Mutagenesis vol. 24 no. 1 pp. 75–83, 2009
Advance Access Publication 3 October 2008
doi:10.1093/mutage/gen054
UV-inducible base excision repair of oxidative damaged DNA in human cells
Shaqil N. Kassam and Andrew J. Rainbow*
Department of Biology, McMaster University, 1280 Main Street West,
Hamilton, Hamilton, Ontario L8S 4K1, Canada
Methylene blue (MB) acts as a photosensitizer and after
excitation by visible light (VL) produces reactive oxygen
species that result in oxidatively damaged DNA. (MB 1
VL) produces predominantly 8-hydroxyguanine as well as
other single base modifications in DNA that are repaired
by base excision repair (BER). We have used a recombinant
non-replicating human adenovirus, Ad5HCMVlacZ, which
expresses the b-galactosidase (b-gal) reporter gene, to
examine the role of the p53 tumor suppressor in
constitutive and inducible BER of MB 1 VL-damaged
DNA in human cells. Host cell reactivation (HCR) of b-gal
activity for MB 1 VL-treated Ad5HCMVlacZ was examined in normal human fibroblasts and several transformed
and tumor cell lines with compromised p53 function using
both non-treated cells and cells pretreated with ultraviolet
light of 200–280 nm wavelength (UVC). Constitutive HCR
of the MB 1 VL-treated reporter gene in untreated cells
did not correlate with wild-type p53 expression levels,
suggesting that factors other than p53 expression levels can
influence constitutive BER of the reporter gene. UVC pretreatment of the normal fibroblast strains resulted in an
enhanced HCR of the MB 1 VL-treated reporter gene
and a concomitant increase in the expression of p53,
suggesting that p53 may be involved in UV-inducible BER
in normal human fibroblasts. In contrast, p53 expression did
not correlate with HCR values for the p53-compromised
cells in UVC-pre-treated cells. In particular, the SKOV-3,
LFS 087 and NF-E6 cells showed no up-regulation of p53
expression following UVC, and yet these cells showed
significant enhancement of HCR following UVC pretreatment. These results indicate that BER of MB 1 VLdamaged DNA is inducible in human cells by pre-UVC
treatment and that the enhancement in BER may result
from both p53-dependent and p53-independent mechanisms.
Introduction
Reactive oxygen species (ROS) are constantly generated in our
cells due to aerobic respiration resulting in damage directed
towards many components of the cell, including the lipid
membrane, proteins and most importantly the DNA, which can
lead to carcinogenesis [as reviewed in ref. (1)]. The main
damage in DNA resulting from ROS is the oxidation of the
purine and pyrimidine bases and oxidation of guanine residues
is the most common giving rise to 7,8-dihydro-8-oxoguanine
(8-OxoG) (2). In addition, the exposure of cells to the UVA
component of sunlight also leads to non-bulky oxidative DNA
base modifications. UVA does not directly excite DNA but
rather indirectly damages DNA by a photosensitized reaction
through the formation of ROS and results in the formation of
non-bulky oxidative DNA base modifications including 8-OxoG
(3,4). The mutagenic effect of 8-OxoG is very well established,
as it can pair efficiently with cytosine or adenine residues (5).
Therefore, if an 8-OxoG is established in the genome, it can
potentially cause a G:C to a T:A transversion initiating the first
step of carcinogenesis [as reviewed in ref. (6)].
Single oxidized DNA base changes produced by UVA and
through aerobic respiration, such as 8-OxoG, are removed by
base excision repair (BER). Abasic sites are generated by
glycosylases, processed by apurinic/apyrimidinic endonucleases
and the oxidatively damaged base is replaced with an undamaged
base. Glycosylases are a group of proteins that can recognize and
excise oxidized bases in the DNA. The hOGG1 is one of the few
glycosylases that are specific for recognizing 8-OxoG. Removal
of the oxidized base results in the creation of an apurinic
nucleotide (AP-Site), which is recognized by an apurinic
endonuclease (APE). APE1 is able to ‘nick’ the DNA 5# to the
damaged site and effectively remove the abasic nucleotide from
the DNA. At this point, DNA polymerase b (Pol-b) fills in the
gap created by APE1, and DNA ligase connects the old and
new strands [for a review of BER see refs (7,8)].
