8/12/2019 ft389

1/9

I nt. J . [ Je . .lI io l. 3 5: 3 89 -3 97 ( 19 91 ) 3~ 9

The F EC cell line as a model for the analysis ofdifferentiation

ANGEL ALONSO', BEATE BREUER, BARBARA STEUER and JURGEN FISCHER eutsches Krebsforschungszentrum Heidelberg Germany

CONTENTS

Characteristics of the F9 cell line 390D ifferentia tion of the F9 cells 390The in d uc tio n o f d iffe re n tia tio n 391Gene expression and differentiation 392Retrovlra l prom oters are negatively regulated in F9 cells 393Somatic cell genetics of the F9 cell line 395Summ ary 395Key W ords 395References 395

------A ddreSl for roprlnt5: Deutsches K rebsforschungszentrum . 1m N euenheim er Feld-280, 0.6900 H eidelberg 1, G erm any. F AX: 6221-401.271.0214-6282/91/503.00( I L OB C f .rPnnlrdmSplin

8/12/2019 ft389

2/9

390 A. A/olISo el al.

Characteristics of the F9 cell lineDifferentiation is a p ro ce ss characterized b y a reo rg aniza tion o f

gene expression which results in the appearance of a new pheno-t yp e. Be tween und if fe re nt ia te d c ells a nd th eir d if fe re nt ia te d c aun.terparts. it is possible to postulate a n in te rm ed iate stag e. thecommitted c ells . In this stag e. th e ce lls have already determinedt he d if fe re nt ia te d pheno type t hey s hou ld adopt, b ut t his p he no ty peis s till n ot re co gn iz ab le . T he ch arac te riza tio n o f th e c omm itm en tstage can only be made. therefore. a posteriori.Mouse teratocarcinomas are tum ors w hich can be experim en-ta lly in du ce d b y e cto pic ally imp la ntin g e mb ryos in the p eriton ea lcavity or the testis. Em bryonal carcinom a cells (EC -cells) are thes te m c ells of te ra to ca rc in om a s. c ells w h ic h s till re ta in th eir c ap ac ityto d iffe re ntia te in to d erivatives o f o ne o r m ore em bryo nic la ye rs. F orthis reason. EC-cells are considered to be similar to embryoniccells. and are able to develop in v it ro so me of th e e arly em bryo nicd ev elo pm en ta l s ta ge s (M artin . 1 98 0). S om e E C-< ::e llsc an d iffe re n-tiate into several tissues and are able to take part in the normalembryo development when injected into blastocysts, whereasothers are unable to do so. The F9 cell line is characterized by thein ab ility to d iffe re ntia te s po nta ne ou sly . a nd is th ere fo re a so -c alle dnullipotent cell line. N evertheless. upon treatm ent w ith severalagents, F9 cells differentiate into endoderm al-like derivatives,de pe nd in g on the cu lture con ditio ns a nd th e typ e of agent added tothe culture m edium (Strickland and M ahdavi. 1978: Strickland etal.. 1980: Hogan et al.. 1983: Steuer ef al., 1990).

T he F 9 c ell lin e w as is ola te d b y B ern stin e e t a l. (1973) as a s ub lineo f t he t er at oc ar cinom a OTT6050. e sta blish ed by im plan ting a six~da y-o ld e mbryo in the testis 01 a 129/J mouse (S te ve ns . 1 97 0).W hen cultured in gelatinized dishes, the cells grow adherentfo rm in g fo ci c om po se d o f tig htly p ac ke d p oly go na l c ells o f s ma ll s izea nd p ro min en t n ucle i (F ig .l).

Th e F 9 c ell lin e h as b ee n w ide ly u se d in m an y la boratorie s du ringthe last decades as a model for the analysis of the molecularmechanisms of d if fe ren tia tion ( Leh tonen et al.. 1989). This hasbeen facilitated by the fact that their karyotype has remain fairlystable during this time. The ce11s c ultured in our laboratory arepseudodiploid, w ith 39 chromosomes. 38 acrocentric and 1m eta cen tric. pro du ce d b y a R obe rtso nia n tra nsloca tio n (R b(2 .8 )).This chromosome is characteristic for the F9 cell line and can beu se d a s a m ark er in e xp erim en ts in vo lv in g ce ll fu sio n. F urth erm ore .th e cells p osse ss o nly o ne se x-ch rom osom e (karyo typ e 39.XQ), amonosamy of chromosome 8 and an insertion of unknown origin atchrom osom e 4 (Fig.2), being therefore slightly different from thecells karyotyped by Hogan e t a /. (1986).The F9 ce lls gro w very ra pid ly w ith a d ou blin g tim e of about 8-10ho urs in th e e xp one ntia lly grow in g p hase . T he ce ll cycle is ch ara c~te rize d b y a very sh ort G l phase a nd an S -ph ase of about 8 hours(Rosenstrauss e t a l.. 1982). Induction of differentiation producesa lengthening of the Gl phase and a decrease in the proliferation

fra ctio n v is ib le fro m 30 h ou rs a fte r a dd itio n o f th e d iffe re ntia tin gagent. This lengthening finally results in growth cessation andd ea th , c ha ra cte ris tic fo r d iffe re ntia te d c ells (L in de r e t a f. . 1 981) .D iffe re ntia tio n o f the F 9 c ells

