Download Retrovirus-mediated gene transfer corrects DNA repair

Gene 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 xeroderma 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 Génétique Moléculaire, UPR 42 CNRS, 94801 Villejuif, France
With the aim to devise a long-term gene therapy protocol
for skin cancers in individuals affected by the inherited
autosomal recessive xeroderma pigmentosum, we transferred the human DNA repair XPA, XPB/ERCC3 and XPC
cDNAs, by using the recombinant retroviral vector LXSN,
into primary and immortalized fibroblasts obtained from two
XP-A, one XP-B (associated with Cockayne’s syndrome)
and two XP-C patients. After transduction, the complete
correction of DNA repair deficiency and functional
expression of the transgenes were monitored by UV
survival, unscheduled DNA synthesis and recovery of RNA
synthesis, and Western blots. The results show that the
recombinant retroviruses are highly efficient vectors to
transfer and stably express the human DNA repair genes
in XP cells and correct the defect of DNA repair of group
A, B and C. With our previous results with XPD/ERCC2,
the present work extends further promising issues for the
gene therapy strategy for most patients suffering from this
cancer-prone syndrome.
Keywords: retroviral vectors; DNA repair genes; skin cancer therapy; xeroderma pigmentosum
Introduction
Xeroderma pigmentosum (XP) is a rare human autosomal
recessive disease characterized clinically by hypersensitivity to ultraviolet (UV) rays, high predisposition for
developing skin cancers (basal and squamous cell carcinomas and melanomas) on sunlight exposed areas,1 and
in some cases, neurological disorders.2–4 XP has a worldwide distribution, with the incidence varying from about
1:250 000 in Europe and the USA to as high as 1:40 000
in Japan, North Africa and in Egypt.5–7 The cellular
phenotype of XP has increased sensitivity to killing following exposure to a wide variety of DNA damaging
agents, including UV radiation and UV mimetic chemicals. The observation that skin fibroblasts in culture from
an XP patient are unable to carry out nucleotide excision
repair (NER) following exposure to UV established the
relationship between DNA repair defect and skin photocarcinogenesis in man.1,8,9
The systematic complementation of DNA repair
defects by cell fusion assay has led to the identification
of seven genetic groups in this disease, designated XP-A
to -G,7 and a variant group, designated XP-V, exhibiting
normal NER.10 So far, most of the relevant human genes,
such as XPA, XPB, XPC, XPD, XPF and XPG, have been
cloned and mapped to different specific chromosomal
locations. A further source of NER defective mutants is a
set of 11 complementation groups of UV-sensitive rodent
cells. The human genes that correct the rodent cell phenotype are designated ERCC (excision repair cross-
Correspondence: M Mezzina
Received 24 March 1997; accepted 23 May 1997
complementing) genes.11,12 Among them, ERCC2, ERCC3,
ERCC4, ERCC5 and ERCC6 were found to be identical to
the genes involved in xeroderma pigmentosum groups
D, B, F and G and Cockayne’s syndrome group B.13–17 A
multiprotein complex of approximately 30 gene products
is involved in the NER pathway, whereby DNA damage
is eliminated and replaced by excision-resynthesis, as has
been demonstrated by using the in vitro repair assay with
purified factors.18 Mutations in one of these genes result
in UV-sensitive disorders: skin cancer-prone XP and
other clinically different syndromes, such as skin cancerfree trichothiodystrophy (TTD) and Cockayne syndrome
(CS), and association of XP with CS.
The NER process is evolutionarily conserved in eukaryotes, and homologue genes of human repair genes have
been identified in many organisms, namely in Saccharomyces cerevisiae.19 In humans, the proteins encoded by
some of these genes are components of the multiprotein
TFIIH complex involved with RNA polymerase II in
basal transcription. Consistently, the yeast homologues of
some of these proteins, the Rad25 and Rad3 (homologue
to ERCC3/XPB and ERCC2/XPD, respectively), have also
been identified as components of TFIIH. Therefore, these
proteins play a dual role in both NER and transcription.20–22
The mean age of XP patients at the time of diagnosis
is 3 years while the mean age of onset of first skin cancer
is 8 years. There is an approximate 30–40 year reduction
in survival. Many patients have died of neoplasms. 2 So
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
from the same patient were performed to resurface the
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,
since skin grafts are still genetically DNA repair defective
and thus cancer prone.24 The genetic correction of XP
cells by retrovirus-mediated transduction with appropriate DNA repair genes may allow possible alternative
long-term therapies.
