MATERIALS AND METHODS

Cells and media

Kps3 is a primary fibroblast designated UV^sS (^{Itoh et al. 1994,1995}). The human primary fibroblast cell strain XP2SA (XP-V) and CS1MO (CS complementation group B (CS-B)) were purchased from the Human Science Research Resources Bank (HSRRB, Osaka, Japan). Mori was a primary normal fibroblast cell strain established in this laboratory (^{Itoh et al. 1994,1995,1996}). WI38VA13 was a normal SV40-transformed cell line. CS2OSSV, SV40-transformed CS-A cell line, was kindly gifted from Dr. M. Ikenaga (Kyoto University). CS1MOSV, SV40-transformed CS-B cell line was established in our laboratory. All cells were cultured in Dulbecco's modified Eagle's Minimum Essential Medium (DMEM; ICN, Irvine, CA) supplemented with 10% fetal bovine serum (FBS; ICN) and antibiotics [Penicillin G (100 units per ml), streptomycin (100 g per ml)] in a humidified incubator in 5% CO₂.

Plasmid DNA

pSVori-8–16, a plasmid defective replication origin of simian virus 40 (SV40), was purchased from the HSRRB (^{Gluzman et al. 1980}). pcDNAI/Neo, an expression vector in mammalian cells, was purchased from Invitrogen (Carlsbad, CA). pCMVEBNA and pSV2neo were purchased from Clontech. pCSA5 (^{Henning et al. 1995}) and pcBlsSE6 (^{Troelstra et al. 1992}) were kindly gifted from Dr. E.C. Friedberg (University of Texas South-western Medical Center) and Dr. J.H.J. Hoeijmakers (Erasmus University), respectively. pCI-neo, a mammalian expression vector, was purchased from Promega (Madison, WI). To construct pCI-ERCC6, both pcBlsSE6 and pCI-neo were digested by Sal I (Roche Molecular Biochemicals, Mannheim, Germany) at 37°C, and subcloned ERCC6 cDNA into pCI-neo.

DNA transfection and immortalization

One day before DNA transfection 5 10⁵-1 10⁶ cells of Kps3 were seeded into 100 mm dishes. Transfection was carried out using the calcium-phosphate precipitation method (Mammalian Transfection Kit; Stratagene, La Jolla, CA). Twenty micrograms of pSVori-8–16 plasmid DNA was added to each dish. Twenty-four hours after transfection, cells were replated, grown, and then maintained at confluence. Several weeks later, transformed colonies were distinguished from the growth-arrested fibroblasts by their morphology and enhanced proliferative activity. The cells of polyclonal cultures were then passaged 1:4 or 1:8, when they had just reached confluency, until they reached crisis and ceased growing. Aliquots of these populations were cryopreserved at different stages of this growth-enhanced phase. When the cells reached crisis, they were maintained by changing media repeatedly. Several colonies emerged out of a population in crisis and were individually cloned. We have recovered two immortalized clones in different transfections. The clones were designated Kps3SVY and Kps3SVI3. Kps3SVY and Kps3SVI3 cells have been propagated in culture for over 6 and 4 y, respectively.

Transfection efficiency was determined by the calcium phosphate precipitation method as described above. Briefly, 1 d before DNA transfection, 1 10⁶ cells of Kps3SVY or Kps3SVI3 were seeded into 100 mm dishes. pcDNAI/Neo (Invitrogen) plasmid DNA was digested with Sfi I (New England Biolabs, Beverly, MA) at 50°C. Thirty micrograms of linearized pcDNAI/Neo was added to each dish. Twenty-four hours after transfection, cells were replated; 72 h after transfection, 150–300 g per ml of G418 (Gibco, Grand Island, NY) was added to the media. Approximately 3 wk after transfection, cells were fixed with 80% methanol and stained with Giemsa.

