An immortalized xeroderma pigmentosum, group C, cell line which replicates SV40 shuttle vectors

185

Mutation Research, 183 (1987) 185-196 DNA Repair Reports Elsevier MTR 06197

An immortalized xeroderma pigmentosum, group C, cell line which replicates SV40 shuttle vectors L. D a y a - G r o s j e a n , M . R . J a m e s , C. D r o u g a r d a n d A. S a r a s i n Laboratory of Molecular Mutagenesis, lnstitut de Recherches Scientifiques sur le Cancer, B.P. No. 8, 94802 Villejuif (France) (Received 21 July 1986) (Revision received 2 September 1986) (Accepted 3 September 1986)

Key words: Xeroderma pigmentosum; Immortalization; SV40 shuttle vectors; DNA replication.

Summary We have established and characterized an immortalized xeroderma pigmentosum (XP), group C, cell line. Transformation of the human fibroblasts was carried out with a recombinant plasmid, pLAS-wt, containing SV40 DNA encompassing the entire early region with a defective origin of DNA replication. The transformed XP cell line, XP4PA-SVwt, and the normal transformed fibroblasts AS3-SVwt, both express SV40 T antigen together with enhanced levels of the transformation-associated cellular protein, p53. XP4PA-SVwt retains the XP UV-repair defective phenotype as demonstrated by low levels of unscheduled DNA synthesis and by the reduced survival of irradiated SV40 virus. Analysis of cellular DNA shows a single major, stable, integration site of pLAS-wt in the XP4PA-SVwt cells. The T antigen in these cells supports efficiently the replication of SV40 based shuttle vectors and should prove suitable for the introduction, expression and selection of genes related to DNA repair and to the study of mutagenesis using defined molecular probes.

The past few years have seen major advances in identifying the genes, and gene products, which are implicated in oncogenesis, nevertheless the course of cellular and molecular events leading to cancer remain unclear. Analysis of human genetic diseases which show a propensity to develop cancer has held the promise of shedding light on the mechanisms of carcinogenesis, analogous to the way in which mutants of lower organisms have allowed the understanding of complex biological phenomena. Correspondence: Dr. L. Daya-Grosjean, Institut de Recherches Scientifiques sur le Cancer, Boite Postale No. 8, 94802 Villejuif (France).

It is approaching 20 years since the first report of the association of a DNA-repair defect with a human cancer-prone syndrome, xeroderma pigmentosum (XP). Disappointingly little understanding, however, has been gained in either the biochemical or genetic aspects of this syndrome or other similar hereditary diseases. Modern molecular biological techniques offer several new approaches to the problems but these methodologies generally require long-lived cell lines amenable to considerable manipulation in cell culture. Such immortal cell lines have proven extremely difficult to obtain as can be judged from the existence of only 9 transformed, immortalized XP fibroblasts

0167-8817/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

186 d e s p i t e persistent efforts in m a n y laboratories. A m o n g these cell lines, only 5 out of the 10 genetic c o m p l e m e n t a t i o n groups, n a m e l y A, C, F, G a n d variant (Yagi a n d T a k e b e , 1983; RoyerP o k o r a et al., 1984; C a n a a n i et al., 1986; Barbis et al., 1986) are r e p r e s e n t e d which shows a severe l i m i t a t i o n a n d f r u s t r a t i o n in the face of the existing genetic a n d p h e n o t y p i c diversity of this syndrome. W e describe here the e s t a b l i s h m e n t of an immortal, t r a n s f o r m e d cell line derived from foetal fibroblasts b e l o n g i n g to X P c o m p l e m e n t a t i o n group C. X P a n d n o r m a l h u m a n foetal fibroblasts were t r a n s f o r m e d b y an SV40 r e c o m b i n a n t that was defective in the viral replication origin ( S V o r i - ) . Both of the resulting cell lines are very stable a n d have b e e n in c o n t i n u o u s culture for 2 years, have high p l a t i n g efficiencies a n d are g o o d recipients for D N A - t r a n s f e c t i o n experiments. Detailed c h a r a c t e r i z a t i o n of the cell lines indicates that the p h e n o t y p i c h a l l m a r k s of XP are retained. I n a d d i t i o n we give p r e l i m i n a r y d a t a which show t h a t SV40-based s h u t t l e - p l a s m i d s replicate efficiently in the cells, a feature which m a y facilitate m o r e efficient, r a p i d a n d s o p h i s t i c a t e d m o l e c u l a r studies into the X P deficiency.