Studies in vitro have shown that DNA repair defects
can be corrected by introducing relevant DNA repair
genes into cells derived from XP patients.25,26 Several
retroviral constructions are efficient tools for gene delivery and stable expression in many human cells and
tissues. 27 We recently devised the first retrovirus harboring a DNA repair gene (XPD/ERCC2), which efficiently
transduces several DNA repair-deficient human primary
skin fibroblasts belonging to the complementation XP-D
group and fully corrects their DNA repair defect.28,29 In
order to validate this investigation with other genes
involved in XP disease and to dispose of basic tools for
gene therapy for a larger population of patients, we
decided to develop additional constructions harboring
other available DNA repair cDNAs. In this paper, we
describe the construction of retrovirus harboring the
genes XPA, XPB and XPC and show that their expression
fully corrects the DNA repair-deficient phenotype of
fibroblasts from two XP-A, one XP-B and two XP-C
patients.
Results
Establishment of recombinant retrovirus
The LXPASN, LXPBSN and LXPCSN vectors were
derived from the Moloney murine leukemia virus
(MoMLV)-based retroviral vector LXSN and were produced after insertion into the polylinker of XPA, XPB and
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 CCRE
packaging cells, subsequent infection of CCRIP 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.
complementation groups, transduced and untransduced
cells were analyzed for:
(1) UV survival: The colony-forming ability was determined in primary and SV40 immortalized cells. Figure
2 shows survival curves of wild-type SV40 immortalized MRC5V1 and diploid 198VI fibroblast compared with those of UV-sensitive untransduced
XP12ROSV and XP24VI (XP-A group, Figure 2a and
b, respectively), XPCS2BASV (XP-B group, Figure 2c)
and XP16VI and XP30VI (XP-C group, Figure 2d).
After transduction with the retrovirus harboring the
appropriate cDNA, all cell lines recovered wild-type
UV resistance, since their survival curves were indistinguishable from those of 198VI and MRC5V1 lines
(Figure 2a–d). When XP cells were cross-transduced
with retrovirus containing other cDNA than that
involved in the genetic group, no correction of UV
survival was observed (Figure 2a, c and d, dotted
lines). This indicates that the retroviral transduction
is gene specific, consistent with our previous observations with LXPDSN.28,29
(2) Unscheduled DNA synthesis (UDS): To correlate the
recovery of UV survival with the ability to perform
repair synthesis, the incorporation of 3H-thymidine
was measured after UV irradiation (Figure 3). Both
XP12ROSV and XP24VI exhibited reduced UDS levels, (5.4 ± 2 and 2.9 ± 1.1 grains per nucleus) at 15
J/m2, corresponding to 26 and 14.6% of normal levels,
respectively. After transduction of the same cells with
LXPASN, the UDS levels were restored to almost normal levels (18 ± 4 and 17 ± 5 grains per nucleus,
respectively, corresponding to 85% of the normal levels, Figure 3a and b). XPCS2BASV cells showed virtually a complete defect of DNA repair with all three
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 1 Scheme of retroviral vector LXSN based on Moloney murine
leukemia virus. LTR: long terminal repeat; C+: packaging signal; polylinker for insertion of DNA repair cDNAs: XPA, XPB and XPC; SV40:
simian virus 40 early promoter; NEO: neomycin phosphotransferase gene.
Arrows show transcription initiation orientation.
Figure 2 UV survival curves in untransduced and transduced XP groups
A, B and C cells. (–l–) MRC5V1 (a and c) and 198VI (b and d) wildtype cells; (–p–) XP12ROSV (a), XP24VI (b), XPCS2BASV (c) and
XP16VI (d) untransduced and (–P–) transduced cells with LXPASN,
LXPBSN and LXPCSN, respectively; (–g–) untransduced and (–G–)
transduced XP30VI cells with LXPCSN (d); (--K--) cells transduced with
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 (,2 grains per nucleus). However, an 87%
of repair synthesis (46.7 ± 8 grains per nucleus) compared 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 association 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
described in Materials and methods, was performed
in XPCS2BASV before and after LXPBSN transduction. Figure 4b shows that a low RRS level was found
(5 ± 2 grains per nucleus) at 24 h after irradiation in
untransduced XPCS2BASV cells, compared with that
of wild-type cells (.250 grains per nucleus,
Figure 4a), while transduced cells showed a wild-type
RRS level (Figure 4c).
