Proc. Natl Acad. Sci. USAVol. 78, No. 10, pp. 6386-6390, October 1981Genetics

Transcription of the simian virus 40 genome in DNA-transformedmurine terato.carcinoma. stem, cells

(cell differentiation/gene regulation/tumor antigen/RNA blotting/SI nuclease duplex analysis)

ALBAN LINNENBACH, KAY HUEBNER, AND CARLO M. CROCEThe Wistar Institute of Anatomy and Biology, 36th Street at Spruce, Philadelphia, Pennsylvania 19104

Communicated by Hilary Koprowski, July 20, 1981

ABSTRACT To study the molecular basis for lack of expres-sion of the simian virus 40 (SV40) early region genes in murineteratocarcinoma-derived stem cells, we introduced a recombinantplasmid consisting of pBR32.2 linked to the herpes simplex virustype 1 thymidine kinase gene and SV40 genome into thymidinekinase-deficient F9 stem cells. The resulting stem cell clone, 12-1, and a retinoic acid-induced differentiated daughter cell clone,12-la, each contain one copy per cell of the entire recombinantplasmid integrated into the cellular genome through a site on thepBR322 genome. Restriction endonuclease analyses indicate thatthere is no difference in integration site or organization of thethree component parts ofthe plasmid genome within cellular DNAofstem and differentiated cells; yet the differentiated cells, 12-la,express SV40 large tumor antigen whereas the stem cells, 12-1,do not. Both stem and differentiated cells produce two size classesof polyadenylylated RNA, 2900 and 2600 bases in length, homol-ogous to the early region of the SV40 genome, detectable by RNAblotting analysis. S1 nuclease analysis ofthe SV40 transcripts pres-ent in stem and differentiated cells indicate that the SV40 mRNAswere identically spliced in the two cell types, in a manner con-sistent with that observed for spliced large and small tumor an-tigen mRNAs in SV40-infected monkey kidney cells. Thus, the fail-ure of 12-1 teratocarcinoma stem cells, containing an integratedSV40 genome, to express SV40 tumor antigen is not due to a lackof transcription of the SV40 early region or to an inability to spliceprimary transcripts.

Developmentally pluripotent (1-4) teratocarcinoma stem cellswhich, by several criteria, seem to be equivalent to cells of theinner cell mass of the mouse blastocyst (5) are able to restrictexpression of oncogenic viral genes (6-11) that are expressedin teratocarcinoma-derived differentiated cells. Because mo-lecular mechanisms operative in regulation ofexpression of viralgenes in this differentiating model system may be analogous tomechanisms involved in gene regulation during embryogenesis(5), it is important to define the molecular basis for-suppressionof expression ofviral genes in teratocarcinoma stem cells. Stud-ies on the resistance of stem cells to infection by simian virus40 (SV40) have shown that the block is not at the level of virusadsorption, penetration, uncoating, or transport to the nucleus(7). When F9 stem cells were infected with SV40, low levels ofunspliced early viral RNA were detected (12, 13). However,investigation of molecular events associated with expression ofviral genes in infected cells is complicated by the lack ofknowl-edge of the number of copies and the state of integration andorganization of the viral genes within stem and subsequent dif-ferentiated cells (14).We have taken advantage of the availability of thymidine

kinase-deficient (TK-) F9 cells (15), a homogeneous stem cellline that differentiates into endodermal cells after exposure to

retinoic acid (16-21), to introduce the SV40 genome into eachcell ofa stem cell line by transfection with a herpes simplex virustype-i thymidine kinase (HSV-1 tk) vector (22). The DNA-transformed F9 stem cells, 12-1, which carry a single integratedcopy of the SV40 genome, are, by criteria thus far tested. (23,24), phenotypically identical to the F9 parental cell except thatthey produce HSV-1 tk. A retinoic acid-induced daughter cellclone, 12-la, which expresses SV40 early gene products (22, 23),has also been isolated and used in conjunction with the 12-1stem cell which does not express SV40 gene products (22, 23),to investigate the molecular basis for the differential expressionof SV40 large tumor antigen (T antigen) in murine teratocar-cinoma-derived stem and differentiated cells.