As a lr ea dy ment io ned, F9d i ff er en ti at e s pont an eous ly only rarely.Nevertheless the addition of retinoic acid (RA) and dibutyryl cAM P(d bcAMP) to ce lls g ro win g u nd er ad he ren t co nd itio ns trig gers theadoption of a new phenotype, with changes in gene expressioni nv o lv ing an incr eas ed synthesis o f tis su e-t yp e p la sm in og en a ctiv a-t or , ex tr ace llular m atrix p ro te in s (Ia min in . e nta ctin a nd co lla gentype IV ), cyto keratin s. lo w le ve ls o f alka lin e p ho sph ata se a ctivity,and the disappearance of the stage-specific em bryonic antigen(S olte r a nd K no wle s. 1 97 8), a ll o f w h ich a re sp ecific m arkers forth eextraembryonic parietal endoderm (Fig. 3: Sherman and Miller.1 97 8: S trick lan d e f al., 1980: Hogan ef a / ..1983). Morphological ly.the cells are now m ore rounded, have m oved apart from each othera nd s om e of th em o cc as io na lly d is pla y la rg e p ro ce sse s re se mb lin gn eu ra l d eriva tive s (R gA )_ T his kin d of differentiation seem s to bed eterm in ed very so on a fter R A tre atm ent, since cu ltu re o f th e ce llsfor 8 hours in the presence of the inducer is sufficient to promoteth eir irr ev ers ib le d iff ere nt ia tio n (L ev in e e t a l.. 1984: Dong e t a l..1 990 ). Tre atm en t w ith R A a lo ne trig ge rs the ce lls to diffe re ntia teinto a p rim itive en dod erm -like sta ge, w hich m ay be subsequentlym odu la te d into e ith er p arie ta l o r visce ra l e nd od erm (H og an e t a l..1981). C are fu l a na ly sis o f th e p rim itive -e nd od erm -like c ells a llo wso ne to d is tin gu ish tw o d iffe re nt c ell populations: one s till c onse rv esa polygonal m orphology and the cells have the tendency to rem ainin small groups attached to each other; the second populations ho w s c ells wi th large cy to pla sm ic p ro ce ss es , s ep ara te d fro m e acho th er a nd a dop ting so me tim es a ne uro n-like m orp ho lo gy (M oore e tal., 1986). Analysis of the expression of the m arkers reveals thatb oth ph eno typ es e xp re ss th e e nd ode rm ~type m arke rs d iffere ntly.C yto ke ra tin s a re demonstrable e arlie r in th e form er po pulatio n.w hich a lso sh ow s la rg e a mo un ts o f intra ce llu la rly loca te d la min in .In con tra st. m ost of th e syn th esize d lam in in is fo und extracellularlyin the second population (Breuer, 1989). W hether both types 01 cellare able to differentiate into visceral endoderm is not known, butaddition of dbcAM P to the culture m edium allow s the distinction ofo nly o ne diffe re ntia te d ce ll-type p opu la tion . su gg estin g th at bo thp rim itive en dod erm -like type s a re a ble to fo llo w th e sa me d ifferen -tia tio n p ath w ay fu rt he r.

C ulture o f sm all clum ps of ce lls in b acterio lo gica l d ish es g en er-a te s th e a pp ea ra nce o f a gg re ga te s g ro win g in su sp en sio n. A dd itio nof RA to the medium leads the cells located at the outer surface ofthe aggregates to differentiate into visceral endoderm, as demon-strated by the synthesis 01 alpha-Ietoprotein A F P ) (Hogan et a /..1981). This seem s to be a reversible process, since after seedingthe aggregates o n g ela tin iz ed d is he s in th e p re se nc e of dbcAMPthesynthesis of AFP ceases with the subsequent appearance of

8/12/2019 ft389

3/9

STell l ce ll s :191

m ark er s t yp ic al for parietal endoderm . T hus. it seem s that whereast he pa ri et al e nd od erm p athway is i rr ev er si ble . th e v is ce ra l p athwaycan be reversed according to the culture c on dit io ns e m plo ye dG r o v e r and A dam son. 19 86).

Treatment w it h 1 06 re tin oic a ci d and 10 -3 dbcAMP d if fe re nt ia te sm ore than 95 of the cells into parietal endoderm . The firstmorphological changes can be observed 24 hours after addition ofboth in du ce rs a nd th e fu lly d if fe re nt ia te d pheno typ e i s obs er ved 72h ou rs a fte r. The d if fe ren ti at ed cells are able to grow further fo rseveral days; this growth slows down in the next days until nop ro life ra tio n is o bs erv ed in t he fu lly differentiated ce ll s. A t th is point,th e d iff er en tia te d c el ls b eg in to b e ove rg ro wn b y a sm all p ro po rtio nof undifferentiated cells. w hich w ere refractory to the action of R Aan d w hich w ill colon ize th e cultu re d ish com ple tely: re tre atm en t o fth ese ce lls w ith R A t rig ge rs th e d iffe re ntia tio n p ro gra m re su ltin g ina large proportion of differentiated cells and again a sm all num bero f re fr ac to ry c ells .The I nduc ti on o f d if fe rentia tio n

B esid es R A/d bcA MP othe r a ge nts h ave b een u sed to d iffe re nti-a te F 9 c e lls in vitro. N -N -d imetyl acetam ide and 5-bromodeoxyuri di neha ve b ee n u se d as d iffe re ntia ting a ge nts (M oo re e t a /.. 1986) an dit h as a ls o b ee n o bs erv ed th at in th e p re se nce o f th e flu ore sc en td yeHoechst 33342 the F9 cells stop growth and express markerscha ra cte ristic of p arie tal en do de rm (S teu er e t at, 1990).

R A-tre ate d F 9 ce lls s om etim es a do pt a n eu ra l-lik e m orp ho lo gy.w ith large processes w hich seem to contact the neighboring cells.A lthough it has been reported that these cell types expresscharacteristics of nerve cells. this has not been confirm ed in taterstu dies u sing th e sa me diffe re ntiatio n p ro to col (L ie si et a /., 1 98 3;Tienari et al.. 1987). F9 cells plated at clonal density in thepresence of STO-conditioned medium and RA differentiate intod erivatives o f a ll th re e g erm laye rs. sug ge stin g th at the y m ay in fa ctbe pluripotent (Koopman and Cotton. 1986. 1987). A sim ilarco nclu sio n ca n b e d ra wn fro m th e e xp erim en ts o f K elle rm an n et a l.(1987). who demonstrated that F 9 c ells tra ns le ete d with arecom binant containing the SV40 large T antigen driven by the E1A

F ig. 1. M orp ho lo gy o f e xp on en tially grow ingF 9 c ells ( >.200),

p ro mo te r a re able to differen tia te in to d eriva tive s o f a ll th re e ge rmlayers w hen injected into anim als. the differentiated cells beingim mortalized. Thus it seem s probable that the F9 cells can differ-e ntia te in to d eriva tive s o f a ll th re e g erm la ye rs , th eir d iffe re ntia tio np ot en tia l th er efo re n ot b ein g re str ic te d t o e nd od erm a l- de riv at iv es .This differentiation pattern seem s to be dependent on the cultureco nditio ns. since on ly o ne p he no type is ob serve d w he n th e ce lls a rep la te d a t n orm al o r h ig h de nsities.

