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Lineage-specific patterns of p21ras proteins in immortalized cell lines derived from mouse teratocarcinoma
Res. Viroi.
(~) INSTITUT PASTEUR/ELSEVIER Paris 1990
1990, 141, 45-55
LINEAGE-SPECIFIC PATTERNS OF p21 ras PROTEINS IMMORTALIZED CELL LLNES DERIVED FROM MOUSE TERATOCARCINOMA
M.-J. Vilarem (1), O. Kellermann (2), M.-P. Gras (a), C. Larsen (1) and M.-H. Buc-Caron (2) O) 02~01 INSERM, Centre Hayem, Hdpital Saint-Louis, 75475 Paris Cea~x I0, and (2) Laboratoire de Diff~renciation cettulaire, UA CNRS 1148, Institut Pasteur, 75724 Paris Cedex 15
SUMMARY The expression of proteins coded by the ras oncogene family was examined in mouse embryonat carcinoma (EC) cells and in immortalized ceil lines derived from EC. These cell lines, which correspond to early stages of differentiation, express the simian virus 40 (SV40) T antigen and still proliferate. By 2-D gel electrophoresis of the immune complexes formed vdth monoclcnal anti-ras antibodies, it was possible to distinguish the products of the Ha-, PC- and Ki-ras genes and to correlate the observed patterns with the differentiation state of the cells. We show in this report (I) that the 2-D gel pattern of ras protein is identical for the various EC tested and is not influenced by SV40 transformation, (2) that p21Kiras is not detected in EC cells, although some EC ceII lines are known to express a Ki-ras transcript, and (3) that the complex patterns of N- and Ha-ras observed in EC cells becomes simpler as differentiation proceeds, with a different, characteristic pattern for neuroectodermal, mesodermal and endodermal derivatives. Such patterns could prove useful as differentiation markers. KEY-WOmbS: Oncogene, Protein p21 r~s, Teratocarcinoma; Mouse, 2-D gel electrophoresis, Differentiation.
Submitted Septem[.~: 28, 1989, accepted November 29, I989.
46
M.J. VILAREM ET AL.
INTRODUCTION
The ras proto-oncogene family members (Ha-ras, N-ras and Ki-ras) encode 21-kDa proteins (p21) which are associated with the inner face of the plasma membrane of the cells via acylation of a cysteine residue by palmitic acid (Buss et al., 1988; Magee et al., 1987). The ras proteins bind guanine nucleotides, exhibit GTPase activity (Der et al., 1988; Gibbs et aL, 1984) and share homologies with the ~-subunits of G proteins, leading to the assumption that they are involved in signal transduction. Ras proto-oncogenes are thought to be involved in proliferation and differentiation. In the case of the pheochromocytoma PC12, microinjection of the activated ras protein induces differentiation into neuron-like ceils (Bar-Sagi et al., 1985; Noda et aL, 1985) and antibody to p21 r a s inhibits nerve-growth-factor-induced differentiation (Hagag et aL, 1986). As for the multipotential embryonal carcinoma (EC) cells (Martin, 1975), it was shown that transfection with Ha-ras does not induce differentiation, but is responsible for the amplification of one among the various differentiated derivatives (Bel~ et aL, 1986). These results might indicate that the effect of ras depends upon the initial differentiation stage of the target cells. High expression of ras RN~ is observed during mouse embryogenesis, with similar levels at all developmer,_tal stages (Adamson, 1987; Muller et al., 1982). Expression of ras RNA was also measured in EC ceils C';Iette et aL, 1987) and in several differentZated cells derived from EC (Sejersen e t al., 1985). The amount of ras RNA was ~hown to slightly decrease in the differentiated cells. These measurements, however, have several drawbacks: Firstly, it is very dif-ficult to separate effects on proliferation and on differentiation. Secondly,, RNA measurements might not reflect the amounts of the corresponding pro-teins, since regulztion can be exerted at the translational or posttranslational levels (Feuerstein et aL, 1985). We have therefore attempted to determine if there exists a differential quantitative and/or qualitative expression of ras proteins during early developmental stages using a recently developed model system in which both undifferentiated (EC) and committed or more differentiated cells continue to proliferate. The method used, 2-D gel electrophoresis, allows distinction on the basis of charge and apparent molecular weight, between the p21 proteins encoded by the Ki-, Ha- and N-ras genes in their various forms generated by post-translational modifications (Feuerstein et aL, 1985). Transformation of EC cells by plasmid pK4, carrying SV40 early genes under the control of
DOC EC NEPHGE PMSF
= = -=
deoxycholate. embryonal carcinoma. non-equilibrium p H gradient. phenylmethylsulphonyl fluoride.
RA SDS SV40 TCA
= = = =
retinoic acid. sodium dodecyl sulphate. simian virus 40. trichtoroacetic acid.
LINEAGE-SPECIFIC
PATTERNS
O F p 2 1 ras P R O T E I N S
47
the adenavirus E la promoter enab!ed us to immortalize teratocarcinomaderived ceil lines corresponding to precursors of the neuroectodermal, mesodermal and endodermaI pathways. These clooes are composed of immature committed ceils with stabie phenotype which can further differentiate upon convenient chemical induction, along a restricted lineage (Kellermann et aL, 1987). Since the differentiated ceils retain their potential for proliferation, any observed change in their ras protein pattern can, ~.nprinciple, be con elated with differentiation. We show in this report that the 2-D gel pattern of ras proteins is identical for the EC cells tested and is not modified by the expression of the SV40 T antigen. In contrast, commitment of the cells toward either the neuroectoderm, endoderm or mesoderm lineages is associated with a reduction in ras protein levels and with a particular pattern of p21H'~-ras. In addition, we noticed that p21 gi'ras, whose transcript is made in EC cells, is not detected.
MATERIALS AND METHODS Cells. E C cones.
The properties of EC clones F9, t003, F9K4b2 and 1003-pK4 are listed in table I. F9K4b2 and 1003-pK4 cells which have integrated the immortalizing vector pK4 cannot be distinguished from the parental clones by their phenetype and their differentiation potentialities. The t003-pK4 cells express the SV40 T antigen at the EC-ceU stage, whereas retinoic acid 0L~_)induction is required for the expression of the T antigen in the case of F9K462. Committed precursors.