MATERIALS AND METHODSCells. TK- F9 cells (15) were transfected with the recom-

binant plasmid pC6 (pBR322/HSV-1 tk/SV40) (22), and a trans-formed stem cell colony, 12-1, was isolated. The differentiatedcell clone, 12-la, was isolated after retinoic acid treatment of12-1 cells. Methods for isolation, maintenance, and character-ization of the stem and differentiated cell lines have been re-ported (22, 23). An SV40-transformed monkey kidney cell lineT22 TK- (ref. 25; unpublished data) was used in immunopre-cipitation experiments.

Immunoprecipitation. Subconfluent cell cultures were washedwith prewarmed methionine-deficient medium supplementedwith 5% dialyzed fetal bovine serum and then incubated in thesame medium for 2 hr. L-[3S]Methionine (50 ACi/ml; 400 Ci/mmol 1 Ci = 3.7 X 1010 becquerels; New England Nuclear) wasadded to each culture, and the cells were incubated for another4 hr. The cells were then chilled and lysed in 0.5% Nonidet P-40/50 mM Tris, pH 8.0/8 mM EDTA/0.6 M NaCI/phenyl-methylsulfonyl fluoride (0. 3 mg/ml) [lysis buffer (26)] for 20 minwith occasional mixing. Lysates were then sonicated four timesfor 5 sec each.

SV40T antigen was immunoprecipitated from the cell lysatesby the double-antibody method described by Hughes and Au-gust (27). Briefly, lysates were clarified by centrifugation at100,000 x g (1 hr; 40C) and supernatants were stored at -70°C;aliquots containing 1.5 x 107 cpm of acid-insoluble label react-ed for 1 hr at 4°C with monoclonal anti-T antigen antibody (28)(1:1000 final dilution) or with nonimmune ascites fluid (1:1000)in a 100-,ul reaction mix containing gelatin (1 mg/ml) and lysisbuffer. Rabbit anti-mouse immunoglobulin (25 ,u1; Bio-Rad) wasthen added and the reaction was allowed to proceed overnightat 4°C. Precipitates were washed three times with 2.5 ml of 20mM Tris, pH 7.6/1 mM EDTA/0.1 M NaCl, 2.5 M KC1/O.5%

Abbreviations: HSV-1 tA, herpes simplex virus type 1 thymidine kinasegene; kb, kilobase(s); kbp, kilobase pair(s); SV40, simian virus 40; Tantigen, large tumor antigen; t antigen, small tumor antigen; TK-, thy-midine kinase deficient.

The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance-with 18 U. S. C. §1734 solely to indicate this fact.

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Proc. NatL Acad. Sci. USA 78 (1981) 6387

Nonidet P40 by centrifugation at 2000 X g for 20 min. Precip -itates were then washed once in 20 mM Tris, pH 7.6/1 mMEDTA/0. 1 M NaCl, 0.5% Nonidet P-40/0.5% sodium deoxy-cholate/0. 1% NaDodSO4 and centrifuged as before. Pelletswere dissolved in 50 A.l ofNaDodSO4 sample buffer (29), boiledfor 1.5 min, and electrophoresed on NaDodSO412% poly-acrylamide gels; molecular weight standards were included oneach gel. Gels were dried and autoradiographed to locate im-munoprecipitated proteins.DNA Transfer and Southern Analysis. Stem and differen-

tiated cellular DNAs were extracted, digested with BamHI andXba I, and analyzed by the Southern transfer method (30) usingnick-translated (31) 32P-labeled SV40 DNA as described (22).