D iffe re ntia tio n h as a ls o b ee n a tta in ed af ter t ransfect ion o f c -f ossequences into undifferentiated F9 cells (Muller and W agner.19 84 ). c-fo s-tran sfe cte d ce lls exp re ss cha ra cte ristics typ ica l fo rp arie ta l e nd od erm a nd h ave lo st th eir p ro life ra tiv e ca pa city . su bse -q ue ntly d yin g sim ila rly to R A-tre ate d ce lls. N everth ele ss, th e factthat only some of the parietal endoderm markers are expressed insome of the cells argues in favour of a different differentiationp ath wa y from th at fo llo we d b y R A . o r f or a m echa nism in trinsic to th eF9 cell line. not directly correlated w ith the process of induction ofd iffe re ntia tio n. T his co nclu sio n is su pp orte d b y re su lts s ho win g th attra nsfe ctio n o f F 9 c e lls w ith a ntis en se c -fo s se qu en ce s o nly p artia llyin hib its d iffe re ntia tio n to e nd od erm (E dw ard s e t a l.. 1 988) ,Furthermore, F9 ce lls tra nsfe cted w ith c-m yc se qu en ces clo ne din th e an ti-se nse p ositio n a lso exp re ss p arie ta l-en do de rm -likecharacteristics. It seem s that transcription of these sequencesallow s neutralization of the endogenous c-m yc RN A sequencesr es ul ting in deve lopm en t of a differentiation p ro gram , in accordancew ith th e fin din g th at c -m yc is d ow n-re gu la te d s ho rtly a fte r tre atin g F9cells with RA/eAMP (Griep and De Luea. 1986. 1988) and that thisd ow n-re gu la tio n m ay p la y an im po rta nt role in the process ofdifferentiation. These results rem ain controversial. as other au-th ors h ave b ee n u na ble to re pro du ce th es e re su lts (N is hiku ra e t a l. ,1990). It s ee ms lik ely th ere fo re th at e xp re ssio n o f s om e o nc og en esknown to have a role in the control of proliferation is sufficient totrigg er th e cells in to a d iffe re ntia te d sta ge . E xpe rim ents in volvingtra ns fe ct io n o f H -r as s eq ue nc es further c or robor at e t his poss ib ili ty( Ya m ag uc hi- Iw a i e t af..1990). C on tro ve rsia l re su lts h av e b ee n a ls oo bta in ed afte r tra nsfe ctio n o f th e E 1a g en e o f a den oviru s. W he re assome authors described a clear effect on differentiation. othersclaim the contrary. the E1a gene products being sufficient to stop

8/12/2019 ft389

4/9

39 ~ A. A/oI lS et a \.

11. . n\ If Iit_ o2 3 4 5

It . . .p f6 8 9

11 12I:. .13 14

I,I I16 17 18 19

marke r ch romosomeFig. 2. G-banded karyotype of F9 cells. Exponenrially growing F9 cellswere treated with Colcemid. spread on glass slices and stained wirhGiemsa stain after Trypsin treatment. Typical for F9 cells is t he p re se nc eof a meta ce ntfl c m ar ke r c hr omos om e an d an Insertion on chromosom e 4(arrow). A sma ll p e rcentage of the ce ll s conta in a m in u te c hr om o so m e ( se ebottom left) of unkn ow n origin.

or even reverse F9 cell differentiation (M ontano and Lane. 1987;Young et af.. 1989). A critica l p oint co mm on to all these experi-m en ts re ma in s the fact that the differentiation stage is deducedfrom th e an alysis o f o nly a fe w m arke rs, m aking it d ifficu lt to asse ssthe tru e differentiated stage of the transfected cells in an unam -b ig uo us m ann er.Gen e exp re ss io n a nd d iffe re ntia tio n

Differentiation of F9 cells is accompanied by a reorganization ofgene expression with the synthesis of new products and the shut-off of others. A lthough a great num ber of gene products w hichchange their expression after differentiation to PE or VE have beenidentified. most if not all of these products are the results of thedifferentiation rather than their cause (W ang e t a l.. 1 98 5)_ S o far.no genes have been isolated w hich can be m ade responsible for thelaunch of the differentiation program . Table 1 show s those genep roduc ts i dent if ie d so far whose expression is modulated duringd if fe re nt ia ti on t o endodermal derivatives.

One strategy fo r identifying genes that m ay b e d ire ctly in vo lv edin the control of differentiation is to search for sequences with ad ifferen tial exp re ssio n a t th e first stag es o f R A-tre atm en t. U nfo rtu -na te ly. ve ry fe w g ene prod ucts ha ve be en id en tified d urin g th e first

10

8 h ou rs of RA-treatment, a time that has been considered as thecom mitm ent period. Transcription of the m yc oncogene has been5 ho wn to d ro p s ho rtly a fte r a dd itio n o fR A (G rie p a nd D e L uc a.1 98 6)but no direct evidence exists that may link the action of the myc-protein to the launch of differentiation. despite the fact thatneu tr al iz in g t he c -myc R NA resu lts in ce ll d if fe re nt ia ti on (Gri ep andDe Luca. 1988). S im ila rly, a s ma ll in cre ase in th e s yn th esis o f c -fo swith a maximum at 60 minutes atter RA-treatment has beenreported (Manson er a/.. 1985). It may very well be that bothoncogenes turn on a cascade of events. one of which is connectedIn some way to channels leading to differentiation but withoutha ving a d irec t, spe cific effec t o n the in itial lau nch of d ifferen tia tion .Also, very e arly a tte r R A-tre atm en t a homeobox .con ta in ing s e-q uen ce, th e E r a 1 gene. show s a strong transcriptional stim ulation(L a R o sa a nd Gud as. 1 98 8). This sequence seem s to be related toth e h om eo bo x-con ta in in g seq uen ces a lrea dy d escrib ed as m od u-lating their expression at late times during F9 d if fe ren ti at ion(Colberg-Poley er al.. 1985a.b). Interestingly. all the5e 5equencesa re k nown b y t h eir D N A-b in din g p ro pe rtie s, a nd th eir ro le . th ere fo re ,m ay rely on the m odulation of transcription of responsible genes,but the question rem ains open w hether these transacting factorsact on the primary target point(s) responsible for the start ofdifferentiation.

The action of R A on differentiation is probably m ediated by theRA-receptors (RARs) present in the nucleus of most cells. Thisaction is carried out through the binding of the receptors to DNAsequences located in the prom oter regions of genes responsive toRA. Thus it seems probable that treatment with RA produces thea ctiv atio n o f a s erie s o f g en es co nta in in g R A-re sp on sive e le me nts.Such elem ents have been located in the prom oter regions of geneswhose transcription is m odulated by R A. such as lam inin, B-RAR orh isto ne H i 0 (A lo ns o e t al.. 1988; V asio s e t al.. 1991: Breuer et al..1989: De The er al.. 1990). The interpretation of these re5ult5 i5n eve rth ele ss co mplica te d b y th e fa ct th at un differen tiated F 9 ce llsc on ta in a lp ha - a nd g am ma -re ce pto rs a nd th at th eir n um be r re ma in sconstant during RA treatment. Only the B - R A R in cre ase s th eirste ad y-sta te in R A-trea te d ce lls. re ach ing a 2 Q..fo ld ind uctio n 4 8h ou rs a fter R A-a dd ition . It m ay b e sp ecu la te d th at trea tm en t w ith R An ot o nly co ntro ls tra nsc rip tio n o f th e R AR s b ut p erh ap s s eco nd arilym odifies pre-existing receptors. allow ing them to becom e activeim me dia te ly a fte r a dd itio n o f th e in du ce r. A no th er p oss ib ility is thatR A a fte r b ein g m eta bolize d in th e cell trig gers th e synthe sis of o th erpro du cts w hich fo llo w a d ifferen t p athw ay tha n th e rece ptors.