Upon RA iiaduction of F9K4b2 ceils, immortalized precursors of the endodermai, mesodermal and neuroectodermai lineages were selected on the basis of loss of EC markers, expression of the SV40 T antigen and the ability to further differentiate along a restricted pathway. Cleric H7 (Keltermann et aL, I987) is an endoderreal stem cell. Clone 86180 is committed to the mesodermal pathway. 1Cll is an immature stem cell committed to serotoninergic differentiation. After exposure to dibut3'ryl cAMP and cyclohexane carboxylic acid, almost t00 % of 1C11 cells, which still divide, acquire a neuron-like phenotype and the ability to synthesize, take up and store serotonin (Buc-Caron et al., 1990). The 1003-pK4 derivatives include a ceil line derived from a mesodermal tumour, 87102, which contains immature mesoblastic ceils, fibroblasts and osteoblasts. C1 is a clone derived from 87102, selected for its expression of osteoblastic markers and its ability to form bone in vivo and in vitro (Kellermann et aL, 1989) (table I).
Culture and labelling conditions, Cells were maintained in ~r~J~ :~s previously described (Feuerstein et aL, lO55).
M.-,1. V I L A R E M
48
ET AL.
TABLE I. - - P r o p e r t i e s o f t e r a t o e a r c i n o m a - d e r i v e d cell lines. Strains CloneF9 Clone F9K4b2 Clone 1003 Clone 1003-pK4 CloneH7 Clone 86180 Tumour87102 CloneCI Clone 1Cll
Cell type in vitro
Turnouts
EC EC Extraembryonicendoderm and multipletypes (*) EC EC: Multipletypes(*) trigerminal (*) EC Yrigerminal Multipletypes(*) EC Trigerminal Multipletypes(*) Endodermal precursor Extraembryonie and embryonic endoderm Mesoderm~precursor Fibrosarcoma Adipocytes Mes0thelium Mesoblastsand Osteofibrosarcomas osteobiasts Fibrosarcoma Osteoblasts Osteosarcomas Bone-formingcells(*) Neuroblastic precursor; Immatureneuroblasts ne~al-like ce~s(*)
T-antigen expression
References
-
Bernstineet al. (1973)
+ (*) -
Keliermann& Kelly(1986); Kellermannet ai. (t987) McBurney (1976)
+
Ke!lermann et aL (t990)
+
Kelle~annet al, (I987)
+ + + +
" Kelhrmann et aL (t990) " Buc-Caron et aL (!990)
(*) Upon induction of differentiation.
Cell labelling. Ceils were incubated for 4 h in methionine-free Dulbecco modified Eagle medium containing 35S-methionine (100 ~Ci/mA, 800 Ci/mmole) and t0 °7o foetal calf serum. Ceils were washed with phosphate-buffered saline and lysed in a buffer containing 0.2 M Tris-HCl p H 7.4, O. 1 M NaCI, 0.005 M MgClz, 0.4 % sodium deoxyeholate (DOC) and 3 m M phenylmethylsulphonyl fluoride (PMSF). Finally, the lysates (15 × IO6 cells) were centrifuged at 100,000 g for 30 m i n at 4°C. p21TM analysis. A pre-dearing of the cell extract was carried out w'th anti-rat antibody (Miles) (5 ~1 o f anti-rat serum added to 0.5 ml o f labelled cell extracts containing 30 × 106 TCA-precipitable cpm). After 60 rain at 4°C, 60 t~l of a 10 % (v/v) suspension of formalin-irLxed Staphylococcus aureus (Staph. A., BRL) were added and the resulting mixture was centrifuged at 10,000 g for I min. The supernatants were incubated overnight at 4°C with the monoclonal anfi-ras antibodies Y13-259 or Y13-238 (Oncogene Science, Inc; F u r t h e t al., I982). The i m m u n e complexes were precipitated by addition of 50 ~ o f the staphylococcal suspension previously coated with anti-rat immunoglobulins for 3 h at 4°C. The immunoprecipitates were colIected by centrifugation, washed 5 times in a buffer containing 0.05 M Hepes buffer p H 7.4, 0.5 % DOC, 1 % Triton-X100, 0.1 M NaCI, 0.00I M EDTA, 0 . 1 % sodium dodecyt sulphate (SDS) a n d 3 m M P M S F . Finally p21r, s proteins were eluted from the
LINEA GE-SPECIFIC PATTERNS
A
O F p21 ras P R O T E I N S
~
4s[
a
Q
i"p
1
I
1
#
z-
45
49
-4qb a
Ph
0
o
':3O b l
[
,i
"!
"~4
52
FIG. 1. - - 2-D gel etectrophoresis analysis (ArEPHGE) of p21 ~ proteins. Two representative fluorographs from the H7 clone (see table I). Cell extracts (30 :~ 106 TCAprecipitable cpm) were immunoprecipitated with non-immune rat serum (top) and with monoclonal Y13-259 antibody (bottom). Box~ delineate the region where Ha-ras and N-ras p21 are located. Arrows point to the expected position of Ki-ras spots. Two spots, actin (a) and polypeptide (p), non-specifically precipitated by the antibocfi~, were used as migration markers.
50
M.-J. VILAREM ET AL.
"Staph. A " pellet with 20 ~tl of electrofocusing buffer (9.5 M urea, 2 °70 ampholines, pH 3.5-10; Pharmacia), 5 % [~-mercaptoethaEol and 2 % "Nonidet-P40" for analysis by 2-D gel electrophoresis. Gel electrophoresis.
A non-equilibrium pH gradient (NEPHGE) was used for the first dimension (Biorad system)., The second dimension was in i 5 % polyacrylamide gels containing 0,1 o70 SDS. Gels were analysed by fluorography. RESULTS 1) Characterization o f p21 ras proteins in teratocarcinoma-derived cells.
Analysis by 2-D gel electrophoresis of p 2 i ras proteins from teratocarcinorna cells is illustrated in figure t. SewraI polypeptides of M W 21-24 kDa were precipitated with Y13-259 anti-p21 antibodies (fig. 1, bottom). Among the proteins non-specifically precipitated in control samples (fig. 1, top), ac-
ill
i
i
I
f
,
,,
, .,,,,,., ....
t
l i i
.
, :.,~ ': :
, ' .
':..,%
. !:
~ . ,
, . . . .
-
...
ill
,~..:-
. • ,
ii ii I
• .:
4
/ FIG. 2. -- Comparative 2-D gel analysis of p21 polypeptides, precipitated with t/13-259 antibody (1 and 2) or Y13-238 antibody (3 and 4). Arrows point to N-ras spots; ! and 3:1C11 clone (see table I); 2 and 4:NIH/3T3 cells.
LINEAGE-SPECIFIC
PATTERNS
5!