Expression of XPA, XPB and XPC proteins
To correlate the correction of UV survival and change of
DNA repair properties in transduced cells with the
expression of the proteins encoded by introduced cDNA,
Western blot analysis was carried out on different cell
lines by using cell extracts obtained from untransduced
and transduced cells. The proteins of these extracts were
resolved by electrophoresis in SDS-polyacrylamide gels,
and analyzed by Western blot, as described in Materials
and methods by using specific antibodies. Figure 5a and
c shows that the XPA and XPC proteins, while clearly
detectable in wild-type, were undetectable in XP-A and
Figure 4 Recovery of RNA synthesis in MRC5V1 (a), untransduced (b)
and transduced (c) XPCS2BASV cells (original magnification × 200).
XP-C cells. After retroviral transduction, the level of the
signal of these proteins became similar to that of wildtype cells. Similar results could be obtained in
untransduced/transduced XPCS2BASV over MRC5V1
cells, with the exception that XPB protein is detectable in
XP-B cell extracts, although the signal is lower than in
wild-type or transduced cells (Figure 5b).
Confirmation of the genetic correction of XP-B cells at
RNA level
Because the monoclonal antibody 1B3 could not differentiate the mutated XPB from the wild-type protein by
Western blot (Figure 5b), in order to confirm that the
phenotypic correction is correlated with the expression of
transgene, we amplified by RT-PCR a 205 bp fragment
of XPB/ERCC3 exon 3 from position 22 to 225 as
decribed in Materials and methods. This fragment, when
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
Figure 5 Detection of the XPA, XPB and XPC proteins by Western blot.
Indicated cell extracts were processed as described in Materials and
methods and membranes were probed with anti-XPA (a), anti-XPB (b)
and anti-XPC (c) antibodies.
HinfI restriction site generated by a T→C transition at
position 62 (Figure 6a), yielding F99S substitution in XPB
protein, which is present in only one allele.35 HinfI cleavage produces two smaller fragments of the expected size
of 185 and 148 bp (Figure 6a) only in untransduced
XPCS2BASV cells (Figure 6b, lane 4). An identical pattern
of 185 bp fragments was observed in MRC5V1 and in
Figure 6 Restriction analysis of RT-PCR amplified fragment of
XPB/ERCC3 exon 3. (a) Diagrams showing HinfI sites in wild-type and
XPCS2BASV cells and the expected size (bp) of the fragments generated.
Between the two diagrams the nucleotide sequence flanking the T→C transition (arrow) is indicated; underlined sequences designate the HinfI site.
Numbers in italic indicate the sequence positions. (b) Restriction pattern
of RT-PCR amplified 205 bp fragment from wild-type, untransduced and
transduced XPCS2BASV cells, before (lanes 1, 3 and 5) and after (lanes
2, 4 and 6) HinfI digestion. The 200 bp marker position is indicated on
the left side of the picture.
In previous reports we described the first retrovirus carrying 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 cancers 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 deleterious 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
(growth and mental retardation, gonadal development
impairment, dwarfism and deafness) could be found
associated with some XP-B, XP-D and XP-G patients.36
This implies that some DNA repair gene products play a
role not only in the mere NER, but also in other pathways
involved in cell differentiation and in development.
Therefore, we developed retroviral vectors carrying
additional DNA repair genes with the aims: (1) to validate the novel transduction technology for other genes
involved in XP; and (2) among them, to transduce cells
from individuals belonging to the XP-A and XP-C
groups, which are the most representative in the XP
patient population (27 and 26%, respectively).36 Furthermore, 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 amino acid (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 carcinogeninduced damaged DNA, and, synergically with other
proteins (belonging to the TFIIH factor), drives other proteins of the complex toward the lesions and allows excision 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
reduced amount of XPA mRNAs and protein,40–42
XP12ROSV cells exhibited no detectable XPA protein,
when revealed by Western blot with anti-XPA antiserum.
The chain termination mutation at codon 207 generally
predicts synthesis of a truncated protein containing the
first 206 of the 273 amino acids of the intact XPA protein.
However, such truncated XPA polypeptide has never
been shown so far. We also observed a similar result for
XP24VI cells, where a similar chain elongation mutation
Correction of inborn DNA repair defect by gene transfer
L Zeng et al
is also present in exon 4 (K Tanaka, personal
communication). This suggests that this portion of the
gene is essential for the stability of XPA gene products,
either at mRNA or protein level, or both. However, following transduction with the recombinant LXPASN
retrovirus, XPA protein could be easily detected by Western blot in XP12ROSV cell extract (Figure 5a). Therefore,
the restoration of XPA protein expression is sufficient to
correct UV sensitivity and DNA repair to normal levels
in both cells.
