Therapy (1997) 4, 1077–1084  1997 Stockton Press All rights reserved 0969-7128/97 $12.00 Retrovirus-mediated gene transfer corrects DNA repair defect of pigmentosum cells of complementation groups A, B and C

L Zeng, X Quilliet, O Chevallier-Lagente, E Eveno, A Sarasin and M Mezzina Laboratoire de Ge´ne´tique Mole´culaire, UPR 42 CNRS, 94801 Villejuif,

With the aim to devise a long-term protocol survival, unscheduled DNA synthesis and recovery of RNA for skin in individuals affected by the inherited synthesis, and Western blots. The results show that the autosomal recessive , we trans- recombinant retroviruses are highly efficient vectors to ferred the human DNA repair XPA, XPB/ERCC3 and XPC transfer and stably express the human DNA repair cDNAs, by using the recombinant retroviral vector LXSN, in XP cells and correct the defect of DNA repair of group into primary and immortalized fibroblasts obtained from two A, B and C. With our previous results with XPD/ERCC2, XP-A, one XP-B (associated with Cockayne’s syndrome) the present work extends further promising issues for the and two XP-C patients. After transduction, the complete gene therapy strategy for most patients suffering from this correction of DNA repair deficiency and functional -prone syndrome. expression of the transgenes were monitored by UV