C uriously, RA-responsive elem ents have been found to date ingene products whose expression takes place rather late duringdifferentiation. w ith the exception of H istone Hr. The reason forthis behavior is completely unknown. It may be argued that theaffin ity o f t he R AR s fo r th e R A-resp on sive e le men ts a na lyzed is ve rylow. and that some time is needed until the receptors increase toa large num ber and are able to bind to the elem ent. N evertheless.analyses of the effect of RA on the number of receptors do notsu ppo rt th is p oin t of vie w. An oth er p ossib ility is tha t th e tra nscrip -tion of the RA-elem ent-containing genes is not only dependent onthe presence of receptors but also on other transcription factorsth at a re s yn th esize d la te r d urin g d iffe re ntia tio n. A lth ou gh n o e xp eri-m e nt al e vid en ce e xis ts to p ro ve th is p oin t. th e tre m en do us c om ple xityo f th e prom ote r re gio ns of so me R A-re gu la te d g en es (like H ie g en e)a rg ue s in fa vo r o f th is in te rp re ta tio n.

M ost o f th e se que nce s diffe re ntia lly tra nscrib ed in R A-trea te dcells and identified so far account for the specific markers of

c5

xo

8/12/2019 ft389

5/9

S I I I I c'l/s 39 3

endoderm . A large num ber of the components of R eichert's m em -b ra ne a re p re se nt i n d if fe re nt ia te d c ells . b ut t he ir e xp re ss io n is n o ta l w a y s homogeneously distributed. independent o f the d if fe re nt ia -tion protocol used. The role of cAMP is not fully understood.b ec au se to ge th er w ith re po rts s ho win g that th e r o le o f cA MP is onlyto enhance the effect of R A. others have b ee n p ub lis he d in dic at in gthat c A M P per se is a ble to differentiate F 9 cells. Nevertheless, its ee m s p ro ba ble that the only effect of cAMP is to m od ulate th erespon se of the cells. Furth erm ore. th e sam e bio logical result canb e o bta in ed w he n th e in tr ac ellu la r concentration o f cA MP is raisedw itho ut addition of exogenou s cA MP . The fact that c A M P is in factable t o modul at e d if fe rent ia ti on e it he r t o par ie ta l o rv is ce ra l endodermmakes it a key point, at least in the pathway to be followed duringdifferentiation in suspension cultures. The cellular response con-cerning the number of markers synthesized is nevertheless relatedto the differentiation protocol used. W hereas treatm ent w ith RA

F ig . 3 . M orphology of i n v itr o differentiated F9cells. F9 cells were seeded at an in iti al d en si ty o f6 00 .0 00 c el/s llO cm plate on gelatinized tissueculture dishes_ Cells were treated either with RAand dbcAMP fA) or with RA alone at final concen-trations of 10- and 10-3 respectively. T he cells w erepassaged every second day during the differentia-tion tim e. D ifA a nd D ifB ga ve th e tw o d ifferen tia tedphenotypes appearing during treatment w ith RAas described in th e te xt (x 200).

alon e triggers the pro duction of a w ell-developed cytoskeleton in F9cells, addition of dbcAMP to the culture medium reduces theproportion of cells expressing cytokeratins 8 and 18. which areresponsible for the production of the intermediate filam ents.S im ila rly , th e c ells d ow n-re gu la te th e in cre as ed s yn th es is o f B -R A Robserved after RA-treatm ent when dbcAM P was included in thedifferentiation protoco1. Thus it is evident that cAMP is able tom od ula te in div id ua lly th e g en e e xp re ss io n o f d iffe re nt s eq ue nc es .R etroviral prom oters are negatively regulated in F9 cells

An interesting property of the F9 cells is their inability to use viralp ro mo te rs e ffic ie ntly . D iffe re ntia tio n o f th e c ells re le as es th is b lo ckand restores transcription of the viral sequences (Kelly andC ondam ine, 1982). This characteristic IS e ven m ore p ron ounced inretroviruses. Although retroviruses efficiently integrate into the

8/12/2019 ft389

6/9

394 A. A/ollso el al.

F ig . 4 .lm mu no lo calizatio n o f cy to keratin s an d lam in in d urin g d ifferen tiatio n. C e l l s wer e tr ea te d f or 3 da vs w it h R A a nd db cA MP(A ,B J o r fo r 5 daysw ith R A a lo ne (C,D) u si ng th e c ul tu re c on dit io ns 85 d es cr ib ed i n F ig . 1. T he c elts w ere fix ed a nd sta in ed w ith a ntib od ie s sp ecific fo r cy to ke ra tin s (A a ndC) or la min in (0 ) u sin g in dire ct im mu no flu ore sce nc e. (B ) i s th e p ha se co ntra st p ic tu re to (A J. ( x 1 50 0).

genome of EC-cells, the proviruses ar e tra nsc riptiona lly inac tiv e.This effect seems to be due to an inefficient function of theenh ancer. since m utations in this region relieve the block an d allowt rans cr ip ti on in undi ff er en ti at ed F 9 cells (LaThan ge a n d Rigby.1987. 1988: s ee a ls o S ilv er e t a l. . 1983). This block relies at leastp artia lly o n th e e xis te nc e o f tra ns ac tin g fa cto rs - absent in F9 butexisten t in th eir differentiated co unte rpa rts-w hich are able to bindto the enhancer region of the LTR (Speck and Baltim ore. 1987).

S im ilarly. other promoters are also very inefficient inundifferentiated F9 cells (Sleigh, 1985: see also Silver et al..1983). For example. the early promoter of SV40 as well as ofpolyom a is only w eakly used in F9 cells, whereas differentiation ofthe cells produces an effect sim ilar to that observed for retroviralpromoters.

