos

The FBJ and FBR murine osteosarcoma virusesrapidly induce osteosarcomas in mice, and a 55 Kprotein encoded by the transforming gene, v-fos, ofthe FBJ virus (J5&a*-fos from FBR-MSV) has beendescribed (Curran & Teich, 1982; Curran & Verma,1984). The cellular homologue, c-fos, also encodes a55 K protein, which differs from the viral protein inthe carboxy-terminal portion, but which, neverthe-less, can transform normal fibroblasts (Miller, Curran6 Verma, 1984). Both proteins are nuclear phospho-proteins and both are turned over rapidly in the cell,\-fos with a half life of 2 h and c-fos about 20 min. c-fosprotein differs in the degree of post-translationalmodifications that can be detected soon after syn-thesis (Curran, Miller, Zokas & Verma, 1984). Seereviews by Miiller & Verma (1984) and Deschamps etal. (1985) for further details.

(8.1) c-fos expression in developing tissues andproliferating cellsA distinguishing feature of c-fos expression is thatalthough transcripts are present at barely detectablelevels in embryos and fetuses, the extraembryonictissues have very high levels (Miiller etal. 1982). Day-7 murine conceptuses consisting predominantly ofextraembryonic tissues have very high levels of ex-pression and, on further examination, a stage- and

tissue-specific pattern is detectable (Muller, Verma &Adamson, 1983c). In the mouse, amnion >visceralyolk sac > placenta, and a similar pattern is found inhuman tissues (Muller etal. 19836). In the mouse thelevel of c-fos mRNA rises to a plateau on the 16th to17th day of gestation in extraembryonic tissues andalso 14th day (haematopoietic) fetal liver, and laterskin and bone/bone marrow contain high levels.Fetal liver and bone marrow probably express c-foslargely because of the population of macrophages andother haematopoietic lineages that express c-foseither constitutively or at some stage in their differen-tiation/maturation processes (Muller, Muller & Guil-bert, 1984; Muller, Curran, Muller & Guilbert, 1985).Macrophages cannot account for the high expressionin very early extraembryonic tissues. In situ localiz-ation of c-fos protein (Adamson, Meek & Edwards,1984) and mRNA (Deschamps et al. 1985) has clearlylocated fos expression in all the cells of the extraem-bryonic tissues. In addition, extraembryonic tissueshave been shown to synthesize p55c"^OJ protein(Mason, Murphy & Hogan, 1985). Therefore, what isthe role of c-fos gene expression in these tissues?

A brief survey follows of the three types of stimulithat induce c-fos expression, including growth, differ-entiation and stress stimuli. Quiescent, serum-starvedmouse or rat fibroblasts in Go, stimulated to 'com-petence to divide' by PDGF, FGF (or serum), acti-vate the fos gene within a few minutes; mRNA levelspeak at 30 min and then fall to low levels in 60 min.This is caused by an increased rate of transcriptionand is accompanied by increased protein synthesis.The increased levels of mRNA and protein synthesisare both transient (Greenberg & Ziff, 1984; Muller,Bravo, Burckhardt & Curran, 1984; Kruijer, Cooper,Hunter & Verma, 1984; Treisman, 1985; Zullo, Coch-ran, Huang & Stiles, 1985; Renz et al. 1985; Green-berg, Hermanowski & Ziff, 1986). In all cases c-fosmRNA accumulations are followed by an increase inthe level of c-myc mRNA, although it is not known ifthese are linked responses. Epithelial and other cellsrespond to their specific mitogens with a similartransient increase in fos mRNA: EGF-stimulatedA431 cells (Bravo, Burckhardt, Curran & Muller,1985); PC12 cells (Greenberg, Greene & Ziff, 1985);and primary hepatocytes (Kruijer etal. 1986); thymo-cytes stimulated with concanavalin A (Moore, Todd,Hesketh & Metcalf, 1986); thyroid cells with thyro-tropin (Colletta, Cirafici & Vecchio, 1986; Tramon-tano, Chin, Moses & Ingbar, 1986); peripheral lym-phocytes with phytohaemagglutinin and calciumionophore (Reed, Alpers, Nowell & Hoover, 1986).Primary amnion cell cultures continue to express c-fosprotein for 2h after culture in vitro but this leveldeclines to zero in 15 h. Addition of undefined factorsin medium conditioned by placental or embryo

456 E. D. Adamson

explants stimulates the re-expression of fos protein(Miiller et al. 1986). Apparently, constitutive ex-pression of fos in amnion may be maintained bystimulatory endogenous factors that are produced byfetal and placental tissues. These examples suggestthat c-fos is transiently expressed whenever mitogensbind to a cell receptor, but the following cases showthat the subsequent response of a cell is not necess-arily correlated with cell division.