DNA transfection and selection of transformants

Kps3SVI3EB3 and CS2OSSVEB were constructed by cotransfection of pCMVEBNA and pSV2neo by the method as described above. Seventy-two hours after transfection, 150 g per ml of G418 was added to the media. Both Kps3SVI3EB3 and CS2OSSVEB cells expressed EBNA-1 protein as confirmed by immunoassay [immunostaining and/or western blotting using antibody EBNA (Ab-1) (Oncogene Science, Uniondale, NY)]. Ten micrograms of pCSA5 was transfected into Kps3SVI3EB3 and CS2OSSVEB cells by the method as described above; 10 g of pCI-ERCC6 was transfected into Kps3SVI3 and CS1MOSV cells by the method as described above. Seventy-two hours after transfection, 150 g per ml of hygromycin B (pCSA5) (Gibco) or G418 (pCI-ERCC6) were added to the media. Approximately 2–4 wk after transfection, several colonies were individually cloned, and grown mass cultures.

Microinjection assay

Microinjection assay was performed as described elsewhere (^{Itoh et al. 1994,1995}). Kps3SVI3, CS2OSSV, or CS1MOSV cells were seeded on coverslips and incubated until they became subconfluent. pCSA5 or pCI-ERCC6 was microinjected into the nuclei of the cells, with glass needles. After incubation for 16–24 h, cells were irradiated with UV light (254 nm) at a dose of 15 J per m^2. Subsequently, RRS was measured as described above. The coverslips were then mounted on glass slides, dipped in Kodak NTB-3 emulsion, and exposed for 24 h at 4°C.

DNA probes and southern hybridization

Variable number of tandem repeat (VNTR) probes (pYNH24, pYNZ32, pYNZ132, and pCMM86) developed originally by^{Nakamura et al. (1987)} were purchased from the HSRRB. Chromosome locations of pYNH24, pYNZ32, pYNZ132, and pCMM86 are 2q, 4p, 6, and 17q, respectively. VNTR probes were labeled with [-³²P] dCTP (6000 Ci per mmol) using Megaprime DNA labeling system (Amersham, Cleaveland, OH). Four different restriction enzymes Bgl II, Hinf I, Pst I, and Msp I (Roche Molecular Biochemicals) were used for the digestion of DNA specimens (5 or 10 g of extracted DNA) and incubated at 37°C for 10–12 h using 50–100 units of enzymes per sample. The digested DNA fragments were loaded on a 25 cm long horizontal 0.8% of 1 Tris-acetate/EDTA (TAE) agarose gel (Pst I and Msp I digested sample probing with pYNH24 and Bgl II and Hinf I digested sample probing with pCMM86) or 1.2% of 1 TAE agarose gel (Pst I digested sample probing with pYNZ32 and pYNZ132). Electrophoresis was carried out in 1 TAE buffer at 1 V per cm of gel for 15–41 h. The DNA fragments were then transferred to a niron membrane (Hybond-N+ Amersham). Prehybridization was carried out at 65°C in a solution containing 1 mM EDTA/7% (wt/vol) SDS/1 M NaHPO₄, pH 7.2 [1 M NaHPO₄ (pH 7.2) stock is composed of 71 g of Na₂HPO₄ and 4 ml of 85% H₃PO₄ per liter] described by^{Church & Gilbert (1984)}. The filters were hybridized with probes at 65°C for 18–21 h in the same solution used for prehybridization. The filters were washed three times at 65°C for 5 min with a solution containing 1% (wt/vol) SDS/40 mM 1 M NaHPO₄ (pH 7.2). The filters were analyzed using a Fujix Bio-Analyzer BAS-2000 (Fuji Photo Film, Tokyo, Japan) for an appropriate time. The images of pYNZ132 and pCMM86 were processed using Adobe Photoshop 5.0-J software.

Calculations of the probability that SV40-transformed cell lines were unrelated to the original Kps3 cells were based on the assumption that genotype frequencies at the genetic marker loci are independent of each other. Allele frequencies were assumed to be those of the most common alleles at each locus, and the Hardy–Weinberg equation was used to determine genotype frequencies. The overall probability that the SV40-transformed cells could have been derived from cells other than Kps3 cells was determined by multiplication of the genotype frequencies at all loci tested.

