Pr i nt e d in Gr eat Br #ai n 59 Characteristics of a Human Cel l Li ne Transformed by DNA from Human Adenovi rus Type 5 By F. L. GRAHAM* AND J. SMI LEYt Departments of Biology and Pathology, McMaster University, Hamilton, Ontario, Canada W. C. RUSSELL AND R. NAIRN;~ Divisions of Virology and Biochemistry, National Institute for Medical Research, Mill Hill, London, NW7 IAA, U.K. (Accepted IO February I977) SUMMARY Human embryonic kidney cells have been transformed by exposing cells to sheared fragments of adenovirus type 5 DNA. The transformed cells (designated 293 cells) exhibited many of the characteristics of transformation including the elaboration of a virus-specific tumour antigen. Analysis of the polypeptides syn- thesized in the 293 cells by labelling with 35S-methionine and SDS PAGE showed a variable pattern of synthesis, different in a number of respects from that seen in other human cells. On labelling the surface of cells by lactoperoxidase catalysed radio-iodination, the absence of a labelled polypeptide analogous to the 25o K (LETS) glycoprotein was noted. Hybridization of labelled cellular RNA with restriction fragments of adenovirus type 5 DNA indicated transcription of a por- tion of the adenovirus genome at the conventional left hand end. I NT RODUCT I ON Transformation of cultured cells by animal viruses has been demonstrated using a variety of DNA viruses and cells. However, most transformation studies have utilized cells of non- human origin, with rodent cells the most commonly used. This is especially true in the case of human adenoviruses; with the exception of one early report (Todaro & Aaronson, I968) transformation of human cells by adenoviruses has not been observed, and isolation of adenovirus transformed human cell lines has not been reported. The difficulty in obtaining transformation of human cells with human adenoviruses is presumably due, in large part, to the fact that exposure of permissive cells to adenoviruses normally leads to a lytic infection with the production of large numbers of infectious virions. However, a technique (the 'calcium technique') has been developed for infecting ceils with virus DNA (Graham & van der Eb, 1973 a; Graham, Veldhuisen & Wilkie, 1973) which appears to be particularly * Research Scholar and "~ Research Student of the Nat i onal Cancer I ns t i t ut e of Canada. :~ Present address : Depart ment of Mi crobi ol ogy and Immunol ogy, Al bert Einstein College of Medicine, I3OO Morris Park Avenue, Bronx, New York, N. Y. ~o46I, USA. 60 r . L. GRAHAM AND OTHERS suitable for transforming cells with DNA (Graham & van der Eb, I973 b; Graham, van der Eb & Heijneker, I974). Of special importance in the context of transformation of permissive cells was the observation that transformation of cells could be induced with fragmented virus DNA (Graham & van der Eb, I973b; Graham et al. I974). Using this approach, trans- formation of human cells has been induced by sheared Ad 5 DNA and an Ad 5 transformed human cell line (293) has been established. This report describes some of the properties of these 293 cells. METHODS Virus and cells. Adenovirus type 5 (strain Ad 75) was grown and purified as described previously (Graham & van der Eb, I973a ) and extraction of DNA from purified virions was carried out according to van der Eb, van Kesteren & van Bruggen 0969). Ad 5 DNA was sheared by forcing DNA solutions 0oo to 2oo/zg DNA/ml in o.t SSC) repeatedly through a 22 gauge needle (inside diam. 0.72 mm). The resulting DNA had a modal sedi- mentation coefficient of 22S as determined by band sedimentation in an analytical ultra- centrifuge, indicating a modal mol. wt. of 8 Io 6 or about I/3 the size of intact Ad 5 DNA. Primary or early passage secondary human embryonic kidney (HEK) cells prepared by standard techniques were grown in Eagle's minimum essential medium (MEM) supple- mented with non-essential amino acids and ~o % calf serum and were used in transformation studies at subconfluency. Exposure of cells to virus DNA was carried out using the calcium technique essentially as described by Graham & van der Eb (I973 