f o r m e d cells derived from the h u m a n foetal fibroblasts were cultured in m i n i m u m essential m e d i u m c o n t a i n i n g 10% FCS. T h e m o n k e y cells were grown in D u l b e c c o ' s m o d i f i e d Eagle m e d i u m s u p p l e m e n t e d with 7% F C S .

Origin-defective recombinant S V40 plasmids T h e large T a q I - E c o R I SV40 D N A fragment, which contains a 13-base-pair deletion at the origin of replication, was o b t a i n e d after cleavage from the p L A S p l a s m i d (gift from M. Ernoult-Lange). This SV40 D N A was ligated to the small T a q I - B a m H I f r a g m e n t of SV40 D N A o b t a i n e d from either w i l d - t y p e (VA 45-54) or temperature-sensitive (tsA58) strains of SV40 virus. The D N A s thus o b t a i n e d , c o n t a i n e d the E c o R l - B a m H I region of SV40 s p a n n i n g p a r t of the late coding region a n d the whole of the early region c o d i n g for either the w i l d - t y p e large T antigen or the tsA58 t e m p e r a ture-sensitive (ts) large T antigen. These SV40 D N A s were inserted into the B a m H I - E c o R I

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Materials and methods

Cells and culture conditions T h e x e r o d e r m a p i g m e n t o s u m X P 4 P A fibroblasts were established in culture from a m n i o t i c fluid o b t a i n e d f r o m a p r e g n a n t female w h o h a d previously given b i r t h to 2 out of 4 c h i l d r e n afflicted b y XP. T h e p r e n a t a l diagnosis of the X P 4 P A f i b r o b l a s t s established t h e m as b e l o n g i n g to c o m p l e m e n t a t i o n group C ( H a l l e y et al., 1979). T h e AS3 h u m a n fibroblasts were established in o u r l a b o r a t o r y a n d were derived f r o m a m n i o t i c fluid o b t a i n e d from a 40-year-old w o m a n exhibiting n o k n o w n clinical or genetic disease a n d w h o h a d a n o r m a l p r e g n a n c y . The established C V 1 P a n d M A - 1 3 4 cell lines of A f r i c a n green m o n k e y k i d n e y cells were used for p l a q u e assay a n d virus p r o d u c t i o n respectively. X P 4 P A a n d A S 3 cells were grown in F10 m e d i u m c o n t a i n i n g 15% foetal calf serum (FCS). Cells were s u b c u l t u r e d once a week, with an add i t i o n a l change of m e d i u m after 4 days. T h e trans-

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Fig. 1. Diagram of plasmids used in this study. (A) pLASwt: SV40-transformation vector. The construction of this plasmid is described in the text. Restriction-enzyme sites are shown for orientation; note that the Taq ! and Msp I sites in SV40 only are indicated. Also shown are: the position of the 13 bp deletion in SV40 which results in loss of the Bgl I site and loss of the viral replication origin, the position of the tsA58 mutation in SV40 T antigen present in the related plasmid pLASts; the mRNA for T antigen is shown with intervening sequence (dotted line) and polyA-tail (wavy line). (B) ~ SVHPplac (trivial name = ~r SV) is based on the ~ VX miniplasmid and is described by Little et al. (1983). It contains a 340 bp SV40 fragment which spans the functional viral replication origin. Other segments of the vector include the plasmid replication origin, a suppressor tRNA (supF), a restriction-site polylinker (plink) and the lactose-operator promoter/repressor binding site (lac 0). Hatched areas represent the SV40 replication origin-promoter region; lines with arrows represent RNA transcripts. The symbol A indicates a deletion.

187 sites of pBR 327 and the appropriate recombinants were named pLAS-wt and pLAS-ts (Fig. 1A).