O F p21 ras P R O T E I N S
tin (a), and an 18-kDa protein (p), provided mobility standards to analyse any significant change in the electrophoretic mobilities of p21 ras from the various cell extracts. Spots corresponding to H a - and N-ras were z',~producibly found in the acidic region of the gel. No spots corresponding to p21 xi-ras (arrows in figure 1 control sample), which are known to migrate in the basic area of the gels (Feuerstein et aL, 1985, and our personal data), were ever detected in EC cells or in any of the derivatives tested (see fig. 1). N-ras and Ha-ras spots were further distinguished by the use of 2 monoctonal antibodies: Y13-259, which exhibits a broad specificity to all p21 ras ( F u r t h e t aL, i982), and Y13-238 whose specificity is restricted to p2I na-ras and p21 ri-r~. As shown in figure 2, the acidic peptide detected by Y13-259 antibodies in clone 1 C l l taken as an example (fig. 2, panel 1) was not recognized by Y13-238 (fig. 2, panel 3) and is therefore likely to have an N - r a s origin. This result was confirmed by the NII-!3T3 pattern (fig. 2, panels 2 and 4) as these cells nave been shown to express only p21N-ras (Feuerstein et al., I985).
EC -
E
-
f2-]
_
_
M
~
NE
=
.....
A
-
" '
J
L[
il ? ' ° j°1
B
FIG. 3. m Comparative 2-D gel analysis o f p21 ra~polypeptides precipitated with Y13-259 antibody.
EC, E, M and NE columns refer to embt3.onal carcinoma, endodermal, mesodermal and neuroectodermat cells, respectively; I to 6 refer to F9 cells and their derivatives; 1 and 2: F9 and FgK4b2 EC ceils; 3 : clone H7 ; 4: clone 86180; 5 : clone 1CI l ; 6: serotoninergic cells derived from ICtl. A to D refer to 1003 cells and their derivatives. A and B: 1003 and 1003-pK4 EC cells; C: clone 87102; D: clone 87102-CI.
52
iVI.-J. V I L A R E I v I E T A L ,
2) Comparative pattern of p21 :in EC cells. The patterns of p2I Nras and p2i Ha-r~ found in F9 (fig. 3-1), 1003 (fig. 3A) and the pK4-transformed clones F9K4b2 (fig. 3-2) and 1003-pK4 (fig. 3B) were characterized by several highly labelled spots. All the patter~s of the EC cells were qualitatively" identical irrespective of the introduction and expression of the SV40 early genes.
3) p21 patterns of committed and differentia~efl cells. In atl teratocarcinoma-deri;ed cell lines tested (fig. 3), the intensity of the p21 spots was markedly reduced compared to that of EC ceils. A major spot identified as N-ras by its absence in immune complexes with Y13-238 antibody was present in each cell extract (marked " 1 " ) . The other spots which were attributed to Ha-ras on the basis of their mobility generated characteristic patterns. Within the same lineage (compare, for instance, ICI 1 (fig. 3-5) and 1C11 cAMPCCA (fig. 3-6) for the neuroectoderm, a:~d 86180 (fig. 3-4) 87102 (fig. 3-C) and clone Cl (fig. 3-D) for mesoderm), the precursor and its more differemia~ed derb'ative shared a common pattern.
DISCUSSION In this report, we analyse the expression of ras proto-oncogene-encoded proteins (p21 ras) in mouse teratocarcinoma-derived cells corresponddng to early stages of commitment and differentiation~ Previous studies have shown that RNA transcription of ras genes is detected with little variation at all stages of mouse embryonic development (Adamson, 1987; M/Jller et al., 1982). These data are not informative as to the possibilit3" of post-transcriptional modifications occurring during differentiation. On the other hand, multiple forms of ras proteins can be detected in normal as well as in transformed ceils using 2-1) gel analysis (Feurstein et al., t985). While the s;~g~';~cance of this heterogeneity is currently unknown, it has been postulated to reflect the existence of subtle charge differences in products encoded by the same gene. In addition, the combined use of anti-ras monoclonaI antibodies with different specificities makes it possible to distinguish between produ~:~c encoded by N-ras, Ha-ras and Ki-ras genes. R a s proto-oncogenes are believed to be mairdy involved in cell proliferation (MiiUer et al., 1982). In the case of PC12 cells, a correlation between ras expression and differentiation has also been reported (Bar-Sng/et aL, 1985; Hagag et aL, 1986; Noda et aI., 1985). We describe here a different situation in which both undifferentiated (EC) and comnfitted or more differentiated cells are maintained in a permanent state of proliferation due to the expres-
L L ' V E A G E - S ' P E C I F I C PATITERb:7~ O F p2Z =as P R O T E I N S
53
sion of the SV40 T antigen. It follows th~. any modification in ras prote!n patterns is likely to be correlated with the differentiation state rather ~han with proliferation. The presence of Ki-ras transcripts has been reported in several EC cells including F9 cells (Sejersen et aL, 1985; Vilette et al., 1.987). In our h a n d s repeated tests failed to detect any traces of p21Ki-r~s proteins in the teratoca.rcinoma cell extracts whereas, under the same experimental condkions: ~21:cras proteins were detected in various control cell lines. These include a cd! line, Calu-1, derived from a lung cancer, S%V488 colopAc cancer ce~s and br: ~.: adenoc~r~bo,~as ~?~!arern e; ~i.~ in ~z~.paration). Tl~,~refore, .our resuks pOii[].t to post-transcriptional control of Ki-~as expression in tera~ocarcino-ma cetl-~ Alternatively, it is not excluded that p21 m - ~ levels in EC cells are b d o w detection levels. Our data show tha" the complex patterns of ?v=ras and Ha-ras promins observed in EC cells are not influenced by SV40 transfon-nation and become simpler as differentiation proceeds. It is noteworthy that neure-ctoderm, mesoderm and endoderm derivatives generate different and char~:cter~s:.[c p21 ras patterns. In addition, for a given lineage, there is no ap~:rec!ab~e change in a part[cular pattern during further differentiatiom These data strongiy suggest that specific patterns of p2i cas are installed at a very ear!y step e f differentiation toward each lineage and are then maintained in full:,- d.iffere~tiated cells. Given. this situation, such patterns could prove useful as differentiation markers. Because of its presence in all cell lineages, N-ras (or a closely related geae product) does not seem to be associated with a specific differentiation s~ in contrast, the other spots, which we attribute to He-ras-gene expression on the basis of their acidic behaviour appear different in ce~s" from differen~ lineages, and might therefore fulfill specific tGn.ctions.