In contrast, nucleotide excision repair (NER) is responsible
for the repair of bulky adducts including UV-induced cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PP)
(9,10). NER can be divided into two interrelated sub-pathways:
(i) transcription-coupled repair (TC-NER) which preferentially
removes DNA damage at a faster rate from the transcribed strand
of actively transcribed genes and (ii) global genomic repair (GGNER) which removes damage more slowly from throughout the
entire genome and from the non-transcribed strand as well as the
transcribed strand of active genes [as reviewed in refs. (11,12)]. In
humans, BER and NER both contribute by varying degrees, to
the removal of DNA damage induced by aerobic respiration and
sunlight. UVC induces DNA damage that is repaired primarily by
NER, whereas oxidative-damaged DNA is repaired by BER.
The p53 protein is a tumor suppressor responsible for the
regulation of genes to prevent uncontrolled cell growth (13,14).
A role for p53 has been implicated in several aspects of the
NER and BER pathways. Besides p53#s role in transcription, it
is also known to interact with DNA, and with proteins that are
involved with DNA repair [as reviewed in ref. (15)]. There is
also evidence for an involvement of p53 in BER [as reviewed
in ref. (16)]. In BER, p53 plays more of protein modulating
role, where it physically and functionally interacts with hOGG1
and APE1 (17). p53 has also been shown to interact with Pol-b,
stabilizing it and facilitating its attachment to DNA (18).
We have reported previously that UVC pre-treatment of
normal human fibroblasts results in enhanced host cell reactivation
*To whom correspondence should be addressed. Tel: þ1 905 525 9140; Fax: þ1 905 522 6066; Email: [email protected]
Ó The Author 2008. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.
All rights reserved. For permissions, please e-mail: [email protected].
75
S. N. Kassam and A. J. Rainbow
(HCR) of a UVC-damaged reporter gene due to an inducible NER
response (19,20). The UVC-enhanced HCR was absent in Li–
Fraumeni syndrome (LFS) cells expressing mutant p53 indicating
that UVC pre-treatment of cells leads to a p53-dependent upregulation of the NER pathway (20). Latonen et al. (21) have
shown that exposure of cells to low doses of UVC results in the
accumulation of p53. UVC causes CPDs and 6-4PPs that are
blocks to transcription and it is the stalling of RNAPII at these
UVC-induced DNA lesions that is thought to trigger the activation
of p53 (22).
The first direct evidence for inducible BER was reported by
Le et al. (23) who showed that low priming doses of X-rays
result in a more rapid removal of thymine glycols in A549
human lung carcinoma cells when given 4 h before a challenging
dose. Subsequently, Offer et al. (24) reported inducible BER
following pre-treatment of mammalian cells with low doses of
gamma rays or cisplatin and Lan et al. (25) reported enhanced
BER of oxidatively damaged DNA in the rat brain in vivo
following ischaemia-induced oxidative injury. In contrast,
Bercht et al. (26) reported an absence of induced BER of
oxidatively damaged DNA in vitro following pre-treatment of
cells with low priming doses of a photosensitizer plus light or
an alkylating agent. However, there are no previous reports
concerning the inducibility of BER by UV.
In the present study, we sought to investigate the inducibility
of BER following UVC pre-treatment. One of the common
in vitro inducers of singlet oxygen-mediated DNA damage is
visible light (VL)-activated methylene blue (MB) (27,28). MB
is one of the few molecules which, when irradiated with VL, is
able to excite molecular oxygen to singlet oxygen (29). MB þ VL
is known to create predominantly 8-OxoG in the DNA of cellfree extracts (30) as well as in whole cells and viruses (27,31).
We have employed a recombinant adenovirus AdHCMVLacZ
(AdCA17), which is a non-replicating virus that expresses the
b-galactosidase (b-gal) reporter gene under the control of the
human cytomegalovirus immediate-early (HCMV-IE) promoter (32,33). We have carried out HCR assays by treating
the AdCA17 virus with MB þ VL and subsequently scoring
for expression of the DNA-damaged reporter gene. HCR of
reporter gene activity requires the repair of transcription
blocking 8-OxoG DNA lesions and reflects repair of DNA
lesions in the transcribed strand. In this way, HCR of the
reporter gene is a measure of the BER ability of the infected
cell. HCR was examined in non-treated and UVC-pre-treated
normal human fibroblasts and several transformed and tumor
cell lines with compromised p53 function. Using this approach,
we were able to examine the role of p53 in constitutive and
UV-inducible BER of MB þ VL-induced DNA damage in
human cells. We report here that BER of MB þ VL-damaged
DNA is inducible in human cells by UVC pre-treatment and that
the enhancement in BER may result from both p53-dependent
and p53-independent mechanisms.