Keywords: retroviral vectors; DNA repair genes; therapy; xeroderma pigmentosum

Introduction complementing) genes.11,12 Among them, ERCC2, ERCC3, ERCC4, ERCC5 and ERCC6 were found to be identical to Xeroderma pigmentosum (XP) is a rare human autosomal the genes involved in xeroderma pigmentosum groups recessive disease characterized clinically by hypersensi- D, B, F and G and Cockayne’s syndrome group B.13–17 A tivity to (UV) rays, high predisposition for multiprotein complex of approximately 30 gene products developing skin cancers (basal and squamous carci- is involved in the NER pathway, whereby DNA damage 1 nomas and ) on sunlight exposed areas, and is eliminated and replaced by excision-resynthesis, as has 2–4 in some cases, neurological disorders. XP has a world- been demonstrated by using the in vitro repair assay with wide distribution, with the incidence varying from about purified factors.18 in one of these genes result 1:250 000 in Europe and the USA to as high as 1:40 000 in UV-sensitive disorders: skin cancer-prone XP and 5–7 in Japan, North Africa and in Egypt. The cellular other clinically different syndromes, such as skin cancer- phenotype of XP has increased sensitivity to killing fol- free (TTD) and lowing exposure to a wide variety of DNA damaging (CS), and association of XP with CS. agents, including UV and UV mimetic chemi- The NER process is evolutionarily conserved in eukar- cals. The observation that skin fibroblasts in culture from yotes, and homologue genes of human repair genes have an XP patient are unable to carry out nucleotide excision been identified in many organisms, namely in Saccharo- repair (NER) following exposure to UV established the myces cerevisiae.19 In humans, the encoded by relationship between DNA repair defect and skin photo- some of these genes are components of the multiprotein 1,8,9 in man. TFIIH complex involved with RNA II in The systematic complementation of DNA repair basal . Consistently, the homologues of defects by cell fusion assay has led to the identification some of these proteins, the Rad25 and Rad3 (homologue of seven genetic groups in this disease, designated XP-A to ERCC3/XPB and ERCC2/XPD, respectively), have also 7 to -G, and a variant group, designated XP-V, exhibiting been identified as components of TFIIH. Therefore, these 10 normal NER. So far, most of the relevant human genes, proteins play a dual role in both NER and transcrip- such as XPA, XPB, XPC, XPD, XPF and XPG, have been tion.20–22 cloned and mapped to different specific chromosomal The mean age of XP patients at the time of diagnosis locations. A further source of NER defective is a is 3 years while the mean age of onset of first skin cancer set of 11 complementation groups of UV-sensitive rodent is 8 years. There is an approximate 30–40 year reduction cells. The human genes that correct the rodent cell pheno- in survival. Many patients have died of .2 So type are designated ERCC (excision repair cross- far, there is no effective long-term treatment available for XP patients. Some protective measures can be taken to keep patients from exposure to sunlight,23 and skin grafts Correspondence: M Mezzina from the same patient were performed to resurface the Received 24 March 1997; accepted 23 May 1997 area with cancer in some patients. However, the latter Correction of inborn DNA repair defect by gene transfer L Zeng et al 1078 therapeutical protocol is efficient only in the short term, complementation groups, transduced and untransduced since skin grafts are still genetically DNA repair defective cells were analyzed for: and thus cancer prone.24 The genetic correction of XP (1) UV survival: The colony-forming ability was determ- cells by retrovirus-mediated transduction with appropri- ined in primary and SV40 immortalized cells. Figure ate DNA repair genes may allow possible alternative 2 shows survival curves of wild-type SV40 immor- long-term therapies. talized MRC5V1 and diploid 198VI fibroblast com- Studies in vitro have shown that DNA repair defects pared with those of UV-sensitive untransduced can be corrected by introducing relevant DNA repair XP12ROSV and XP24VI (XP-A group, Figure 2a and genes into cells derived from XP patients.25,26 Several b, respectively), XPCS2BASV (XP-B group, Figure 2c) retroviral constructions are efficient tools for gene deliv- and XP16VI and XP30VI (XP-C group, Figure 2d). ery and stable expression in many human cells and After transduction with the retrovirus harboring the tissues.27 We recently devised the first retrovirus harbor- appropriate cDNA, all cell lines recovered wild-type ing a DNA repair gene (XPD/ERCC2), which efficiently UV resistance, since their survival curves were indis- transduces several DNA repair-deficient human primary tinguishable from those of 198VI and MRC5V1 lines skin fibroblasts belonging to the complementation XP-D (Figure 2a–d). When XP cells were cross-transduced group and fully corrects their DNA repair defect.28,29 In with retrovirus containing other cDNA than that order to validate this investigation with other genes involved in the genetic group, no correction of UV involved in XP disease and to dispose of basic tools for survival was observed (Figure 2a, c and d, dotted gene therapy for a larger population of patients, we lines). This indicates that the retroviral transduction decided to develop additional constructions harboring is gene specific, consistent with our previous obser- other available DNA repair cDNAs. In this paper, we vations with LXPDSN.28,29 describe the construction of retrovirus harboring the (2) Unscheduled DNA synthesis (UDS): To correlate the genes XPA, XPB and XPC and show that their expression recovery of UV survival with the ability to perform fully corrects the DNA repair-deficient phenotype of repair synthesis, the incorporation of 3H-thymidine fibroblasts from two XP-A, one XP-B and two XP-C was measured after UV irradiation (Figure 3). Both patients. XP12ROSV and XP24VI exhibited reduced UDS lev- els, (5.4 ± 2 and 2.9 ± 1.1 grains per nucleus) at 15 Results J/m2, corresponding to 26 and 14.6% of normal levels, respectively. After transduction of the same cells with Establishment of recombinant retrovirus LXPASN, the UDS levels were restored to almost nor- The LXPASN, LXPBSN and LXPCSN vectors were mal levels (18 ± 4 and 17 ± 5 grains per nucleus, derived from the Moloney murine respectively, corresponding to 85% of the normal lev- (MoMLV)-based retroviral vector LXSN and were pro- els, Figure 3a and b). XPCS2BASV cells showed vir- duced after insertion into the polylinker of XPA, XPB and tually a complete defect of DNA repair with all three XPC cDNAs as described in Materials and methods and depicted in Figure 1. The expression of the introduced sequence was under the control of the LTR promoter and the polyadenylation signal. After transfection of ⌿CRE packaging cells, subsequent infection of ⌿CRIP cells and selection with G418,30–32 among all clones tested, the best virus titers obtained were 3 × 105 c.f.u./ml for LXPASN, 3 × 106 c.f.u./ml for LXPBSN and 2 × 106 c.f.u./ml for LXPCSN. The difference of virus titers might be due to either different expression levels of neo marker or the cytotoxic effect of the transgene, or both.