:A~.~ALY~E C O M P A K % T I V E E N G E L B[~LVENSI05 }~7[ DES PROTE[NSS p~l r~S DAN£. DES L[ON~ES [.~,[Z-~.[ORTA~ISEESDEKiVEES D U TERA'; 0 C ,~[ C iINO~ [~. D [ [ 0 ~ ]E[~:
L'expression des prot6ines cod4es par Ia famili.e d'oncog~n:s ra:~ a ~t6 ar/,al}?s,~-~e clans los carcinomes embryonnaires (EC) et dans des Iign6e.sce~iulaires immo~a!~s@s qui en d~rivent. Cos li~6es qui correspondent ~ des s.tades pr6cozes de diffBrendatiom expriment l:antig~ne T de SV.40et continuen'~ ~ prolif&er. L'4le~rophorBs¢ Ndimensiennelle (2-D) d'immuncomplexes form6s avee des anticorps monoci~;na~; anti-ras, a permis de distinguer les produits des g~r~es rgs .e~ de corr~Ier ~e~.:~ro,SJs observ4s ave¢ I'6tat de diff~renciaticn des cellules. Nos r4sultats indiqucnt (1) que la distribution en eTectrophorBse 2.D des pro~b nes ras est identique dans plusieurs Iign~es EC et n'est pas modifi6e pa~ I'expresskm des oncog6nes de SV40, (2.) que Ia prot~ine p2t ~ s n~es~ pas d@6I&t dans los ceHu~ los EC alors que ie transcrit correspondant a 6t6 mis en ~vidence darts certamcs d.e cos tign~es et (3) queta ~rtographie des prot~ines Ha- and ?~Cras, complexe dans
54
M..-J. V I L A R E M E T A L .
les EC, se simplifie dans los d6riv~s diff6renci6s, et que la distribution est diff6rente darts les Iign6es neuroectodermiques, m6sodermiques et endodermiques. L'analyse biochimique des p21 ~aspourrait contribuer/t l'identification des stades pr6coces de la diff6renciation. MOTS-CLES: Oncogbne, Prot6ine p2t r,s T6ratocarcinome; Souris, Electrophor~se bidimensionnelle, Diff6renciationo
ACKNGWLEDGEMi~NTS This work has been aided by grants from the ARC, Centre National de la Recherche Scientifique and Ligue Natienale Fran~aise contre le Cancer.
REFERENCES
ADAMSON,E.D. (1987), Oncogenes in development. Development, 99, 449-471. BAR-SAGI,Do & FEgAMISCO,J.R. (1985), Microinjection of the ras oncogene protein into PC12 cells induces morpiaological differentiation. Cell, 42, 841-848. BELL, J.C., JARDINE,K. & McBumqEV,M.WS. (1986), Lineage-specific transformation after differentiation of mu!tipotential murine stem cells containing a human oncogene. MoL Cell n2oL, 6, 617-625. BERNSTtNE, E.G., HOOPER, M.L., GRANCHAMr',S. & EPHgUSSt, B. (1973), Alkaline phosphatase activity in mouse teratoma. Proc. nat. Acad. Sci. (Wash.), 70, 3899-3903. Buc-CARON, M.H., LAUNAY,J.M., LAMBLIN,D. & KELLEgMANN,O. (1990), Serotonin uptake, storage and synthesis in an immortalized committed cell line derived from mouse teratocarcinoma. Proc. nat. Acad. Sci. (Wash.) (in press). Buss, J.E., DER, E.J. & SOLSKI, P.A. (I988), The six amino-terminal amino acids of p60 src are sufficient to cause mycistylation of p21 v-ras. MoL Cell. BioL, 8, 3969-3963. D~R, C.J., WEISSMAN,B. & MACDONALD,M.J. (1988), Altered guanine nucleotide binding and H-ras transforming and differentiating activities. Oncogene, 3, 105-112. FEUERSTEIN, N. & AL~, I.U. (1985), Comparative analysis of p21 p;oteins from various cell types by two-dimensional gel etectrophoresis. J. Cell. Bioehem., 29, 253-263. FURTH, M.E., DAVIS, L.J., FLEtJrtDELYS, B. & SCOLNIC~, E.H. (1982), Monocional antibodies to the p21 products of the transforming gone of Harvey murine sarcoma virus and of the cellular ras gone family. J. Virol., 43, 294-304. GIBBS,J.B., SIGAL,i.S., PoE, M. & SCOLNICK,E.M. (1984), Intrinsic GTPase activity distinguishes normal and oncogenic ras p21 molecules. Proc. nat. Acad. Sci. (Wash.), 81~ 5704-5708. HAGAG,N., HALEGOUA,S. & VIOIA, M. (1986), Inhibition of growth factor-induced differentiation of PC12 cells by microinjectionof antibody to ras p21. Nature (Lond.), 319, 680-682. KELLEgMANN, O. & Ed~LLY, F. (1986), Immortalization of early embryonic cell derivatives after the transfer of the early region of simian virus 40 into F9 teratocarcinoma cells. Differentiation, 32, 74-81. KELLERMANN,O., BuC-CARON, M.H. & G~.ILLARD, J. (1987), Immortalization of precursors of endodermal, neuroectodermal and mesodermal lineages, following the introduction of the slw_ianvirus (SV40) early region into F9 cells. Differentiation, 35, 197-205.
LINEAGE-SPECIFIC PATTERNS
O F p 2 I ras PROTEi'~qS
55
KELLERMANN)O., Buc-CARON, M.H., MARIE,P.J., LAMBLIN,D. & JACOI3~F. (I990), An immortalized osteogenic cell line derived from mouse teratocarcinoma is able to mineralize in vivo and in vitro. Z Cell Biol., !10 (in press). McBoRNEY, M. k1976), Clonal lines of teratocarcinoma cells in vitro: differentiation and cytogenetic characteristics. J. Cell Physiol., 89, 441-456. MAGEE, A.Y., GUTIERREZ,L., McKoY, I.A., MARSHALL,C.J. • HALL, A. (lq87), Dynamic fatty acylation of p21N-ras. E M B O J., 6, 3353-3357. MARTIN, G.R. (J975), Teratocarcinoma as a model system for the study of embryogenesis and neoplasia. Cell, 5, 229-243. I~gII]LLER,R,, SLAMON,D.J., TREMBLAY,J.M., CLINE, M.J. & VERMA,I. (1982), Differential expression of cellular oncogenes during pre- and postnatal development of the mouse. Nature (Lond.), 299, 640-644. NODA, M., Ko, M., OGURA, A., LIU, D., AMANO, T., TAKANO,T. & IKAWA,Y. (1985), Sarcoma vir)Jses carrying ras oncogenes induce differentiationassociated properties in a neuronal cell line..Nature (Lond.), 317, 73-75. SEJERSEN, T., SOMEGI,J. & RINCERTZ, N.R. (1985), Expression of cellular oncogenes ;.r~ reratoma-derived cell lines. Exp. Cell Res., 160, 19-30. V1LETTE, D., EMANOIL-RAVIER,R., BtreFE, D., RIMBAUT,C. (~ PERtES, J. (i987), YCl-ras gene amplification and malignant behavior in _rnurineembryonal carcinoma cell lines. Cancer Res., 47, 867-873.
