Cancer Gene Therapy (2005) 12, 389–396
All rights reserved 0929-1903/05 $30.00
r 2005 Nature Publishing Group
www.nature.com/cgt
Restoring DNA repair capacity of cells from three distinct
diseases by XPD gene-recombinant adenovirus
Melissa Gava Armelini,1 Alysson Renato Muotri,2 Maria Carolina Nasser Marchetto,1
Keronninn Moreno de Lima-Bessa,1 Alain Sarasin,3 and Carlos Frederico Martins Menck1
1
Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, 05508900 SP, Brazil; 2Laboratory of Genetics, The Salk Institute, 10010 North Torrey Pines Road, La Jolla,
California 92037, USA; and 3Laboratory of Genetic Instability and Cancer, UPR 2169, Institut Gustave
Roussy (IGR), 94805, Villejuif, France.
The nucleotide excision repair (NER) is one of the major human DNA repair pathways. Defects in one of the proteins that act in this
system result in three distinct autosomal recessive syndromes: xeroderma pigmentosum (XP), Cockayne syndrome (CS) and
trichothiodystrophy (TTD). TFIIH is a nine-protein complex essential for NER activity, initiation of RNA polymerase II transcription
and with a possible role in cell cycle regulation. XPD is part of the TFIIH complex and has a helicase function, unwinding the DNA
in the 50 -30 direction. Mutations in the XPD gene are found in XP, TTD and XP/CS patients, the latter exhibiting both XP and CS
symptoms. Correction of DNA repair defects of these cells by transducing the complementing wild-type gene is one potential
strategy for helping these patients. Over the last years, adenovirus vectors have been largely used in gene delivering because of their
efficient transduction, high titer, and stability. In this work, we present the construction of a recombinant adenovirus carrying the
XPD gene, which is coexpressed with the EGFP reporter gene by an IRES sequence, making it easier to follow cell infection.
Infection by this recombinant adenovirus grants full correction of SV40-transformed and primary skin fibroblasts obtained from
XP-D, TTD and XP/CS patients.
Cancer Gene Therapy (2005) 12, 389–396. doi:10.1038/sj.cgt.7700797
Published online 14 January 2005
Keywords: DNA repair; recombinant adenovirus; xeroderma pigmentosum; ultraviolet light
ll living organisms are constantly threatened by series
of exogenous and endogenous agents that cause
A
damage to DNA. In humans, these DNA lesions may lead
to cell death, tissue degeneration, aging and cancer.1
Ultraviolet (UV) light is one of such damaging agents,
inducing photoproducts on DNA, mainly cyclobutane
pyrimidine dimers (CPD) and (6–4) pyrimidine pyrimidone (6–4 PPs), which cause considerable distortions in
the structure of the double helix. These lesions need to be
removed from DNA, this being performed by an efficient
machinery of DNA repair called nucleotide excision
repair (NER). The removal of these photoproducts is
not equally handled by NER through the genome.
Lesions in actively transcribed DNA strands are repaired
faster and more efficiently by transcription-coupled repair
(TCR) than those in the nontranscribed DNA, repaired
by global genomic repair (GGR).2,3
Received April 25, 2004.
Address correspondence and reprint requests to: Dr Carlos
Frederico Martins Menck, Department of Microbiology, Institute of
Biomedical Sciences, University of São Paulo, Av. Prof. Lineu
Prestes, 1374, São Paulo, SP 05508-900, Brazil.