Isolation of RNA. Total cellular RNA was prepared by cen-trifugation through cesium chloride (32) or by hot phenol ex-traction (33). Cytoplasmic RNA was obtained by 0.65% NonidetP40 lysis of cells in the presence of ribonucleoside-vanadylcomplexes, followed by five extractions with phenoVchloro-form/hydroxyquinoline (34). RNAs were dissolved in distilledwater (15 mg/ml) and stored at -200C. Poly(A)+ mRNAs wereobtained from total RNA preparations by two cycles ofoligo(dT)-cellulose chromatography (35, 36).RNA Transfer and Blotting Analysis. Stem and differen-

tiated cell RNAs and marker DNAs were denatured with deion-ized glyoxal and dimethyl sulfoxide, electrophoresed on an agar-ose gel, and blotted onto nitrocellulose filters by usingconditions described by Thomas (37). 32P-Labeled SV40 DNAprobes (specific activity, 1.5 X 108 cpm/,Ag) were prepared bynick-translation (31); reactions were terminated by addition ofNaOH to 0.3 M and boiling for 2 min prior to Sephadex G-50chromatography. Formamide was deionized (38) and stored at-200G. Nitrocellulose filters were hybridized with the probefor 36 hr at 37C and prepared for autoradiography as described(37).

SI Nuclease-Resistant Duplex Analysis of RNAs. SplicedSV40 early mRNAs were detected by the method of Berk andSharp (39) which was adapted to include Southern transfer andhybridization with nick-translated 32P-labeled SV40 DNAprobes. Aliquots (0.2 pig) of unlabeled EcoRI-digested SV40DNA were mixed with 100 pug of total cellular RNA in a 1.5-mlEppendorf tube, brought to 0.15 M in sodium acetate (pH 6.0),and ethanol precipitated. The pellets were dissolved in 16 1Iof deionized formamide, to which 4 A.l of a 5-fold concentratedhybridization buffer (39) was added. Denaturation was carriedout by complete submersion in an 850C water bath for 15 min(40) followed by immediate submersion in a 490C bath for a 3-hr hybridization (39). Twenty volumes of chilled S1 nucleasebuffer (41) containing 100 units of S1 nuclease (Sigma, type III)was added to each tube as described (40), followed by incubationat 37C for 30 min. Reactions were terminated by addition of0.2 vol of 0.5 M Tris, pH 9.5/0.1 M EDTA (42), and the mix-tures were divided into two parts. Carrier tRNA (20 ,g) wasadded to each sample before precipitation with 2.5 vol ofethanol. One set of samples was prepared for fractionation onan alkaline agarose gel; the other was prepared for a neutral gel(39). The gels were prepared for transfer to nitrocellulose filtersin the same manner described for Southern DNA transfer (30),except that it was unnecessary to denature the alkaline gel afterelectrophoresis. The nitrocellulose filters were hybridized to32P-labeled SV40 DNA, washed, and autoradiographed.

RESULTSImmunoprecipitation of SV40 T Antigen from 12-1 and 12-

la Cells. The F9-derived teratocarcinoma stem cell line waspreviously found not to express SV40 T antigen by an indirectimmunofluorescent assay using monoclonal and polyclonal anti-

SV40 T-antigen antibodies (22, 23). The retinoic acid-induceddifferentiated daughter cell line was found to be 100% positivefor SV40 T antigen by the same assay (22, 23). Similarly, SV40-specific tumor-associated surface antigen was detected on 12-la differentiated cells but not on 12-1 stem cells (23). In orderto confirm these previous results, monoclonal antibody to SV40T antigen was used to immunoprecipitate protein from cell ly-sates of 12-1 and 12-la cells. Immunoprecipitated products fromthe two teratocarcinoma-derived cell lines and an SV40-trans-formed monkey kidney cell line, T22 TK-, were separated bypolyacrylamide gel electrophoresis. The 94,000-dalton T anti-gen was precipitated from the T22 TK- SV40-transformed mon-key kidney cells and the 12-la differentiated cells by the anti-SV40 T antigen monoclonal antibody but not by the nonimmuneserum (Fig. 1). Neither SV40 T antigen nor any other proteinwas precipitated from 12-1 stem cells by anti-SV40 T-antigenmonoclonal antibody.