(8.2) c-fos induction associated with celldifferentiationWhen PC12 cells are stimulated with nerve growthfactor (NGF), they sprout neurites and becomedifferentiated, non-dividing, neurone-like cells. Thec-fos gene is rapidly activated with similar kinetics aswhen proliferation is stimulated with EGF or FGF(Greenberg et al. 1985; Kruijer, Schubert & Verma,1985; Milbrandt, 1986). In contrast, c-fos is notactivated when PC12 cells differentiate to chromaf-fln-like cells when treated with glucocorticoids(Deschamps et al. 1985). In the case of PC12 cells,c-myc is also activated, whereas in other differen-tiating systems c-myc mRNA levels usually fall pre-ceding or accompanying falling levels of prolifer-ation. Other cell types that express elevated c-fosmRNA levels when stimulated to differentiate in-clude HL-60 and U-937 human leukemia cells whentreated with the phorbol ester TPA (Mitchell, Zokas,Schreiber & Verma, 1985; Miiller et al. 1985) andWEHI-3B cells induced with granulocyte-colonystimulating factor (Gonda & Metcalf, 1984). In thelatter case, c-fos mRNA accumulates to high levelsonly after 2-3 days, when differentiation to mono-cytes occurs. In cell lines that differentiate to mono-cyte/macrophages,/as mRNA levels remain modera-tely high and therefore differ from growth factorresponses. The activation of c-fos appears to belineage-specific since HL-60 cells differentiating togranulocytes do not activate fos (Miiller et al. 1985;Mitchell et al. 1985). c-fos expression is constitutivelyhigh in a mast cell precursor line (PB-3c) grown in thepresence of interleukin 3 (IL-3) (Conscience, Verrier& Martin, 1986). When IL-3 is withdrawn, the c-foslevel falls and is rapidly induced when growth factor isadded back. This cell line and macrophages expresshigh levels of fos while actively proliferating asdifferentiated cells. It cannot be concluded thatcertain lineages of differentiation require fos acti-vation, since an HL-60 cell line resistant to TPA canbe induced to differentiate to macrophages in theabsence of detectable c-fos mRNA expression (Mit-chell, Henning-Chubb, Huberman & Verma, 1986).Neither can it be concluded that fos is not requiredsince it is possible that a stable form of fos protein ispresent.

(8.3) c-fos activation by other stimuli such as heatshockFrom the above examples, it can be seen that manydifferent external stimuli seem to activate the tran-sient expression of c-fos mRNA. When quiescentHeLa cells are changed from culture at 37 °C to 40 °Cto 44°C, a temperature-dependent, 5- to 20-foldincrease in c-fos mRNA and protein levels follows inabout lh (Andrews, Harding, Calbet & Adamson,1987). Ionic signals such as calcium ionophore, ben-zodiazepines and elevated K+ (Curran & Morgan,1985; Morgan & Curran, 1986), the cell divisioninhibitor mitomycin C and ultraviolet light (unpub-lished observations of S. Edwards and E. Adamson)all induce c-fos mRNA expression. Partial hepa-tectomy and even a sham operation elevate c-fosmRNA levels in rat liver (Kruijer et al. 1986).Wounding a fibroblast monolayer results in rapidinduction of c-fos (Verrier, Miiller, Bravo & Miiller,1986). /3-adrenergic stimulation of mice in vivo pro-duces hyperplasia in parotid and submandibular sali-vary glands and stimulates transient c-fos expression(Barka, Gubits & Vander Noen, 1986). In summary,many kinds of external stimuli achieve a rapid cellularresponse in the form of c-fos gene induction andstrongly suggests that fos is important in relayingextracellular signals to the nucleus, but the mechan-isms remain unknown.