E-mail: [email protected]
NER is a highly conserved mechanism4 that involves
more than 30 proteins acting in a tightly regulated
manner.5 Briefly, the NER pathway consists mainly of
six steps: recognition of the lesion carried out basically by
the XPC-hHR23B complex (for GGR) or RNA pol II
stalling (for TCR); opening of the double helix at the
lesion site by the concerted action of the two DNA
helicases XPB and XPD; demarcation of the lesion
requiring the activity of the XPA and RPA proteins;
dual incision of the damaged strand by the XPF and XPG
endonucleases; synthesis of DNA in the gap left by the
removal of a 24mer-32mer oligonucleotide by the
replicative DNA polymerases and PCNA; and ligation
to the parental strand by DNA ligase I.6,7
The XPD protein is a subunit of the TFIIH complex
(transcription factor IIH) with a molecular weight of
86.9 kDa and comprising 760 amino acids.8 It has an
intrinsic 50 -30 helicase activity that is absolutely required
for NER.9–11 Regardless of its participation in NER, the
TFIIH complex is also found in the activation of cdk
kinases involved in the phosphorylation of several
substrates such as RNA polymerase II, transcription
activators and nuclear hormone receptor.12,13
Mutations in a single gene involved in NER may
lead to three different human disorders: xeroderma
XPD complementation by recombinant adenovirus
MG Armelini et al
390
pigmentosum (XP), Cockayne syndrome (CS) and
trichothiodystrophy (TTD) that show distinct clinical
phenotypes.5,14 XP is an autosomal recessive syndrome in
which patients have severe sunlight sensitivity that leads
to progressive degeneration of sun-exposed regions of the
skin and eyes, usually leading to various forms of
cutaneous malignancy.15,16 A significant number of
patients present progressive neurological degeneration.14,17 There are seven complementation groups of
XP, designated XP-A to XP-G, each of them corresponding to a specific gene defect in DNA repair, and a variant
group, designated XPV, that presents normal NER but a
postreplicative repair deficiency.
CS is characterized by cutaneous photosensitivity, and
CS cells display increased sensitivity to UV light due to a
defect in TCR.18 Surprisingly, CS patients are apparently
not predisposed to develop skin cancer. Furthermore, CS
is a very pleiotropic disorder with physical and mental
retardation.19,20 The main hallmark of TTD is sulfurdeficient brittle hair, caused by a greatly reduced content
of cysteine-rich matrix proteins in the hair shafts. This is
accompanied by mental retardation, unusual facies,
ichthyotic skin and reduced stature.21 Many, but not all
patients, with TTD are sensitive to sunlight, although
they do not have any unusual pigmentation changes, and
there are no reports of cancer in TTD patients.22
Interestingly, mutations in three XP genes (XPB, XPD
and XPG) are also found in TTD and XP/CS patients
(exhibiting both XP and CS symptoms) giving rise to very
specific phenotypes associated with each syndrome.23
Thus, the XP/CS patients mutated in either one of the
XPB, XPD and XPG genes present a combination of the
cutaneous abnormalities of XP with the severe neurological and developmental anomalies typical of CS
patients.24 The majority of TTD patients carry mutations
in the XPD gene and present only a TTD phenotype.25,26
Despite the fact that XPD and TTD/XPD patients have
mutations in the same XPD gene and both present
extreme sensitivity to sunlight, only XPD patients are
predisposed to skin cancer.23
Some of the clinical features of these syndromes, such
as UV sensitivity and predisposition to skin cancer
(characteristic for XP), may be due to defective functioning of TFIIH in NER, whereas others symptoms, such
as severe growth retardation, neurodysmyelination
(XP/CS and TTD) and brittle hair (TTD), may be caused
by a subtle defect in the transcriptional activity of
TFIIH.27,28
Correction of DNA repair defects of these cells by
transducing the complementing gene is one potential
strategy for helping XP patients. In vitro, cell complementation has been achieved following stable or transient
expression of the specific XP gene.29 In 1995, Carreau
et al29 developed a retrovirus carrying the XPD gene and
in 1996, Quilliet et al30 showed an efficient and long-term
complementation with this vector. However, complementation of XP fibroblast primary cells by retroviral
transduction would only be possible after selection in
culture.31 Contrary to the retrovirus, adenoviral vectors
can be easily obtained at high titers, and do not require
antibiotic selection or clone isolation due to the highly
efficient machinery of infection.32 Furthermore, a limitation to the usefulness of C-type retrovirus vectors is that
they can only access the cell nucleus if the nuclear
membrane breaks down; therefore, they can only transduce dividing cells.33,34 In addition, there is always the
potential for a randomic integration event of retrovirus
that could either silence gene expression or trigger cells to
cancer initiation.35,36 Recently, Muotri et al37 showed a
full correction of SV40-transformed and primary skin
fibroblasts obtained from XP-A and XP-C patients by
infection with recombinant adenovirus carrying the XPA
and XPC genes. In addition, protein expression showed to
be stable for at least 2 months after infection.
In this work, we present the construction of a
recombinant first-generation adenovirus harboring the
XPD cDNA and the EGFP gene (AdSHIRES-XPD) and
its ability to complement SV40-transformed and primary
skin fibroblasts obtained from XP-D, XP/CS and TTD
patients after infection with this adenovirus.