The methylation degree of some promoter sequences alsose em s to playa ro le in con trolling gene e xpression. M eth ylation hasbeen associated with a repressed state of the gene. sequencesw hich are actively tran scribed being u nderm ethylated in rela tion totheir inactive cou nterparts. Tra nsfected retroviral seque nces arerapidly m ethylated in early m ouse em blYos and in ES cells. Thism ethylated stage is accom panied by an inactivation of transcrip-tion. Nevertheless. whether this m ethylation is the cause of the

rep ression still rem ains to be elucidated. E xperim ental evidenceindicates that m ethylation accom panies repression but is not itsprim ary cause (S tew art et al 1982; G au ts ch a nd W ils on , 1983).Th e aforem entioned results su pport the g eneral conclu sion thatthe F9 cells are restricted in their transcriptional properties andm im ic th e situa tion foun d in early em bryonic cells. This conclusionis supported by the finding that several transcription factors areabsent in undifferentiated cells. in a sim ilar manner to earlye mbryonic cells. but app ear after d ifferentiation . S o, a P E A 2 and aP EA 1 activity (sim ilar to the hum an A Pi activity) h ave been fo und ind iffe re ntia te d b ut n ot in s te m c ells . d es pite th e fa ct th at fo s-p ro te inhas been identified in untreated F9 cells (Kryszke et a/.. 1987;Caceres e t a /., 1990). In this context, it should be m entioned thattransfection of c-jun sequences into P19 teratocarcinom a stemcells trigg ers differentiation, but w hether th is is also the case in F 9cells rem ains to be dem onstrated (De G root e t a /., 1 99 0). A s erie so f o ct-b in din g a ctiv itie s h as a ls o b ee n d es crib ed in F 9 c ells a nd th eirs yn th es is h as b ee n s ho wn to b ed ev elo pm en ta lly re gu a te d (L en ard oe t al.. 1 98 9; S ch ole r et a/.. 1991 and references therein). Inter-estingly, w hereas so me gene ral transcription factors are p rese nt inF9 cells. like S P1 or A P2 activity, oth ers like N F1, are m issing. T hus,F9 cells also represent a well-suited system for experiments

8/12/2019 ft389

7/9

F9 F9 dittoA lkaline ph oshatase m ern stin e et al.. 197 3) +Apo li po pr ote in E ( Ba as he rr ud dl n e e a l. . 19an + -/++.C lon es w ith re du ce d e xp re ssio n (L evin e e t a t., 1 98 4) +Cytokeratins 8 and 18 (Oshima. 1981) +EGF receptors (Adamson and Hogan. 19841 + ++E nt ac t n ( Co op er et af.. 19 83) +Era-l L a R os a and Gudas. 1988) +F eto mo dulln (Im ad a ee af.. 1 990) +H -2Kd (Kn ow le s et a l., 198 0) +H ox -l.3 (M urp hy ee a l.. 1 98 8) + ++L am in in (Gr ov er e e a l., 1 98 7) + ++Lamins A and C (Lebel e e a l.. 1 98 71 +Onc og en e e xp re ss io n (S eje rs en e t a l. , 1985:Locket and Sleigh, 19871 +/-. +/-*Parathyroid horm one-like protein C han et al., 199m +P ro te og ly can -1 9 IG ro ve r et al.. 19 871 +R etin ol- bin din g p ro te in (S op ra no ee a l.. 1988 ) +R ex -l (H os le r et a l.. 198 91 +Spare (Mason et af., 986) +SSEA 1 (Solter and Knowles, 19781 +f ,-Interleron (Francis and Lehman, 1989) +TG m2 (M um mery et al., 1 990 ) +Tissue type plasminogen activator (Sherman et a l. , 1 97 m +Transferrin receptors (Adamson and H ogan, 1984) + ++T ran sth yre tin (S op ran o etal.. 1 988) +Ty pe IV co llagen (G ro ver et a l., 198 7) +

T A B L E 1ID EN TIF IE D G EN E P RO DU CT S W IT H R EG UL AT ED G EN EE X PR E S SI O N D U R IN G F 9 D I FF E R EN T IA T IO N

F 9 d iff. co rre sp on ds to c ells d iffe re ntiated eith er to p arie ta l o r to visc era lendoderm.+. presence of a specific gene productabsence of a specific gene productva ry ing amoun ts

c on ce rn in g t he mo le cu la r mec ha nisms c on tr olli ng g en e e xp re ss io na t th e tra ns crip tio nal lev el.Som atic cell genetics of the F9 cell line

A v ery interesting analysis of the properties of the F9 cells canbe obtained from the results of cell fusion experim ents. H ybridsb etw ee n F 9 a nd a mou se immortaliz ed . p arie ta l e nd od erm -lik e c ellline. the P FH R9 cells. display a d ifferentiated phenotype and arealso im mortalized (H ow e and O shim a. 19 82). T hus the differen ti-a te d p hen oty pe is d om in an t o ver th e EC-ch ara cteristics. T he sameh olds true for fusio n ex perim ents in volv ing PY S.2 cells. ano therm ouse parietal-like perm anent cell line. O n the contrary, fusionexperiments involvingRA/dbcAMP-treatedF9cel ls (4-daytreatment)a nd th eir u nd iffe re ntia te d c ou nte rp arts a lw ay s d is pla ye d anundifferentiated phenotype and w ere im mortalized. This can beinterp reted either as rescue o f the differentiated phenotype by theF9 cells (Littlefield and Felix. 1982) or as the result of the non-v ia bil ity oft he f us io n p ro du cts b etwee n RA-tr ea te d a nd unt re ate d F9cells. O ur results support the second possibility . suggesting thatfu sio n p ro du cts b etw ee n R A-tre ate d a nd u ntre ate d F 9 c elis d ies ho rtly a fte r fu sio n. s im ila rly to d iffe re ntia te d F 9 c ells (W re de .

Srl'1II ails 3951989). F urth erm ore . m ic ro nu cle i p re pa re d fro m PFHR9 c ells w h ic hha ve b ee n su perin fe cte d w ith re tro viru se s ca rryin g th e N eo mycinresistance gene w ere fused w ith undifferentiated F9 cells and theph en otyp e o f the re su ltin g N eom ycin resista nt fu sio n p rod ucts w asanalyzed. Som e of the resulting clones displayed all markersc ha ra cte ris tic fo r p arie ta l e nd od erm a nd k ary oty pin g d em on stra te da ch ro mo so me 4 triso my i n a ll o f th em . T his trisom y w as ne ve r fo un din the undifferentiated fusion products so far analyzed (Steuer.1991). Furtherm ore. quantitation experim ents follow ing a m ath-em atical m odel dem onstrated that one chrom osom e alone cannotbe suff ic ien t fo r co nfe rrin g the d iffe re ntia te d p he no typ e a nd tha t a tle ast o ne o th er. a s ye t u nid en tifie d. ch ro mo so me m ust b e in vo lve d.Thus these results suggest that differentiation of F9 cells isprobably not accomplished by a single sequence and that thisprocess can be sw itched on in hybrids in the absence of m ost of thed iff er en tia te d c ell c hro m os om e s.Summary