Organization of the SV40 Genome in 12-1 and 12-la Cells.The pC6 recombinant plasmid (22) was constructed by insertionof a BamHI cleaved SV40 genome into one of the two BamHIsites ofthe pBR322/HSV-1 tk plasmid, pHSV-106. Because thepHSV-106 plasmid (43) was constructed by insertion of the 3.4-kilobase (kb) HSV-1 BamHI fragment, containing the HSV-1tk gene, into the BamHI site ofpBR322, BamHI cleavage ofpC6cleaves the tripartite plasmid genome into its three componentparts, the 4.3-kilobase-pair (kbp) pBR322 genome, the 3.4-kbpHSV-1 tk gene, and a 5.2-kbp linear SV40 genome. We pre-viously reported (22) that the 12-1 stem cell line contains onecopy per cell of the pC6 DNA integrated into the cellular ge-nome through a site on the pBR322 genome. Thus, the 3.4-kbpHSV-1 tk and 5.2-kbp SV40 genomes remain intact and adjacentto each other with pBR322 sequences flanking them on eitherside within the 12-1 cellular genome.

In order to determine whether this arrangement of the in-tegrated plasmid genome remained unchanged during the pro-cess ofdifferentiation, we cleaved cellular DNA from 12-1 cells,

M S D1P3 o[P3 &tJ[ CTfl

. -94

-54

1 2 3 4 5 6

FIG. 1. Immunoprecipitation of SV40T antigen from 12-1 and 12-la cells. [3S]Methionine-labeled extracts from SV4O-transformedmonkey kidney cells (M), 12-1 stem cell (S), and 12-la differentiatedcells (D) were reacted with monoclonal anti-SV4O T antigen antibody(aT) or with nonimmune antibody (P3) and precipitated by additionof rabbit anti-mouse immunoglobulins. The 12% polyacrylamide gelwas fluorograrhed, dried, and exposed for 7 days. Size markers aredaltons x10- .

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6388 Genetics: Linnenbach et aL

q-- V-

I- q- I-

I- V-I _N4 CMCN- i Ce

I-

15.0 --_

5.2-

1 2 3 1 2 3FIG. 2. Arrangement of the SV40 genome in stem and differen-

tiated cells. CellularDNA from 12-1 cells, from 12-1 cells after 12 daysof exposure to retinoic acid, and from 12-la cells was cleaved with XbaI (Left) and BamHI (Right), electrophoresed, transferred to nitrocel-lulose filters, and hybridized to SzP-labeled SV40 DNA. Sizes areshown in kbp.

12-1 cells exposed to retinoic acid for 12 days, and 12-la cellswith Xba I (an enzyme that does not cut within the pC6 plasmidgenome) and with BamHI. The cleaved DNAs were separatedelectrophoretically, transferred to nitrocellulose filters, andhybridized to 32P-labeled SV40 DNA. DNA from the three celltypes cleaved by Xba I (Fig. 2 Left) exhibited a single 15-kbpband containing DNA homologous to SV40. We previouslydemonstrated (22) that the pBR322 and HSV-1 tk sequences arepresent in this same 15-kbp Xba I fragment. After BamHI cleav-age of the DNA from the same three cell types (Fig. 2 Right),a single 5.2-kbp band, the same length as BamHI linearized

S D

Bases

'_ 2900I 2600

1 2 3 4FIG. 3. Total SV40 RNA in 12-1 and 12-la cells. Poly(A)+- and

poly(A)-RNA from 12-1 stem cells (S) and 12-la differentiated cells(D) were denatured, separated electrophoretically, transferred to ni-trocellulose filters, and hybridized to 3zPlabeled SV40 DNA. Lanes 1and 3, 10 pZg of poly(A)-RNA; lanes 2 and 4, 1 jg of poly(A)+RNA.