Materials and methods
Cell culture conditions
Cells used in this work are listed in Table 1. SV40transformed and primary skin fibroblasts were isolated
from skin biopsies of XP complementation group D
patients (XP22VI and XP6BE-SV), XP combined with CS
patients (XPCS2 and XPCS2-SV) and TTD patients
(TTD1VI and TTD1VI-SV). MRC5-V1 and NHF, which
Table 1 Characteristics and origins of the cell lines
Cell names
Cellular status
Mutation in DNA repair gene
Clinical phenotype
Laboratory of origin
NHF
MRC5-VI
HEK 293
XP22VI
XP6BE
TTD1VI
TTD1VI-SV
XPCS2
XPCS2-SV
Diploid
SV40-transformed
Human Ad5-sheared DNA-transformed
Diploid
SV40-transformed
Diploid
SV40-transformed
Diploid
SV40-transformed
wt
wt
wt
XPD
XPD
XPD
XPD
XPD
XPD
wt
wt
wt
XP
XP
TTD
TTD
XP/CS
XP/CS
CFP Lotfi
AR Lehmann
Clontech
A Sarasin
A Sarasin
A Sarasin
A Sarasin
A Sarasin
EC Friedberg
Cancer Gene Therapy
XPD complementation by recombinant adenovirus
MG Armelini et al
are normal for DNA excision repair, were used as positive
controls. The MRC5-V1 cell line was derived from the
normal lung tissue of a 14-week-old male fetus, and is also
SV40-transformed. The NHF primary cell line is derived
from normal skin. HEK 293 cells were used for the
recombinant virus production. Cells were routinely grown
at 371C in a 5% CO2 humidified atmosphere in
Dulbecco’s modified Eagle’s medium for transformed
cells (DMEM; Life Technologies, Inc., Carlsbad, CA) and
minimum essential medium for primary and HEK 293
cells (MEM; Life Technologies, Inc., Carlsbad, CA).
Culture media were supplemented with heat-inactivated
10% fetal calf serum (FCS; Cultilab, Campinas), and
antibiotics (0.1 mg/ml each of penicillin and streptomycin
and 0.25 mg/ml of fungizone).
In Figure 1a, we show a scheme illustrating the XPD
gene and the site of the mutations found in the cells used
in this work. This illustration was adapted from Itin
et al.38 All of the cell lines used have mutations in the Cterminal domain of the protein, where there is the
interaction domain between the XPD helicase and the
p44 protein, both components of the TFIIH factor.12,39
The mutation found in the XPCS2 cell line occurs in the
helicase V domain, corresponding also to the DNAbinding domain.14
Adenovirus production and infection
Briefly, we have constructed a recombinant adenovirus
(AdSHIRES-XPD, based on adenovirus type 5), deleted
in extensive portions of E1/E3 regions, carrying the
cDNA of XPD and the EGFP (enhanced green fluor-
a
escent protein) reporter gene linked by an IRES sequence,
Internal Ribosome Entry Site.40A scheme of AdSHIRESXPD is illustrated in Figure 1b. This construct permits
both the gene of interest and the EGFP gene to be
translated from a single bicistronic mRNA. Plasmids were
obtained from Clontech Laboratories Inc. (Palo Alto,
CA). XPD and EGFP cDNAs are fragments of 2.8 and
0.72 kbp, respectively. The cassette containing the XPDIRES-EGFP, extracted from pIRES2-EGFP, was cloned
into appropriate restriction sites inside the polylinker of a
vector called pShuttle (3.9 kbp) The expression of these
genes is under the control of the strong cytomegalovirus
immediate-early promoter/enhancer (PCMV IE) and the
polyadenylation signal from the bovine growth hormone
gene (BGH Poly A). The recombinant virus was produced
according to the protocol described in Muotri et al.37
Recombinant adenovirus infection for all cells tested
was performed according to Graham and Prevec.41
Briefly, approximately 106 cells in 6-cm diameter dishes
were infected with 0.3 ml of a concentrated viral solution
in 1 ml of MEM or DMEM (depending on the cell type)
for 1 hour at 371C, before the addition of 3 ml of complete
culture medium to the dish. Quantification of the
adenovirus vector was performed by optical absorbance.42 The titer of virus stock was 2.36 1012 particle
units/ml and the multiplicity of infection (MOI) used in
the experiments was 590 infection units per cell.