(8.4) c-fos expression in teratocarcinoma modelsystems of developmentc-fos mRNA is expressed at very low levels inproliferating EC cells (Miiller, 1983; Vilette,Emanoil-Ravier, Tobaly & Peries, 1985; Edwards &Adamson, 1986) and increases modestly during F9aggregate differentiation to embryoid bodies, peak-ing on day 3 after retinoic acid addition (S. Edwardsand E. Adamson, unpublished data; Miiller, 1983).P19 EC aggregates stimulated with DMSO to differ-entiate into a mixture of cell types, including cardiacmuscle express c-fos mRNA in increasing amountspeaking on the 12th day (Edwards & Adamson,1986). These results indicate that c-fos expression canaccompany teratocarcinoma cell differentiation andsupport the findings of Miiller & Wagner (1984) andRuther, Wagner & Miiller (1985) who showed thatthe integration and expression of normal exogenousc-fos genes in F9 cells stimulates differentiation. P19EC cells are less affected, however, and PC13 cellsare not stimulated to differentiate by c-fos expression.Thus c-fos expression alone is not sufficient to triggerdifferentiation. Apparently contrary to the hypoth-esis that c-fos expression may be necessary (but notsufficient) for differentiation to occur, Mason et al.(1985) did not find elevated c-fos mRNA levels duringF9 differentiation in monolayer cultures stimulated


with retinoic acid and cAMP, but did detect atransient slight induction 60min after RA addition.

An effective way to determine the function of agene is to introduce complementary RNA into cellsand determine the effect of the prevention of theexpression of the protein product of that gene. Theintroduction of 'anti-sense' fos DNA into 3T3 cellshas already shown to be effective in reducing growthrates and in reducing the frequency of such clones(Holt, Gopal, Moulton & Nienhuis, 1986). Ourunpublished results indicate that anti-sense fos DNAexpressed in F9 EC cells inhibits their ability todifferentiate in response to RA. This experimentalapproach together with antibody injections, willlikely become prominent in the near future. So far,the data support a hypothesis that fos may be import-ant to development in two ways: one, to act as the'second messenger' for a variety of external stimuliand second, to act in some part of the differentiationprocess, perhaps a step that is connected with growthrestriction or with initiating a new programme of generegulation associated with differentiated function.

(9) c-myb

c-myb is the homologue of the viral transforming genein E26 and other avian viruses that produce myelo-blastosis in birds, c-myb expression is restricted to,and is developmentally regulated, in haematopoietictissue and is therefore thought to play a specific rolein haematopoiesis (Duprey & Boettiger, 1985). c-mybis expressed in a differentiation stage-specific mannerin pre-B cells lines (DeCino, Herbst, Lernhardt &Raschke, 1987). Five percent of the haematopoieticcells of the chick yolk sac contain all the detectablec-myb mRNA of that tissue. These cells were ident-ified as M-CFC or the committed progenitors to themacrophage lineage. As the cells differentiate, thelevel of c-myb falls more than 100-fold. A similar fallaccompanies WEHI-3B cell differentiation to macro-phages (Gonda & Metcalf, 1984). Proto-oncogenec-myb expression is not restricted to macrophagessince both T cells in the murine thymus and cells ofthe erythroid lineage also express c-myb, and this fallstenfold with increasing age (Sheiness & Gardinier,1984). In situ hybridization confirms these lineagesand demonstrates the presence of c-myb mRNA inrapidly proliferating precursors of the myeloid anderythroid lines in human bone marrow cells (Emilia etal. 1986).

(10) The myc gene family

c-myc is the genomic counterpart of the transforminggene of the avian myelocytomatosis virus (MC29)

and, like c-myb and c-fos, its protein product movesrapidly to the nucleus after synthesis and is short-lived. Other homologous genes have been termedL-myc and N-myc corresponding to their activitydetected in lung and neural tumours, respectively.The examples of c-myc expression in response tomitogens of many kinds have been listed in thesection on c-fos and include thrombin, with insulinacting on Chinese hamster fibroblasts (Blanchard etal. 1985). In contrast, there are some exceptionswhere growth-stimulating factors do not induce c-mycmRNA levels: insulin (Kelly, Cochran, Stiles &Leder, 1983); B cell growth factor (Smeland et al.1985), and adenovirus infection (Liu, Baserga &Mercer, 1985). In spite of the large variations seen inc-myc mRNA levels, its rate of transcription increasesonly modestly when cells move from Go to Gi andthen remains at the same lower level in all phases ofthe cell cycle (Thompson, Challoner, Neiman &Groudine, 1985; Rabbitts et al. 1985; Kaczmarek,1986) and differentiation (Dean, Levine & Campisi,1986). Even more than for c-fos, post-transcriptionalregulation is the major means by which the level ofexpression of c-myc is modulated. However, forgrowth stimulation of fibroblast cells by growth fac-tors, an elevated transcription rate can occur depen-ding on the cell type and the growth factor.