a, b). About 3 days after exposure to DNA the cultures were switched to low calcium ion medium (Freeman et aL I967) and examined periodically for the presence of colonies of transformed cells. After establishment of the cell line the cells were grown routinely in Eagle's medium containing I o /o calf serum supplemented with tryptose phosphate broth (ETC) (see text). Fluorescent antibody techniques. Fluorescent antibody techniques using the indirect method have been described previously (Hayashi & Russell, I968). Antisera. A rabbit P antiserum was prepared as previously described (Russell et al. I967). Hamster tumour sera were obtained from hamsters bearing turnours induced by transplant- ing hamster cells transformed by sheared virus DNA (Graham e t a r. I974) into baby hamsters. Sera were also obtained from hamsters bearing tumours induced by similarly transplanting hamster cells (H I4a) transformed by an adenovirus temperature sensitive mutant (Williams, t973). In this latter case it is known that these hamster cells only contain about 35 % of the left hand end of the virus genome (Sambrook et al. I974). Cell labelling and polyacrylamide gel electrophoresis (PAGE). Monolayers of cells in I oz glass bottles were labelled by removing medium and replacing with 0"5 ml of medium lacking methionine but containing 35S-methionine (2o #Ci/ml, 20o to 350 Ci/mmol, Radiochemical Centre, Amersham, Bucks.) and incubating at 38"5 C for I h or as appropriate. Cells were then scraped off and washed in phosphate buffered saline prior to suspension in a small volume of hypotonic buffer (5 mM-tris/HC1, pH 7"8). Samples were denatured by boiling in sodium dodecyl sulphate (SDS) and analysed by PAGE using the discontinuous buffer system containing SDS and stained with Coomassie or submitted to autoradiography as described previously (Russell & Blair, I977). Iodination procedure. Cells were grown in Glasgow-modified Eagle's medium containing Io% foetal calf serum supplemented with a non-essential amino acid mixture (Gibco Biocult) as monolayers in 6o mm plastic Petri dishes and iodinated using the lactoperoxidase catalysed method as described elsewhere (Meager, Nairn & Hughes, i975). Adenovirus transJormed human cells 6t Cytochem&try. Cells were grown as monol ayers on coverslips in plastic Pet ri dishes, fixed in et hanol and stained with Gi emsa in t he st andard manner or with phenant hr enequi none as indicated elsewhere (Russell, Br odat y & Armst rong, I97I). Restriction enzyme digestion and electrophoresis. Endonucl ease endo R.Hin d 111was pur- chased f r om Bethesda Research Laborat ori es. Ad 5 DNA was digested with excess endo- nuclease at 37 C for 3 h in 20 mM-tris-HC1, pH 7"4, 60 mM-NaC1, and 7 mM-MgC12. Samples were t hen adjusted t o cont ai n o' 5 % SDS, o' oo5 % br omophenol blue, 5 % glycerol and 2o/ d samples cont ai ni ng 2/zg virus DNA were l oaded on to I6 mm cylindrical 1% agarose gels. The gels were el ect rophoresed at 2 V/ cm f or I2 h at r oom t emper at ur e in the el ect rophoresi s buffer of Sharp, Sugden & Sambr ook 0973), lacking et hi di um bromi de. Gels were stained with et hi di um bromi de and the DNA bands were visualized and phot o- graphed under shortwave ul t ravi ol et illumination. RNA- DNA hybridization. Monol ayer s were labelled f or ~ h with 50 #Ci / ml 3H-uridine (New Engl and Nucl ear, 20 Ci / mmol ) and whole cell RNA was ext ract ed by t he hot phenol SDS- met hod (Warner et al. I966 ). Af t er et hanol preci pi t at i on the RNA was dialysed against o. I x SSC, t hen hybri di zed t o unlabelled Ad 5 DNA restriction fragment s which had been previ ousl y t ransferred f r om agarose gels to nitrocellulose strips by t he met hod of Sout her n (i975). Hybr i di zat i on was done at 65 C in 2 SSC plus 0 " I ~o SDS and Ioo #g/ ml yeast RNA f or 2o h. Washing, ribonuclease digestion and fl uorography were done as des- cri bed by Sout her n (I975) except t hat t he X- r ay film was flash hypersensitized (Laskey & Mills, I975) before use. The resulting negatives were scanned with a Joyce- Loebl mi cro- densitometer. R E S UL T S