Flow cytometry analysis (FACS)
After viral stocks preparation, XP6BE-SV cells were
infected with different concentrations of AdSHIRES-XPD
XP6BE2
36-61
35/51
69/88
I
Ia
I
35/57
XP6BE1
XP22VI1,2
R683W
XPCS21
G602D
225/239
455/468
533/554
III
VI
IV
II
II
III
IV
V
87/103 118/132 228/251 253/264
587/613 654/671
V
VII
346/353
760
VI
557/602
R722W
TTD1VI1
716-730
TTD1VI2
ψ
b
ITR
Amp
∆E1/E3
Swa I
ITR
Pac I
Pac I
PA
PI-Sce I
EGFP IRES
EGFP
XPD
XPD
CMV
I-Ceu I
Figure 1 XPD gene mutations and scheme of the AdSHIRES-XPD vector. (a) Mutations found in cells used in this work are indicated as the
number and the type of the amino acid changed (solid black line indicates deletion). The seven domains DNA/DNA helicase are indicated by
white boxes with the number of amino acids involved, and the seven domains DNA/RNA helicases are indicated by boxes below the XPD protein.
Adapted from Itin et al.38 (b) Recombinant XPD-adenovirus construct. ITR, inverted terminal repeat; c, packaging signal; CMV, cytomegalovirus
immediate-early promoter/enhancer; PA, bovine growth hormone gene polyadenylation signal; Amp, ampicillin-resistance gene. Arrows indicate
transcription orientation. Some restriction sites used for construction are shown (PacI, PI-SceI, I-CeuI).
Cancer Gene Therapy
391
XPD complementation by recombinant adenovirus
MG Armelini et al
392
for EGFP detection. Cell samples were trypsinized
and resuspended in DMEM. Cells were immediately
analyzed in a FACSCalibur flow cytometer (Becton
Dickinson, San Jose, CA) equipped with a 488 nm
argon laser for excitation of the reporter protein, and a
530/30 nm bandpass filter for monitoring fluorescent
emissions. For each sample, 10,000 events were collected
by list-mode data, which consisted of forward scatter
(FCS), side scatter (SSC) and fluorescent emissions
(FL-1).
Fluorescence microscopy analysis
Microscopic evaluation of EGFP-expressing cells was
performed with a fluorescence microscope (Leica DM
LB) equipped with a standard B/G/R filters set (excitation: 400/20, 495/15, 570/50 nm). Microphotographs were
recorded on a KP-D581U digital color video camera
(Hitachi-USA) using the software Leica EWS 2100
Capture Station (Leica, Wetzlar).
Western blot
The cellular lysis and SDS-PAGE were performed
according to standard procedures.43 In all, 30 mg of total
protein samples per lane were transferred to Hybond-C
membrane (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) and probed with the specific antibodies, antiXPD polyclonal and anti-GFP (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The secondary antibody, antigoat HRP-conjugated IgG, was obtained from Santa
Cruz Biotechnology, Inc. (CA) and anti-rabbit peroxidase-conjugate IgG was from Sigma-Aldrich Chemical
CO (St Louis, MO).
Evaluation of cell survival after UV irradiation
Cells were infected 48 hours before irradiation by a
germicidal lamp, predominantly at 254 nm. Cell survival
was measured 5 days later (for primary cells) or 3 days
later (for SV40-transformed cell lines) by the addition of
the tetrazolium salt XTT (final concentration 0.12 mg/ml)
to the culture medium. Surviving cells, with active
mitochondria, cleave the XTT substrate into an orange
formazan dye. The amount of formazan dye formed after
1-hour incubation was measured by a Genesys 5 spectrophotometer (Spectronic Instruments) at OD 450 and
650 nm. Cell survival was calculated as the percentage of
absorbance in relation to the absorbance of untreated
cells. For determination of colony forming ability,
approximately 1000 cells (infected or not) were plated in
60 mm Petri dishes 14–16 hours before UV irradiation.
After irradiation, cells were maintained in culture for 15
days, then being fixed with 10% formaldehyde and
stained with 1% violet crystal. Colonies with the minimal
number of 15 cells were scored. Survival values were
obtained as the ratio of the number of colonies from
irradiated cells to nonirradiated cells.
