MOLECULAR AND CELLULAR BIOLOGY, Jan. 1987, p. 556-559 Vol. 7, No. 10270-7306/87/010556-04$02.00/0Copyright © 1987, American Society for Microbiology

Induction of c-Ha-ras Transcription in Rat Cells by Simian Virus 40Large T Antigen

KAORU SEGAWAt* AND NOBUO YAMAGUCHIDepartment of Virology, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokane-dai, Minato-ku,

Tokyo 108, Japan

Received 18 June 1986/Accepted 13 October 1986

Rat 3Y1 cells expressing simian virus 40 large T antigen under the control of the mouse mammary tumorvirus long terminal repeat were established. The amount of c-Ha-ras mRNA in those cells was elevated by about20 times in parallel with large T antigen after exposure to dexamethasone for 48 h. In chloramphenicolacetyltransferase assays with a plasmid containing the c-Ha-ras-1 promoter the increase in c-Ha-ras mRNA wasshown to occur at the transcriptional level.

Transformation of cells by simian virus 40 (SV40) involvesmany complex alterations in biological and biochemicalproperties (19). These changes in cellular growth control andbehavior are presumably mediated by perturbations in nor-mal patterns of metabolism and gene expression. A numberof individuals have analyzed the changes between trans-formed and untransformed parental cells (reviewed in refer-ence 19). However, it was not clear whether these changeswere the main cause of transformation by SV40 or secondaryevents induced after the transformation. If one could regu-late the amount of large tumor (T) antigen (Ag) in cell clonesand analyze the changes induced in parallel with the amountsof large T Ag, these cell clones could be a good tool forexamining the phenomena closely related to the transform-ing function of SV40 large T Ag. For this purpose, weconstructed a plasmid (pMMWT-LN) which can expresslarge T Ag in a hormone-dependent manner (Fig. 1). Byusing rat cells carrying plasmid pMMWT-LN, we examinedcellular oncogene expression as a first step in analyzing thechanges induced by SV40 large T Ag.Rat 3Y1 cells (8) were transfected with the constructed

plasmid pMMWT-LN, and G418-resistant colonies werepicked 14 days after transfection. For isolation of cell linesexpressing large T Ag in response to dexamethasone (Dx),dot blot analysis ofRNA from the above-mentioned colonieswas carried out as described previously (10). Some coloniesexpressed the SV40 large-T-Ag gene irrespective of thepresence of Dx (data not shown). Clones which couldexpress the large-T-Ag gene in a Dx-dependent manner werenamed MM3Y cells. MM3Y cells grew to slightly highersaturation densities in monolayer cultures than did parental3Y1 cells. However, they could not form colonies in theagarose medium (Fig. 2C). Semiconfluent MM3Y cultureswere incubated with or without 1 ,uM Dx for 48 h, andpoly(A)+ RNAs were prepared. Northern blot analyses withSV40 DNAs as probes are shown in Fig. 2A. The main bandswere doublets of about 2.8 and 2.5 kilobases (kb), corre-sponding to the sizes of mRNAs for small and large T Agsstarted in the mouse mammary tumor virus long terminalrepeat (LTR), respectively. The amounts of large plus smallT Ag mRNAs in MM3Y clone 8 (MM3Y-8) and MM3Y clone12 (MM3Y-12) cells in the presence of Dx were 25 and 30

* Corresponding author.t Present address: Department of Microbiology, Keio University

Medical School, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan.

times greater, respectively, than those in the control culturein the absence of Dx, as measured by counting the radioac-tivities of the corresponding bands. For immunoprecipita-tion of large T Ag (14a), semiconfluent cells were cultivatedwith or without 1 puM Dx for 48 h and then labeled with[35S]methionine for 6 h. The expression of SV40 large T Agin the presence of Dx was increased by 30 to 40 times, asmeasured by counting the radioactivities (Fig. 2B). p53 wasalso immunoprecipitated proportionally with increasingamounts of large T Ag. Small T Ag could not be detected inthis experiment. MM3Y-8 cells could form colonies in theagarose medium containing Dx (Fig. 2C). The number ofcolonies with diameters greater than 0.05 mm was more than10 times as large in the presence than in the absence of Dx(Fig. 2C). Similar growth properties were observed forMM3Y-12 cells (data not shown). In contrast, parental 3Y1cells could not grow in the agarose medium, irrespective ofthe presence of Dx (Fig. 2C). These results indicate thatMM3Y cells show a conditional transformed phenotype in