(~) INSTITUT PASTEUR/ELSEVIER Paris 1990
1990, 141, 45-55
LINEAGE-SPECIFIC PATTERNS OF p21 ras PROTEINS IMMORTALIZED CELL LLNES DERIVED FROM MOUSE TERATOCARCINOMA
M.-J. Vilarem (1), O. Kellermann (2), M.-P. Gras (a), C. Larsen (1) and M.-H. Buc-Caron (2) O) 02~01 INSERM, Centre Hayem, Hdpital Saint-Louis, 75475 Paris Cea~x I0, and (2) Laboratoire de Diff~renciation cettulaire, UA CNRS 1148, Institut Pasteur, 75724 Paris Cedex 15
SUMMARY The expression of proteins coded by the ras oncogene family was examined in mouse embryonat carcinoma (EC) cells and in immortalized ceil lines derived from EC. These cell lines, which correspond to early stages of differentiation, express the simian virus 40 (SV40) T antigen and still proliferate. By 2-D gel electrophoresis of the immune complexes formed vdth monoclcnal anti-ras antibodies, it was possible to distinguish the products of the Ha-, PC- and Ki-ras genes and to correlate the observed patterns with the differentiation state of the cells. We show in this report (I) that the 2-D gel pattern of ras protein is identical for the various EC tested and is not influenced by SV40 transformation, (2) that p21Kiras is not detected in EC cells, although some EC ceII lines are known to express a Ki-ras transcript, and (3) that the complex patterns of N- and Ha-ras observed in EC cells becomes simpler as differentiation proceeds, with a different, characteristic pattern for neuroectodermal, mesodermal and endodermal derivatives. Such patterns could prove useful as differentiation markers. KEY-WOmbS: Oncogene, Protein p21 r~s, Teratocarcinoma; Mouse, 2-D gel electrophoresis, Differentiation.
Submitted Septem[.~: 28, 1989, accepted November 29, I989.
46
M.J. VILAREM ET AL.
INTRODUCTION
The ras proto-oncogene family members (Ha-ras, N-ras and Ki-ras) encode 21-kDa proteins (p21) which are associated with the inner face of the plasma membrane of the cells via acylation of a cysteine residue by palmitic acid (Buss et al., 1988; Magee et al., 1987). The ras proteins bind guanine nucleotides, exhibit GTPase activity (Der et al., 1988; Gibbs et aL, 1984) and share homologies with the ~-subunits of G proteins, leading to the assumption that they are involved in signal transduction. Ras proto-oncogenes are thought to be involved in proliferation and differentiation. In the case of the pheochromocytoma PC12, microinjection of the activated ras protein induces differentiation into neuron-like ceils (Bar-Sagi et al., 1985; Noda et aL, 1985) and antibody to p21 r a s inhibits nerve-growth-factor-induced differentiation (Hagag et aL, 1986). As for the multipotential embryonal carcinoma (EC) cells (Martin, 1975), it was shown that transfection with Ha-ras does not induce differentiation, but is responsible for the amplification of one among the various differentiated derivatives (Bel~ et aL, 1986). These results might indicate that the effect of ras depends upon the initial differentiation stage of the target cells. High expression of ras RN~ is observed during mouse embryogenesis, with similar levels at all developmer,_tal stages (Adamson, 1987; Muller et al., 1982). Expression of ras RNA was also measured in EC ceils C';Iette et aL, 1987) and in several differentZated cells derived from EC (Sejersen e t al., 1985). The amount of ras RNA was ~hown to slightly decrease in the differentiated cells. These measurements, however, have several drawbacks: Firstly, it is very dif-ficult to separate effects on proliferation and on differentiation. Secondly,, RNA measurements might not reflect the amounts of the corresponding pro-teins, since regulztion can be exerted at the translational or posttranslational levels (Feuerstein et aL, 1985). We have therefore attempted to determine if there exists a differential quantitative and/or qualitative expression of ras proteins during early developmental stages using a recently developed model system in which both undifferentiated (EC) and committed or more differentiated cells continue to proliferate. The method used, 2-D gel electrophoresis, allows distinction on the basis of charge and apparent molecular weight, between the p21 proteins encoded by the Ki-, Ha- and N-ras genes in their various forms generated by post-translational modifications (Feuerstein et aL, 1985). Transformation of EC cells by plasmid pK4, carrying SV40 early genes under the control of
DOC EC NEPHGE PMSF
= = -=
deoxycholate. embryonal carcinoma. non-equilibrium p H gradient. phenylmethylsulphonyl fluoride.
RA SDS SV40 TCA
= = = =
retinoic acid. sodium dodecyl sulphate. simian virus 40. trichtoroacetic acid.
LINEAGE-SPECIFIC
PATTERNS
O F p 2 1 ras P R O T E I N S
47
the adenavirus E la promoter enab!ed us to immortalize teratocarcinomaderived ceil lines corresponding to precursors of the neuroectodermal, mesodermal and endodermaI pathways. These clooes are composed of immature committed ceils with stabie phenotype which can further differentiate upon convenient chemical induction, along a restricted lineage (Kellermann et aL, 1987). Since the differentiated ceils retain their potential for proliferation, any observed change in their ras protein pattern can, ~.nprinciple, be con elated with differentiation. We show in this report that the 2-D gel pattern of ras proteins is identical for the EC cells tested and is not modified by the expression of the SV40 T antigen. In contrast, commitment of the cells toward either the neuroectoderm, endoderm or mesoderm lineages is associated with a reduction in ras protein levels and with a particular pattern of p21H'~-ras. In addition, we noticed that p21 gi'ras, whose transcript is made in EC cells, is not detected.
MATERIALS AND METHODS Cells. E C cones.
The properties of EC clones F9, t003, F9K4b2 and 1003-pK4 are listed in table I. F9K4b2 and 1003-pK4 cells which have integrated the immortalizing vector pK4 cannot be distinguished from the parental clones by their phenetype and their differentiation potentialities. The t003-pK4 cells express the SV40 T antigen at the EC-ceU stage, whereas retinoic acid 0L~_)induction is required for the expression of the T antigen in the case of F9K462. Committed precursors.