Transformation of HEK cells Using procedures similar to those used successfully t o t r ansf or m r at and hamst er kidney cells ( Gr aham et al. I974) a t ot al of 8 t r ansf or mat i on experiments have been carri ed out with, on average, eo cultures of HEK cells per experiment. For reasons which are not com- pletely clear, t r ansf or mat i on of human cells by Ad 5 DNA was ext raordi nari l y inefficient compar ed to results with rat or hamst er cells even t hough the DNA had been sheared t o eliminate infectivity. Onl y t wo experi ment s wi t h HE K cells were successful and in each of these a single morphol ogi cal l y t r ansf or med col ony was observed approxi mat el y I mont h af t er exposure of t he cultures t o Ad 5 DNA. At t empt s t o isolate these colonies failed but in one case the original dish was ret ai ned and eventually (about day 75) a few t r ansf or med cells coul d again be observed in t he area of the dish where the col ony first developed. Since t he cul t ure still cont ai ned a rat her dense monol ayer of nor mal cells t he serum cont ent was reduced t o z % in an at t empt t o select t he t r ansf or med cell phenot ype. At day 99 the culture was t ransferred t o a large plastic bot t l e in medi um cont ai ni ng 2 % cal f serum (CS) and by this time a significant pr opor t i on of the cells now assumed t he t r ansf or med cell phenot ype. The cul t ure was again split (I t o 2) at days I2o and I42 and at day I5I enough cells were obt ai ned t o freeze 3 ampoul es in liquid ni t rogen (passage 4). At each t ransfer and subse- quent i ncubat i on in 2 % CS the pr opor t i on of t r ansf or med cells (based on t hei r charact er- istic mor phol ogy) increased. The nor mal cells were not compl et el y eliminated until about passage 6, aft er which t he serum concent r at i on was increased t o ~o % CS. At passage 13, t he cells ent ered a ' crisis' phase which lasted until passage ~ 6 (from day 300 to about day 4oo) and which was charact eri zed by little or no growt h. The growt h rate increased r at her sharply aft er passage I6 and t he cells coul d t hereaft er be split I in 3 about twice weekly (Fig. I). 62 F. L. GR AHAM AND OT HE R S ?. 0 0 _o I 1 8 - 16 14 1 2 - 1 0 - 8 - 6 - 4 - 2 - ] / 0 _ / . - 2 / - 4 ~ I I ] l I . ? l i
P
,g
P / . / g / / ' o' I I [ I I 200 400 600 Days Fig. I. Gr owt h curve of 293 cells. The points on the curve represent the area of the 293 cell monol ayer at each consecutive passage (taking into account removal of cells for freezing, experiments, etc.) except that the first poi nt was calculated assuming an area of Io -~ cm 2 for a single cell. Thi s gr owt h pat t er n appear s t o be reproduci bl e f or these cells in the sense t hat , in t wo addi t i onal instances, sublines st art ed f r om ampoul es of cells frozen at passage 6 ent ered a crisis ar ound passage x3 or 14. I n one case t he subline failed t o recover, in t he other, gr owt h r esumed 6o t o 8o days later. The cells have since been mai nt ai ned in Eagl e' s medi um (or medi um cont ai ni ng reduced cal ci um i on concent rat i on) suppl ement ed wi t h Io % cal f serum and t r ypt ose phosphat e br ot h (ETC). Hor se serum can be subst i t ut ed fore' calf ser um and appear s t o be equally suitable f or cell growt h. The cells have been subcul t ured mor e t han ~oo t i mes and can pr es umabl y be consi dered an est abl i shed line (293 cells). Adenovirus transformed human cells 63 Fig. 2. St ai ni ng propert i es of 293 cells. (a) Gi ems a st ai ni ng: 220. (b) Phenant hr enequi none st ai ni ng: x t I00. (c) Fl uor escent ant i body st ai ni ng wi t h t ur nout ser um: x I000. (d) Fl uor escent ant i body st ai ni ng wi t h P ant i ser um: 35oo. 5 VlR 3 6 64 F. L. GRAHAM AND OTHERS Morphology of 293 cells The cells exhibited a typical adeno- t r ansf or med cell phenot ype with a t endency t o con- t i nue dividing aft er reaching confl uency and to grow in islands or clumps. Like adeno- t r ansf or med r at and hamst er cells, 293 cells were grossly epithelioid in charact er (as was t he ot her t r ansf or med col ony observed