Gene-specific correction of DNA repair defect To examine whether the transferred XPA, XPB and XPC correct the DNA repair defect of cells of the three XP

Figure 2 UV survival curves in untransduced and transduced XP groups A, B and C cells. (–„–) MRC5V1 (a and c) and 198VI (b and d) wild- Figure 1 Scheme of retroviral vector LXSN based on Moloney murine type cells; (–ć–) XP12ROSV (a), XP24VI (b), XPCS2BASV (c) and leukemia virus. LTR: long terminal repeat; ⌿+: packaging signal; poly- XP16VI (d) untransduced and (–b–) transduced cells with LXPASN, linker for insertion of DNA repair cDNAs: XPA, XPB and XPC; SV40: LXPBSN and LXPCSN, respectively; (–̅–) untransduced and (–̆–) simian virus 40 early promoter; NEO: neomycin phosphotransferase gene. transduced XP30VI cells with LXPCSN (d); (--࡯--) cells transduced with Arrows show transcription initiation orientation. LXPCSN (a and c) and LXPBSN (d, for XP30VI cells only). Correction of inborn DNA repair defect by gene transfer L Zeng et al 1079

Figure 3 Unscheduled DNA synthesis of transduced and untransduced XP-A (a and b), XP-B/CS (c) and XP-C (d) cells.

UV doses (Ͻ2 grains per nucleus). However, an 87% of repair synthesis (46.7 ± 8 grains per nucleus) com- pared with normal cells (54 grains per nucleus) at 15 J/m2 was observed in cells transduced with LXPBSN (Figure 3c). The DNA repair synthesis in XP-C cells was recovered at least by 80% of normal cells 198VI (37 ± 6 grains per nucleus) for XP16VI + LXPCSN (30 ± 11 grains per nucleus) and by 92% for XP30VI + LXPCSN (34 ± 7 grains per nucleus), whereas untransduced XP-C cells displayed few grains (0.6 to approximately 4) per nucleus (Figure 3d). (3) Recovery of RNA synthesis (RRS): Because of the associ- ation of XP with CS in the XPCS2BA patient, we decided to measure RRS level, which is dramatically impaired in cells from CS and XP/CS.33,34 Therefore, the incorporation of 3H-uridine, measured as Figure 4 Recovery of RNA synthesis in MRC5V1 (a), untransduced (b) described in Materials and methods, was performed × in XPCS2BASV before and after LXPBSN transduc- and transduced (c) XPCS2BASV cells (original magnification 200). tion. Figure 4b shows that a low RRS level was found (5 ± 2 grains per nucleus) at 24 h after irradiation in XP-C cells. After retroviral transduction, the level of the untransduced XPCS2BASV cells, compared with that signal of these proteins became similar to that of wild- of wild-type cells (Ͼ250 grains per nucleus, type cells. Similar results could be obtained in Figure 4a), while transduced cells showed a wild-type untransduced/transduced XPCS2BASV over MRC5V1 RRS level (Figure 4c). cells, with the exception that XPB is detectable in XP-B cell extracts, although the signal is lower than in Expression of XPA, XPB and XPC proteins wild-type or transduced cells (Figure 5b). To correlate the correction of UV survival and change of DNA repair properties in transduced cells with the Confirmation of the genetic correction of XP-B cells at expression of the proteins encoded by introduced cDNA, RNA level Western blot analysis was carried out on different cell Because the monoclonal antibody 1B3 could not differen- lines by using cell extracts obtained from untransduced tiate the mutated XPB from the wild-type protein by and transduced cells. The proteins of these extracts were Western blot (Figure 5b), in order to confirm that the resolved by electrophoresis in SDS-polyacrylamide gels, phenotypic correction is correlated with the expression of and analyzed by Western blot, as described in Materials transgene, we amplified by RT-PCR a 205 bp fragment and methods by using specific antibodies. Figure 5a and of XPB/ERCC3 3 from position 22 to 225 as c shows that the XPA and XPC proteins, while clearly decribed in Materials and methods. This fragment, when detectable in wild-type, were undetectable in XP-A and amplified from XPCS2BASV DNA, contains an additional Correction of inborn DNA repair defect by gene transfer L Zeng et al 1080 transduced XPCS2BASV cells (Figure 6b, lanes 2 and 6). The 37 and 19 bp fragments are too short to be visible in the gel conditions used.