XPB
XPB protein is an 89 kDa species belonging to the TFIIH
complex and it possesses a 3′–5′ ATP-dependent helicase
activity. In connection with the 5′–3′ helicase activity of
XPD protein, it releases DNA damaged strand after excision.43 It is required for the transcriptional activity of
TFIIH complex. Therefore, it plays a dual role in both
NER and transcription. Only three families belong to the
XP-B group: two presenting a combination of XP and CS
(XP11BE and XPCSBA) and one presenting mild symptoms of TTD. Although the precise role of causative
mutations in patients has not yet been established, different mutations, such as phenylalanine-to-serine (F99S) and
threonine-to-proline (T119S) substitutions, lead to different cellular phenotypes (in terms of DNA repair) and
clinical symptoms in patients, ie XP/CS combination and
TTD, respectively.35,44 Our results showed a high sensitivity of XPCS2BASV cells to the cytotoxic effect of UV
in colony-forming assays, and a poor level of residual
UDS (,1% of normal), indicating that the F99S mutation
causes virtually complete inactivation of the DNA repair
function of the XPB protein. Furthermore, Western blots
showed a reduced amount of 89 kDa polypeptide (Figure
5b), according to the hypothesis that in XPCS2BA only
the paternal allele is expressed.35 Nevertheless, the
reduced level of ERCC3 transcript could also be
explained by alteration of the expression of ERCC3/XPB
by the mutation. Transduction with LXPBSN confers,
however, wild-type expression level of the protein and
only wild-type mRNA species are expressed (Figure 5b),
suggesting that dominant expression of the transgene
over the endogenous mutant one occurs.
XPC
The XPC protein is a 125 kDa polypeptide45 and its
activity is supposed to drive repair proteins to damage
DNA in nontranscribed strands, since XP-C cells display
defective repair of lesions localized only in nontranscribed strands. Furthermore, mutations in the p53 gene
in skin tumors from these patients were detected only in
nontranscribed strands.46 All mutations in the XPC gene
(single aa substitutions, insertion or chain terminating)
identified in several XP-C individuals, were accompanied
by a many-fold reduction of XPC mRNA levels.47 In
XP30VI and XP16VI cells used here, the low UV survival
rate and reduced UDS levels (Figures 2d and 3d) are also
accompanied by undetectable XPC protein by Western
blots (Figure 5c). Although mutations in our cell lines are
not yet identified, we can deduce that alterations in the
XPC gene may yield unstable transcripts and altered
forms of the protein might not be detectable with conventional Western blotting procedure. Our results have
proved that the wild-type XPC protein corrects the repair
defect and restores the UV survival rate and the
expression of XPC protein to normal levels in our XP-C
cell lines. Therefore, this biochemical feature is consistent
with the clinical features of XP-C patients, since XP
patients with neurological abnormalities are generally
found in groups A, B and D, but not in group C.
Materials and methods
Clinical and genetical data of patients
XP donors are listed in Table 1. The XP12RO and the
XP24VI patients have skin and neurological abnormalities and both cell lines exhibited UV hypersensitivity.
Patient XP12RO carries a C→T transition at nucleotide
619 of exon 5 in the XPA gene. This mutation alters the
Arg-207 codon (CGA) to a nonsense codon (TGA) in both
alleles.48 XP24VI was assigned to the XP-A group by
somatic fusion and microinjection experiments. The
XP24VI patient came from a consanguinous marriage and
presented photosensitivity from 5 months after birth, and
was diagnosed as XP at the age of 5 years with an apparent facial dysmorphosis and difficulty in walking. However, no cutaneous tumor was found at the time of diagnosis. XPCS2BASV cell line came from one of two
brothers with relatively mild clinical symptoms of combined XP and CS and with a virtually complete deficiency
of NER. This line was assigned to the XP-B group by
microinjection experiments. The sequence analysis on
PCR-amplified mRNA revealed a single base substitution
(T→C transition), resulting in a phenylalanine-to-serine
substitution at position 99 (F99S), and this mutation was
found in only one allele. 35 The XP30VI patient was diagnosed as classical XP at the age of 2 years without tumor
and the XP16VI patient was diagnosed at the age of 7
years with multiple facial carcinoma. Both were confirmed as XP complementation group C by cell fusion
experiments.