Materials and methods
Cells
The repair-proficient primary human skin fibroblasts, GM 9503, GM 38A, GM
969, along with the SV40-transformed normal skin fibroblasts, GM637, were
obtained from National Institute of General Medical Sciences (Camden, NJ).
The other SV40-transformed normal lung fibroblasts AG02804D was obtained
from The National Institute on Aging (Camden, NJ). A normal neonatal foreskin
(NF) fibroblast strain (established by Dr D. A. Galloway, Fred Hutchinson
Cancer Research Centre, Seattle, WA), and the E6-expressing transformants of
this normal strain were both obtained from Dr B. C. McKay, Centre for Cancer
76
Therapeutics, Ottawa Regional Cancer Centre, Ottawa, Ontario, Canada. The
immortalized LFS fibroblasts LFS 041 and LFS 087 were generously provided
by Dr Micheal A. Tainsky, Barbara Ann Darmanos Cancer Institute, Wayne
State University, 110 East Warren Avenue, Detroit, MI. SKOV-3 human
ovarian cancer cells were obtained from the American Tissue Culture
Collection cell repository (Rockville, MD). Passage numbers of the primary
human fibroblasts used in the experiments were 18-22 for GM9503, GM969
and GM38A and 26-29 for NF. All cell cultures were grown at 37°C in
a humidified incubator in 5% CO2 and cultured in Eagle’s a-minimal essential
media (a-MEM) supplemented with 10% foetal bovine serum and antimycotic–
antibiotic 100 lg/ml penicillin, 100 lg/ml streptomycin and 250 ng/ml
amphotericin B (Gibco BRL, Grand Island, NY). Media for the E6 transformants
were supplemented with 250 lg/ml geneticin (G418; Sigma–Aldrich Canada,
Oakville, Ontario, Canada).
Virus
The recombinant adenovirus vector Ad5HCMVlacZ (AdCA17) (34) was
obtained from Dr F.L. Graham, McMaster University. This vector contains the
lacZ gene under the control of the HCMV-IE promoter (299 to þ72 relative
to transcription start site) inserted into the deleted E1 region of the adenovirus
genome in the left to right orientation. Deletion of the E1 region of the genome
renders the adenovirus unable to replicate in most mammalian cells. The virus
was propagated, collected and titred as described previously (35).
Treatment of virus with MB þ VL
Preparation of MB was as described previously (36). A total of 80 ll of
Ad5HCMVLacZ virus was added to 3.6 ml of phosphate buffered saline (PBS)
containing 20 lg/ml MB in 35-mm Petri dishes on ice. With continuous
stirring, the virus suspensions were irradiated (or mock irradiated) with VL. VL
irradiation of virus employed a General Electric 1000-W halogen lamp (GE
R1000) at a distance of 70 cm from the bulb. After each time point, 400 ll of
irradiated virus was removed, diluted appropriately in unsupplemented a-MEM
and used to infect the cell monolayers.
HCR experiments
SV40-transformed cells, LFS cells and tumor cells were seeded at 3.8 104
cells per well and primary human fibroblasts were seeded at 1.9 104 cells per
well in 96-well plates (Falcon, Franklin Lakes, NJ). Cells were incubated for
18–24 h, pre-treated with UVC or left untreated and subsequently infected with
40 ll of untreated or MB þ VL-treated virus for 90 min at a multiplicity of
infection of 20 or 40 plaque-forming units per cell, overlaid with 160 ll of
complete a-MEM and incubated for a further 44–48 h prior to harvesting.
Pre-treatment of cells with UVC
For the HCR assays in which cells were pre-treated with UVC, overlaying
media were aspirated from the cell monolayers and 40 ll of warmed PBS was
added to each well prior to exposure to UVC. The source of UVC was a
General Electric germicidal lamp (model G8T5) emitting predominantly at 254 nm
and cells were exposed to UVC at a fluence of 1 J/m2/s.