Upon RA iiaduction of F9K4b2 ceils, immortalized precursors of the endodermai, mesodermal and neuroectodermai lineages were selected on the basis of loss of EC markers, expression of the SV40 T antigen and the ability to further differentiate along a restricted pathway. Cleric H7 (Keltermann et aL, I987) is an endoderreal stem cell. Clone 86180 is committed to the mesodermal pathway. 1Cll is an immature stem cell committed to serotoninergic differentiation. After exposure to dibut3'ryl cAMP and cyclohexane carboxylic acid, almost t00 % of 1C11 cells, which still divide, acquire a neuron-like phenotype and the ability to synthesize, take up and store serotonin (Buc-Caron et al., 1990). The 1003-pK4 derivatives include a ceil line derived from a mesodermal tumour, 87102, which contains immature mesoblastic ceils, fibroblasts and osteoblasts. C1 is a clone derived from 87102, selected for its expression of osteoblastic markers and its ability to form bone in vivo and in vitro (Kellermann et aL, 1989) (table I).
Culture and labelling conditions, Cells were maintained in ~r~J~ :~s previously described (Feuerstein et aL, lO55).
M.-,1. V I L A R E M
48
ET AL.
TABLE I. - - P r o p e r t i e s o f t e r a t o e a r c i n o m a - d e r i v e d cell lines. Strains CloneF9 Clone F9K4b2 Clone 1003 Clone 1003-pK4 CloneH7 Clone 86180 Tumour87102 CloneCI Clone 1Cll
Cell type in vitro
Turnouts
EC EC Extraembryonicendoderm and multipletypes (*) EC EC: Multipletypes(*) trigerminal (*) EC Yrigerminal Multipletypes(*) EC Trigerminal Multipletypes(*) Endodermal precursor Extraembryonie and embryonic endoderm Mesoderm~precursor Fibrosarcoma Adipocytes Mes0thelium Mesoblastsand Osteofibrosarcomas osteobiasts Fibrosarcoma Osteoblasts Osteosarcomas Bone-formingcells(*) Neuroblastic precursor; Immatureneuroblasts ne~al-like ce~s(*)
T-antigen expression
References
-
Bernstineet al. (1973)
+ (*) -
Keliermann& Kelly(1986); Kellermannet ai. (t987) McBurney (1976)
+
Ke!lermann et aL (t990)
+
Kelle~annet al, (I987)
+ + + +
" Kelhrmann et aL (t990) " Buc-Caron et aL (!990)
(*) Upon induction of differentiation.
Cell labelling. Ceils were incubated for 4 h in methionine-free Dulbecco modified Eagle medium containing 35S-methionine (100 ~Ci/mA, 800 Ci/mmole) and t0 °7o foetal calf serum. Ceils were washed with phosphate-buffered saline and lysed in a buffer containing 0.2 M Tris-HCl p H 7.4, O. 1 M NaCI, 0.005 M MgClz, 0.4 % sodium deoxyeholate (DOC) and 3 m M phenylmethylsulphonyl fluoride (PMSF). Finally, the lysates (15 × IO6 cells) were centrifuged at 100,000 g for 30 m i n at 4°C. p21TM analysis. A pre-dearing of the cell extract was carried out w'th anti-rat antibody (Miles) (5 ~1 o f anti-rat serum added to 0.5 ml o f labelled cell extracts containing 30 × 106 TCA-precipitable cpm). After 60 rain at 4°C, 60 t~l of a 10 % (v/v) suspension of formalin-irLxed Staphylococcus aureus (Staph. A., BRL) were added and the resulting mixture was centrifuged at 10,000 g for I min. The supernatants were incubated overnight at 4°C with the monoclonal anfi-ras antibodies Y13-259 or Y13-238 (Oncogene Science, Inc; F u r t h e t al., I982). The i m m u n e complexes were precipitated by addition of 50 ~ o f the staphylococcal suspension previously coated with anti-rat immunoglobulins for 3 h at 4°C. The immunoprecipitates were colIected by centrifugation, washed 5 times in a buffer containing 0.05 M Hepes buffer p H 7.4, 0.5 % DOC, 1 % Triton-X100, 0.1 M NaCI, 0.00I M EDTA, 0 . 1 % sodium dodecyt sulphate (SDS) a n d 3 m M P M S F . Finally p21r, s proteins were eluted from the
LINEA GE-SPECIFIC PATTERNS
A
O F p21 ras P R O T E I N S
~
4s[
a
Q
i"p
1
I
1
#
z-
45
49
-4qb a
Ph
0
o
':3O b l
[
,i
"!
"~4
52
FIG. 1. - - 2-D gel etectrophoresis analysis (ArEPHGE) of p21 ~ proteins. Two representative fluorographs from the H7 clone (see table I). Cell extracts (30 :~ 106 TCAprecipitable cpm) were immunoprecipitated with non-immune rat serum (top) and with monoclonal Y13-259 antibody (bottom). Box~ delineate the region where Ha-ras and N-ras p21 are located. Arrows point to the expected position of Ki-ras spots. Two spots, actin (a) and polypeptide (p), non-specifically precipitated by the antibocfi~, were used as migration markers.
50
M.-J. VILAREM ET AL.
"Staph. A " pellet with 20 ~tl of electrofocusing buffer (9.5 M urea, 2 °70 ampholines, pH 3.5-10; Pharmacia), 5 % [~-mercaptoethaEol and 2 % "Nonidet-P40" for analysis by 2-D gel electrophoresis. Gel electrophoresis.
A non-equilibrium pH gradient (NEPHGE) was used for the first dimension (Biorad system)., The second dimension was in i 5 % polyacrylamide gels containing 0,1 o70 SDS. Gels were analysed by fluorography. RESULTS 1) Characterization o f p21 ras proteins in teratocarcinoma-derived cells.
Analysis by 2-D gel electrophoresis of p 2 i ras proteins from teratocarcinorna cells is illustrated in figure t. SewraI polypeptides of M W 21-24 kDa were precipitated with Y13-259 anti-p21 antibodies (fig. 1, bottom). Among the proteins non-specifically precipitated in control samples (fig. 1, top), ac-
ill
i
i
I
f
,
,,
, .,,,,,., ....
t
l i i
.
, :.,~ ': :
, ' .
':..,%
. !:
~ . ,
, . . . .
-
...
ill
,~..:-
. • ,
ii ii I
• .:
4
/ FIG. 2. -- Comparative 2-D gel analysis of p21 polypeptides, precipitated with t/13-259 antibody (1 and 2) or Y13-238 antibody (3 and 4). Arrows point to N-ras spots; ! and 3:1C11 clone (see table I); 2 and 4:NIH/3T3 cells.
LINEAGE-SPECIFIC
PATTERNS
5!