Discussion In previous reports we described the first retrovirus car- rying a DNA repair gene (LXPDSN) which efficiently transduces diploid fibroblasts from DNA repair-deficient patients belonging to the XP-D group.28,29 This first result allowed us to envisage a novel gene therapy for skin can- cers in XP patients. However, unlike other monogenic human disorders, several genes are involved in XP. They encode for the multi-protein complex which, in normal individuals, removes DNA damage and prevents del- eterious consequences of UV radiation (skin cancers and other pathologies, including neurological degenerations). This heterogeneity of symptoms in XP patients reflects the genetic heterogeneity of the disease. In fact, severe neurological degenerations are often accompanied by skin tumors in patients belonging to XP-A and XP-D groups, and other metabolic dysfunctions typical of CS Figure 5 Detection of the XPA, XPB and XPC proteins by Western blot. (growth and mental retardation, gonadal development Indicated cell extracts were processed as described in Materials and impairment, dwarfism and deafness) could be found methods and membranes were probed with anti-XPA (a), anti-XPB (b) associated with some XP-B, XP-D and XP-G patients.36 and anti-XPC (c) antibodies. This implies that some DNA repair gene products play a role not only in the mere NER, but also in other pathways HinfI restriction site generated by a T→C transition at involved in cell differentiation and in development. position 62 (Figure 6a), yielding F99S substitution in XPB Therefore, we developed retroviral vectors carrying protein, which is present in only one allele.35 HinfI cleav- additional DNA repair genes with the aims: (1) to vali- age produces two smaller fragments of the expected size date the novel transduction technology for other genes of 185 and 148 bp (Figure 6a) only in untransduced involved in XP; and (2) among them, to transduce cells XPCS2BASV cells (Figure 6b, lane 4). An identical pattern from individuals belonging to the XP-A and XP-C of 185 bp fragments was observed in MRC5V1 and in groups, which are the most representative in the XP patient population (27 and 26%, respectively).36 Further- more, unlike most XP-A, XP-B and XP-D patients, XP-C individuals suffer only from skin tumors (without the above mentioned complex clinical features) and, thus, they are the most probable candidates for gene therapy protocol. The precise role of each DNA repair protein has not yet been established. However, biochemical data of purified proteins and/or (aa) sequence data allowed us to suggest a specific role in the NER pathway for each protein.