Harvesting and scoring
Infected cells were scored for b-gal as described previously (36). Incubation
medium was aspirated from each well and 60 ll of chlorophenolred b-Dgalactopyranoside (prepared in 0.01% Triton X-100, 1 mM MgCl2 and 100 mM
phosphate buffer at pH 8.3; Boehringer-Mannheim, Indianapolis, IN) was
added to the infected cells. To assay for the expression of the b-gal levels from
the lacz gene, optical density (OD) readings were taken using a 96-well plate
reader (Bio-Tek Instruments EL340 Bio Kinetics Reader) at several time
intervals at 570 nm. The OD of wells infected with untreated virus was plotted
as a function of time. Plates with maximum OD readings just under the
saturation plateau were analysed in further detail. For plotting the b-gal survival
curves, the average background level of b-gal activity was subtracted from each
averaged point from the measurements taken from a minimum of triplicate
wells.
Western blot analysis of p53 expression
All cells were seeded in 100-mm cell culture dishes (Falcon, Franklin Lakes,
NJ) at a density that would achieve confluence. Between 18–24 h after seeding,
medium was replaced with 5.9 ml of PBS and then UVC irradiated at 1 J/m2/s
for 25 sec or left untreated and then re-fed with the complete a-MEM. The cells
were then allowed to incubate for 24 h at which point they were scraped off
the dishes into centrifuge tubes and centrifuged for 10 min at 1000 r.p.m. The
cells were then re-suspended in 10 ml PBS and re-centrifuged for 10 min at
1000 r.p.m. The cells were suspended in lysis buffer (50 mM Tris, 150 mM
NaCl and 1% NP40) containing protease inhibitors. After centrifugation (1 min
at 13 000 r.p.m.), the supernatants were isolated and protein concentrations
UV-inducible base excision repair
were measured in duplicate using the Bio-Rad protein reagent in a Bradford
assay (Bio-Rad, Richmond, CA). Protein aliquots were prepared and 30–40 lg
of each protein sample was loaded and separated by sodium dodecyl sulphate–
polyacrylamide gel electrophoresis (10%). Proteins were then transferred onto
a nitrocellulose membrane and blocked overnight at 4°C in 10% skim milk in
Tris-buffered saline with 0.05% Tween 20. Blots were then probed with
a mouse monoclonal antibody to p53 conjugated to horseradish peroxidase
(Santa Cruz Biotechnology, Santa Cruz, CA). Protein loading was verified by
monitoring actin levels in each lane. After the addition of ECL staining reagent
(Western Lightning Chemiluminescence Reagent, PerkinElmer Life Sciences),
blots were visualized by exposure to Kodak X-Omat AR film.
Results
Relative β-Galactosidase Activity
Constitutive HCR of the MB þ VL-treated reporter gene in
untreated normal fibroblasts and p53-compromised cells
In order to determine the role of p53 in constitutive BER of
MB þ VL-induced DNA damage, we examined HCR of an
MBþVL-treated reporter gene in normal human fibroblasts as
well as in human cells with compromised p53. The normal
human fibroblast strains were GM9503, GM38A, GM969 and
NF. The cell lines with compromised p53 were GM637,
AG02804, NF-E6, LFS 087, LFS 041 and SKOV-3. NF and
NF-E6 is a matched isogenic pair of cell strains in which the
NF-E6 cells have been transfected with the papilloma E6 protein
resulting in abrogation of p53 (37). The SV40-transformed cell
lines GM637 and AG02804D have their p53 abrogated by the
SV40 large T antigen (38). The LFS cells LFS 087 and LFS 041
each have a mutation in the p53 gene and the human ovarian
cancer SKOV-3 cells express no p53 at all (39,40).
Representative survival curves of b-gal activity for MB þ
VL-treated AdCA17 are shown for the normal fibroblasts in
Figure 1 and for the various p53-compromised cell lines in
Figure 2. Each figure shows results for the GM9503 normal
fibroblasts used in the same experiment for comparison. The
VL exposure in seconds required to reduce b-gal activity to
37% of that for non-VL-exposed virus (D37) was used as
a measure of HCR. In order to account for the variation in D37
values between individual experiments, we calculated the D37
value for each of the various fibroblast strains and human cell
lines relative to the D37 value of the normal GM9503 strain
obtained in the same experiment. The D37 values relative to the
GM9503 strain for each cell line were then averaged over
several experiments and are shown in Table I, column 2. For
the p53-compromised cell lines, the HCR value was significantly greater than that for GM9503 cells in NFE6, GM637,
LFS 087 and LFS 041 but not in AG02804D and SKOV-3
cells. However, the HCR value in the normal NF strain was
also significantly greater than that of the GM9503 strain and
none of the p53-compromised cell lines had an HCR value
significantly different from the normal NF strain. This indicates
a significant difference in HCR among the normal strains, such
that none of the p53-compromised cell lines can be considered
significantly different compared to normal.