SV40 DNA, was detected in each of the three cell types afterhybridization with 32P-labeled SV40 DNA. We concluded thatno gross rearrangement in the plasmid genome occurred inthese cells during the process of differentiation.

Transcription of the SV40 Genome in 12-1 and 12-la Cells.Total RNA was extracted from 12-1 stem and 12-la differen-tiated cells, selected by oligo(dT)-cellulose chromatography,glyoxalated, separated by agarose gel electrophoresis, andtransferred to nitrocellulose filters (37). Hybridization with 3P-labeled SV40 DNA detected a 2.9-kb and a 2.6-kb mRNA in thepoly(A)+mRNA fractions of both stem and differentiated cells(Fig. 3). The observed sizes of these two SV40 transcripts arein agreement with those found early after infection of monkeykidney cells with SV40 DNA (44). When total cytoplasmic RNAfrom stem and differentiated cells was analyzed, the same twoSV40 RNAs were detected in the 12-1 stem cell and, as ex-pected, in the 12-la differentiated cell (Fig. 4). Interestingly,the 2.9-kb RNA species, which is the size of small tumor (t an-tigen) mRNA, is present in stem cells in larger amounts thanthe 2.6-kb species, which is the size of T antigen mRNA; thereverse is true for the differentiated cells.

SI Nuclease Analysis of SV40 Transcripts. The sizes of theSV40 RNAs we detected in the stem cells (Figs. 3 and 4) weresuggestive of mature SV40 transcripts. A direct demonstrationof spliced SV40 RNAs was carried out according to the tech-nique developed by Berk and Sharp (39) modified as outlinedin Fig. 5.

Alkaline agarose gel electrophoresis of S1 nuclease-resistantduplexes, Southern transfer to nitrocellulose filters, and hy-bridization with 32P-labeled SV40 DNA, revealed a 1.9-kb frag-ment in the 12-1 stem cells (Fig. 6a, lane 2 and 6b, lane 1) and

N CNT- qr-

Bases

, _.-2900* ~-2600

1 2FIG. 4. Cytoplasmic SV40 RNA (10 ,Hg/lane) from 12-1 stem and

12-la differentiated cells, electrophoresed, blotted, and hybridized to32P-labeled SV40 DNA.

Proc. Nad Acad. Sci. USA 78 (1981)

Proc. Natd Acad. Sci. USA 78 (1981) 6389

transformed cellRNA

denaturation

hybrid

EcoRl SV40DNA

Ization 1

large T-antigenmRNA

autoradiography

FIG. 5. Outline of method used for analysis of 51 nuclease-resis-tant duplexes obtained after hybridization of 12-1 and 12-la cellularRNAs with linear SV40 DNA. Sizes of the resistant SV40 duplexesshown are those described by Berk and Sharp (39).

in the 12-la differentiated cells (Fig. 6a, lane 3). The 650- and350-base leader fragments were not detected under the ex-perimental conditions used. The 1.9-kb fragment is common

M S D SBases

5226 -

4010 -

3040-

21861768 -

1216-i 691101

to both T and t antigen mRNAs (39) and can occur only if thecellular RNA contained spliced SV40 RNA.

Splicing of the SV40 RNA in teratocarcinoma-derived stemcells was confirmed by neutral gel electrophoresis of intact S1nuclease-digested DNARNA hybrids. The observed 2250- and2550-base-pair duplexes (Fig. 6c, lane 2) are identical to thoseobserved for spliced T and t antigen mRNAs in SV40-infectedmonkey kidney cells (39).