FCS and seeded into 30-ram petri dishes. Each day after seeding, two cultures were trypsinised and the cells counted.

DNA transfection and transformation Plasmid D N A was linearised by restriction enzyme Sal I, which has a single recognition site in the pBR region of the SV40 recombinant molecules. The linearised DNA was transfected into either XP4PA or AS3 cells using the calciumphosphate-mediated precipitation technique described by Wigler et al. (1978). Cells were plated 20 h before transfection at 5 × 105 cells per 60-mm petri dish. DNA-calcium phosphate precipitates were prepared so that each dish received 0.5 ml containing 1.0 /~g of plasrnid and 10 /xg of high molecular weight salmon sperm D N A as carrier. Cells were exposed to DNA for 5-6 h, then refed with complete medium. Transformed cell fines were isolated using the mass culture procedure described by Huschtscha and Holliday (1983). In brief, newly transfected cells were subcultured when they reached confluence and maintained in culture under normal conditions until they entered crisis. A series of transformed cell fines were obtained from XP4PA and AS3 fibroblasts after transfection with either pLAS-wt or pLAS-ts DNA.

Plating efficiency Cells were seeded in liquid medium at 100 and 1000 cells per 60-mm dish. The plating efficiency in liquid medium was calculated after 15 days of culture when cell colonies were stained with Giemsa and counted. Plating efficiency in soft-agar was evaluated after seeding 104 and 105 cells per 60-mm dish using the method of MacPherson and Montagnier (1964).

T antigen assay by indirect immunofluorescence Cells were examined for the presence of SV40 T antigen during the early passages by the indirect imnmofluorescence procedure (Daya-Grosjean and Monier, 1978). Radioimmunoprecipitation assay of T antigen and p53 Proteins were labelled in actively growing cell cultures by either [32p]phosphate or [35S]methionine. Cells were lysed and radioactive protein extracts were immunoprecipitated by normal or anti-SV40 tumour serum using the method described by Kress et al. (1978). The immunoprecipitates were analysed by SDS-PAGE using the discontinuous system of Laemmli (1970). Growth kinetics Confluent cultures were trypsinised, suspended in growth medium containing either 10% or 0.5%

Isolation of cellular DNA Total cell D N A was isolated from transformed cells by a modification of the procedure described by Gross-Bellard et al. (1973). Cells were lysed with 1 ml per 10-cm petri dish of 10 mM Tris-HC1 p H 7.8, 1 mM EDTA, 5 mM NaC1, 0.6% SDS and 100 # g / m l of proteinase K (Merck) and incubated at 60°C for 3 h. The lysate was then extracted once with phenol and once with chloroform/isoamyl alcohol (24:1). The DNA was then precipitated with alcohol and the pellet of D N A dissolved in 10 mM Tris-HC1 (pH 7.8) 1 mM EDTA. The DNA solution was treated with 50/~g/ml of pancreatic RNAase for 2 h at 37°C. The DNA solution was then redigested with proteinase K and extracted with phenol and chloroform/isoamyl alcohol as before. Purified DNA was obtained by precipitation with alcohol and finally dissolved in Tris-HC1-EDTA. Analytical electrophoresis and Southern blot hybridization Restriction enzyme digestions were under the conditions specified by the suppliers, New England Biolabs (Beverly, MA, U.S.A.) and Boehringer (Mannheim, F.R.G.). 10 /~g of digested genomic DNA was fractionated in 1% agarose horizontal slab gels in TBE buffer (0.089 M Tris-borate, 0.089 M boric acid, 0.002 M EDTA). SV40 DNA was loaded on parallel lanes of the gel as a reconstruction mixture containing 10 /~g of salmon sperm D N A with 1 or 10 copies of SV40 per genome equivalent to serve as markers. Southern transfer and hybridization were by standard

188 procedures (Southern, 1975; Maniatis et al., 1982). Probes were prepared by nick-translation of plasmid or SV40 D N A using [32p]deoxynucleotides from New England Nuclear (Boston, MA) and DNA-polymerase I from Boehringer or the nick-translation kit from Amersham (U.K.).