*1

PBR

aNEDH3

FMMWT-LN

MMTVH3

' TR

TAg MuSBhFIG. 1. Map of pMMWT-LN. Two Hindlll-BamHl fragments of

pMTl containing the SV40 early gene (17) and pMDSG (9) contain-ing the mouse mammary tumor virus (MMTV) LTR (M) wereligated. The resulting plasmid was named pMMWT. pMMWT-LNwas made by ligation of (i) the BamHI fragment containing the TnSgene conferring G418 resistance (15; indicated by "NEO") linked tothe Rous sarcoma virus LTR (pLTR-neo) with (ii) the linearizedpMMWT DNA BamHI fragment. In this plasmid, the transcriptionof SV40 large T Ag starts in the MMTV LTR region and terminatesat the regular position in the inserted HindIII-BamHI fragment ofthe SV40 genome. B1, H3, Ni, and RI, BamHI, HindlIl, NdeI, andEcoRI cutting sites, respectively. PBR, pBR322 DNA in theplasmid.

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FIG. 2. Expression ofSV40 largeNorthern blot analysis ofSV40 T Ag m]MM3Y-8 (lanes 1 and 2) and MM3Ycultivated in the presence (+) or absencwere prepared. To establish the relatimRNA, we fractionated 0.5,ug from eaccontaining formaldehyde and transferrecnitrocellulose filter (18). Hybridizationcontaining 10,uM N-tris(hydroxymesulfonic acid (TES) (pH 7.4), 0.2% sodiEDTA, and 300 mM NaCl at 65°C for 2washed in 2x SSC (lx SSC is 0.15 Mcitrate) at 37°C. The major bands corresp(2.8 kb) and large (2.5 kb) T Ag mRNArThe positions of the HindIlI fragmentscated by closed circles. The sizes of thes23.1, 9.4, 6.6, 4.4, 2.3, and 2.0 kb. Thefor 2 days. (B) Immunoprecipitation of1 and 2) and MM3Y-12 (lanes 3 andpresence (lanes 2 and 4) or absence (lan48 h were labeled with [35S]methioniniextracts were prepared as described eestablish the relative amount of large TXeach extract containing equal numb(precipitable [35S]methionine counts (10wtion with sera from hamsters bearing SThe samples were analyzed on 129%polyacrylamide gels. LT and p53 standtumor antigen p53, respectively. Molec

parallel with the amounts of large T Ag, at least in terms of1 2 3 4 B colony formation in the soft agarose medium. We did not

check whether MM3Y cells could induce tumors in- I ~ ~syngeneic rats with continuously administered Dx.

As a first step in identifying the genes under the control ofincreasing amounts of large T Ag in MM3Y cells, weexamined cellular oncogene expression. The runoff tran-

*f *- IT scribed mRNAs from MM3Y-8 cells cultivated in the pres-ence or absence of Dx were hybridized with plasmid DNAscontaining p53 and 19 viral oncogene DNA fragments: abl,_

u_-n53 erbA, erbB, fes, fgr, fms, fos, fps, myb, myc, mos, raf,_ _ 5r Ha-ras, Ki-ras, rel, ros, sis, src, and yes. Only the amount ofc-Ha-ras mRNA was found to be increased after exposure toDx (data not shown). The amount of c-Ha-ras mRNA inMM3Y cells was increased in parallel with that of large T AgmRNA, depending upon the dose of Dx (Fig. 3A). Theamount of ras p21 immunoprecipitated was also increased ina Dx-dependent manner (data not shown). In contrast, aDx-dependent increase in c-Ha-ras mRNA was not observedin parental 3Y1 cells (Fig. 3A). The level of c-Ha-ras mRNAbegan to increase at 24 h after exposure to 1 ,uM Dx andalmost reached a plateau 48 h after exposure (Fig. 3B). Toeliminate backgrounds in dot blot analyses, we carried outNorthern analyses (Fig. 3C). The results clearly indicatedthat the amount of c-Ha-ras mRNA was increased in Dx-treated MM3Y cells by about 20 times, as measured by