Measurement of UDS and RRS after UV irradiation

UDS and RRS after UV irradiation were measured by a method described elsewhere (^{Itoh et al. 1994}) with some modifications. Briefly, to minimize variations between different preparations and to facilitate comparison, 20 l aliquots (1–2 10⁴ cells) of the patient's and control (WI38VA13) cell suspensions were plated with micropipettes in two rows on the same coverslip (18 mm 18 mm) (^{Itoh et al. 1994}). The cells were incubated for 5–7 d. They were then washed with phosphate-buffered saline (PBS), irradiated with UV (254 nm) at a dose of 30 J per m², immediately labeled with [³H] thymidine (40 Ci per ml) for 2.5 h, and fixed. Coverslips were mounted on glass slides, dipped in NTB-3 emulsion (Kodak), and exposed for 24 h at 4°C. Grains above 50 nuclei were counted under a microscope.

RRS was measured as described elsewhere (^{Itoh et al. 1994}). One to two 10⁴ cells were plated on a coverslip as described above and incubated for 1–2 d to teach the growth phase. They were then washed with PBS and irradiated with UV (254 nm) at a dose of 15 J per m^2. Cells were then incubated for 23 h and labeled with [³H]uridine (40 Ci per ml) for 1 h. Autoradiography was performed by the method described above.

Growth in soft agarose

Kps3SVY and Kps3SVI3 cells were tested for their ability to grow in 0.1% or 0.3% soft agarose. The method for preparation of soft agarose was described by^{Volpe & Cleaver (1995)}. Briefly, agarose was dissolved in boiling PBS, cooled to 46°C, and diluted to 0.1% or 0.3% in medium containing serum, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and cell suspensions. The cellular agarose suspension was plated and hardened on ice before being placed in a 37°C incubator in 5% CO₂.

Doubling time

One 10⁴ cells of Kps3SVY and Kps3SVI3 were seeded into 60 mm dishes. Cells were harvested and counted at regular intervals using trypan-blue staining. Each point shows mean values from three independent experiments for each cell line.

UV survival assay and plating efficiency

UV survival was measured according to^{Itoh et al. (1994)}. Briefly, appropriate numbers of cells were inoculated onto 60 mm dishes and left to attach for 10 h. Subsequently, cells were rinsed with PBS and exposed to UV light (254 nm) at a fluence rate of 0.7 J per m per s. After 1–2 wk, the plates were washed with PBS, fixed with 80% (vol/vol) methanol and stained with Giemsa, and colonies were counted. For each dose, three dishes were used per experiment. The relative survival was plotted versus the UV dose on semilogarithmic paper. To determine the effect of caffeine on UV survival, caffeine was added at a final concentration of 1 mM just after UV irradiation and the plates were incubated for 2 wk. Plating efficiency was determined by colony numbers at 0 J per m² of UV survival.

UV survival using trypan-blue staining was as follows: 1 10⁵ cells were seeded onto 60 mm dishes and left to attach for 10 h. Subsequently, cells were rinsed with PBS and exposed to UV light as described above. After 3 d, viable cells were counted by using trypan-blue staining. For each dose, three dishes were used per experiment.

RESULTS AND DISCUSSION

Establishment of immortal SV40-transformed UV^sS cells

To isolate immortal cells from a primary cell strain, Kps3, we transfected the SV40 DNA containing the large T antigen with a defective origin of DNA replication into Kps3 cells by the calcium-phosphate precipitation method. After replating the cultures once, we allowed them to become confluent and watched for the emergence of foci of transformed cells. These cells were passaged 1:4 or 1:8. They exhibited increased growth rates, compared with the parental cells, and significantly extended life spans. After a phase of vigorous growth, however, the growth rate began to decline until proliferation ceased and the cultures entered crisis (Figure 1). With this approach, we recovered Kps3SVY and Kps3SVI3 cells, as SV40-transformed Kps3 fibroblasts in different experiments. Kps3SVY cells are derived from the original experimental plate. In contrast, Kps3SVI3 cells are derived from a rare colony emerging from a culture in crisis and being isolated (Figure 1). The remaining cells of the original plates showed a finite life span. Both Kps3SVY and Kps3SVI3 cells express the SV40 large T antigen as demonstrated by immunostaining (data not shown).