Cell culture conditions
Cells were grown at 37°C in 5% CO2 humidified atmosphere in Eagle’s minimal essential medium (GIBCO,
Inchinnan, UK) supplemented with 15% fetal calf serum
(Dominique Dutscher, Strasbourg, France) and antibiotics
at 1 mg/ml each of penicillin and streptomycin and 2.5
mg/ml of fungizone (GIBCO), while 10% FCS was used
for transformed cells. NIH3T3 and retrovirus packaging
CCRE and CCRIP cells were grown at 37°C in 10% CO2
in DMEM medium supplemented with fetal bovine
serum and antibiotics.
Construction of vectors and production of recombinant
retrovirus
The retroviral LXSN vector is based on MoMLV. 31,32 Figure 1 depicts LXSN containing a selectable neomycin (G418) marker which is under the control of the SV40 early
promoter. XPA and XPB genes are the fragments of 0.91
kb and 2.85 kb full-length cDNA, respectively.49,50 XPC
gene is a fragment of 3.55 kb full-length cDNA.45 These
genes were inserted into appropriate restriction sites in
LXSN polylinker. The recombinant vectors were named
according to the order of genetic elements in the vectors:
LXPASN for XPA, LXPBSN for XPB and LXPCSN for
XPC. To reduce the potential for helper virus production,
we chose two retrovirus-packaging cell lines, CCRE and
CCRIP, to produce recombinant retrovirus.30 CCRE cell
1081
Correction of inborn DNA repair defect by gene transfer
L Zeng et al
1082
Table 1 Donors and cell lines
Donors
Phenotype
Cells derived
198VI
MRC5V1
wild type
diploid fibroblasts
SV40-transformed
XP24VI
XP12RO
XP
diploid fibroblast
SV40-transformed
XPCS2BA
XP/CS
SV40-transformed
XP-B
JHJ Hoeijmakers (Rotterdam, The Netherlands)
XP16VI
XP30VI
XP
diploid fibroblasts
XP-C
Villejuif, France
line was cultured in a 6-cm Petri dish at 5 × 105 for 12 h
and then the cells were transfected with 5 mg of DNA of
each of the three recombinant plasmids using the standard calcium phosphate method. When the cells reached
confluency, the culture supernatant was harvested, filtered and used to transduce the CCRIP cell line in the
presence of polybrene (Sigma-Aldrich Chimie, St Quentin
Fallavier, France) at 8 mg/ml. CCRIP cell line was cultured in the presence of neomycin (GIBCO) at 1 mg/ml
for selection and neo-resistant clones obtained were
tested for retrovirus titer by transducing NIH3T3 fibroblasts under neomycin selection (1 mg/ml). For every
recombinant retrovirus at least 15 G418-resistant colonies
were expanded and their titers were determined. These
titers may vary from 103 to 106 c.f.u./ml according to the
different genes cloned and, for the same construction, to
different individual clones. Clones producing the retrovirus with higher titers were expanded. Primary fibroblasts and transformed cells were transduced with the
recombinant retrovirus according to the genetic complementation groups and were selected with 1 mg/ml of
neomycin and grown in the presence of neomycin for at
least 8 weeks.
UDS and RRS
Analysis of repair synthesis was carried out in untransduced and transduced primary and transformed fibroblasts as previously described.51 Briefly, cells were grown
on glass coverslips for 2 days. After 2 more days in
serum-deprived medium (0.5%) they were UV irradiated
with doses from 0 to 15 J/m2 and then incubated with
3
H-thymidine at 10 mCi/ml (specific activity of 50
Ci/mmol; DuPont de Nemors, Les Ulis, France) for 3 h,
followed by a chase of 1 h with cold thymidine.
Coverslips with the cells were mounted on to glass slides,
dipped in Kodak NTB-2 emulsion (Kodak, New York,
NY, USA), and exposed for 1 week at 4°C. The mean
number of grains per nucleus was obtained by counting
at least 30 non-S-phase nuclei. RRS was measured as follows: cells were grown as for UDS experiments and UV
irradiated with 15 J/m2. Cells were then incubated for 23
h and then labeled for 1 h in a medium containing 3Huridine at 10 mCi/ml (specific activity of 50 Ci/mmol).
Autoradiography was performed as described above for
measurement of UDS except that the exposure time was
24 h.