Unscheduled DNA synthesis 105 cells were seeded onto 2-cm 2 coverslips placed in 30-mm petri dishes in normal medium. When cells were just confluent the medium was replaced with fresh medium containing 0.5% FCS and the cultures were incubated a further 16-20 h. 1 h before UV-irradiation, cells were prelabelled with 10 tlCi [3H]thymidine (77 C i / m m o l e , N E N ) added to the growth medium. For UV-irradiation, the medium was removed, and the coverslip cultures washed twice with PBS. Irradiation was carried out under a germicidal lamp (mainly at 254 nm) with a dose rate of 0.12 J m - 2 sec 1 at the doses indicated. The cells were then labelled in growth medium containing [3H]thymidine for 2 h. A 1-h chase incubation with cold thymidine (10 5 M) followed the radioactive labelling, and coverslip cultures were subsequently washed and fixed in methanol. The dried, fixed coverslips were mounted on slides, dipped in Ilford Nuclear Research emulsion and exposed for 1 week at 4°C. After development the cells were stained with Giemsa and the average number of grains over the nucleus scored, cells undergoing replication synthesis being excluded. Virus survival assay Suspensions of SV40 tsA58 virus were UVirradiated on ice, using a germicidal lamp emitting mainly at 254 nm at a fluence of 1.4 Jm--2 sec 1. Transformed cells were infected and adsorption carried out for 2 h. The cells were then washed with PBS, fresh medium added and incubation was carried out for 10 days at 33°C, or 8 days at 41°C. Titres of the viral progeny obtained were determined on CV1-P cells using a standard plaque assay procedure as previously described (Sarasin and Benoit, 1980). Shuttle-vector replication D N A transfection was performed in 10-cm culture dishes which were about 50% c o n f u e n t at the

time of transfection. Transfection was with 0.1 or I /Lg plasmid in 4 ml per dish of 200 /zg/ml D E A E - d e x t r a n for 3 4 h followed by a 2-min 10% glycerol-shock. Some of the human cells have proved to be sensitive to this procedure and in this case 1 ml per dish of a calcium phosphate precipitate containing 5 t~g plasmid D N A without carrier D N A was left on the cells overnight. Hirt extracts were prepared at the times indicated (Hirt, 1967). Results

Establishment of transformation After transfection of XP4PA and AS3 with pLAS-wt the cells were maintained as mass cultures and subcultured when they reached confluence. After 2 - 3 subcultures, changes in morphology of the cells were observed. Cells began to lose their regular fibroblastic appearance and showed high mitotic activity and increased growth rate. Upon further subculturing a loss of contact inhibition was observed and large numbers of cells were continually shed into the medium. Both cell cultures entered crisis periods, i.e. a degenerative phase during which many cells tended to shed into the medium leaving granulated irregular cells. After subculturing at higher densities these eventually established into permanent cultures. The AS3-SVwt culture remained in crisis over a longer period, 4 - 6 passages, compared to the X P 4 P A SVwt cell line which emerged from crisis more quickly. This nomenclature corresponds to the p L A S - w t transformed counterparts of XP4PA and AS3 fibroblasts. Growth characteristi~ Fig. 2 shows the growth kinetics of these cell lines relative to those of their parental cells. The transformed cell lines grow more quickly and have also lost the serum dependence for cell division exhibited by the parental cells. As is clearly seen in this figure, the XP4PA-SVwt cells (Fig. 2A) grow much faster than the AS3-SVwt cell line (Fig. 2B). The plating efficiencies of these transformed cell lines were measured by seeding at low density in normal liquid medium. The X P 4 P A SVwt cells had a plating efficiency of 40% and the AS3-SVwt 30%, slightly less, which is in good

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agreement with the other characteristics. Only X P 4 P A - S V w t cells show the ability to produce colonies in soft-agar but no growth was seen below a threshold seeding density of 105 cells per 60-ram plate. The difference in characteristics exhibited by these two cell lines was further demonstrated when metaphase preparations were examined. Estimates of chromosome numbers showed that both the parental cells had a modal number between 38 and 42. The X P 4 P A - S V w t showed heteroploidy with a modal number between 80 and 100 chromosomes while the AS3-SVwt normal fibroblast cell line retained a modal number of 38-42 chromosomes. Finally, in Fig. 2 it is interesting to note that the untransformed XP4PA fibroblasts grew rather better than the normal AS3 foetal fibroblasts; however, senescence was observed in these cells and they eventually died.