Cancer Gene Therapy
UDS – unscheduled DNA synthesis
This assay was performed according to the protocol of
Cleaver and Thomas44 with some modifications. Briefly,
104 cells were grown on glass cover slips for 24 hours.
After 24 hours of culture in a serum-deprived medium
(1% FCS), 10 mCi/ml of 3H-methyl thymidine (86.0 Ci/
mmol, Amersham Pharmacia Biotech) was added to the
medium for 1 hour. The cells were washed with PBS and
then UV irradiated with 10 J/m2. After 3 hours in the
presence of 3H-methyl thymidine, followed by a chase of 1
hour with cold thymidine (100 mM), the cells were fixed
with methanol-acetate (3:1). The cells mounted onto glass
slides were washed three times with 5% trichloroacetic
acid for 15 minutes each, then rinsed twice with 70%
ethanol and once with absolute ethanol. The slides were
dipped into an EM-1 (Amersham Pharmacia Biotech)
emulsion and exposed for 4 days at 41C. After development, the mean number of grains per nucleus was
obtained by counting at least 30 non-S-phase nuclei.
Results
Recombinant adenovirus construction
The AdSHIRES-XPD was derived from adenovirus type
5, deleted in E1/E3 regions. The construction was checked
for the presence of the XPD and EGFP genes by PCR and
restriction analysis (data not shown). The XPD and
EGFP genes were under the control of the strong
cytomegalovirus immediate-early promoter/enhancer
(PCMV IE). The construction has an IRES sequence that
permits both the gene of interest and the EGFP gene to be
translated from a single bicistronic mRNA. Owing to the
expression of EGFP protein, infection in the target cells
could be followed by visualization of this protein under a
fluorescence microscopy. The results show that 100% of
cells were infected (Fig 2a and b). The absence of
contamination by a wild-type virus was confirmed by
the lack of cell lysis when cultures, other than HEK 293
cell line, were infected with the recombinant adenovirus
preparation.
Protein expression
In order to confirm XPD protein production in the cells
infected with AdSHIRES-XPD, a Western blot analysis
was performed. The recombinant adenovirus was able to
infect all primary and transformed cell lines (Fig 2c).
Normal and mutated fibroblasts, noninfected, showed a
faint protein signal, confirming the protein expression
with missense mutations. The expression of XPD in
infected cells is much stronger than what is detected in
control cells (MRC5-V1 and NHF). In fact, even 24 hours
after infection, it was already possible to detect the
overexpression of both proteins (Fig 2d). Despite cell
division and protein dilution, the expression continued to
increase up to at least 72 hours after infection. Comassieblue-stained gels confirmed similar protein loading for the
various samples (data not shown).
XPD complementation by recombinant adenovirus
MG Armelini et al
393
a
Visible Light
UV Light
b
Marker % Gated
All
100
M1
96.82
Marker % Gated
All
100
M1
0.26
c
+ AdSHIRES-XPD
1
2
3
4
5
6
7
8
80 kDa
Anti-XPD
32 kDa
Anti-EGFP
6B
E
XP
MR
C5
-V
I
d
XP6BE+ AdSHIRES-XPD
24
48
72
80 kDa
Anti-XPD
32 kDa
Anti-EGFP
Figure 2 Efficient infection of AdSHIRES-XPD in XPD primary fibroblasts. (a) Viral infection can be followed in XPCS2 cells by visualization of
the EGFP protein in a fluorescence microscope ( 100). (b) FACS analysis of XP6BE cells before and after AdSHIRES-XPD infection. M1:
percentage of the total counted cells that express the EGFP protein. (c) Detection of XPD and EGFP protein expression by Western blot. Primary
fibroblasts were infected with AdSHIRES-XPD and collected after 72 hours. (1) NHF (normal fibroblast); (2) XPCS2 (XP/CS); (3) XP22VI (XPD);
(4) TTD1VI (TTD). (d) Kinetics of transgene expression in XP6BE cells infected with AdSHIRES-XPD. Cells were harvested at the indicated
times after infection. Protein extracts on membranes were probed with anti-XPD and anti-EGFP as indicated. XPD and EGFP have the expected
molecular weight in infected cells (indicated on the left), 30 mg of total protein in each well.
Cellular complementation of DNA repair defect
In order to quantify the efficiency of complementation
using the AdSHIRES-XPD, phenotypical analysis was
carried out in deficient cells infected or not with the
recombinant adenovirus and irradiated with UV light.