(10.1) c-myc expression and cell proliferationc-myc, until recently, has been well correlated tovarious aspects of cell proliferative states (Birnie,Burns, Clark & Warnock, 1984). In transgenic mice,where levels of c-myc are elevated by introduction ofthe c-myc proto-oncogene coupled to the immuno-globulin [i or K enhancer, frequent occurrence oflymphomas occurs within a few months of birth(Adams et al. 1985). Translocation of the c-myc geneto a chromosomal location that endows altered ex-pression has been found in many leukemias in mouseand human. Although induction of the c-myc geneoccurs when erythroleukemic cells are stimulated todifferentiate, expression is transient and, in general,low levels of c-myc are present in differentiatinghaematopoietic cells (Gonda & Metcalf, 1984; Lach-man & Skoultchi, 1984). In view of the association ofc-myc with proliferation, it is not easy to understandwhy both mitotic and meiotic phases of germ cellshave very low levels of c-myc transcripts (Stewart,Bellve & Leder, 1984). For somatic cells likequiescent Swiss 3T3 fibroblasts, DNA synthesis isstimulated by c-myc protein injected into the nuclei(Kaczmarek et al. 1985). Here c-myc acts like acompetence growth factor to initiate the cell cycle sothat cells progress into S phase. It now appears that atleast one way that c-myc may act is by participating inthe process of DNA synthesis, since the addition of

458 E. D. Adamson

affinity-purified anti-c-myc antibodies to isolated nu-clei from several types of human cells reversiblyinhibits DNA synthesis and DNA polymerase activityof the nuclei (Studzinski, Brelvi, Feldman & Wall,1986).

(10.2) c-myc in developmentNot surprisingly, the c-myc gene is highly activeduring development. Stage-specific expression ofc-myc mRNA was found by Pfeifer-Ohlsson et al.(1984) in human placenta with 30-fold variation inlevel. Four- to five-week placenta was the highest,and this declines to much lower levels by the end ofthe first trimester. The mRNA is located predomi-nantly in the cytotrophoblast cells where [3H]thymi-dine labelling also shows these cells to be rapidlydividing. The nondividing syncytiotrophoblast of theterm placenta has 40-fold lower levels. Interestingly,not every cytotrophoblast cell contains c-myc tran-scripts, and it was suggested that a wave of mitoticactivity moving down the placental villi may correlatewith accumulated levels of c-myc mRNA. A mostrelevant observation is that coexpression of c-sis andc-myc occurs in the early placenta (Goustin et al.1985). Since c-sis codes for a PDGF-like mitogen,secretion of this activity into the medium of culturedtrophoblasts of first trimester placenta may not becoincidental (see section 11).

The human fetus proper also displays stage- andcell-specific expression of c-myc with highest levels inthe rapidly proliferating epithelial tissues. These highlevels are sustained throughout the first trimester incontrast to declining levels in the placenta during thesecond month of gestation. The distribution of c-myclevels implies more than mere association with pro-liferation; in 3- to 4-week embryos, c-myc expressionis low, while in extraembryonic tissue c-myc mRNAlevels are high (Pfeifer-Ohlsson et al. 1985). Further-more, predominantly normal development occurs intransgenic mice that express varying levels of c-myc(Leder et al. 1986).

During murine development, c-myc mRNA hasbeen observed at high levels throughout, but a relatedgene, N-myc, is highly expressed at very early stages(7-5 days of gestation) and continues at this level until11-5 days when it declines thereafter (Jakobovits,Schwab, Bishop & Martin, 1985). Zimmerman et al.(1986) examined the expression of all three myc-reiated genes in murine postnatal development andobserved that c-myc expression is present at all stagesand in all tissues, c-myc levels decline to low levels inolder adults in most tissues except adrenals, thymus,spleen, intestine and heart. Tissue- and stage-specificexpression was demonstrated for L-myc and N-myc.L-myc expression is highest in newborn forebrain,hindbrain and kidney, at lower levels in lung and

intestine, and absent in other tissues. L-myc ex-pression is still present in adult lung but decreases inall other tissue so that a 17-day-old brain no longerexpresses L-myc. N-myc was thought to be neuroecto-derm-specific but in fact is found in a variety ofnewborn tissues. It is highest in newborn brain,kidney and intestine, and declines rapidly with in-creasing age. In addition, a striking differential distri-bution is observed in various B cell lines. N-myc ispresent in pre-B cells but not B cells or plasma cells,while c-myc is expressed in all of these lines, and L-myc is never expressed. Very high c-myc expressionoccurs in late fetal mouse cerebellum that declinesand is succeeded by a second peak in postnatal days 3to 10 (Ruppert, Goldwitz & Wille, 1986). The latter islocalized in the mitotically active external granularlayer and accompanies a change in the ratio of the twomyc mRNAs to adult ratios. In general, the ex-pression of myc family genes correlates with commit-ted but still proliferating cells as well as with cells thatare rapidly differentiating along the neural pathway.