T antigen and p53 protein When cells were examined for the presence of SV40 T antigen, positive nuclei were observed by indirect immunofluoresence and 100% T antigen

positive cells were obtained in cultures by the sixth sub-culture. Radioactively labelled protein extracts from the transformed cells were incubated with either serum from Syrian hamsters bearing SV40-induced tumours or monoclonal antibodies against large T antigen. The latter serum immunoprecipitated the large T antigen together with a cell coded protein, p53 (Fig. 3 A-B). Both these proteins are highly phosphorylated as seen in the immunoprecipitate of the 32p-labelled protein extract (Fig. 3B).

Unscheduled DNA synthesis The DNA-repair capacity of the X P 4 P A - S V w t cell line was measured after UV-irradiation and compared to that of the parental cells. Fig. 4 shows UDS values as a function of UV dose and shows that XP4PA and its isogenic transformed derivative have around 30% repair ability relative to the normal cell line. Clearly the presence of the SV40 viral D N A has not modified the repair capacity of the XP4PA cells (Fig. 4). The AS3 cell line and its transformant show normal unscheduled D N A synthesis after UV-irradiation (Fig. 4).

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Virus survival Fig. 5 shows the viral progeny o b t a i n e d after

infection of XP4PA-SVwt cells with UV-irradiated tsA58 SV40 virus. The tsA58 SV40 mutant has a single base-pair substitution ( G : C to A : T ) which gives rise to a large T antigen temperaturesensitive for the initiation of viral D N A synthesis (Bourre and Sarasin, 1983). After infection, cells A 30 |

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were maintained either at the permissive temperature of 33°C for 10 days or at the restrictive temperature of 41°C for 8 days. The survival of the viral progeny was measured in CV1P cells by plaque assay at 33°C. The survival of the viral progeny from the XP4PA-SVwt cells is markedly decreased by UV-light when compared with survival in the permissive monkey CV1P cells or in the SV40 transformed normal human foetal fibroblasts AS3-SVwt and confirms that these transformed XP cells retain the deficiency in the repair of UV-damaged viral probes. The virus survival measured in XP4PA-SVwt and AS3SVwt after a lytic cycle at 41°C implies that in these cells the T antigen is functional for viral replication because it complements for growth of the tsA58 mutant at the restrictive temperature. In non-transformed cells the tsA58 mutant does not give rise to viral progeny at 41 ° C (data not shown). It should be noted that the decrease in viral progeny, with increasing UV dose, obtained in the XP transformed cells compared to that in normal transformed cells is accentuated at 41 ° C.

T'ANTIGEN

Organisation X P 4 P A - S Vwt

Fig. 6. Restriction enzyme mapping of the integrated SV40 genome in XP4PA-SVwt cells. (A) Total cellular D N A was digested with the indicated enzymes and analysed in agarose gels as described. Lane 1 shows supercoilcd (FI) and linear (FIII) SV40 D N A and Lane 2 is a reconstruction mixture containing 10/~g of cartier salmon sperm D N A with 10 pg of linear SV40 which approximates one copy per genome equivalent. Molecular weight markers (kbp) are indicated on the tight of the figure. Lanes 3 - 1 0 are digests with the enzymes shown of 10 /lg genomic DNA. The gel was Southern transferred and probed with SV40 as described in Methods. (B) M a p of the integrated SV40 determined from the data in (A) and other similar gels not shown. Open boxes represent SV40, hatched areas represent cell D N A and thin lines represent pBR327. Restriction enzyme sites determined to be present are shown in addition to the K p n I site included to aid the discussion. Only those Taq I and Msp I sites that are detectable with the SV40 probe are shown. A n interpretation of the blotting data is given in the text. The left hand junction with cellular D N A has been determined to be between the K p n I and EcoR I sites. The right hand junction (indicated by ?)