Figure 1.

Pattern of proliferation of pSVori-8–16 transfected clonal populations of UV^sS fibroblasts. Cumulative population doublings after the start of transfection were determined from the number of cells recovered from culture plates at the indicated times. Figure indicates the pattern of Kps3SVI3 cells.

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VNTR analyses

To confirm whether these immortal clones are derived from the parental Kps3 cells, we conducted VNTR analyses (Figure 2). We used the VNTR probes for markers on chromosomes 2q (Figure 2a–c), 4p (data not shown), 17q (Figure 2d), and 6 (Figure 2e). Figure 2 shows the results of VNTR analyses in Kps3, Kps3SVY, Kps3SVI3, and three unrelated human primary cell strains (Mori, XP2SA, and CS1MO). Kps3SVY and Kps3SVI3 cells displayed genotypes identical to those in the parental Kps3 cells, but distinct from those in Mori, XP2SA, or CS1MO cells (Figure 2a–e); however, Kps3SVI3 cells have lost one of the two chromosomes 2q and 6 on the pYNH24 and pYNZ132 locus, respectively (Figure 2a,e). We further examined Kps3SVI3 cells at several stages in the transforming process using the pYNH24 probe (Figure 2b,c). One of the two chromosomes 2q in Kps3SVI3 cells at approximately 100 population doublings (PDL), which immediately emerged following crisis, remained at the same position as in the parental Kps3 cells (Figure 2b, lane 2; Figure 2c, lane 3). That in Kps3SVI3 cells over 200 PDL was lost completely (Figure 2b, lane 4; Figure 2c, lane 2). Thus, it seems that Kps3SVI3 cells lost one of the two chromosomes 2q with increased passages. Based on these results, we concluded that both cells were derived from the parental Kps3 cells, as the probability that Kps3SVY and Kps3SVI3 cells derived from a cell type other than the parental Kps3 cells was less than 1.3 10^-5.

Figure 2.

VNTR analyses of SV40-transformed UV^sS cells to compare the genotypes of different cells and the parental UV^sS cells. (a) Ten micrograms per lane of genomic DNA, digested by Pst I, was loaded on 0.8% of 1 TAE agarose gel. Electrophoresis was carried out at 1 V per cm for 41 h. The DNA fragments were detected using pYNH24 probe. Lane numbers indicate DNA from Kps3SVY (1), Kps3SVI3 (2), Kps3 (UV^sS) (3), Mori (normal) (4), and XP2SA (XP-V) (5), respectively. (b) Ten micrograms per lane of genomic DNA, digested by Pst I, was loaded on 0.8% of 1 TAE agarose gel. Electrophoresis was carried out at 1 V per cm for 15 h. The DNA fragments were detected using pYNH24 probe. Lane numbers indicate DNA from Kps3 (1), Kps3SVI3 (200 PDL) (2), Kps3SVY (3), and Kps3SVI3 (4), respectively. (c) Ten micrograms per lane of genomic DNA, digested by Msp I, was loaded on 0.8% of 1 TAE agarose gel. Electrophoresis was carried out at 1 V per cm for 24 h. The DNA fragments were detected using pYNH24 probe. Lane numbers indicate DNA from Kps3SVY (1), Kps3SVI3 (2), Kps3SVI3 (200 PDL) (3), Kps3 (4), Mori (5), CS1MO (CS-B) (6), and XP2SA (7), respectively. (d) Five micrograms per lane of genomic DNA, digested by Hinf I, was loaded on 0.8% of 1 TAE agarose gel. Electrophoresis was carried out at 1 V per cm for 26 h. The DNA fragments were detected using pCMM86 probe. Lane numbers indicate DNA from Kps3SVY (1), Kps3SVI3 (2), Kps3SVI3 (200 PDL) (3), Kps3 (4), Mori (5), CS1MO (6), and XP2SA (7), respectively. (e) Five micrograms per lane of genomic DNA, which was digested by Pst I, was loaded on 1.2% of 1 TAE agarose gel. Electrophoresis was carried out at 1 V per cm for 26 h. The DNA fragments were detected using pYNZ132 probe. Lane numbers indicate DNA from Kps3SVI3 (1), Kps3 (2), Mori (3), XP2SA (4), and Kps3SVY (5), respectively.