The only non-neural tissue in which a relativelyhigh level of expression of N-myc and c-myc isobserved is fetal and newborn kidney. If the mycfamily genes can all be stimulated by growth factors,possibly either the production and activity of prepro-EGF accounts for myc activation, or more likely, theconstant exposure of the kidney to blood-bornegrowth factors stimulates high levels of expression ofthese oncogenes. N-myc is also expressed in mouseand human teratocarcinoma stem cells and in adultmouse testis (Tainsky, Cooper, Giovanella & VandeWoude, 1985; Jakobovits et al. 1985).

The myc gene is highly conserved through evol-ution and even Drosophila melanogaster genome con-tains sequences that hybridize with the v-myc probe,although there is little amino acid sequence homologyin the products. Nevertheless, hybridizing transcriptsare found in embryos, pupae, adults and in a Droso-phila cell line (Kc), and stage-specific expression ofseveral different-sized transcripts were recorded. Theresults also suggest that the transcripts found in earlyembryos are of maternal origin since they are foundonly in the ovaries (Madhavan, Bilodeau-Wentworth& Wadsworth, 1985).

(10.3) c-myc expression in teratocarcinoma cellsThe steady-state levels of c-myc found in proliferatingcells have been shown to decrease drastically (Dony,Kessel & Gruss, 1985) in F9 EC cells at a stage evenpreceding overt differentiation induced by retinoicacid 72 h later (Griep & DeLuca, 1986). Post-tran-scriptional mechanisms are apparently wholly respon-sible for reduced c-myc mRNA levels in F9 cells. InF9 cells stimulated to differentiate, the steady state ofc-myc is reduced by as much as 50 % within 3 h (Griep


& DeLuca, 1986). However, if F9 cells are blockedfrom differentiating in response to RA by the ad-dition of 5 mM-sodium butyrate to the medium(Levine, Campisi, Wang & Gudas, 1984) the cells stillrespond to RA by decreased levels of c-myc. Itappears that reduced levels of c-myc are not sufficientfor differentiation but may precede differentiation orreduced growth rates. The undermethylation of thec-myc gene in F9 EC cells relative to differentiatedteratocarcinoma cells and mouse liver DNA mayaccount for its high level of expression and for itsregulation (Griep & DeLuca, 1986). The second exonof the c-myc gene becomes methylated during F9differentiation and this is remarkable in the face ofglobal demethylation of every other gene tested(Young & Tilghman, 1984; Razin et al. 1984).

(11) c-sls

The predicted sequence of p28v~J", the transformingprotein of the simian sarcoma virus (SSV), is strik-ingly homologous to the B chain of PDGF (Water-field et al. 1983; Robbins et al. 1983; Chiu et al. 1984)or PDGF-2 (Rao et al. 1986). In SSV-transformedcells, p28VJ" is proteolytically processed to generate adisulphide-linked dimer (Johnson, Betsholtz, Heldin& Westermark, 1986), and its presence and activity inthe culture medium can be detected and neutralizedby antibodies to PDGF. Using \-sis probes, tran-scripts of c-sis have been detected in many cell lines,tumours and tissues (Eva etal. 1982). Transformationof cells by a wide range of oncogenic agents appearsto activate a cellular gene encoding a PDGF-likemolecule and the induced expression of the c-sis geneby TGF/3 has been suggested as the intermediary stepin the transformed phenotype induced by TGF/3(Leof, Proper, Getz & Moses, 1986). Mouse embryofibroblast cells stimulated with TGF/J express acti-vated the c-sis gene after 4 h with a peak at 12-16 hand release a PDGF-like activity into the culturemedium, c-fos is also induced maximally at 4h withthe appearance of PDGF activity, while c-mycmRNA levels peak at 8-12 h suggesting that PDGFmay have activated both genes. Since TGF/? is pres-ent in most normal tissues (Roberts et al. 1981) andalso in the placenta, serum (Stromberg & Twardzik,1985) and blood platelets (Assoian et al. 1983), it ispossible that fetal growth may be modulated, at leastlocally, by the release of TGF£ and PDGF.