These phenomena in Dx-treated MM3Y cells could beexplained by the following mechanisms. First, c-Ha-rasgenes are amplified. Second, the transcription of c-Ha-rasgenes is enhanced. Third, the stability of c-Ha-ras mRNA isincreased. The first effect could be eliminated because the

+ copy number of the c-Ha-ras gene was the same before andafter exposure to Dx (data not shown). To check transcrip-tional activity, we cotransfected MM3Y-8 cells with plasmid

* pras-CAT-1, which contains six Spl-binding sites of thec-Ha-ras-1 promoter region (6), and plasmid pSV2gpt. Cellsresistant to the inhibitors (aminoputerin and mycophenolicacid) were isolated. The cell extracts from cell mixturescultivated in the presence or absence of Dx were subjectedT Ag in MM3Y cells. (A) to a chloramphenicol acetyltransferase (CAT) assay. The

RNA. Poly(A)+ RNAs from CAT activity in Dx-treated cell extracts was clearly higher'-12 (lanes 3 and 4) cells, than that in control extracts (Fig. 4, lanes 1 and 2). No CATce (-) of 1 lM Dx for 48 h, activity was detected in extracts transfected with the pro-,pive amount of large T Ag moter-less plasmid pSVO-cat (4) (Fig. 4, lanes 3 and 4). Ad the separated mRNAs toa higher level of CAT activity was detected in extracts fromwas carried out in buffer SV40 virion-transformed3Y1 cells (Fig. 4, lane 5), but there

thyl)methyl-2-aminoethane- was very weak CAT activity in parental 3Y1 cell extractsium dodecyl sulfate, 10 mM (Fig. 4, lane 6). These results indicated that the promoter-M4 h (5). The filter was then enhancer activity of the c-Ha-ras gene is enhanced in Dx-NaCl plus 0.015 M sodium treated MM3Y cells. Therefore, the second possibility seemsvond to the sizes of the small to be a major mechanism, although we cannot completely

started in the LTR region, eliminate the additional contribution of the third possibility.se(in order from thetop) are We isolated clonal cell lines which showed a conditionalfilterwas autoradiographed transformed state (colony formation in soft agarose medium)large T Ag. MM3Y-8 (lanes in a hormone-dependent manner (Fig. 2). The transcription4) cells cultivated in the

ies 1 and 3) of 1,M Dx fore (30 jCi/ml) for 6 h. Thelsewhere (13, 14, 14a). ToAg, we subjected aliquots ofers of trichloracetic acid-6cpm) to immunoprecipita-iV40-induced tumors (14a).sodium dodecyl sulfate-

for large T Ag and cellularcular weight markers (open

circles) are phosphorylase b (92,500), bovine serum albumin(67,000), ovalbumin (46,000), carbonic anhydrase (30,000), andcytochrome c (12,500). Small T Ag was not detected in thisexperiment. The expected position of small T Ag is indicated by aclosed circle. (C) Growth of MM3Y and3Y1 cells in the agarosemedium. MM3Y-8 (panels 1 and 2) and3Y1 (panels 3 and 4) cellswere seeded in 0.35% agarose medium in the presence (+) orabsence (-) of Dx and cultivated for 14 days.