UV sensitivity was measured by XTT cleavage in the
culture medium for both primary and SV40-transformed
cells and by colony forming ability only for SV-40transformed cells. All cells with different mutations in the
XPD gene (from patients with distinct syndromes) are
more sensitive to UV than normal human cells (Fig 3a–c).
Infection of these cells with AdSHIRES-XPD complements their sensitivity to UV, as resistance is recovered to
levels close to normal cell line (MRC5-V1 and NHF).
The assay to measure UDS was performed 72 hours
after viral infection. The aspect of the nuclei of irradiated
(10 J/m2) and nonirradiated cells is illustrated in Figure 4a.
Nonirradiated XP22VI, XPCS2, TTD1VI and NHF cell
lines have exhibited a low number of grains (XP22VI:
3.271.8; XPCS2: 2.571.8; TTD1VI: 0.870.9 and NHF:
2.172.3). Damage caused by UV light induces, in normal
fibroblasts, an increase in the number of grains, due to
DNA repair activity (56.777.4). Under similar conditions, XP22VI, XPCS2 and TTD1VI cell lines also present
an increase in UDS (XP22VI: 23.674.3; XPCS2:
25.076.7; TTD1VI: 41.476.6), but these numbers are
inferior when compared to control cells. After infection
with AdSHIRES-XPD, the UDS level was restored
in these cells to levels comparable to the DNA
Cancer Gene Therapy
XPD complementation by recombinant adenovirus
MG Armelini et al
394
a
b
100
c 100
100
% survival
10
10
NHF
10
MRC5-VI
XP6BE-SV
TTDVI-SV
XPCS2-SV
XP22VI
TTD1VI
XPCS2
XP22VI+Ad
XPCS2+Ad
XP6BE-SV
2
4
6
8
TTDVI-SV
XP6BE-SV+Ad
TTDVI-SV+Ad
0.1
1
0
MRC5-VI
XP6BE-SV+Ad
TTDVI-SV+Ad
XPCS2-SV+Ad
TTD1VI+Ad
1
1
0
2
4
6
8
0
2
4
6
8
UV dose (J/m²)
Figure 3 Complementation of UV-sensitivity by the XPD recombinant adenovirus. Cells were infected or not with AdSHIRES-XPD, and exposed
to UV irradiation at increasing doses. Cell survival was measured (a) 3 days (transformed cells) or (b) 5 days (primary cells) after UV irradiation
by the detection of XTT cleavage, as described in Materials and methods. (c) Cell survival was determined by colony-forming ability for SV40transformed cells.
repair proficient control (XP22VI: 48.379.4; XPCS2:
48.5714.1; TTD1VI: 53.877.7). These data are shown in
Figure 4b.
Discussion
XPD protein is a helicase and a subunit of the TFIIH
complex.6–8 This complex is necessary for the initiation of
transcription and the completion of NER.6,7,11 Mutations
in the XPD gene can lead to three different diseases: XP,
XP/CS and TTD.5,14,45 There is no extended treatment for
these patients, but the techniques based on gene
complementation have been largely studied in the hope
of prolonging survival and relieve the carcinogenic effects.
One efficient way of delivering genes into mammalian
cells is the use of a recombinant adenovirus vector,
particularly when a high-level expression of transgene
products in cultured cells is required.32,37
In the present work, we describe the construction of a
recombinant adenovirus carrying the XPD cDNA and its
ability to complement SV40-transformed and primary
fibroblasts belonging to patients mutated on this gene, but
with different clinical symptoms (XP, XP/CS and TTD).
The construction developed here is interesting due to
the internal ribosome entry site (IRES) sequence , which
links the cDNA of XPD and EGFP reporter gene. This
construction permits both the gene of interest and the
EGFP gene to be translated from a single bicistronic
mRNA. Thus, infection of the target cells could be
followed by visualization of the EGFP protein in a
fluorescence microscope and FACS analysis. This allowed
us to verify that 100% of cells were infected without any
trace of contamination by a wild-type virus. The
recombinant adenovirus obtained was able to infect
normal and XPD mutated fibroblasts, both SV40transformed and primary cells. No cytotoxic effects were
observed in the cells after infection, indicating that no
wild-type virus is produced during virus production.