(11.1) c-sis in developmentThe developmental^ regulated expression of the c-sisgene has been mentioned in section 10 since c-mycand c-sis expression occur together in the highlyproliferative cytotrophoblast cells of the first trimes-ter cytotrophoblast shell. What is interesting about

this study (Goustin et al. 1985) is that placentalexplants secrete a PDGF-like activity into the me-dium in a similar developmental time course. Inaddition, cytotrophoblast-like cell lines establishedfrom early placentae display PDGF receptors withsimilar affinity for PDGF as fibroblast cells. Thesecells also respond to PDGF by tenfold elevation ofc-myc mRNA levels in 2 h and by increased synthesisof c-myc protein (several-fold stimulation within 7h).Autocrine stimulation of cytotrophoblast cells by thec-sis product that then results in c-myc and c-fosexpression is an attractive hypothesis. However, theplacenta produces a number of other growth factorssuch as IGFs, FGF and TGF/3, and these could alsocontribute.

PDGF is thought to mediate the proliferation ofsmooth muscle cells in injured arteries and may beinvolved in the pathogenesis of atherosclerosis.Although in injury, the main source of PDGF is theplatelet, other cell types produce PDGF-like activity.For instance, rat aortic smooth muscle cells isolatedfrom 13- to 18-day-old rat pups secrete a PDGFactivity into the medium, and could account forautocrine stimulation of growth of these cells inculture. This activity is developmentally controlledsince the same cell type isolated from adult rats doesnot secrete PDGF (Seifert, Schwartz & Bowen-Pope,1984). c-sis transcripts have been detected at moder-ate levels in cultured human and bovine endothelialcells, at low levels in in vivo endothelium from humanumbilical vein and at very low levels in bovine aorticendothelium in vivo (Barrett et al. 1984). Theseresults also suggest that the sis gene is activated incertain cell types when removed from in vivo to invitro conditions.

Model systems using murine EC cells have sugges-ted that early embryonic stem cells may produce aPDGF-like activity (Gudas, Singh & Stiles, 1983;Rizzino & Bowen-Pope, 1985) but fail to bind thisgrowth factor. It has not been definitively determinedif these cells lack PDGF receptors, or if they arewholly down-regulated by the secreted PDGF. How-ever, when F9, PC13 and PSA1 cells differentiate,they form cell types that are able to bind and respondto PDGF. If the corresponding embryonic cells,namely early embryonic ectoderm cells, producePDGF-like factors, these could then stimulate thegrowth of the adjacent, more differentiated celltypes, such as mesoderm cells which arise on the 7thday of gestation in mouse. PDGF also has a potentchemoattractant activity that stimulates the motilityof certain types of cells and this could play a role inthe migration of embryonic cells. Embryonic ecto-derm cells could also produce other growth factorsshown to be secreted by EC cells (Rizzino, 1982;Heath, Mahadevan & Foulkes, 1986).

460 E. D. Adamson

(12) Other proto-oncogenes

The latest of the viral oncogenes to be identified witha cellular protein is v-erb-A, which has 89% hom-ology to the thyroid hormone (T3) receptor (Wein-berger et al. 1986). Thyroid hormone is known to beessential for tadpole metamorphosis and the switch toadult-type gene activities (Knowland, 1984). Thisreceptor occurs widely in mammalian tissues butplays important roles in the liver and brain (reviewedby Oppenheimer, 1979). The receptors have higheraffinity for T3 than T4, exist in two molecular forms(Casanova et al. 1984) and appear to be largelynuclear (Erkenbrack & Rosenberg, 1986). Since thy-roid hormone is synthesized throughout gestation,presumably the receptor is also an early product indevelopment.

The c-erb-A gene is related (22 % at the amino acidlevel) to the glucocorticoid receptor (Weinberger etal. 1985; Hollenberg et al. 1985) which is also partlynuclear and partly cytoplasmic. The glucocorticoidreceptor is expressed in many cell types and is activeduring embryogenesis in fetal liver, visceral yolk sac(G. Andrews, personal communication) and the sec-ondary palate (Diewart & Pratt, 1981; Kim, Lauder,Joh & Pratt, 1984).

Two proto-oncogenes originally identified asmouse genomic sequences adjacent to integratedproviruses of the mouse mammary tumour virus(MMTV) in a large proportion of tumours in mice,int-1 and int-2 are unrelated genes, int-2 is transcribedin embryos from day 8-5 to 12-5 and also is seen inadult testis but not other adult tissues (Jakobovits,Shackleford, Varmus & Martin, 1986). This limiteddistribution should be helpful in determining thefunction of the int gene.