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The arrangement of the transforming viral DNA was determined by digestion of genomic DNA with restriction enzymes which cut pLASwt in each of the 3 possible ways: (i) Firstly, digestion with enzymes which do not cut at all (i.e. no sites in pLASwt) cleave in the flanking genomic DNA both sides of the integrated SV40 and will produce one band on agarose gel electrophoresis for each different site of integration; in addition the size of the band delimits the total number of copies of SV40. The no-cutting enzymes Xba I and Sac I each produce single bands (Fig. 6) indicating a single site of insertion. The sizes of the bands are approximately 18 kb and 13 kb respectively which places an upper limit of 2-3 copies of pLASwt. (ii) Secondly, enzymes which cut once within pLASwt should cleave out unit-length plasmid if there is more than one copy arranged in a tandem cannot be determined because apparently it is between pBR and cell D N A and is not detected by the SV40 probe. Unitlength 7.4 kbp pLASwt is cleaved out by EcoR I digestion as shown. The m R N A for SV40 T-antigen is indicated.

192

array, plus one or two extra fragments which would include flanking genomic D N A (it should be noted that the Southern blot has been probed

with SV40 not with pLASwt and therefore will not reveal fragments containing only pBR sequences). The enzymes EcoR I and BamH I cleave the

Fig. 7. Analysis of Hirt extracts. Cells were transfected with ~r SV plasmid and Hirt extracts prepared between 24 and 120 h after transfection as described in Methods. Portions of each extract were electrophoresed on agarose gels without (A) or with (B) Mbo I digestion, the gels were Southern transferred and probed with nick-translated ~r SV. In (A) undigested plasmid (amount indicated in nanograms) was run as marker; in the left most lane (a long exposure of the 1.0 ng marker lane) and in the cell extracts, forms I and II of the multimeric plasmid species can be seen and are indicated by n, 2n etc. In (B) the marker bands are Sau 3AI-digested ~r SV (1 ng) and the two main product fragments are indicated by the arrows. In both (A) and (B) 0.1 ng of BamHI digested form III qr SV is indicated by the diagonal arrow. These results show that the majority of plasmid extracted from all the cells as shown in (A) is sensitive to Mbo I digestion (B). Each gel was loaded with the same amount of Hirt extract. While replication of the shuttle vector in XP4PA-SVwt occurs to a similar extent and peaks at about the same time (48-72 h) as COS7 cells, the AS3-SVwt cells do not show comparable levels of replication until late times approaching 120 h; however these data have not been corrected for variations in cell number or transfection efficiency.

193 plasmid once and at the junctions of SV40/pBR. EcoR I generates a strongly hybridizing band of unit length (7.4 kb) plus a much fainter band of approximately 14 kb. This means that there is at minimum one copy of pLASwt in a head-to-tail relationship with a terminal, partial copy. In contrast BamH I does not produce a unit length band but gives a strongly hybridizing band of about 15 kb and a weakly hybridizing band of about 13 kb. This means that there are less than 2 complete copies. A comparison of the strengths of hybridization of the two BamH I and EcoR I bands suggests that the partial copy contains between one fifth to one tenth of SV40 sequences and this places the probable junction of SV40 and chromosomal D N A between the Kpn I - E c o R I region of SV40 as shown in the diagram at the bottom of Fig. 6. The other junction cannot be determined because apparently it is within pBR sequences and therefore will not be revealed by the SV40 probe. (iii) Thirdly, enzymes such as Msp I, Bgl I and Taq I cleave pLASwt in many positions. Fig. 6 shows that these digestions reveal the expected internal fragments, consistent with the analyses given above. These data are interpreted to indicate that XP4PA-SVwt contains 1.1-1.2 copies of the SV40 sequences in pLASwt (and at most 1.6 copies of pLASwt), arranged in a head-to-tail partial-dimer, integrated at a single chromosomal site (see diagram, bottom Fig. 6). This organization of the viral DNA has remained stable with long-term propagation of XP4PA-SVwt (data not shown). Replication of SV40-shuttle vector