Cancer Gene Therapy
High levels of protein production were detected in
target cells by Western blot analysis 24 hours after
infection. Apparently, overexpression of XPD protein
does not interfere with human cell viability, contrary to
what has been observed for Drosophila S2 cell lines.46
These authors observed that an excess of XPD titrates
CAK activity, resulting in decreased Cdk T-loop phosphorylation, mitotic defects and lethality. In this work,
primary fibroblasts infected with AdSHIRES-XPD remained alive, in a good state and expressing XPD and
EGFP proteins for at least 15 days (data not shown).
Phenotypic analysis was carried out on different XP-D
cell lines, infected or not with recombinant adenovirus, in
order to check for the biological activity of transgene
expression. Responses to UV irradiation were evaluated
by cell survival measured by XTT cleavage and colonyforming ability. The virus could rescue UV resistance in
all the cell lines tested. Moreover, expression of active
XPD protein leads to a very homogeneous capacity to
perform DNA repair synthesis (UDS). Both results
indicate complementation for the XPD gene in the
mutated cell lines after UV exposure. Curiously, overexpression of this protein does not lead to cell survival or
UDS levels raised above DNA repair proficient cells. This
has been observed before for XPA and XPC proteins37,47
and clearly indicates that these proteins (including XPD,
this work) are not limiting for the control of DNA repair.
Despite of the fact that XPD and XP/CS cells have
mutations in distinct sites in the XPD gene, and the
patients show characteristic clinical features, the response
to UV irradiation was similar to control cells after
infection with the recombinant virus. XP and XP/CS
cells have presented similar levels of UDS after UV
irradiation, confirming previous observations.48,49 Nevertheless, a spurious DNA degradation, not related to DNA
repair, has been described for XP/CS cells, after UVirradiation.5 The reason how these specific mutations in
the XPD gene result in the peculiar properties of the XP/
CS cell lines remains obscure.5 Anyway both cells, XP and
XP/CS, show similar recovery of survival and UDS after
XPD complementation by recombinant adenovirus
MG Armelini et al
a
+AdSHIRES-XPD
395
Acknowledgments
This work was supported by the Fundac¸ão de Amparo à
Pesquisa do Estado de São Paulo – FAPESP (São Paulo,
Brazil), Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq, Brası́lia, Brazil) and CAPESCOFECUB (Brasilia; Brazil/Aix en Provence, France).
MGA and KMLB have PhD fellowships from FAPESP.
We thank Drs CFP Lofti (University of Sao Paulo, SP,
Brazil), A Lehmann (MRC, Brighton, UK) and EC
Friedberg (University of Texas Southwestern Medical
Center at Dallas, TX) for providing some of the cell lines
used in this work.
NHF
XP22VI
References
b
70
NHF
grains per nuclei
60
XP22VI
50
XPCS2
40
TTD1VI
30
XP22VI+Ad
20
XPCS2+Ad
10
TTD1VI+Ad
0
0
10
UV dose (J/m²)
Figure 4 DNA repair measured by UDS in different XP-D cell lines.
The number of labeled grains per nucleus is proportional to the repair
activity of cells. (a) Microscope analysis showing the aspect of UVirradiated nuclei (10 J/m2) cells typically seen in UDS experiments
(magnification: 1000). Heavily marked nuclei indicate S-phase
cells. (b) The UDS activity is expressed by grains per nucleus. Data
correspond to mean values from 30 nuclei.
virus complementation. It has been suggested that XP
syndrome results from mutations that only affect DNA
repair, while TTD symptoms result from mutations that
cause subtle abnormalities in transcription.50,51 This
explains why mutations found in XP and TTD patients
result in major repair deficiency while having only minor,
if any, effects on transcription, thus permitting cells to
remain viable.22
Adenovirus vectors represent a powerful technology for
better understanding of the phenotypic reversion of NERdeficient cells and cellular UV responses in vitro.
Furthermore, the recombinant adenovirus technology
represents a hope in gene therapy development for these
syndromes. An interesting perspective is the construction
of ‘‘all deleted’’ (or helper-dependent, HD) adeno vectors.
These vectors are less immunogenic, keeping their
transgene expression quite stable in vivo. Moreover, XP
knockout mice52 represent a good model for potential
gene therapy studies, and they may be valuable to test the
in vivo efficacy of adenovirus vectors.
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