(13) Proto-oncogene expression interatocarcinoma cells

Insufficient data have been accumulated to evaluateteratocarcinoma cells as model systems for studyingproto-oncogene mechanisms in growth and differen-tiation, but the potential is great since the stem cellsmay be engineered to express experimentally intro-duced genes. The effect on the stem cell and on thepattern of differentiation produced should be highlyinstructive. The studies of Muller & Wagner (1984)and Ruther et al. (1985) have clearly suggested a rolefor c-fos in differentiation, for example. Once an ECcell line has been established that can express indi-vidual oncogenes, the cells may be aggregated withnormal embryonic cells at the morula stage, ormicroinjected at the blastocyst stage, to follow itseffect on the development of the resulting chimaericanimals. The proportion of engineered cells in each

tissue of the chimaera will vary in individual animalsand may allow a titration of dose versus effect.

To date, Table 2 summarizes what we know of thelevels of oncogene expression in teratocarcinomacells as transcripts that hybridize with DNA probes.As expected, the oncogenes most closely associatedwith proliferation are strongly expressed: these in-clude c-src, c-abl, c-rasHa, c-ras1^, c-myc, and N-myc.When rates of proliferation decline as differentiationoccurs, several oncogenes decline in mRNA level:c-abl and c-ras decline modestly; c-myc and N-mycdecline drastically. Neuronal differentiation is ac-companied by significant increases in c-src (Sejersenet al. 1985; Lynch et al. 1986), but N-myc activitydecreases to low levels (Sejersen et al. 1986). Thenuclear oncogene, c-fos, is expressed transiently atsome point during differentiation that may indicate arole in some general step of that process (Edwards &Adamson, 1986).

Some studies have centred on introducing viral oractivated oncogenes into cell lines to observe effectson cell phenotype and differentiation. For instance,an activated ras gene, c-ras^3, introduced into P19 ECcells appears to have little or no effect and does notprevent the induction of differentiation in response toretinoic acid (Bell, Jardine & McBurney, 1986).However, viruses expressing v-fos (especially FBJ-MSV) are able to immortalize or transform mouseembryo-derived primary cell cultures of fibroblasts,myoblasts, and other cell types (Jenuwein, Muller,Curran & Muller, 1985), thus linking \-fos expressionwith increased growth potential in embryonic cells.Since c-fos can also transform established cell linesunder the right conditions (Miller et al. 1984), thedistinction, if any, between the properties of v-fos andc-fos proteins is not clear. The specific cell typeexpressing the c-fos protein may modulate the out-come. The specific processed forms of oncogeneproducts such as c-fos, c-src and c-erb-A may differamong tissues and may give rise to differences infunction.

(14) Conclusions

For most oncogenes, a specific function or activity hasnot been assigned, and data are still too sparse tohypothesize a mechanism in oncogenesis or to predicta role in development. In the cases of c-erb-B/EGFreceptor, c-fms/CSF-1 receptor, c-sis/PDGF, anobvious niche in growth regulation can be deduced.Although these components are not direct generegulatory molecules, they do have wide-rangingeffects through their interconnections with a family ofprotein kinases and through their effects on phos-phoinositide metabolism, ion fluxes and nutrient

int-1

int-2


Table 2. Expression o/c-oncs in teratocarcinoma cells

c-onc

c-erb-B/EGF-R

c-fms

c-src

c-mosc-fesc-mybc-erb-A

c-abl

c-ras

c-myc

N-myc

c-fos

c-sis/PDGF-like factor

CeU line

F9, PC13OC15

P19

HT-H

PCC7

F9P1951801A1

PC13, F9, 3TDMPYS-2, PSA5E, C110


OC15, F9, HR9


PCC4


F9

human ECCmouse ECC

PCC7, PCC3,PCC4, F9

F9, OC15

F9

F9

P19

PCC4

PCC3 etc.F9, PC13

Exp" in ECC

-Intracellular

autophos. acty

-

-

+

-+

-

low

++ +

+ + + geneamplified

+

+ +

+ +

+ +

low

low

low

low

+

lowlow

Exp" indifferentiated cells

++ +

+

trophoblast-likecells +

3—5x inc. duringneuronal diff '

-+ + +

change in mobility

-

low

++

lower

-

Post-transcriptionaldown-regulation within

24 h of inductionof differentiation

85 % reduction duringnerve differentiation

low

5x inc. by day 3 withRA. Aggregates

No inc. with RA &cAMP. Monolayers

20 x inc. by day 12 withDMSO. Aggregates

lower

low-

Reference

Reese/ al. 1979;Adamson & Hogan, 1984;Welter er al. 1987.

Mummery er al. 1985.

Izhar er al. 1986.

Sejersen er al. 1985.


Lynch er al. 1986.

Sejersen ex al. 1985.


Muller, 1983.


Vilette er al. 1985.


Dony era/. 1985;Sejersen er al. 1986.