XPA XPA protein is a zinc metalloprotein consisting of 273 amino acids which, in connection with XPE protein, binds preferentially to UV- or chemical carcinogen- induced damaged DNA, and, synergically with other proteins (belonging to the TFIIH factor), drives other pro- teins of the complex toward the lesions and allows exci- sion of damaged DNA strands. This suggests that it is involved in the recognition step of several types of DNA damage.37–39 According to previous results, showing a 40–42 Figure 6 Restriction analysis of RT-PCR amplified fragment of reduced amount of XPA mRNAs and protein, XPB/ERCC3 exon 3. (a) Diagrams showing HinfI sites in wild-type and XP12ROSV cells exhibited no detectable XPA protein, XPCS2BASV cells and the expected size (bp) of the fragments generated. when revealed by Western blot with anti-XPA antiserum. Between the two diagrams the nucleotide sequence flanking the T→C tran- The chain termination at codon 207 generally sition (arrow) is indicated; underlined sequences designate the HinfI site. predicts synthesis of a truncated protein containing the Numbers in italic indicate the sequence positions. (b) Restriction pattern first 206 of the 273 amino acids of the intact XPA protein. of RT-PCR amplified 205 bp fragment from wild-type, untransduced and transduced XPCS2BASV cells, before (lanes 1, 3 and 5) and after (lanes However, such truncated XPA polypeptide has never 2, 4 and 6) HinfI digestion. The 200 bp marker position is indicated on been shown so far. We also observed a similar result for the left side of the picture. XP24VI cells, where a similar chain elongation mutation Correction of inborn DNA repair defect by gene transfer L Zeng et al 1081 is also present in exon 4 (K Tanaka, personal expression of XPC protein to normal levels in our XP-C communication). This suggests that this portion of the cell lines. Therefore, this biochemical feature is consistent gene is essential for the stability of XPA gene products, with the clinical features of XP-C patients, since XP either at mRNA or protein level, or both. However, fol- patients with neurological abnormalities are generally lowing transduction with the recombinant LXPASN found in groups A, B and D, but not in group C. retrovirus, XPA protein could be easily detected by West- ern blot in XP12ROSV cell extract (Figure 5a). Therefore, the restoration of XPA protein expression is sufficient to Materials and methods correct UV sensitivity and DNA repair to normal levels in both cells. Clinical and genetical data of patients XP donors are listed in Table 1. The XP12RO and the XPB XP24VI patients have skin and neurological abnormali- XPB protein is an 89 kDa species belonging to the TFIIH ties and both cell lines exhibited UV hypersensitivity. → complex and it possesses a 3′–5′ ATP-dependent Patient XP12RO carries a C T transition at nucleotide activity. In connection with the 5′–3′ helicase activity of 619 of exon 5 in the XPA gene. This mutation alters the XPD protein, it releases DNA damaged strand after exci- Arg-207 codon (CGA) to a nonsense codon (TGA) in both 48 sion.43 It is required for the transcriptional activity of alleles. XP24VI was assigned to the XP-A group by TFIIH complex. Therefore, it plays a dual role in both fusion and microinjection experiments. The NER and transcription. Only three families belong to the XP24VI patient came from a consanguinous marriage and XP-B group: two presenting a combination of XP and CS presented photosensitivity from 5 months after birth, and (XP11BE and XPCSBA) and one presenting mild symp- was diagnosed as XP at the age of 5 years with an appar- toms of TTD. Although the precise role of causative ent facial dysmorphosis and difficulty in walking. How- mutations in patients has not yet been established, differ- ever, no cutaneous tumor was found at the time of diag- ent mutations, such as phenylalanine-to-serine (F99S) and nosis. XPCS2BASV cell line came from one of two threonine-to-proline (T119S) substitutions, lead to differ- brothers with relatively mild clinical symptoms of com- ent cellular phenotypes (in terms of DNA repair) and bined XP and CS and with a virtually complete deficiency clinical symptoms in patients, ie XP/CS combination and of NER. This line was assigned to the XP-B group by TTD, respectively.35,44 Our results showed a high sensi- microinjection experiments. The sequence analysis on tivity of XPCS2BASV cells to the cytotoxic effect of UV PCR-amplified mRNA revealed a single base substitution → in colony-forming assays, and a poor level of residual (T C transition), resulting in a phenylalanine-to-serine UDS (Ͻ1% of normal), indicating that the F99S mutation substitution at position 99 (F99S), and this mutation was causes virtually complete inactivation of the DNA repair found in only one allele.35 The XP30VI patient was diag- function of the XPB protein. Furthermore, Western blots nosed as classical XP at the age of 2 years without tumor showed a reduced amount of 89 kDa polypeptide (Figure and the XP16VI patient was diagnosed at the age of 7 5b), according to the hypothesis that in XPCS2BA only years with multiple facial . Both were con- the paternal allele is expressed.35 Nevertheless, the firmed as XP complementation group C by cell fusion reduced level of ERCC3 transcript could also be experiments. explained by alteration of the expression of ERCC3/XPB by the mutation. Transduction with LXPBSN confers, Cell culture conditions ° however, wild-type expression level of the protein and Cells were grown at 37 Cin5%CO2 humidified atmos- only wild-type mRNA species are expressed (Figure 5b), phere in Eagle’s minimal essential medium (GIBCO, suggesting that dominant expression of the transgene Inchinnan, UK) supplemented with 15% fetal calf serum over the endogenous one occurs. (Dominique Dutscher, Strasbourg, France) and antibiotics at 1 ␮g/ml each of penicillin and streptomycin and 2.5 XPC ␮g/ml of fungizone (GIBCO), while 10% FCS was used The XPC protein is a 125 kDa polypeptide45 and its for transformed cells. NIH3T3 and retrovirus packaging ⌿ ⌿ ° activity is supposed to drive repair proteins to damage CRE and CRIP cells were grown at 37 C in 10% CO2 DNA in nontranscribed strands, since XP-C cells display in DMEM medium supplemented with fetal bovine defective repair of lesions localized only in nontran- serum and antibiotics. scribed strands. Furthermore, mutations in the gene in skin tumors from these patients were detected only in Construction of vectors and production of recombinant nontranscribed strands.46 All mutations in the XPC gene retrovirus (single aa substitutions, insertion or chain terminating) The retroviral LXSN vector is based on MoMLV.31,32 Fig- identified in several XP-C individuals, were accompanied ure 1 depicts LXSN containing a selectable neomycin (G- by a many-fold reduction of XPC mRNA levels.47 In 418) marker which is under the control of the SV40 early XP30VI and XP16VI cells used here, the low UV survival promoter. XPA and XPB genes are the fragments of 0.91 rate and reduced UDS levels (Figures 2d and 3d) are also kb and 2.85 kb full-length cDNA, respectively.49,50 XPC accompanied by undetectable XPC protein by Western gene is a fragment of 3.55 kb full-length cDNA.45 These blots (Figure 5c). Although mutations in our cell lines are genes were inserted into appropriate restriction sites in not yet identified, we can deduce that alterations in the LXSN polylinker. The recombinant vectors were named XPC gene may yield unstable transcripts and altered according to the order of genetic elements in the vectors: forms of the protein might not be detectable with conven- LXPASN for XPA, LXPBSN for XPB and LXPCSN for tional Western blotting procedure. Our results have XPC. To reduce the potential for helper virus production, proved that the wild-type XPC protein corrects the repair we chose two retrovirus-packaging cell lines, ⌿CRE and defect and restores the UV survival rate and the ⌿CRIP, to produce recombinant retrovirus.30 ⌿CRE cell Correction of inborn DNA repair defect by gene transfer L Zeng et al 1082 Table 1 Donors and cell lines