Full figure and legend (209K)

Figure 6.

UV survival of SV40-transformed UV^sS cells transfected with CSA or CSB cDNA. One 10⁵ cells were inoculated onto 60 mm dishes. After UV irradiation at a fluence rate of 0.7 J per m² per s, cells were incubated for 3 d, and counted by using trypan-blue staining. Each point represents an average of the following independent experiments: WI38VA13 cells () were used as a normal control (two experiments). (a) CS2OSSVEB cells ( four experiments); CS2OSSVEB-pCSA5 ( three experiments). (b) CS1MOSV cells ( four experiments); CS1MOSV-pCI-ERCC6 ( three experiments). (c) Kps3SVI3EB3 cells ( two experiments); Kps3SVI3EB3-pCSA5 cells ( four experiments). (d) Kps3SVI3 cells ( two experiments); Kps3SVI3-pCI-ERCC6 cells ( six experiments). Error bar, SEM.

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Morphology and cellular characteristics of SV40-trans- formed UV^sS cells

The morphology of Kps3SVY and Kps3SVI3 cells is shown in Figure 3. Cells of both lines were slightly smaller than Kps3 cells. Kps3SVY cells are likely to make a mass formation compared with Kps3SV13 cells during their growing processes (Figure 3). The doubling time of these cells is shown in Figure 4 and Table 1. The growth rate of Kps3SVY cells was lower than that of Kps3SVI3 cells. The plating efficiency of Kps3SVY cells was also lower than that of Kps3SVI3 cells (Table 1). Moreover, although Kps3SVY cells did not grow in soft agar, Kps3SVI3 cells grew sparsely in soft agar (Table 1). The UV sensitivity, Kps3SVI3 cells were similar to Kps3 cells, but Kps3SVY cells were more sensitive than Kps3 cells (Figure 5). A diminution by caffeine on UV survival was not found in both cells (Figure 5), whereas that in SV40-transformed XP variant cells was found (^{Volpe & Cleaver 1995}). Both cells showed the defect in RRS (Table 2). Thus, in spite of using the same method to immortalize Kps3 cells, Kps3SVY and Kps3SVI3 cells exhibited slightly different phenotypes. These cellular differences of Kps3SVY and Kps3SVI3 may be caused by the loss of the one allele of several chromosomes as described above.

Figure 3.

Morphology of SV40-transformed UV^sS cells. Appropriate numbers of cells were seeded and incubated for 2–3 d. (a) Kps3SVY, (b) Kps3SVI3, (c) Kps3 cells. Scale bar: 200 m.

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Figure 4.

Growth Curves of SV40-transformed UV^sS cells. Kps3SVY cells (); Kps3SVI3 cells (). Cells were seeded in 60 mm dishes, and then counted at regular intervals (24, 48, 72, 120, and 144 h) using trypan-blue staining. Each point shows mean values from three independent experiments for each cell line. The SEM is smaller than the symbols for most points.

Full figure and legend (15K)

Figure 5.

