Transcription simian DNA-transformed .Transcriptionofthesimianvirus40genomeinDNA-transformed murine
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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 -70C;aliquots containing 1.5 x 107 cpm of acid-insoluble label react-ed for 1 hr at 4C 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 4C. 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.
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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 nitroce