Donors Phenotype Cells derived Genetic group Source

198VI wild type diploid fibroblasts Villejuif, France MRC5V1 SV40-transformed C Arlett (Brighton, UK) XP24VI XP diploid fibroblast XP-A Villejuif, France XP12RO SV40-transformed D Bootsma (Rotterdam, The Netherlands) XPCS2BA XP/CS SV40-transformed XP-B JHJ Hoeijmakers (Rotterdam, The Netherlands) XP16VI XP diploid fibroblasts XP-C Villejuif, France XP30VI

line was cultured in a 6-cm Petri dish at 5 × 105 for 12 h cell numbers in function of UV doses (approximately and then the cells were transfected with 5 ␮g of DNA of 1 × 103 to 8 × 103 and 2 × 104 to 2 × 106 for primary and each of the three recombinant plasmids using the stan- transformed cells, respectively). Irradiation was perfor- dard calcium phosphate method. When the cells reached med as in UDS experiments and cells were maintained confluency, the culture supernatant was harvested, fil- in medium supplemented with 20% and 10% FCS for pri- tered and used to transduce the ⌿CRIP cell line in the mary and transformed fibroblasts, respectively, for 14 presence of polybrene (Sigma-Aldrich Chimie, St Quentin days. The relative survival was calculated as the number Fallavier, France) at 8 ␮g/ml. ⌿CRIP cell line was cul- of colonies obtained in UV irradiated over unirradiated tured in the presence of neomycin (GIBCO) at 1 mg/ml cells. for selection and neo-resistant clones obtained were tested for retrovirus titer by transducing NIH3T3 fibro- Detection of (Western blot) blasts under neomycin selection (1 mg/ml). For every The proteins encoded by the introduced genes were ana- recombinant retrovirus at least 15 G418-resistant colonies lyzed by 6 or 8% (according to the molecular mass of were expanded and their titers were determined. These proteins to be analyzed) acrylamide-SDS gel electro- titers may vary from 103 to 106 c.f.u./ml according to the phoresis of cellular extracts obtained from different cell different genes cloned and, for the same construction, to lines. Protein samples were transferred into Hybond+ different individual clones. Clones producing the retro- membranes (Amersham, Les Ulis, France) and probed virus with higher titers were expanded. Primary fibro- with specific antibodies, according to the manufacturer’s blasts and transformed cells were transduced with the procedure: anti-XPA antiserum (kindly provided by Dr K recombinant retrovirus according to the genetic comp- Tanaka, IMCB, Osaka University, Japan); the monoclonal lementation groups and were selected with 1 mg/ml of anti-XPB antibody (1B3) raised against the N-terminal of neomycin and grown in the presence of neomycin for at human XPB protein (AA 80–480) was a gift from Dr J- least 8 weeks. M Egly (Strasbourg, France); and the polyclonal antibody raised against human XPC protein was kindly provided UDS and RRS by Dr F Hanaoka (IMCB, Osaka University, Japan). Analysis of repair synthesis was carried out in untrans- duced and transduced primary and transformed fibro- Reverse transcription PCR amplification blasts as previously described.51 Briefly, cells were grown Total RNA was isolated from approximately 1 × 108 cells on glass coverslips for 2 days. After 2 more days in from MRC5V1 (normal), XPCS2BASV (XP-B/CS) and serum-deprived medium (0.5%) they were UV irradiated XPCS2BASV transduced by recombinant retrovirus with doses from 0 to 15 J/m2 and then incubated with LXPBSN, using the RNA Isolation Kit (Bioprobe Systems, 3H-thymidine at 10 ␮Ci/ml (specific activity of 50 Montreuil-sous-Bois, France). RNA (5 ␮g) was used for Ci/mmol; DuPont de Nemors, Les