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FIG. 3. Expression of the c-Ha-ras gene in MM3Y cells. (A) Dotblot analysis of c-Ha-ras and T Ag mRNAs. Poly(A)+ RNAs wereprepared from MM3Y-8 and 3Y1 cells treated with 0 ,uM (spots 1),0.05 p.M (spots 2), 0.5 p.M (spots 3), and 1 p.M (spots 4) Dx for 48 h.To establish the relative amounts of c-Ha-ras and large T AgmRNAs, we denatured 5.0 ,ug of each preparation in formaldehydeand applied it with suction to a 4-mm-diameter spot on a nitrocel-lulose sheet (BA45, 0.45-p.m-pore diameter) by using a 96-wellMinifold apparatus (20). The nitrocellulose sheets were baked andhybridized with the nick-translated, 450-base-pair EcoRI fragmentof plasmid pBS-9 which contains the v-Ha-ras gene-specific frag-ment of Harvey murine sarcoma virus (1) (indicated by H-ras) orSV40 DNA (indicated by TAg). Hybridization was carried out asdescribed in the legend to Fig. 2. The filter was autoradiographed for4 days. (B) Time course of the expression of large T Ag and c-Ha-rasmRNAs. MM3Y-8 cells were cultivated in the presence of 1 pM Dxfor 0 (dot 1), 24 (dot 2), 48 (dot 3), and 96 (dot 4) h. The same amount(5.0 p.g) of poly(A)+ RNA from the cultures mentioned in panel Awas spotted on a nitrocellulose membrane. The filter was processedas described in panel A. (C) Northern blot analysis of c-Ha-rasmRNA. Poly(A)+ RNA (5.0 ,ug) from each preparation mentioned inFig. 2A was run on a 1% agarose gel and transferred to a nitrocel-lulose filter. The filter was analyzed as described in the legend toFig. 2 by using the nick-translated, 450-base-pair fragment ofplasmid pBS-9 (1) as a probe. The lanes are the same as in Fig. 2A.The filter was autoradiographed for 7 days. The size of the majorband is 1.4 kb, calculated from marker X HindlIl fragments dena-tured by formaldehyde (closed circles). The sizes of these (in orderfrom the top) are 23.1, 9.4, 6.6, 4.4, 2.2, and 2.0 kb.

of the c-Ha-ras gene was enhanced by about 20 times inDx-treated MM3Y cells (Fig. 3 and 4). It is quite probablethat the enhanced transcription of the c-Ha-ras gene hassome role in the colony formation in soft agar medium of 3Y1cells induced by SV40 large T Ag, since transcription wasinduced shortly after exposure to 1 p.M Dx in parallel withthe induction of SV40 large T Ag (Fig. 3). In this context, itis interesting to note that Pulciani et al. reported that NIH3T3 cells can be transformed by c-Ha-ras p21 at elevatedlevels of expression (11). Although our results suggest thatthe enhanced expression of c-Ha-ras mRNA is closelycorrelated with the transformed state (colony formation insoft agarose medium) of MM3Y cells, it remains to beelucidated whether this enhancement is the primary targetfor the transforming function of SV40 large T Ag. To answerthis question, we must survey many SV40 transformantsstatistically. In our laboratory, similar overexpressions of

1 2 3 4 5 6

+~--

FIG. 4. Stable expression of the CAT gene directed by thec-Ha-ras promoter. Plasmid pras-CAT-1 containing the putativepromoter region of the c-Ha-ras-1 gene (6) and plasmid pSV2gptwere used to transfect MM3Y-8 cells. Colonies resistant to theinhibitors (mycophenolic acid and aminoputerin) were replated inthe presence (+, lane 1) or absence (-, lane 2) of 1 p.M Dx for 48 h.Cell extracts were prepared by freezing-thawing three times and bysonication. After a brief centrifugation to remove cell debris, thesame amounts of extracts, as measured by the optical density at 280nm, were incubated with [14C]chloramphenicol and acetyl coenzymeA for 30 min, and CAT activity was measured by thin-layerchromatography as described previously (4). pSVO-cat (4), whichhas no promoter, was also used for transfection as described above,and cell extracts from drug-resistant cells cultivated with (+, lane 3)or without (-, lane 4) Dx were subjected to a CAT assay. The CATassay was also performed with extracts from SV40 virion-transformed 3Y1 cells (lane 5) and parental 3Y1 cells (lane 6)transfected with pras-CAT-1.

c-Ha-ras mRNA have been observed among tested SV40transformants of 3Y1, rat brain, and BALB 3T3 cells. InSV40-transformed 3Y1 cells, high CAT activity levels wereobserved after transfection with the pras-CAT-1 gene (Fig.4).