Jakobovitz er al. 1985.


Muller, 1983.

Edwards & Adamson(unpublished data)

Mason er al. 1985.

Edwards & Adamson,1986.

Vilette era/. 1985.

Sejersen er al. 1985.Gudas er al. 1983;Rizzino & Bowen-Pope,1985.

F9, PSA

F9, PSA

Jakobovits ef al. 1986.

low

Footnotes: RA = retinoic acid; cAMP = dibutyryl cyclic AMP; DMSO = dimethyl sulphoxide.

transport. Nevertheless, the role of these growth-related proteins is poorly understood. Largely this isdue to their complicated interconnections with manycellular metabolic processes. Indeed, with a fewexceptions such as PDGF, the oncogenes seem to belargely concerned with general metabolic processes as

much as (if not more than) growth regulation per se.The proto-oncogenes that are known to have enzy-matic activity, such as c-src, c-abl, the ras family, alsofit into this metabolic framework. c-erb-A/thyroidhormone receptor is even more directly concernedwith overall metabolic rates.

462 E. D. Adamson

The nuclear-located proto-oncogenes are betterplaced for roles in growth/differentiation/gene regu-lation. They bind to DNA to various degrees,although specific binding sites have not been ident-ified. The c-fos protein appears to bind to chromatin,particularly at deoxyribonuclease I-sensitive sites,thus indicating a role in the regulation of geneexpression (Sambucetti & Curran, 1986; Renz,Verrier, Kurz & Muller, 1987). c-myc production issimilarly activated but usually at a slightly later time.This has given rise to the possibility of chains ofcommand leading to gene-stimulating events gener-ated by signals from the environment, c-fos and c-mycmay then activate sets of genes that lead to DNAsynthesis and progression through the cell cycle, or tosets of genes that regulate differential cell-lineagepathways.

TGF0

IPDGF-—PDGF-R—-c-fos-"C-myc—Tas •*.

TPA- pK-C—*-c-fos-~-c-myc-~-c-fms

EGF -EGF-R—-c-fos-^c-myc—»• J

proliferation

differentiation

TGFa- ras

In reviewing the data here it becomes clear thatalmost without exception, the proto-oncogenes havebeen shown to play roles in differentiation as wellas proliferation. Is this because directing the celltowards proliferation limits the cell's resources tocontinue the previous commitment or directiontowards a differentiated phenotype (e.g. c-mycl). Oris it a more direct process that facilitates a differentia-tive pathway (e.g. c-fos?). Only in one case is thefunction clear, that is, c-fms and this product appearslate and may only be a marker and not a regulator ofdifferentiation.

For considerations of the roles of proto-oncogenesin development, some specificity of action may residein c-src since a neurone-specific isotype has beenidentified in brain during proliferation, commitmentand expression of the neuronal phenotype. Otherproto-oncogenes are less well defined to specifictissues. The placenta and other extraembryonic tis-sues (VYS, amnion, and chorion), however, do holda special place in the high levels of expression ofseveral c-oncs. In addition to c-myc, c-abl, c-ras, andc-src, very high levels of c-sis, c-fos, and c-fms occur,and these levels vary between the tissues mentionedand between the temporal stages of the gestational

period. This makes it less likely that the undermeth-ylated state of the DNA in these tissues (Rossant,Sanford, Chapman & Andrews, 1986) broadly dic-tates the levels of proto-oncogene expression. It isstill possible that specific sites of methylated basescontrol the expression of these or other genes in atemporal and tissue-specific manner. The undermeth-ylated state of the placenta could be necessary for thederepression of a wide range of growth-related genesthat are known to be active in the placenta. In thepresence of secreted 'autocrine' growth factors suchas TGFa", TGF0, PDGF, IGF-I, and IGF-II, recep-tors are continually down-regulated, synthesized andactivating the chain of command. Although thesetissues have been recognized as 'pseudomalignant,'they are not tumorigenic in nude mice and havesomehow controlled the signal to proliferate in paral-lel to the needs of the growing fetus. Studies onmodes of oncogene activation in carcinogenesis showthat a quantitative change of expression is one mech-anism of such activation (reviewed by Klein & Klein,1984). Understanding the mechanisms of control inthe placenta may therefore be instructive for futureclinical applications (Conway, 1983).

Limitations of space did not allow recognition of allrelevant references; for these omissions my apologies areoffered. I am grateful to Drs C. Van Beveren, C. Der, andS. Edwards for comments on the manuscript. This work wassupported by grants CA 28424, P30 CA 30199 and HD21957 from the National Institutes of Health.

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