in 8 at t empt s t o t r ansf or m HEK cells) but were, on average, not i ceabl y larger t han most ot her adeno- t r ansf or med cells, showing a wide vari- at i on in cell size (Fig. 2a). I n some cases, t he cell nuclei appeared swollen and f r equent het er okar yons were not ed. Anot her similarity was the ability of 293 cells t o grow equal l y well in medi um cont ai ni ng or lacking calcium ions or t o grow in medi um cont ai ni ng reduced serum concent rat i ons. A characteristic feat ure of many of t he cells was a dense ovoi d body whi ch coul d readily be detected by staining wi t h phenant hr enequi none and which generally seemed t o be l ocat ed at or near t he nucl ear membr ane (Fig. 2 b ) . The human charact er of t he cells was confi rmed by the chr omosome bandi ng pat t er n with qui nacri ne must ar d and were quasi t et rapl oi d when tested at passage 8 (P. Pearson, personal communi cat i on). Chr omo- some spreads were again pr epar ed f or cells at passage 38 as well as f or a subline of 293 cells (293 N) established f r om a t umour i nduced in a nude mouse. I n bot h cases t he resulting kar yot ype was t hat of human cells t hough t he average number of chr omosomes in 293 N cells appear ed t o be significantly l ower t han f or t he parent al line. (Similar observat i ons have been made by P. Gal l i more, personal communi cat i on. ) Superinfeetion with adenoviruses One characteristic of 293 cells which makes t hem of considerable usefulness f or studies with adenoviruses is t hat t hey can be superinfected by Ad 5 as well as ot her human adeno- virus serotypes. Indeed, 293 cell monol ayers were excellent substrates f or pl aque t i t rat i ons of adeno serotypes ~, 2, 5 and 7 as well as t ype I2 which we have been unabl e t o pl aque on ot her established human cell lines. Pl aque assays with Ad 5 resulted in pl aques appeari ng several days earlier on 293 monol ayers t han on HeLa or KB monol ayers t hough t he final number s of plaques were similar. Anot her interesting pr oper t y of 293 cells is t hat Ad 5 DNA t i t rat ed on these cells (using t he cal ci um technique) is Io t o 5o times mor e infectious t han when t i t rat ed on KB cells or pr i mar y HE K cells (F. L. Gr aham, unpubl i shed observations). The reasons f or this are unknown, but coul d possibly be related t o t he f act t hat , as discussed in following sections, 293 cells express one or mor e Ad 5 specific product s. Oncogenicity The ability of 293 cells t o induce t umour s was tested by injection of I t o 2 t o 7 cells sub- cut aneousl y i nt o 5 t o 6-week-old ' nude ' mice. Aft er 6 to 8 weeks no t umour s were visible and t he animals were re-injected. Aft er approx. 15 t o 2o weeks f r om t he t i me of t he first injection, t umour s became appar ent in a small pr opor t i on (3/2o) of t reat ed animals. (An out br eak of muri ne hepatitis prevent ed t he course of t he experi ment f r om being fol l owed l onger t han 25 t o 3o weeks). I n cont rast , Ad 5 DNA t r ansf or med r at or hamst er cells i nduced t umour s in 9o t o Ioo % of injected mice within 3 t o 6 weeks. One animal exhibiting a 293 cell-induced t umour was sacrificed at 16 weeks and the t umour was removed, mi nced and put i nt o culture. The resulting cells (line 293 N) were somewhat similar t o t he original 293 cells but grew slightly faster and reached great er cell densities. The 293 N cells like t he parent al cells, were capabl e of gr owt h in medi um with low concent rat i ons of calcium ions. Adenori rus t r a mf o r me d human cells 65 I n addi t i on the 293 N cells cont i nued t o express Ad 5 specific T antigen(s) as detected by indirect i mmunofl uorescence. Fluorescent antibody experiments Monol ayer s of 293 cells on coverslips were react ed with hamst er t umour antisera and with rabbi t ant i serum against t he adenovi rus t ype 5 P antigen. The latter ant i serum reacts wi t h earl y antigens but mai nl y against the maj or earl y 72 K single-stranded DNA bi ndi ng prot ei n (Russell et al. 1967; Russell & Skehel, I972; Russell & Blair, 1977). Wi t h t he t