UV survival of SV40-transformed UV^sS cells. Appropriate numbers of cells were inoculated onto 60 mm dishes. After UV irradiation at a fluence rate of 0.7 J per m² per s, cells were incubated for 14 d, fixed with 80% methanol, and stained with Giemsa. To determine the effect of caffeine, caffeine was added at a final concentration of 1 mM just after UV irradiation. Each point represents an average of three or four experiments: error bar, SEM. WI38VA13 cells () were used as a normal control. Kps3 cells (); Kps3SVY cells (); Kps3SVY cells with 1 mM caffeine (); Kps3SVI3 cells (); Kps3SVI3 cells with 1 mM caffeine ().

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Table 1 - Cellular characteristics of SV40-transformed UV^sS cells.

Full table

Table 2 - Levels of unscheduled DNA synthesis and recovery of RNA synthesis in SV40-transformed cells.

Full table

Complementation test

To confirm that SV40-transformed UV^sS cells are distinct from CS cells, we performed a further complementation test by transfection. We transfected CSA or CSB cDNA into Kps3SVI3EB3 or Kps3SVI3 cells, and measured UV survival (Figure 6). Although CSA or CSB cells were complemented with CSA or CSB cDNA, respectively (Figure 6a,b), Kps3SVI3 cells were not complemented with both cDNA (Figure 6c,d). Moreover, to confirm these results, we performed a microinjection assay using CSA or CSB cDNA. RRS levels of Kps3SVI3 cells were not recovered by microinjection of these cDNA (data not shown). These results confirmed that Kps3SVI3 cells were distinct from CS cells.

The preferential repair of the active genome compartment is accounted for largely by the faster repair of the transcribed strand (^{Mellon et al. 1987}). This specialized strand-directed form of NER is designated transcription-coupled repair (TCR). CS cells are deficient in enhanced repair of the transcribed strand of the active genes, i.e., deficient in TCR, and having normal global genome repair (^{Venema et al. 1990;van Hoffen et al. 1993}). The defective factors of CS have been cloned (^{Troelstra et al. 1992;Henning et al. 1995}). CSB protein might possess a helicase function (^{Troelstra et al. 1992}). CSA protein is a member of the tryptophan-aspartic acid (WD)-repeat family of regulatory proteins and interacts with the p44 protein, a subunit of basic transcription factor TFIIH, and with CSB protein (^{Henning et al. 1995}). Recently, the interactions between CSB and XPA, and CSB and XPB were reported (^{Selby & Sancar 1997}). XPB and XPD have been shown to carry mutations in subunits of TFIIH (^{Schaeffer et al. 1993,1994;Van Vuuren et al. 1994}). These results suggest that CS is involved in transcription and NER. Moreover, it was hypothesized that CS was deficient in two processes: impaired TCR and an inability to release trapped transcription (^{Van Gool et al. 1997}). Because UV^sS patients showed the biochemical characteristics of CS (^{Itoh et al. 1994,1995}) and SV40-transformed UV^sS cells showed CS phenotype (Table 2; Figure 5, 6), the defective factor in UV^sS may be a TCR factor and/or transcription factor. UV^sS patients, however, showed mild clinical manifestations without neurologic abnormalities compared with CS patients. We consider that this discrepancy may be caused by the differences of TCR capacity against oxidative damages. Recently, it was reported that CS cells were deficient in TCR of thymine glycols (^{Leadon & Cooper 1993}). If oxidative damages such as thymine glycols induced the demyelination of neurons in CS patients, UV^sS patients may show a normal level of TCR against oxidative damages. These cell lines offer the opportunity to utilize transfection studies on cells with the UV^sS defect in DNA repair and transcription.

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Acknowledgments

We thank Dr. Naohiro Tsuyama for technical advice on establishing immortal clones; Dr. Mitsuo Ikenaga, Dr. Errol C. Friedberg, and Dr. Jan H.J. Hoeijmakers for gifts of cells or plasmids; and Dr. Graciela Spivak for helpful discussion of the manuscript. We also thank Mrs. Yuka Itoh for preparation of the manuscript. This work was supported partly by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan (to T.I. and M.Y.), by the Lydia O'Leary Foundation (to T.I.), by the Naito Foundation (to T.I.), and by the Kao Foundation for Arts and Sciences (to T.I.).