The shuttle vector used in these studies was ~r SVHPplac (referred to here as tr SV; Fig. 1B) and was obtained from P.F.R. Little, Chester Beatty Institute (Little et al., 1983). It contains the 340 bp Hind I I I - P v u II fragment of wild-type SV40, which spans the viral replication origin, cloned into the vector of Seed (see Maniatis et al., 1982; Little et al., 1983). As ~r SV does not contain any coding sequence of the SV40 T antigen the replication of the vector in mammalian cells is dependent upon T antigen provided in trans. Our data indicate that ~r SV does replicate in both XP4PA-SVwt and AS3-SVwt cells. D N A from

Hirt extracts, prepared at various times after transfection with ~r SV, was electrophoresed on agarose gels without enzyme digestion (Fig. 7A) or after digestion with the restriction enzyme Mbo I (Fig. 7B). From Fig. 7B it is clear that the majority of the ~r SV in the Hirt extracts is sensitive to Mbo I, an enzyme that will not digest any unreplicated adenine-methylated input plasmid (the basis of this assay is described by Calos et al., 1983). The XP4PA-SVwt cells support replication of the vector with comparable kinetics and magnitude as monkey COS-7 cells which have been shown to replicate similar vectors very efficiently (Calos et al., 1983). AS3-SVwt shows less plasmid and the replication of the vector apparently peaks at a later time though, rather than a difference in kinetics of vector replication per cell, this may reflect a continuing increase in cell number due to initially fewer cells per dish and the sensitivity of these cells to transfection procedures. The plasmid D N A used in this experiment shows multimeric species which arise during propagation in the recA ÷ bacterial host (Maniatis et al., 1982; Little et al., 1983); that the many bands seen on agarose gel electrophoresis represent multimeric head-totail arrays is demonstrated by the production of unit-length linear plasmid by digestion with a single-cut enzyme (e.g. Bam H I; indicated by the arrow in Fig. 7A). It is interesting to note that all species appear to replicate with equal efficiency in the mammalian cells. Experiments performed simultaneously with those described above (data not shown) have given negative results in several other SV40 (ori ÷)-transformed cell lines including MRC5-V2 (Huschtscha and Holliday, 1983), AT5BI-VA (Green et al., 1985) and XP12RO-SV (Royer-Pokora et al., 1984). Discussion For detailed molecular biological studies of human genetic mutants, permanent cell lines are a prerequisite that has often represented a serious problem. While human lymphoblasts are efficiently immortalized following infection with Epstein Barr virus (Andrews et al., 1974), these suspension cell cultures are less amenable to some manipulations; for example they are difficult to

194 DNA-transfect. In contrast most other human cell types are extremely resistant to establishment as vigorous immortal cell lines. Small et al. (1980) demonstrated that the use of a replication origindefective SV40 was efficient in transformation of human fibroblasts. However, these authors did not assess the progression of the transformed loci to permanent immortal cultures. In practise this second step does not appear to be very efficient as there are few reports of successful establishment of cell fines using ori SV40 (Murnane et al., 1985; Barbis et al., 1986; Canaani et al., 1986). This would appear to reflect the low frequency of one or more secondary event(s) needed to acquire the fully transformed phenotype. One may draw a parallel here with the ineffectiveness of combinations of oncogenes used to attempt to transform human cells compared to rodent cells (Sager et al., 1983). Clearly a more complete understanding of the multiple genetic or epigenetic events involved in transformation of human cells is required before any given fibroblast cell line may b e immortalized with assured success. An alternative means of establishing permanent cell lines has been described recently (Mayne et al., 1986). Although this method used ori + SV40, the novel feature was that the SV40 transforming sequences were in a vector that also carried a dominant selectable marker. Selection for the marker after transfection of diploid fibroblasts produced many DNA-transformants which also showed morphological changes characteristic of SV40 transformants and immortal lines were eventually derived. The selection for the dominant marker may have a similar effect as using o r i - SV40, by imposing chromosomal stability of the SV40 sequences which are tightly linked to the marker gene. Other effects may be operating however, for example selection for DNA-transformants allows much improved logistics because a larger seed population may be transfected yet only the rare (10 4 to 10 -5) D N A recipients remain in culture. Nevertheless, the SV40 ori which we have used in this report (and also used by Murnane et al., 1985; Barbis et al., 1986; C a n a a n et al., 1986), has certain advantages which improves the overall success rate of immortalization. It is believed that the inability of the SV40 o r i - sequence to respond to T antigen stimulation of replication leads to a