umour ant i serum fluorescent nucl ear dots were not ed in about 2o % of t he cells; however, t he most charact eri st i c finding was a large fl uorescent fleck apparent l y in association with t he nucl ear membr ane (Fig. ze). The same feat ure was also not ed on reacting the cells with t he rabbi t P antiserum. In this case (Fig. 2 d) t he staining was less intense and onl y about 5 % of the cells showed nucl ear dots. On staining HeLa cells infected with adenovi rus type 5 in t he presence of cytosine arabi nosi de with these sera, t he t umour ant i serum exhibited mi ni mum nuclear fluorescence and occasional cyt opl asmi c flecks in association with the nucl ear membrane. On t he ot her hand t he P ant i serum showed intense characteristic staining as fibres and dots on all t he cells as previously described (Hayashi & Russell, I968). The fluorescent staining of t he 293 cells coul d be significantly reduced by adsorbi ng bot h t he t ur nout and P antisera with extracts of t he 293 cells but not with extracts of cont rol cells. No fluorescent antigens coul d be detected in t he 293 cells when t he cells were react ed with adenovi rus t ype 5 hexon or fibre antisera. Polypeptide synthesis in 293 cells Monol ayer s of 293 cells were pulse labelled with 35S-methionine f or r h and f or l onger periods. In some experi ment s t he I h pulses were ' chased' f or about 18 h. The extracts were submi t t ed t o analysis by SDS pol yacryl ami de gel electrophoresis fol l owed by aut oradi o- graphy. The labelling pat t erns were compar ed to those obt ai ned f r om ot her human cell lines labelled in parallel in the same way. In the course of about t went y experiments carri ed out at a number of different passage levels the 293 cells were compar ed with pr i mar y and secondary human embr yoni c ki dney cells, with ot her epithelioid cells such as He La and KB cells and with human fibroblast lines such as WI-38 and MRC-5. The labelling pat t erns seen with the cont r ol cell lines remai ned substantially const ant whereas t he labelling pat t erns seen with the 293 cells showed considerable vari at i on f r om experi ment t o experiment. This vari at i on di d not seem t o be a reflection of the growt h state of t he cell (i.e. whet her in log or lag phase) or of t he passage number. The differences f r om the ot her human cell lines were evident bot h as a di mi nut i on in labelling of some polypeptides and the appearance of ' ne w' bands in the aut oradi ograms. I t was also not ed t hat the labelling pat t erns changed significantly aft er ' chasi ng' t he 293 cells, suggesting t hat some processing of t he pol ypept i des was occurring. Fig. 3 shows a typical aut or adi ogr am obt ai ned f r om labelled extracts of 293 cells and infected and uni nfect ed HeLa cells. When compar ed t o t he uni nfect ed cell pat t er n it will be seen t hat t here is some di mi nut i on in t he labelling of high mol. wt. pol ypept i des part i cul arl y at mobilities correspondi ng t o t he 25o K/ LETS (large, ext ernal t r ansf or mat i on sensitive) gl ycoprot ei n (Hynes, 1973). It is also clear t hat t here is no detectable synthesis of the 72 K pol ypept i de in t he 293 cells. The latter pol ypept i de is t he maj or pol ypept i de synthesized earl y in infected cells and is heavily phosphor yl at ed (Russell & Skehel, ~97z; Russell & Blair, 1977). The maj or difference in the labelling pat t erns is the appar ent synthesis in the 293 cells of a pol ypept i de of tool. wt. about 68 K which can also be seen earl y in adenovi rus-i nfect ed cells. As yet, however, t here is no clear i ndi cat i on whet her this is a virus- 5-2 66 F. L. GRAHAM AND OTHERS 293 LETS? . - . - - ~ 35 S 32p 1z5 l ,A, 16v 7v c 16v v 72K 1 n base 1 (V) 2 (VII) Fig. 3- Autoradiograms after SDS PAGE of extracts of HeLa and 293 cells after labelling with 35S-methionine for ~ h at the post infection (V) times shown. Uninfected HeLa cells (C) and 293 cells were similarly labelled for i h. Iodinated type 5 virus (12~I V) and extracts of a2P-labelled infected ceils (a2p I6 V - see Russell & Blair, I977) were