HCR of the MB þ VL-treated reporter gene is increased in
UVC-pre-treated compared to untreated cells
We also examined the HCR of the MB þ VL-treated reporter
gene in UVC-pre-treated cells in order to determine if prior
UVC treatment of cells can enhance the BER of an oxidatively
damaged reporter construct. Representative survival curves of
b-gal activity for MB þ VL-treated AdCA17 in UVC-pre-treated
compared to non-treated cells are shown for the normal fibroblast
strains in Figure 3 and for the p53-compromised cells in Figure 4.
It can be seen that UVC pre-treatment of all the normal
fibroblast strains resulted in a substantially enhanced HCR of
the MB þ VL-treated reporter suggesting that BER of MB þ
VL-induced DNA damage is inducible by UVC pre-treatment
in normal human fibroblasts. In contrast, the enhanced HCR in
UVC-pre-treated p53-compromised cells was generally less
than that obtained in the normal fibroblasts or absent. The
relative D37 value obtained in UVC-pre-treated cells compared
to that in non-treated cells was determined for each experiment
and the mean relative D37 values for several experiments are
shown in Table I, column 3. It can be seen that three of the four
normal cell strains (GM9503, GM969 and GM38A) show
a significant enhancement of HCR following low-dose UVC
pre-treatment to cells and three of the six p53-compromised
cell lines (NF-E6, LFS087 and SKOV-3) similarly showed a
significant enhancement following UVC treatment to cells. The
remaining three cell lines (GM637, AG02804D and LFS 041)
1
1
0.1
0.1
GM9503
GM38A
NF
0
30
60
GM9503
GM969
90
120
150
180
0
30
60
90
120
150
180
Visible Light Exposure to MB Treated Virus (s)
Fig. 1. HCR of b-gal activity for MB þ VL-treated Ad5HCMVlacZ virus in normal human fibroblasts. Each panel shows the results of a single representative
experiment that included the GM9503 normal human fibroblast strain. Each point is the average standard error of triplicate determinations.
77
S. N. Kassam and A. J. Rainbow
1
Relative β-Galactosidase Activity
1
GM9503
GM637
GM9503
AG02804D
SKOV-3
0.1
0.1
Relative β-Galactosidase Activity
0
30
60
90
120 150 180
0
1
1
0.1
0.1
30
60
90
120 150 180
GM9503)
0
LFS087
GM9503
LFS041
NF-E6
30
60
90
120
150
180
0
30
60
90
120 150 180
Visible Light Exposure to MB Treated Virus (s)
Fig. 2. HCR of b-gal activity for MB þ VL-treated Ad5HCMVlacZ virus in p53-compromised cells. Each panel shows the results of a single representative
experiment that included the GM9503 normal human fibroblast strain. Each point is the average standard error of triplicate determinations.
Table I. HCR capability for b-gal activity of MB þ VL-treated Ad5HCMVlacZ
virus in untreated and UVC-treated normal and p53-compromised cell lines
Cell line
Relative
D37 SE in
untreated cells
(number of
experiments)
Relative
D37 SE
UVC-treated/
no-UVC (number
of experiments)
GM9503
GM38A
GM969c
NF
NF E6
GM637F
AG02804D
LFS087
LFS041
SKOV-3
1.00
1.05
0.91
1.17
1.18
1.54
1.11
1.23
1.26
0.92
1.56
1.45
1.57
1.25
1.32
1.06
1.27
1.15
1.20
1.45
(18)
0.08
0.12
0.17
0.13
0.20
0.22
0.11
0.28
0.29
(5)
(4)
(4)a
(3)a
(4)a
(3)
(4)a
(3)a
(2)
0.09
0.15
0.13
0.12
0.08
0.13
0.22
0.04
0.27
0.05
(15)a
(4)a
(4)a
(6)
(5)a
(4)
(3)
(6)a
(3)
(2)a
Relative
D37 SE
in UVC-treated
cells (number of
experiments)
1.0 (15)
1.08 0.09
0.97 0.09
0.96 0.06
0.91 0.16
0.94 0.09
0.90 0.29
0.62 0.13
0.89 0.07
0.90 0.32
(4)
(4)
(4)
(3)
(3)
(2)
(6)b
(3)b
(2)
Column 2 shows the D37 standard deviation (SE) for each cell line relative to
the GM9503 line in untreated cells averaged over a number of independent
experiments. Column 3 shows the D37 SE in UVC-pre-treated relative to that
in untreated cells averaged over a number of independent experiments. Column
4 shows the D37 SE for each cell line relative to the GM9503 line in UVCpre-treated cells averaged over a number of independent experiments.
a
Significantly .1 by a two sample independent t-test (P , 0.05).
b
Significantly ,1 by a two sample independent t-test (P , 0.05).