Ulis, France) for 3 h, reverse transcription reaction. Briefly, RNA was dis- followed by a chase of 1 h with cold thymidine. solved in 10 ␮l of reverse transcriptase buffer (250 mm m m Coverslips with the cells were mounted on to glass slides, Tris-HCl (pH 8.3), 375 m KCl, 15 m MgCl2), in the dipped in Kodak NTB-2 emulsion (Kodak, New York, presence of RNase inhibitor (Boehringer Mannheim, NY, USA), and exposed for 1 week at 4°C. The mean Meylan, France), 5 ␮l of 0.1 m DTT, 5 ␮lof10mmdNTP, number of grains per nucleus was obtained by counting 1 ␮g of poly-dT and 400 units of MoMLV reverse tran- at least 30 non-S-phase nuclei. RRS was measured as fol- scriptase (GIBCO) were added, and then incubated at lows: cells were grown as for UDS experiments and UV 37°C for 30 min. PCR was performed to amplify the frag- irradiated with 15 J/m2. Cells were then incubated for 23 ment from position 22 to 225 in exon 3 of XPB/ERCC3 h and then labeled for 1 h in a medium containing 3H- containing the T→C transition (Figure 6a) using primers: uridine at 10 ␮Ci/ml (specific activity of 50 Ci/mmol). 5′-TTGGAAGCCTTCTCTCCAG TTTACAAATATGC-3′ Autoradiography was performed as described above for and 5′-GCATAATTCCATCAGGGACTCCAGTCTTG-3′ measurement of UDS except that the exposure time was (GENSET, Paris, France). To 1 ␮g of cDNA, PCR reaction 24 h. contained 10 ␮lofTaq reaction buffer (100 mm Tris-HCl, pH 8.8, 15 mm MgCl2, 500 mm KCl, 1% Triton X-100), 1 UV survival ␮l (1 pmole/␮l) of each primer, 10 ␮l of dNTPs (2 mm), The colony-forming ability was determined in primary and 1.5 U of Taq polymerase (Bioprobe Systems). Ampli- and transformed fibroblast cells by seeding increasing fication was performed by 30 cycles in 100 ␮l total vol- Correction of inborn DNA repair defect by gene transfer L Zeng et al 1083 ume, 1 min denaturing at 95°C, 1 min annealing at 55°C, 14 Flejter WL et al. Correction of xeroderma pigmentosum comp- and 1 min extension at 72°C. Finally, the amplified DNA lementation group D mutant cell phenotypes by fragments were digested by HinfI (New England Bio- and gene transfer: involvement of the human ERCC2 DNA Labs, Distributor: Ozyme, Montigny-le-Bretonneux, repair gene. Proc Natl Acad Sci USA 1992; 89: 261–265. 15 Sijbers AM et al. Xeroderma pigmentosum group F caused by France) and then analyzed by agarose gel electrophoresis. a defect in a structure-specific DNA repair . Cell 1996; 86: 811–822. Acknowledgements 16 O’Donovan A, Wood RD. Identical defects in DNA repair in xeroderma pigmentosum group G and rodent ERCC group 5. This work was supported by grants from the Association Nature 1993; 363: 185–188. Franc¸aise contre les Myopathies (AFM, Evry, France), the 17 Troelstra C et al. ERCC6, a member of a subfamily of putative Association pour la Recherche sur le Cancer (ARC, Ville- , is involved in Cockayne’s syndrome and preferential juif, France), the Fe´de´ration Nationale des Groupements repair of active genes. Cell 1992; 71: 939–953. des Entreprises Franc¸aises dans la Lutte Contre le Cancer 18 Aboussekhra A et al. Mammalian DNA nucleotide excision (FEGEFLUC, Marseille, France) and the Ligue Nationale repair reconstituted with purified protein components. Cell 1995; Contre le Cancer (LNCC, Versailles, France). L Zeng and 80: 859–868. X Quilliet are fellows from AFM and the Institut de For- 19 Weeda G, Hoejimakers JHJ. Genetic analysis of nucleotide exci- sion repair in mammalian cells. Semin Cancer Biol 1993; 4: mation Supe´rieure Biome´dicale (IFSBM, Villejuif, France), 105–107. respectively. 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