The teratocarcinom a stem cell line F9 has been widely used asa m odel for the analysis of m olecular m echanism s associated w ithd iffe re ntia tio n. T his c ell lin e h as b ee n co ns id ere d t o be nul li po tentan d a ble to d iffe re ntia te in to en do de rm al-lik e d eriv ativ es u po ntre atm en t w ith re tin oic a cid . Nev erth ele ss. u nd er d efin ite cu ltu reconditions. F9 cells are able to differentiate into derivatives of allthree germ layers. The F9 cells express characteristics of earlym ouse em bryonal cells and possess all repression factors know nto b e p re sen t in ce lls o f the e arly m ou se e mb ryo ge ne sis. Inductionof differentiat ion can be achieved not only by a dd in g c hemic alagents to the culture m edium but also by transfection o f s ev era loncogenic s eq uen ce s. In somatic c ell g en etic e xp erim en ts. immor-tal ized. d i fferent ia ted F9-like cells have been shown to expressdominantly g enes responsible fo r the ap pearance of the differen ti-a te d p he no ty pe .K EY W ORD S: l i' ra fo ( (f 1 r; no J / o a l . \. I 'I I/ In ) 'o /: /' I/ /' .\ il . t ln 'I 'w /H I /I 'I II .all cullun '. ;/11'ilm tlJj/t'n'lIlialio ll. ;1/(/ ,.1;01 / (if (hffi'n'lIl;al;O I/.Acknowledgments

This work has been supported by a grant from the DeutscheF or sc hu ng sg em ein sc ha ft to A ng el A lo ns o.ReferencesA D A M SO N E . O . a n d H O G AN B .L .M . 1 9 8 4 ) . E x p r e s s io n o f E G F re c e p to r a n d t r a n s f e r r in

b y F 9 a n d P 1 3 te r a to c a rC in o m a c e ll s . D i f f e r e n t i a t i o n 2 7: 1 52 -1 57 .A L O N S O . A . , B R E U E R , B . . B O U T E R F A . H . a n d D O E N E C K E . D . 1 9 8 8 ) . E a r l y in c r e a s e in

h is to n e H lo m RN A d u r in g d i f fe r e n t i a t io n o f F 9 c e l ls t o p a r ie ta l e n d o d e rm . E M B D J .7 : 3 0 03 -3 0 08 .

B A A S H E E R U D O IN . K . . S T E IN . p . . S T R IC K L A N D . S . a n d W IL L IA M S . D . L . 1 9 8 7 ) . E .-p r e s s io n o f th e m ur in e a li p o p r o te m F g en e is c o u p le d t o t h e d i f fe r e n t i a te d s t a t e o fF 9 e m br - ,o n a l c a r c in o m a c e ll s . P r o c . N a t . A c a d . S c i . , U S A 8 4: 7 09 -7 13 .

B ER NS T IN E. E .G ., H O O PE R , M . L G R A N D C H A M P . S . a n d E P H R U S S I , B . 1 9 7 3 ) . A lk a -l in e p h o s p h a t a s e a c t iv i ty in m ou s e te r a to m as . P r o c . N a t l . A c a d . S c i . U SA 7 0 : 3 8 9 9 -3 9 0 3 .

B RE U ER , B . 1 9 8 9 ) . A na ly s e d e r G en re g u la t l o n I n d e r F 9 T e ra to k a r z in o m az e l l l n i c imu n d l f f e r e n z t i e r te n s o w ie d l f f e r e n z t i e r te n Z u s t a n d . R e t r o w a le V e k to r e n u n d d a se n d o g e n e H ls to n H 1 G e n . P h . D . T h e s I s . Unive r si ta t He ide lbe rg .

BREUER.B.. F lSCHER. J . a n d A L O N S O . A . 1 9 8 9 1 . C lo n in g a n d c h a r a c te r i z a t io n o f th em o u s e h Is to n e H r p r o m ot e r r e g io n . G e n e 8 1: 3 07 .3 14 .

C H AN . S .D .H .. STREWlER, G .1 . . K IN G , K .L a nd N IS S E N S O N . R.A. (1990) . upre ssiono f a p ar ath yr oi d h o rm on e - l i k e p r o te m a n d I ts r e c e p to r d u r in g d i f f e r e n t i a t io n o fe m b r y o n a l c ar ci no ma c el ls . Mol. EndOCfl no l . 4 : 6 38- 646 .

-~

8/12/2019 ft389

8/9

396 A. A/III/so ct al.COLBERG-POLEY,A ,M . .VOSS . S ., CHOWDHURY,K . a nd GRUSS. P . ( 1985a ). Structural

a na ly sis o f m urin e g en es c on ta in in g homeobox sequ en c es a nd th eir e xp re ss io n ine m br yo n al c ar ci no m a cells. Nature 3 14 : 7 31 -7 38 .

C O L B ER G -P OL E Y, A .M ., VOSS . S .D . a nd GRUSS . P . ( 1 98 5b ). S tr uc tu re and exp ressionof two classes of m am malian h om eo bo x- co nt ai ni ng g en es . C ol d S pr in g H ar bo rSymposia o n Qua nti ta ti ve B io lo gy . Vo l. 5 0. C ol d S pr in g Har bo r. N e wYo r k. p p. 2 85 -290.

C O O P E R , A,R.. TAYLOR,A. and HOGAN,B.L .M. (1983). C hang es in the rate of lam in inand en tac ti n synthe sis in F9 e mbryon al carcinom a cells treated wi th re ti no i c a c i da nd c yc li c AMP. De l '. B ;o l. 99 : 510- 516 .

C AC ER ES . J ., G LiK IN . G .. B RA V O. R . a nd W E IN M AN N . R. ( 19 90 ). c -f os m e di at edstimulation o f s n A P-l D N A b in di ng a ct iv it y in u nd iff er en tia te d t era to ca rc in om a c elll ines. Oncogene 5 : 5 9- 67 .

De GROOT, R .P .. K RU YT . F .A .E .. v an d er S AR G. P .T . a nd K RU IJ ER . W . (1 99 0). E cto pice~pression of c-jun leads to differentiation of P19 em bryonal carcinom a cells.EMBO J . 9 : 1 83 1- 18 37 .

D e T HE . H .. V IV AN CQ -R UIZ. M .M .. TlO LLA IS . P .. S TU NN EN BE RG . H . and D EJE AN . A .(1990). Identification of a retinoid acid responsive elem ent in the retinoic acidreceptor R gene. Nature 3 43 : 1 77 -1 80 .

D O NG . J .M .. L I. F . a n d C HI U. J .F .( 19 90 ). I nd uc tio n o f F 9 c ell d iff er en tia tio n by transiente ~p os ure to re tin oic a cid . B io che m. B iop hys. R es. C om mun. 17 0: 14 7-15 2.

E DW AR DS . S A. R UN DR EL L. A .Y .K . an d A DA MS ON . E .A . (198 8). E ~pression o f c-fosa ntis en se m RN A in hib its d iffe re ntia tio n o f F 9 c ells to p arie ta l e nd od erm . D e v. B io i.129: 91-102.