O F p21 ras P R O T E I N S
tin (a), and an 18-kDa protein (p), provided mobility standards to analyse any significant change in the electrophoretic mobilities of p21 ras from the various cell extracts. Spots corresponding to H a - and N-ras were z',~producibly found in the acidic region of the gel. No spots corresponding to p21 xi-ras (arrows in figure 1 control sample), which are known to migrate in the basic area of the gels (Feuerstein et aL, 1985, and our personal data), were ever detected in EC cells or in any of the derivatives tested (see fig. 1). N-ras and Ha-ras spots were further distinguished by the use of 2 monoctonal antibodies: Y13-259, which exhibits a broad specificity to all p21 ras ( F u r t h e t aL, i982), and Y13-238 whose specificity is restricted to p2I na-ras and p21 ri-r~. As shown in figure 2, the acidic peptide detected by Y13-259 antibodies in clone 1 C l l taken as an example (fig. 2, panel 1) was not recognized by Y13-238 (fig. 2, panel 3) and is therefore likely to have an N - r a s origin. This result was confirmed by the NII-!3T3 pattern (fig. 2, panels 2 and 4) as these cells nave been shown to express only p21N-ras (Feuerstein et al., I985).
EC -
E
-
f2-]
_
_
M
~
NE
=
.....
A
-
" '
J
L[
il ? ' ° j°1
B
FIG. 3. m Comparative 2-D gel analysis o f p21 ra~polypeptides precipitated with Y13-259 antibody.
EC, E, M and NE columns refer to embt3.onal carcinoma, endodermal, mesodermal and neuroectodermat cells, respectively; I to 6 refer to F9 cells and their derivatives; 1 and 2: F9 and FgK4b2 EC ceils; 3 : clone H7 ; 4: clone 86180; 5 : clone 1CI l ; 6: serotoninergic cells derived from ICtl. A to D refer to 1003 cells and their derivatives. A and B: 1003 and 1003-pK4 EC cells; C: clone 87102; D: clone 87102-CI.
52
iVI.-J. V I L A R E I v I E T A L ,
2) Comparative pattern of p21 :in EC cells. The patterns of p2I Nras and p2i Ha-r~ found in F9 (fig. 3-1), 1003 (fig. 3A) and the pK4-transformed clones F9K4b2 (fig. 3-2) and 1003-pK4 (fig. 3B) were characterized by several highly labelled spots. All the patter~s of the EC cells were qualitatively" identical irrespective of the introduction and expression of the SV40 early genes.
3) p21 patterns of committed and differentia~efl cells. In atl teratocarcinoma-deri;ed cell lines tested (fig. 3), the intensity of the p21 spots was markedly reduced compared to that of EC ceils. A major spot identified as N-ras by its absence in immune complexes with Y13-238 antibody was present in each cell extract (marked " 1 " ) . The other spots which were attributed to Ha-ras on the basis of their mobility generated characteristic patterns. Within the same lineage (compare, for instance, ICI 1 (fig. 3-5) and 1C11 cAMPCCA (fig. 3-6) for the neuroectoderm, a:~d 86180 (fig. 3-4) 87102 (fig. 3-C) and clone Cl (fig. 3-D) for mesoderm), the precursor and its more differemia~ed derb'ative shared a common pattern.
DISCUSSION In this report, we analyse the expression of ras proto-oncogene-encoded proteins (p21 ras) in mouse teratocarcinoma-derived cells corresponddng to early stages of commitment and differentiation~ Previous studies have shown that RNA transcription of ras genes is detected with little variation at all stages of mouse embryonic development (Adamson, 1987; M/Jller et al., 1982). These data are not informative as to the possibilit3" of post-transcriptional modifications occurring during differentiation. On the other hand, multiple forms of ras proteins can be detected in normal as well as in transformed ceils using 2-1) gel analysis (Feurstein et al., t985). While the s;~g~';~cance of this heterogeneity is currently unknown, it has been postulated to reflect the existence of subtle charge differences in products encoded by the same gene. In addition, the combined use of anti-ras monoclonaI antibodies with different specificities makes it possible to distinguish between produ~:~c encoded by N-ras, Ha-ras and Ki-ras genes. R a s proto-oncogenes are believed to be mairdy involved in cell proliferation (MiiUer et al., 1982). In the case of PC12 cells, a correlation between ras expression and differentiation has also been reported (Bar-Sng/et aL, 1985; Hagag et aL, 1986; Noda et aI., 1985). We describe here a different situation in which both undifferentiated (EC) and comnfitted or more differentiated cells are maintained in a permanent state of proliferation due to the expres-
L L ' V E A G E - S ' P E C I F I C PATITERb:7~ O F p2Z =as P R O T E I N S
53
sion of the SV40 T antigen. It follows th~. any modification in ras prote!n patterns is likely to be correlated with the differentiation state rather ~han with proliferation. The presence of Ki-ras transcripts has been reported in several EC cells including F9 cells (Sejersen et aL, 1985; Vilette et al., 1.987). In our h a n d s repeated tests failed to detect any traces of p21Ki-r~s proteins in the teratoca.rcinoma cell extracts whereas, under the same experimental condkions: ~21:cras proteins were detected in various control cell lines. These include a cd! line, Calu-1, derived from a lung cancer, S%V488 colopAc cancer ce~s and br: ~.: adenoc~r~bo,~as ~?~!arern e; ~i.~ in ~z~.paration). Tl~,~refore, .our resuks pOii[].t to post-transcriptional control of Ki-~as expression in tera~ocarcino-ma cetl-~ Alternatively, it is not excluded that p21 m - ~ levels in EC cells are b d o w detection levels. Our data show tha" the complex patterns of ?v=ras and Ha-ras promins observed in EC cells are not influenced by SV40 transfon-nation and become simpler as differentiation proceeds. It is noteworthy that neure-ctoderm, mesoderm and endoderm derivatives generate different and char~:cter~s:.[c p21 ras patterns. In addition, for a given lineage, there is no ap~:rec!ab~e change in a part[cular pattern during further differentiatiom These data strongiy suggest that specific patterns of p2i cas are installed at a very ear!y step e f differentiation toward each lineage and are then maintained in full:,- d.iffere~tiated cells. Given. this situation, such patterns could prove useful as differentiation markers. Because of its presence in all cell lineages, N-ras (or a closely related geae product) does not seem to be associated with a specific differentiation s~ in contrast, the other spots, which we attribute to He-ras-gene expression on the basis of their acidic behaviour appear different in ce~s" from differen~ lineages, and might therefore fulfill specific tGn.ctions.