greater stability of the SV40 sequences. Upon integration into the host genome, these sequences are maintained in a stable manner with no detectable extrachromosomal viral D N A (see Results, this paper). This is in contrast to long-established SV40 virus transformed cell lines in which variable levels of free SV40 D N A are often detected and the integrated D N A tends to show an instability upon further cell manipulation (Green et al., 1985; Daya-Grosjean et al., 1984; Norkin et al., 1985). Also we have observed extrachromosomal SV40 sequences in one cell line, 46BRSV3, described by Mayne et al. (James, unpublished) which suggests the persistence of replication-competent T-antigen and SV40 origin of replication. While systematic studies to compare these two approaches have not been performed, one conclusion from the observations of ourselves and Mayne et al. is that a combination of the two strategies may lead to more consistent success in immortalisation of human fibroblasts. To that end we have constructed a derivative of pLASwt which includes a selectable gene (G418 resistance) and we are assessing its transforming abilities. A similar vector has been described which uses an inducible promoter in place of the SV40 replication origin early promoter (Gerard and Gluzman, 1985). In our current work described here, we have obtained a SV40 o r i - transformed derivative of xeroderma pigmentosum group C cells. The cell line, XP4PA-SVwt, is immortal with a single integration site which has remained stable over two years. It has retained the phenotypic characteristics of the parental fibroblasts and of xeroderma pigmentosum as seen by their reduced repair capacity after UV damage, measured by unscheduled D N A synthesis. The UV-repair deficiency in X P 4 P A - S V w t is further demonstrated by the reduced survival of irradiated SV40 in these cells. SV40 large T antigen is expressed in these cells together with elevated levels of phosphorylated cellular p53 protein. Phosphorylated p53 is not detected in normal human cells and there may be a correlation between p53 function and its phosphorylation during tumorigenesis. Moreover, an important contribution of the SV40 large T antigen in this process may be its ability to bind the p53 thus considerably stabilising the protein by increasing its effective concentration (Milner and

195 G a m b l e , 1985; M o n t e n a r h et al., 1985). O u r observations c o n c e r n i n g the p53 p r o t e i n have b e e n s u b s t a n t i a t e d b y the recent reports showing its direct i n v o l v e m e n t i n the establishment of transf o r m a t i o n a n d its ability to immortalize cells (Jenkins et al., 1984; Eliyahu et al., 1984, 1985; P a r a d a et al., 1984). Finally, the T a n t i g e n expressed i n the X P 4 P A - S V w t cells supports the replication of SV40-based shuttle vectors. D a t a to be presented elsewhere e x a m i n e i n detail the replication a n d plasmid-rescue in bacteria of SV40 a n d B K V - b a s e d shuttle-vectors. These cells are efficiently transfected as d e t e r m i n e d b y assays for c h l o r a m p h e n i col acetyl transferase (CAT) after transient transfection with C A T expression vectors a n d they are efficiently t r a n s f o r m e d using d o m i n a n t l y selectable markers ( m a n u s c r i p t in preparation). Thus these cells will be useful for the i n t r o d u c t i o n , expression a n d selection of genes related to D N A repair a n d to the study of mutagenesis using defined molecular probes.

Acknowledgements W e t h a n k Dr. J. Bou6 for giving us the a m n i o t i c fluids a n d Dr. G. R e n a u l t for helping us with the repair assay. M.R. James was supported b y a fellowship from I n t e r n a t i o n a l Agency for Research o n Cancer, Lyon. This work was supported b y the c o m m i s s i o n of E u r o p e a n C o m m u n i t i e s (Brussels, Belgium) a n d the " A s s o c i a t i o n p o u r le D r v e l o p p e m e n t de la Recherche sur le C a n c e r " (Villejuif, France).

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