F RA NC IS . M .K . a nd L EH MA N J .M . (1 98 9). C on tro l o f8 -in te rie ro n e ~p rc ss io n in m urin ee mb ry on al c arc in om a F 9 c ells . M o l. C ell . 8 io l. 9 : 3 55 3- 35 56 .

GAUTSCH. J.W. and W ILSON. M.C. (1983). Delayed de novo m ethylation inte ra to ca rc in om a s ug ge sts a dd itio na l tis su e-s pe cific m ec ha nis ms fo r c on tro llin gg e ne e ~ pr es si on . N at ur e 3 01 : 32-37.

G RIE P, A .E . a nd D e LU CA , H .F . (19 86). D ecre ased c-m yc e ~p ressio n is an ea rly eve ntin r eti no ic a cid .i nd uc ed d iff er en tia tio n o fF 9t er at oc ar cin om a c el ls . P ro c . N a t/ . A c ad .Scl. US A 8 3 : 5 5 39 -5 5 43 .

G R IE P, A .E . a nd D e L UC A, H .F . ( 19 88 ). A nt is en se M yc s eq ue nc es i nd uc e d iff er en tia ti ono f F 9 c ells . P roc. N atl. A ca d. S ci. U SA 8 5 : 6 8 06 -6 81 0 .

GROVER. A. and ADAMSON. E.D. (1986). Evidence for the e~istence of an earlycom mon biochem ical pathway in the differentiation of F9 cells into visceral orpa rietal en dod erm : m od ula tion b y cA MP . Dev. 8iol. 114: 492. 503 .

G RO VE R. A .. B OU RD ON . M .E . a nd A DA MS ON . E .D . (1 98 7). P ro te og ly ca n-1 9, la min inand collagen type IV production is corre lated w ith levels of m R N A in F9 cella gg re ga te s d iffe re ntia tin g in th e p re se nc e o f c yic lic AMP. Differentiation 3 6: 1 38 -144.

HOGAN.B.L .M. , BARLOW.D .P . a nd T IL LY , R . ( 19 83 ). F9 te ra to ca rc in om as a s a m od elfor the differentiation of parieta l and visceral endoderm in the m ouse em bryo.C an cer S utY . 2 : 1 15 -1 40 .

HOGAN.B.L .M. . CONSTANTINI.F. and LACY. E. (1986). M anipulating the m ouse em -b ryo. C old S prin g H arbo r L abo ratory, N ew Y ork.

HOGAN. B .L .M . . TAYLOR , M . and ADAMSON, E . (1981) . C ell in te ra ctio n m od ula tee mb ry on al c ar ci no ma c ell d if fe re nti at io n i nt o p ar ie ta l o rv is ce ra l e nd od er m. Nature2 91 : 2 35 .2 37 .

HOSLER , B .A . . L a ROSA.G . J. , GR IPPO. J.F. an d G UD AS , L . ( 198 9). E ~pre ssion of R e~ -1. a gene containing zinc finger m otifs. is rapidly reduced by retinoic acid in F9te ra to ca rc in om a c ells . M ol. Cell. 8iol. 9 : 5623.5629 .

H OW E. W .E . a nd OSHIMA. R .G . (1 98 2). C oo rd in ate e ~p re ss io n o f p arie ta l e nd od erm alfu nc tio ns in h yb rid s o f e mb ry on al c arc in om a a nd e nd od erm al c ells . Mol. Cel l. Bioi.2 : 3 31 .3 37 .

IMADA.S. . YAMAGUCHIH . a nd IMADA .M . ( 19 9 0) . D if fe re n ti al e ~ pr es si on o ff et om o du li na nd ti ss ue p la sm in og en a cti va to r t o c ha ra ct er iz e p ar ie ta l e nd od er m d if fe re nti at io nof F 9 em bryona l carcin om a cells. D e v. B io i. 1 41 : 4 26 .4 30 .

KE LL ERM ANN ,0 ., BUC-CARON , M .H . a nd GA IL LARD.J. (1 98 ?). Im mortalization ofp re cu rs ors o f e nd od erm al. n eu ro ec to de rm al a nd m es od erm al lin ea ge s, fo llo win gth e in tro du ctio n o f th e s im ia n v iru s (S V4 0). e arly re gio n in to F 9 c ells . Differentiation3 5: 1 97 -2 05 .

KE LL Y , F. a nd CONDAM iNE , H . ( 19 82 ). Tum or viruses and early m ouse em bryos.B io ch im . B io p hy s. Acta 6 51 : 1 05 -1 41 .

K NO WL ES . B .B .. PA N, S., S OL TE R. D ., L lN NE NB AC H. A .. C RO CE . C . and H UEB NER . K .(198 0). E ~pression o f H -2. lam in in and SV40T and TASA on differen tiation oftra ns fo rm ed m urin e te ra to ca rc in om a c ells . N atu re 288: 615. 617 .

KOOPMAN,P. and COTTON.R.G.H. (1986) . T he re sp on se o f e mb ry on al c arc in om a c ellsto retino ic acid de pen ds o n co lon y size. Differentiation 3 1: 5 5- 60 .

KOOPMAN, P . and COTTON. R .G ,H . ( 1987) . P lu ri po te nt d if fe re nt ia ti on of single Fe m br yo na l c ar cin om a c el ls . E xp . C ell R es . 1 68 : 5 67 -5 71 .

KRYSZKE. M .H ., P IE TT E, J . a nd YANIV.M. (1987). in du ctio n o f a fa cto r that b in ds tthe polyoma virus A enhancer on differentiation o f e mb ry on al c arc in om a c ellsNature 328: 254- 256 .

L a R OS A. G .J . a nd G UD AS , U . (1 98 8) . Earl y re ti no i c ac id - induced F9 teratocarcinomastem cell g ene ERA-I: a ltern ate splicing creates tra nscrip ts for a ho meob o~ -c on ta in in g p ro te in a nd o ne la ck in g th e h om eo bo ~. M ol . C el l. B io i. 8 : 3906-3917 .

La ROSA, G.j. and GUDAS, U. (1988). An early effect of retinolc acid: cloning of anmRNA (Era.l ) e ~h ib itin g ra pid a nd p ro te in s yn th es is -in de pe nd en t in du ctio n d urin gt er at oc ar ci no m a s te m c el l d if fe re n ti at io n . P ro c. N atl. A ca d. S ci. U SA : 8 5 : 3 2 9- 33 3 .

La THANGUE. N .8 . a nd RIGBY,W . J. ( 1987) . An a de nov ir us E la -l ik e t ra n sc ri pt io n f ac tois regulated d ur in g t he differentiation o f m u ri ne e mb ry on al c ar ci nom a s te m c ell sC e l l 4 9: 5 07 -5 13 .