78
also showed an increased HCR in pre-UVC-treated cells,
although the enhancement was not significant.
HCR of the MB þ VL-treated reporter gene in UVC-pretreated cell lines, relative to UVC-pre-treated GM9503 cells
UVC-induced DNA damage is known to activate p53 via posttranslational modifications and increase the transcription of
p53-dependent DNA repair genes (15,21,41). It was therefore
considered of interest to compare HCR of the MB þ VL-treated
reporter gene in normal fibroblasts with that in the p53compromised cells with UVC pre-treatment. To do this, we
determined the D37 value of each of each cell line for survival
of b-gal activity for MB þ VL-treated AdCA17 in UVC-pretreated cells. In order to account for the variation in D37 values
between individual experiments, we calculated the D37 value
for each of the various fibroblast strains and human cell lines
relative to the D37 value of the normal GM9503 strain obtained
in the same experiment. The D37 values relative to the GM9503
strain for each cell line were then averaged over several
experiments and are shown in Table I, column 4. It can be seen
that HCR of the MB þ VL-treated reporter gene was similar in
the four different UVC-pre-treated normal fibroblasts. In contrast
to the results for constitutive HCR in non-treated cells, HCR of
UV-inducible base excision repair
1
Relative β-Galactosidase Activity
1
0.1
0.1
GM38A UVC
GM38A No UVC
GM9503 UVC
GM9503 No UVC
Relative β-Galactosidase Activity
0
30
60
90
120
150
0
180
1
1
0.1
0.1
NF UVC
NF No UVC
0
30
60
30
60
90
120
150
180
120
150
180
GM969 UVC
GM969 No UVC
90
120
150
180
0
30
60
90
Visible Light Exposure to MB treated virus (s)
Fig. 3. HCR of b-gal activity for MB þ VL-treated Ad5HCMVlacZ virus in UVC-pre-treated (open symbols) and untreated (closed symbols) normal fibroblasts.
Cells were either irradiated with 15 J/m2 of UVC or mock irradiated, then infected at a MOI of 40 pfu per cell with untreated or MB þ VL-treated Ad5HCMVlacZ
and subsequently harvested 44–48 h after infection. Each panel shows the results of a single representative experiment. Each point is the average standard error of
triplicate determinations.
the MB þ VL-treated reporter gene in UVC-pre-treated cells
was similar or less in the p53-compromised cells compared to
that in the normal strains. In particular for the p53-compromised
cell lines, the LFS 041 and LFS 087 lines were significantly less
proficient in HCR compared to GM9503 and the LFS 087 line
showed a significantly reduced HCR compared to all the normal
strains tested. Although the other p53-compromised cells also
showed a reduced HCR compared to GM9503 cells, this
reduction was not significant.
Effect of low-dose UVC treatment on p53 expression levels in
normal and p53-compromised cells
To confirm that UVC pre-treatment of cells increased the p53
expression levels, Western blot analysis was performed in
untreated and UVC-treated cells. Representative Western blots
are shown in Figure 5. It can be seen that basal p53 levels in the
untreated human fibroblasts (GM9503, GM969 and NF) are
extremely low or undetectable and all show up-regulation of
p53 expression following UVC treatment. NF-E6, LFS 041 and
SKOV-3 cells also showed extremely low or undetectable p53
protein expression in untreated and UVC-pre-treated cells as
reported by others (39,42,43). High expression levels of p53
were detected in both untreated and UVC-pre-treated LFS 087
cells. High p53 expression levels have been reported previously for LFS087 cells (39) and this is consistent with the
accumulation of p53 in cells with mutant p53 (44). Both SV40transformed cell lines, GM637 and AG02804D, also showed
high p53 expression levels in both untreated and UVC-pretreated cells with no p53 up-regulation following UVC. This is
consistent with previous reports showing that abrogation of
p53 by SV40 Tag results in accumulation of p53 (45).