run in parallel as markers. On the right of the figure the labelled bands corresponding to the major structural polypeptides are indicated. On the left the mobilities corresponding to LETS and 72 K polypeptides are marked. The remaining arrows indicate the differences in mobilities between the 293 cells and the uninfected HeLa ceils (C). Origin of electrophoresis is at the top of the figure. coded or an i nduced cel l ul ar pol ypept i de (cf. Sabor i o & Oberg, I976). The ot her not abl e di fference bet ween t he 293 cells and uni nfect ed HeLa ceils is t he appar ent synt hesi s of a number of smal l er mol . wt. pol ypept i des. At t empt s t o det ect differences in phos phor yl at ed pol ypept i des bet ween t he 293 cells and ot her cells by l abel l i ng wi t h 3zp- or t hophosphat e (Russel l & Blair, I977) were unsuccessful . Cell surface labelling patterns On l abel l i ng surface component s of t he 293 cells by l act oper oxi dase cat al ysed 1~5I i odi na- t i on a number of differences coul d be seen when compar ed to t he pat t er n obt ai ned wi t h nor mal human embr yo fi brobl ast s ( HEF; Fi g. 4)- Mos t of t he hi gh tool. wt. i odi nat ed pol y- pept i des of t he HEF cells are absent f r om t he 293 cells and concomi t ant l y ot her smal l er Adenovirus transformed human cells I I I I I I 250 K 2~ ~t2 2.0 1.5 7 1,0 X -~ o.5 o 2.0 1.5 1.0 0.5 2.5 293 I I I I I I 0 2 4 6 8 10 12 Distance migrated (cm) Fig. 4. Cell surface l abel l i ng profile of 293 cells i n compar i s on t o h u ma n embr yo f i br obl ast cells (HEF). Cells were i odi nat ed, denat ur ed i n SDS, a nd equal pr ot ei n sampl es r un on adj acent t racks of a di scont i nuous SDS-pol yacryl ami de sl ab gel. Purified reovi rus was el ect rophoresed i n paral l el as a mar ker . The gels were sliced i nt o z mm sections f or gamma count i ng i n a Packar d Aut o- gamma spect romet er. On t he x-axis, o represent s t he st art of t he 7"5 ~ resol vi ng gel. El ect rophoresi s is f r om left t o ri ght . A 1 a nd t'2 are t he reovi rus st r uct ur al pol ypept i des of 155 K a nd 7z K mol . wt. respectively. 67 labelled polypeptides appear to be more prominent in the transformed cells. In particular the absence of a polypeptide probably analogous to the 25o K/LETS glycoprotein of other systems is noted and this is consistent with results obtained with certain other adenovirus transformed cell lines (Meager et al. I975). RNA- DNA hybridization In order to determine which region(s) of the Ad 5 genome are transcribed in 293 cells, the total cellular RNA labelled with SH-uridine was hybridized to unlabelled Ad 5 DNA fragments generated by endo R.Hin d III, which had previously been transferred from agarose gel to nitrocellulose strips by the blotter method of Southern 0975). Fig. 5 shows 68 F. L. GRAHAM AND OTHERS H G F E D C B A i I ! I ~ . . . . . . . . . . . . . . . . . . . . i i i 1~ ~' (a) (b) | I I I I I I I I I I I I I I I I I I (c) Fig. 5. Hybridization of labelled 293 cellular RNA to Ad 5 DNA fragments generated by endo R. Hi nd Il L Ad 5 DNA (2 #g) was digested with endo R. Hi nd III and electrophoresed t hrough a I ~ agarose gel as described in Methods. The DNA bands were phot ographed under ultraviolet light in the presence of o' 5 ~ #g/ml ethidium bromide. The fragments were transferred to nitro- cellulose strips, and then hybridized to 5 IO7 ct/min of 293 cellular RNA labelled with aH-uridine. Radioactivity was detected by fluorography, and the resulting negatives scanned with a Joyce- Loebl microdensitometer. (a) Phot ograph of the ethidium bromi de fluorescence of Ad 5 Hi nd l l I fragments separated in a ] % agarose gel. (b) Fl uorogram of the nitrocellulose strip produced from the gel displayed in (a) after hybridization to labelled 293 cellular RNA. (c) Microdensitometer tracing of the fluorogram in (b). A d e n o v i r u ~ t r a n s f o r m e d h u m a n c e l l s 69 t hat 293 RNA hybridizes to the H i n d I I I G and E fragments, which together constitute the extreme left hand 17 % of the genome (C. Mulder, personal communication). In order to