Recently, Winberry et al. reported that the mRNAs of 10proto-oncogenes but not of the mos gene were equallyexpressed in SV40-transformed and untransformed rat F-111cells (21). Several points can be made about this discrep-ancy. First, they analyzed total RNA but not poly(A)+ RNAby dot blot analyses, in which backgrounds are often prob-lematic. Second, their SV40-transformed F-111 cells couldnot grow well in the agarose medium (2), leading to thepossibility that more malignant transformants would expressmuch larger amounts of c-Ha-ras mRNA. Third, the originallevel of c-Ha-ras mRNA in their F-111 cells might have beenhigher than that in our 3Y1 cells. Actually, we could detectonly a trace amount of sis and erbB mRNAs in 3Y1 cells, incontrast to their F-111 cells (21).At present, it is not clear whether SV40 large T Ag itself

can modulate directly the transcription of the c-Ha-ras geneby binding to the promoter region. In view of the recentfindings by Franza et al. that the adenovirus Ela gene couldalso enhance the expression of the transfected ras gene byapproximately 10-fold (3), it might well be that some cellularfactor(s) induced or modified (or both) by SV40 large T Ag isinvolved in the enhancement of transcription. To clarifythese two possibilities, we are carrying out footprint exper-iments with purified SV40 large T Ag.

It is reasonable to assume that the expression of somecellular genes (other than the c-Ha-ras gene) is also modu-lated by large T Ags. By using hybridization experimentswith runoff transcribed mRNAs as mentioned above, wefound that the amount of c-fos mRNA (2.2 kb) was de-creased in the presence ofDx (data not shown). We have not

A 1 2 3 4

* 0 * I* TAg

MM3Y

*&* *.* H-ras

3Y1 * * e 1 H-ras

B 1 2 3 4

Q* * 'TAg

MM3Y

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analyzed whether this repression occurred at the transcrip-tional level. Recently, the amounts of transforming growthfactors a and d were found to be elevated in the conditionedmedium from Dx-treated MM3Y cells (manuscript in prepa-ration), as observed in many cells transformed by the rasgene family (7, 12, 16). Therefore, it will also be interestingto analyze the relationship between c-Ha-ras gene expres-sion and the production of transforming growth factors a and,B, since both are closely related to the cellular propertiesthat lead to growth in the agarose medium.

We thank S. Ishii for providing pras-CAT-1 and for stimulatingdiscussions.

This work was partly supported by grants-in-aid for cancerresearch from the Ministry of Education, Science, and Culture.

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E. H. Chang, D. R. Lowy, and E. M. Scolnick. 1980. Dualevolutionary origin for the rat genetic sequences of Harveymurine sarcoma virus. J. Virol. 36:408-420.

2. Fluck, M., and T. L. Benjamin. 1979. Comparisons of two earlygene functions essential for transformation in polyoma virus andSV40. Virology 96:205-228.

3. Franza, B. R., Jr., K. Maruyama, J. I. Garrels, and H. E. Ruley.1986. In vitro establishment is not a sufficient prerequisite fortransformation by activated ras oncogene. Cell 44:409-418.

4. Gorman, C. M., L. F. Moffat, and B. H. Howard. 1982.Recombinant genomes which express chloramphenicol acetyl-transferase in mammalian cells. Mol. Cell. Biol. 2:1044-1051.

5. Greenberg, M. F., and E. B. Ziff. 1984. Stimulation of 3T3 cellsinduces transcription of c-fos proto-oncogene. Nature (London)311:433-438.