:A~.~ALY~E C O M P A K % T I V E E N G E L B[~LVENSI05 }~7[ DES PROTE[NSS p~l r~S DAN£. DES L[ON~ES [.~,[Z-~.[ORTA~ISEESDEKiVEES D U TERA'; 0 C ,~[ C iINO~ [~. D [ [ 0 ~ ]E[~:
L'expression des prot6ines cod4es par Ia famili.e d'oncog~n:s ra:~ a ~t6 ar/,al}?s,~-~e clans los carcinomes embryonnaires (EC) et dans des Iign6e.sce~iulaires immo~a!~s@s qui en d~rivent. Cos li~6es qui correspondent ~ des s.tades pr6cozes de diffBrendatiom expriment l:antig~ne T de SV.40et continuen'~ ~ prolif&er. L'4le~rophorBs¢ Ndimensiennelle (2-D) d'immuncomplexes form6s avee des anticorps monoci~;na~; anti-ras, a permis de distinguer les produits des g~r~es rgs .e~ de corr~Ier ~e~.:~ro,SJs observ4s ave¢ I'6tat de diff~renciaticn des cellules. Nos r4sultats indiqucnt (1) que la distribution en eTectrophorBse 2.D des pro~b nes ras est identique dans plusieurs Iign~es EC et n'est pas modifi6e pa~ I'expresskm des oncog6nes de SV40, (2.) que Ia prot~ine p2t ~ s n~es~ pas d@6I&t dans los ceHu~ los EC alors que ie transcrit correspondant a 6t6 mis en ~vidence darts certamcs d.e cos tign~es et (3) queta ~rtographie des prot~ines Ha- and ?~Cras, complexe dans
54
M..-J. V I L A R E M E T A L .
les EC, se simplifie dans los d6riv~s diff6renci6s, et que la distribution est diff6rente darts les Iign6es neuroectodermiques, m6sodermiques et endodermiques. L'analyse biochimique des p21 ~aspourrait contribuer/t l'identification des stades pr6coces de la diff6renciation. MOTS-CLES: Oncogbne, Prot6ine p2t r,s T6ratocarcinome; Souris, Electrophor~se bidimensionnelle, Diff6renciationo
ACKNGWLEDGEMi~NTS This work has been aided by grants from the ARC, Centre National de la Recherche Scientifique and Ligue Natienale Fran~aise contre le Cancer.
REFERENCES
ADAMSON,E.D. (1987), Oncogenes in development. Development, 99, 449-471. BAR-SAGI,Do & FEgAMISCO,J.R. (1985), Microinjection of the ras oncogene protein into PC12 cells induces morpiaological differentiation. Cell, 42, 841-848. BELL, J.C., JARDINE,K. & McBumqEV,M.WS. (1986), Lineage-specific transformation after differentiation of mu!tipotential murine stem cells containing a human oncogene. MoL Cell n2oL, 6, 617-625. BERNSTtNE, E.G., HOOPER, M.L., GRANCHAMr',S. & EPHgUSSt, B. (1973), Alkaline phosphatase activity in mouse teratoma. Proc. nat. Acad. Sci. (Wash.), 70, 3899-3903. Buc-CARON, M.H., LAUNAY,J.M., LAMBLIN,D. & KELLEgMANN,O. (1990), Serotonin uptake, storage and synthesis in an immortalized committed cell line derived from mouse teratocarcinoma. Proc. nat. Acad. Sci. (Wash.) (in press). Buss, J.E., DER, E.J. & SOLSKI, P.A. (I988), The six amino-terminal amino acids of p60 src are sufficient to cause mycistylation of p21 v-ras. MoL Cell. BioL, 8, 3969-3963. D~R, C.J., WEISSMAN,B. & MACDONALD,M.J. (1988), Altered guanine nucleotide binding and H-ras transforming and differentiating activities. Oncogene, 3, 105-112. FEUERSTEIN, N. & AL~, I.U. (1985), Comparative analysis of p21 p;oteins from various cell types by two-dimensional gel etectrophoresis. J. Cell. Bioehem., 29, 253-263. FURTH, M.E., DAVIS, L.J., FLEtJrtDELYS, B. & SCOLNIC~, E.H. (1982), Monocional antibodies to the p21 products of the transforming gone of Harvey murine sarcoma virus and of the cellular ras gone family. J. Virol., 43, 294-304. GIBBS,J.B., SIGAL,i.S., PoE, M. & SCOLNICK,E.M. (1984), Intrinsic GTPase activity distinguishes normal and oncogenic ras p21 molecules. Proc. nat. Acad. Sci. (Wash.), 81~ 5704-5708. HAGAG,N., HALEGOUA,S. & VIOIA, M. (1986), Inhibition of growth factor-induced differentiation of PC12 cells by microinjectionof antibody to ras p21. Nature (Lond.), 319, 680-682. KELLEgMANN, O. & Ed~LLY, F. (1986), Immortalization of early embryonic cell derivatives after the transfer of the early region of simian virus 40 into F9 teratocarcinoma cells. Differentiation, 32, 74-81. KELLERMANN,O., BuC-CARON, M.H. & G~.ILLARD, J. (1987), Immortalization of precursors of endodermal, neuroectodermal and mesodermal lineages, following the introduction of the slw_ianvirus (SV40) early region into F9 cells. Differentiation, 35, 197-205.
LINEAGE-SPECIFIC PATTERNS
O F p 2 I ras PROTEi'~qS
55
KELLERMANN)O., Buc-CARON, M.H., MARIE,P.J., LAMBLIN,D. & JACOI3~F. (I990), An immortalized osteogenic cell line derived from mouse teratocarcinoma is able to mineralize in vivo and in vitro. Z Cell Biol., !10 (in press). McBoRNEY, M. k1976), Clonal lines of teratocarcinoma cells in vitro: differentiation and cytogenetic characteristics. J. Cell Physiol., 89, 441-456. MAGEE, A.Y., GUTIERREZ,L., McKoY, I.A., MARSHALL,C.J. • HALL, A. (lq87), Dynamic fatty acylation of p21N-ras. E M B O J., 6, 3353-3357. MARTIN, G.R. (J975), Teratocarcinoma as a model system for the study of embryogenesis and neoplasia. Cell, 5, 229-243. I~gII]LLER,R,, SLAMON,D.J., TREMBLAY,J.M., CLINE, M.J. & VERMA,I. (1982), Differential expression of cellular oncogenes during pre- and postnatal development of the mouse. Nature (Lond.), 299, 640-644. NODA, M., Ko, M., OGURA, A., LIU, D., AMANO, T., TAKANO,T. & IKAWA,Y. (1985), Sarcoma vir)Jses carrying ras oncogenes induce differentiationassociated properties in a neuronal cell line..Nature (Lond.), 317, 73-75. SEJERSEN, T., SOMEGI,J. & RINCERTZ, N.R. (1985), Expression of cellular oncogenes ;.r~ reratoma-derived cell lines. Exp. Cell Res., 160, 19-30. V1LETTE, D., EMANOIL-RAVIER,R., BtreFE, D., RIMBAUT,C. (~ PERtES, J. (i987), YCl-ras gene amplification and malignant behavior in _rnurineembryonal carcinoma cell lines. Cancer Res., 47, 867-873.