resolve fragments F and G well it was necessary to run fragment i (corresponding to the extreme right hand 4 % of the genome) out of the gel. Consequently, it remains possible t hat 293 cells cont ai n RNA derived from H i n d I I I I. It is of interest t hat we can detect no RNA derived from the H i n d I l l A fragment, which should cont ai n the sequences coding for the 72 K DNA binding protein (Lewis e t al. I976), assuming t hat the gene for the protein is located at the same position on bot h the Ad 2 and Ad 5 genomes. Presumabl y the RNA species detected are analogous to the left hand early strand specific RNAs detected in Ad 2 and Ad 5 infected and t ransformed cells (Sharp, Gallimore & Flint, I974; Flint, Berget & Sharp, I976; Fl i nt et al. I976). DI SCUSSI ON These studies have shown t hat Ad 5 DNA fragments can t ransform human cells wi t h an efficiency which is at least 2 logs lower t han t hat which is routinely obt ai ned with rat or hamster cells. Furt her, a line of human cells t ransformed by Ad 5 could be established by careful mai nt enance of cell cultures following exposure of HEK cells to sheared virus DNA. The reasons for the difficulty in t ransformi ng human cells with Ad 5 DNA are not known. One difficulty which did arise in t ransformat i on studies with HEK cells which was not a probl em with rat ki dney cells was t hat normal HEK cells t ended t o persist in DNA-t reat ed cultures for long periods following exposure and formed rat her dense monolayers. This could have resulted in growt h inhibition of any t ransformed cells which mi ght have been induced. Other differences between human and rat cells are the slow growth rate and clear ' cri si s' phase exhibited by the Ad 5 t ransformed human cells, and their extremely low oncogenicity in nude mice. The 293 cells are similar to Ad 5 t ransformed rat and hamst er cells in their epithelioid morphol ogy, their ability to grow in medi um containing low con- centrations of calcium ions, and their ability to elaborate virus-specific T antigen. The 293 cells were f ound to cont ai n a rather unusual feature in the presence of large ovoid bodies in the region of the nuclear membrane clearly seen on staining by phenant hrenequi none. Thi s cytochemical technique has been used to detect basic proteins and appears to react speci- fically with arginine residues (Russell e t al. I97I). Whet her these inclusions bear any relationship to the T antigen containing entities detected by fluorescence is not clear. Stain- ing by the DAPI technique (Russell, Newman & Williamson, 1975) which can readily detect DNA in the cytoplasm indicates t hat these bodies do not cont ai n detectable amount s of DNA (unpublished observations). Their nat ure and their relationship (if any) t o the mai nt enance of the t ransformed state will require furt her study. The fluorescent ant i body results indicate t hat the cells probabl y cont ai n i nformat i on from the conventional left hand end of the adenovirus genome since a positive reaction was obt ai ned with t umour serum from animals bearing t umours induced by cells which are known to cont ai n only t hat port i on of the genome (Sambrook e t al. I974). This was con- firmed by the RNA- DNA hybridization studies which detected the presence in 293 cells of mRNA compl ement ary to the extreme left end of the Ad 5 genome. The results with the P antiserum suggest t hat these cells do not elaborate significant amount s of the maj or early adenovirus antigen (Russell, ~974) associated with the 72 K single stranded DNA binding protein, consistent with our failure in three separate experiments to detect any virus-specific mRNA compl ement ary to the region (around 6o to 7o %) of the adeno genome known to code for the DNA binding protein (Lewis e t al. I976 ). These studies also suggest t hat the P 70 F. L. GRAHAM AND OTHERS ant i serum, while reacting pri nci pal l y with t he 72 K pol ypept i de (Russell & Skehel, I972), also reacts with the ot her early, so called, ' T' antigen(s). The analyses of t he pol ypept i de synthesis in t he t r ansf or med cells were somewhat