UV survival
The colony-forming ability was determined in primary
and transformed fibroblast cells by seeding increasing
Genetic group
Source
Villejuif, France
C Arlett (Brighton, UK)
XP-A
Villejuif, France
D Bootsma (Rotterdam, The Netherlands)
cell numbers in function of UV doses (approximately
1 × 103 to 8 × 103 and 2 × 104 to 2 × 106 for primary and
transformed cells, respectively). Irradiation was performed as in UDS experiments and cells were maintained
in medium supplemented with 20% and 10% FCS for primary and transformed fibroblasts, respectively, for 14
days. The relative survival was calculated as the number
of colonies obtained in UV irradiated over unirradiated
cells.
Detection of gene expression (Western blot)
The proteins encoded by the introduced genes were analyzed by 6 or 8% (according to the molecular mass of
proteins to be analyzed) acrylamide-SDS gel electrophoresis of cellular extracts obtained from different cell
lines. Protein samples were transferred into Hybond+
membranes (Amersham, Les Ulis, France) and probed
with specific antibodies, according to the manufacturer’s
procedure: anti-XPA antiserum (kindly provided by Dr K
Tanaka, IMCB, Osaka University, Japan); the monoclonal
anti-XPB antibody (1B3) raised against the N-terminal of
human XPB protein (AA 80–480) was a gift from Dr JM Egly (Strasbourg, France); and the polyclonal antibody
raised against human XPC protein was kindly provided
by Dr F Hanaoka (IMCB, Osaka University, Japan).
Reverse transcription PCR amplification
Total RNA was isolated from approximately 1 × 108 cells
from MRC5V1 (normal), XPCS2BASV (XP-B/CS) and
XPCS2BASV transduced by recombinant retrovirus
LXPBSN, using the RNA Isolation Kit (Bioprobe Systems,
Montreuil-sous-Bois, France). RNA (5 mg) was used for
reverse transcription reaction. Briefly, RNA was dissolved in 10 ml of reverse transcriptase buffer (250 mm
Tris-HCl (pH 8.3), 375 mm KCl, 15 mm MgCl2), in the
presence of RNase inhibitor (Boehringer Mannheim,
Meylan, France), 5 ml of 0.1 m DTT, 5 ml of 10 mm dNTP,
1 mg of poly-dT and 400 units of MoMLV reverse transcriptase (GIBCO) were added, and then incubated at
37°C for 30 min. PCR was performed to amplify the fragment from position 22 to 225 in exon 3 of XPB/ERCC3
containing the T→C transition (Figure 6a) using primers:
5′-TTGGAAGCCTTCTCTCCAG TTTACAAATATGC-3′
and 5′-GCATAATTCCATCAGGGACTCCAGTCTTG-3′
(GENSET, Paris, France). To 1 mg of cDNA, PCR reaction
contained 10 ml of Taq reaction buffer (100 mm Tris-HCl,
pH 8.8, 15 mm MgCl2, 500 mm KCl, 1% Triton X-100), 1
ml (1 pmole/ml) of each primer, 10 ml of dNTPs (2 mm),
and 1.5 U of Taq polymerase (Bioprobe Systems). Amplification was performed by 30 cycles in 100 ml total vol-
Correction of inborn DNA repair defect by gene transfer
L Zeng et al
ume, 1 min denaturing at 95°C, 1 min annealing at 55°C,
and 1 min extension at 72°C. Finally, the amplified DNA
fragments were digested by HinfI (New England BioLabs, Distributor: Ozyme, Montigny-le-Bretonneux,
France) and then analyzed by agarose gel electrophoresis.
Acknowledgements
This work was supported by grants from the Association
Française contre les Myopathies (AFM, Evry, France), the
Association pour la Recherche sur le Cancer (ARC, Villejuif, France), the Fédération Nationale des Groupements
des Entreprises Françaises dans la Lutte Contre le Cancer
(FEGEFLUC, Marseille, France) and the Ligue Nationale
Contre le Cancer (LNCC, Versailles, France). L Zeng and
X Quilliet are fellows from AFM and the Institut de Formation Supérieure Biomédicale (IFSBM, Villejuif, France),
respectively. We are endebted to: Drs K Tanaka, F
Hanaoka (Osaka University, Japan), G Weeda (Erasmus
University, Rotterdam, The Netherlands) and J-M Egly
(CNRS/INSERM, Strasbourg, France) for providing antibodies and cDNA-containing plasmids; Drs C Arlett
(MRC, Brighton, UK), D Bootsma and JHJ Hoeijmakers
(Erasmus University, Rotterdam, The Netherlands) for
providing cell lines and Drs O Danos (AFM-GENETHON, Evry, France) and J-M Heard (Institut Pasteur,
Paris, France) for providing packaging cells and helpful
discussion.
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