6. Ishii, S., G. T. Merliro, and I. Pastan. 1985. Promoter region ofthe human Harvey ras proto-oncogene: similarity to the EGFreceptor proto-oncogene promoter. Science 230:1378-1381.

7. Kaplan, P. L., W. C. Topp, and B. Ozane. 1981. Transforminggrowth factor(s) production enables cells to grow in the absenceof serum: an autocrine system. Proc. Natl. Acad. Sci. USA78:485-489.

8. Kimura, G., A. Itagaki, and J. Summers. 1975. Rat cell line 3Y1and its virogenic polyoma- and SV40-transformed derivatives.Int. J. Cancer 15:694-706.

9. Lee, F., R. Mulligan, P. Berg, and G. Ringold. 1981.Glucocorticoids regulate expression of dihydrofolate reductasecDNA in mouse mammary tumor virus chimaeric plasmids.Nature (London) 294:228-232.

10. Oda, K., M. Masuda-Murata, K. Shiroki, and H. Handa. 1986.Mitogenic activity of adenovirus type 12 ElA gene induced byhormones in rat cells. Virology 58:125-133.

11. Pulciani, S., E. Santos, L. K. Long, V. Sorrentino, and M.Barbacid. 1985. ras gene amplification and malignant transfor-mation. Mol. Cell. Biol. 5:2836-2841.

12. Salomon, D., J. A. Zwiebel, M. Noda, and R. H. Bassin. 1984.Flat revertants derived from Kirsten murine sarcoma virus-transformed cells produce transforming growth factor. J. Cell.Physiol. 121:22-30.

13. Segawa, K., and Y. Ito. 1982. Differential subcellular localiza-tion of in vivo-phosphorylated and nonphosphorylated middle-sized tumor antigen of polyoma virus and its relationship tomiddle-sized tumor antigen phosphorylating activity in vitro.Proc. Natl. Acad. Sci. USA 79:6812-6816.

14. Segawa, K., and Y. Ito. 1983. Enhancement of polyoma virusmiddle T antigen tyrosine phosphorylation by epidermal growthfactor. Nature (London) 304:742-747.

14a.Segawa, K., and N. Yamaguchi. 1986. Characterization of thechimeric SV40 large T antigen which has a membrane attach-ment sequence of polyoma virus middle T antigen. Virology155:334-344.

15. Southern, P. J., and P. Berg. 1982. Transformation of mamma-lian cells to antibiotic resistance with a bacterial gene undercontrol of the SV40 early region promoter. J. Mol. Appl. Genet.1:327-341.

16. Stern, D. F., A. B. Roberts, N. S. Roche, M. B. Sporn, and B. A.Weinberg. 1986. Differential responsiveness of myc- and ras-transfected cells to growth factors: selective stimulation ofmyc-transfected cells by epidermal growth factor. Mol. Cell.Biol. 6:870-877.

17. Sugano, S., and N. Yamaguchi. 1984. Two classes of transfor-mation-deficient, immortalization-positive simian virus 40 mu-tants constructed by making three base insertions in the T-antigen gene. J. Virol. 52:884-891.

18. Thomas, P. S. 1980. Hybridization of denatured RNA and smallDNA fragments transferred to nitrocellulose. Proc. Natl. Acad.Sci. USA 77:5201-5205.

19. Topp, W. C., D. Lane, and R. Poliack. 1981. Transformation bySV40 and polyomavirus, p. 205-296. In J. Tooze (ed.), Molec-ular biology of DNA tumor viruses, part II. Revised. ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.

20. White, B. A., and F. C. Bancroft. 1982. Cytoplasmic dothybridization: simple analysis of relative mRNA levels in mul-tiple small cell or tissue samples. J. Biol. Chem. 257:8569-8572.

21. Winberry, L., C. Priehs, K. Friderici, M. Thompson, and M.Fluck. 1985. Expression of proto-oncogenes in normal andpapovavirus-transformed or -infected rat fibroblasts. Virology147:154-168.

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