variable and t he pat t erns obt ai ned were quite unlike ot her t r ansf or med cell lines exami ned (Russell, I974). Whet her this i nherent instability in t he pat t er n of synthesis is rel at ed t o lack of cont r ol of gene expression or perhaps t o het erogenei t y in the cell popul at i on can onl y be decided by mor e detailed investigations and by cloning of the cells. Some of the pol ypept i des synthe- sized are similar in mobi l i t y in t he el ect ropherograms to those seen in cells infected earl y in the lytic system but i mmunopr eci pi t at i on experiments using bot h the P and T antisera have so far yielded inconclusive results. I n all these studies t here has been no evidence of t he pro- duct i on of either capsid antigens or capsid polypeptides. Thus t here is no i ndi cat i on of whet her t he variable results in these analyses reflect variable expression of virus or cellular genes. Thr ee experiments on the virus RNA t ranscri pt i on pat t er n all gave consistent results, however, suggesting t hat the differences in pol ypept i de synthesis are not due t o differences at t he level of t ranscri pt i on of virus genes. The i odi nat i on experiments gave results similar t o those described in ot her adenovi rus t r ansf or med cell systems (Meager et al. 1975), the large 250 K pol ypept i de (LETS glyco- prot ei n) not being accessible t o the i odi nat i on react i on in the 293 cells. A similar finding has been r epor t ed by Chen, Gal l i more & McDougal l (I976) using an i mmunofl uorescent t echni que and it has been shown by these aut hor s t hat t here is a correl at i on bet ween t umour ogeni ci t y and t he lack of the LETS gl ycoprot ei n on t he cell surface. It is also not abl e t hat t he synthesis of a large mol. wt. pol ypept i de of similar tool. wt. t o LETS was sub- stantially reduced compar ed t o He La cells. Thus, in the case of the 293 cells the lack of radi o-i odi nat i on of t he large 250 K pol ypept i de may result f r om an i nhi bi t i on of its synthesis r at her t han its non- appear ance at t he surface of the t r ansf or med cell. Investigations elsewhere (R. Wei nmann, personal communi cat i on) using DNA- DNA hybri di zat i on t echni ques have i ndi cat ed t hat the 293 cells cont ai n 4 t o 5 copies of the left hand ~2% of t he Ad 5 genome and about t copy of t he right l o %. The RNA- DNA hybri di zat i on studies described here are consi st ent with these findings in t hat virus RNA specific f or t he left end of t he virus genome was detected. However, at present we are unabl e t o say whet her the virus sequences f r om t he ri ght 4% of t he genome are t ranscri bed. Studies with adenovi rus t ype 2 t r ansf or med r at cells have indicated t hat some cell lines can cont ai n DNA sequences (correspondi ng t o ' l a t e ' regions) which are apparent l y unt ranscri bed ( Sambr ook et al. I974; Fl i nt & Sharp, I976). The 293 cell line described here is, t o our knowledge, t he onl y adenovi rus t r ansf or med human cell line established t o date. Our prel i mi nary char act er i zat i on of some of t he propert i es of these cells suggests t hat t hey will be quite useful in studies wi t h adeno- viruses. I n part i cul ar, t he 293 cells can be readily superinfected with human adenovi rus and recently have been utilized t o select host range mut ant s of adenovi rus type 5- Recombi nat i on tests have indicated t hat these host range mut at i ons map within t he left hand region of t he genome and pr obabl y very close t o t he left hand end (Harri son, Gr aham & Williams, ~976), findings whi ch are consistent with t he propert i es of the cells described in this communi cat i on. This wor k was suppor t ed in par t by the Nat i onal Cancer Inst i t ut e of Canada. One of us ( FLG) is i ndebt ed t o t he Eur opean Mol ecul ar Biology Organi zat i on f or a short t er m Fellowship which allowed some of t he wor k t o be carried out at Mill Hill. Anot her ( RN) was in receipt of an MRC scholarship f or t rai ni ng in Research Met hods. 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