La THANG UE. N.B. and RIG 8Y. W .J. (19 88 ). T rans-actin g protein factors and theregulation ofeukaryotictranscription.ln Transcr ip t ion andSp l icmg(Eds . B .D . H a m esa nd D .M . Glove r ). IRLPre ss . Oxford. pp . 1-42.

L EB EL . S ..L AM PR ON . C ., ROYAL.A. a n d R AYMON D .Y. (1987) . La min s A an d C ap pea rd u ri ng r et in o ic a ci d- in d uc e d d if fe re nt ia ti on o fm o u se e mb ry on al c ar cin om a c el ls . JC e l l B io i. 1 05 : 1 09 9- 11 04 .

L EH TO N EN , E .. L AA SO N EN . A . a nd T lE NA RI . J. (1989). Teratocarcinoma stem cells asa m od el for differe ntiatio n in the m ouse e mbryo . Int. J. D ev . 8 10 1. 33 : 105- 116 .

LENARDO. M . J . , STAUDT.L . . ROBBINS, P. ,KUANG.A. . MULLIGAN. R .C . a nd BALTIMORE, D. ( 1989) . Re pr es si on of the IgH enhancer i n t er at oc ar ci nom a c el lsa ss oc ia te d w it h a n ov el o c ta m er f ac to r. Science 243: 544. 546 .

LEVIN E. R.A.. CAM PISI, J.. W ANG . S. and GU DAS. L. (1984). B uty ra te in hib its th ere tin olc a cid -in du ce d d iffe re ntia tio n o f F 9 t er at oc ar ci nom a s te m c ell s. Dev. B io1 05 : 4 43 -4 50 .

L EV IN E . R .A .. L a R O SA . G .J . a nd G U DA S, L. J. (1984). lso la t ion ofcDNAclones f or g e ne se ~h ib itin g re du ce d e ~p re ss io n a fte r d iffe re ntia tio n o f m urin e te ra to ca rc in om a s te mcells. M ol. C ell. B io i. 4 : 2142-2150 .

L lE SI, P .. R EC HA RD T, L . a nd W AR TIO WA AR A, J . ( 19 83 ). N erv e g ro wth fa cto r in du ce sa dr en er gic n eu ra l d if fe re nt ia ti on i n F 9 t er at oc ar cin om a c ells . Nature 306: 265- 26 7

L INDER.S.. KRONDAHL.U. . SENNERSTAM.R. and RINGERTZ.N.R. (1981). Retinoica cid .in du ce d d iffe re ntia tio n o f F 9 e m bry on al c arc in om a c ells . E x p. C e ll . R es . 1 32 :453-460.

LITTLEFIELD.J.W . and FELIX. J.S. (1982). Rescue of term inally differentiatingteratocarcinoma cells b yf us io n t o u nd iff er en tia te d p ar en ta l c el ls . Somatic Cell Genet.6 : 7 43 -7 57 .

L O C K E T T , R .J . a nd S LE IG H, M .J . (1 98 7). O nc og en e e ~p re ss io n in d iff er en tia tin g F 9m o us e e m br yo na l c ar cin om a cells. Exp . C e ll R e s. 173: 370- 378 .

M AN SO N. I., M UR OH Y, D . an d H OG AN . B .l.M . (19 85). E ~p ressio n o f c.fos in parietale nd od erm . a mn io n a nd d iffe re ntia tin g F 9te ra to ca rc in om a c ells . Di ff eren ti a ti on 30:76-81.

M A RT IN , G .R . ( 19 80 ). T er ato ca rc in om as a nd m a mm ali an e mb ry og en es is . SCience 209:768-776.

M AS ON , U ., M UR PH Y. D .. M ON KE . M ., F RA NC K E. U .. E LL IO T T. R .W . a nd H O GA N. B .L .M .(1 98 6). D ev elo pm en t a nd tra ns fo rm atio n-s en sitiv e e ~p re ss io n o f th e Spare geneo n m o us e c hr om o so me 11 . EMBD J . 5 : 1 83 1- 18 37 .

MONTANO ,X.and LANE.D.P. (1987). T he a de no vir us E la g en e in du ce s d iff er en tia tio no f F 9 te ra to ca rc in om a c ells . M ol. C ell. B io i. 7 : 1782-1790 .

M OO RE . E .E ., M IT RA , N .S . a nd M OR IT Z, E.A. (1986) . Di ffe ren ti at ion o f F 9 e mb ry on alc ar ci no ma c ells . D if fe re nc es i n t he e ff ec t o f r eti no ic a cid . 5 -b ro m od eo ~iu ri dln e an dN-N~imethylacetamide. Differentiation 31 : 183- 190 .

M U MM E RY , C .L .. S LA G ER . H ., K R UI JE R. W . F EI JE N, A ., F RE UN D . E ., K OO R NN EE F, I . a ndV an d en E IJ ND EN -V AN R AA IJ , A .J.M. (1990). El\pression of tr an sf or min g g ro wt hfa cto r 8 2 during th e d if fe re nti ati on o f m ur in e e mb ry on al c ar cin om a a nd e mb ry on ics te m c el ls . D e v. B io i. 1 37 : 1 61 -1 70 .

M U RP H Y. S .P .. G A RB ER N . J .. O D EN W AL D. W .F .. L AZ ZA RI NI , R .A . a nd L IN NE Y, E . ( 19 88 ).D iffe re ntia l e xp re ss io n o f th e h om eo bo ~ g en e Hox-1.3 i n F 9 e m br yo na l c ar cin om acells. P roc. N at . A ca d. S ci. U SA 8 5 : 5 5 87 -5 5 91 .

MOLLER, R . a nd W AGNE R ,E.F . (1984 ). D i ff e ren ti at ion o fF 9t er ato ca rc in om a s te m c el lsa ft er t ra ns fe r o f c -f os p ro to -o nc og en es . Nature 3 : 438-442.

N IS HIK U RA . K .. K IM , U . a nd M U RR A Y, J.M . (1 99 0) . D if fe re ntiatio n o f F 9 c ells isI nd ep en de nt o f c -m yc e ~p re ss io n. Oncogene 5 : 9 8 1- 98 8.

OSHIMA, R.G. (1981 ). I den t if ica ti on a nd immunop re ci pi ta ti on o f c yt osk el et al p ro te in sfrom murine e~tra-i mbryonic e nd od er ma l c ell s. J . B i oi . Chem. 256: 8124 -8133 .

R O S E N S TR A US S, M .J ., SUNDELl. C .L . a nd LlS KA Y, R .M . (1 982 ). Cell-

8/12/2019 ft389

9/9