DISCUSSIONTaking advantage of advances in the understanding of controlof teratocarcinoma stem cell differentiation, coupled with thepowerful tools of molecular biology, we have exploited the F9cell line, which does not undergo significant spontaneous dif-ferentiation but can be induced to differentiate into endodermalcells, to investigate molecular events involved in the control ofSV40 gene expression in stem cells. Transfection ofthe F9 TK-cell line with a recombinant plasmid consisting of the SV40 ge-nome, the HSV-1 tk gene, and the pBR322 genome has resultedin isolation of a stem cell clone, 12-1, which has a single copyper cell of the SV40-containing plasmid integrated into chro-mosomal DNA (22). SV40 gene products are undetectable inthis stem cell line (refs. 22 and 23; this report) whereas, afterinduction with retinoic acid, the differentiated cells expressSV40 early gene products (refs. 22 and 23; this report) in parallelwith major histocompatibility antigens H-2 and /32-microglob-ulin (23, 24) and basement membrane proteins (23). Using the12-1 stem cell line and a differentiated daughter cell line, 12-la, we have investigated transcription of the SV40 genome.Although SV40 T antigen is undetectable in the stem cell clone,12-1, and is easily detectable in the differentiated clone, 12-la,by immunofluorescence and immunoprecipitation, the SV40genome is transcribed in both cells. Two SV40-specific RNAsof the sizes expected for SV40 T and t antigen mRNAs are pro-duced in both cell types. In addition, SV40 splicedpoly(A)+mRNAs, also of the sizes expected for T and t antigens,are present in the cytoplasm of both cell types.The simplest interpretation of these results is that stem cells

that carry integrated SV40 genomes are capable ofsynthesizing

M SBases

a

.. _~~~~~~~~~~~~~~~.---

.w . - 1900

1 2 3 1a

Alkalineb

5226 -

4010- -

3040- -

2186----1768-1216-1169 ----1101

-4-. - 2550-2250

1 2

c

Neutral

FIG. 6. Spliced SV40 mRNA in 12-1and 12-la cells. Total cellular RNA from12-1 stem (S) and 12-la differentiated (D)cells was hybridized to EcoRI-cleaved SV40DNA, treated with S1 nuclease, electro-phoresed on alkaline or neutral gels, blot-ted, and hybridized with 32P-labeled SV40DNA. (a and b) Results after alkaline gelelectrophoresis; (c) results after neutralgel electrophoresis. Marker lanes (M) con-

tained SV40 DNA fragments prepared bydigestions of SV40 DNA with BamHI, PstI, HindIu, and BamHI/Hpa II; lanes la-beled S contained duplexes derived from12-1 RNA; lanes marked D contained du-plexes derived from 12-la RNA.

EcoRI SV40 DNA

EcoRlSV40 DNA

650 1900

small T-antigenmRNA

660 1900 1900- SI nucleuse

Alkaline gel Neutral gel

I I- 1900

660-39

denaturation

Ineutralization neutralization

transfer to nitrocellulose

=P-SV4O DNA hybridization

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6390 Genetics: Linnenbach et aL

authentic early SV40 mRNAs and that lack ofexpression ofSV40early region genes is due to a posttranscriptional event. In vitrotranslation of SV40-specific mRNAs from stem cells will deter-mine whether the block to SV40 T antigen expression in stemcells occurs at the translational level or if the mRNAs producedin stem cells are defective. Segal et a. (12) have reported thatlack of expression of SV40 T antigens in SV40-infected F9 cellswas due to posttranscriptional control. In their case, however,they detected a small amount ofunspliced RNA homologous tothe SV40 early region and no spliced SV40 mRNA. Becausemost of the exogenously added SV40 DNA in infected F9 cellsis probably not integrated into chromosomal DNA, it is possiblethat the expression of free viral DNA is regulated differentlyfrom that of integrated SV40 genomes.We have shown previously, using the same methods for de-

tection of RNA and the same 12-1 stem cells, that no RNAshomologous to H-2 and ,f 2-microglobulin genes are detectablein stem cells, it is clear that the teratocarcinoma-derived stemcells exert control over gene expression by at least two differentmechanisms: one transcriptional and one posttranscriptional.We thank Lynette Miles for excellent technical assistance. This work

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