A comparison of the relative HCR values in UVC-pre-treated
compared to untreated cells (Table I, column 3) with the p53
expression levels shown in Figure 5 indicated that increased p53
expression in the UVC-pre-treated cells correlated with
enhanced HCR of the MB þ VL-treated reporter gene in the
UVC-pre-treated compared to untreated normal human fibroblast strains. This suggested some involvement of p53 in the
inducible BER of the MB þ VL-damaged reporter gene in
normal human fibroblasts. In contrast, p53 expression did not
79
Relative β-Galactosidase Activity
S. N. Kassam and A. J. Rainbow
1
1
1
0.1
0.1
0.1
LFS041 UVC
LFS041 No UVC
LFS087 UVC
LFS087 No UVC
Relative β-Galactosidase Activity
0
30
60
90
120
150
180
1
0
30
60
90
120
SKOV-3UVC
SKOV-3NoUVC
150
180
0
30
60
90
120 150 180
1
1
GM637 UVC
GM637 No UVC
AG02804D UVC
AG02804D No UVC
0.1
NF-E6 UVC
NF-E6 No UVC
0.1
0.1
0
30
60
90
120 150 180
0
30
60
90
120 150 180
0
30
60
90
120 150 180
Visible Light Exposure to MB treated virus (s)
Fig. 4. HCR of b-gal activity for MB þ VL-treated Ad5HCMVlacZ virus in UVC pre-treated (open symbols) and untreated (closed symbols) p53-compromised
cells. Cells were either treated with 15 J/m2 of UVC or mock treated then infected at an MOI of 40 pfu per cell with untreated or MB þ VL-treated Ad5HCMVlacZ
and subsequently harvested 44–48 h after infection. Each panel shows the results of a single representative experiment. Each point is the average standard error of
triplicate determinations.
correlate with enhanced HCR of the MB þ VL-treated reporter
gene in the UVC-pre-treated compared to untreated cells for the
p53-compromised cells. In particular, the SKOV-3, LFS 087
and NF-E6 showed no up-regulation of p53 expression following
UVC, and yet these cells show significant enhancement of HCR
following UVC pre-treatment (Figure 4, Table I, column 3).
Discussion
A role for p53 has been implicated in several aspects of the
BER pathway (46). Achanta et al. (17) have shown that p53
physically associates with the hOGG1 glycosylase and APE1
proteins that are involved in the sensing and excision steps of
BER. In addition, a functional interaction of p53 and the
3-methyladenine DNA glycosylase, the first enzyme acting in
the BER pathway, has also been reported (47). p53 has been
reported to regulate BER in G1 (48,49) and p53 was shown to
stimulate BER, even when added as a recombinant protein,
suggesting a direct role for p53 in BER (50). In an in vitro
assay using extracts from cells expressing p53 temperature
sensitive mutants that acquire wild-type activity at the
permissive temperature, Offer et al. (48) show that the mutant
80
protein not only did not induce BER activity but also rather
seemed to reduce the p53-dependent BER activity, suggesting
that this mutant-associated activity may indicate a negative
‘gain of function’. The p53 protein interacts with Pol-b (50)
and may also modulate the activity of Pol-b, as some p53
mutant cells have very low levels of this enzyme (18). It has
also been shown that oxidative stress can up-regulate Pol-b
levels, as well as those of p53, indicating a possible link
between the two (51). Although p53 appeared not to act as
a transcription factor for Pol b (18), this does not mean that the
transcription activation function of p53 is not involved in BER.
One role of p53 in NER is its role as a transcription factor
that facilitates the expression of proteins required for NER
(15,52–54). In NER, activated p53 acts as a transcription factor
for the XPC gene (53). There is evidence for the involvement
of the XPC protein in the recognition of oxidative DNA
damage (55) and more recent reports indicate that the XPC
protein is involved in the BER of oxidative DNA damage
(56,57). This suggests that the regulation of XPC transcriptional activity by p53 may play a role in BER.
HCR of the MB þ VL-treated reporter gene in untreated
cells did not correlate with wild-type p53 expression levels
UV-inducible base excision repair
Fig. 5. Western blots of p53 protein expression in normal GM9503, GM969
and NF fibroblasts as well as p53-compromised NF-E6, SKOV-3, LFS 041,
LFS 087, AG02804D and GM637 cell lines. Cells were either pre-treated with
25 J/m2 of UVC or untreated, and lysates were collected 24 h later. Blots were
probed with anti-p53 antibody, stripped and re-probed with anti-actin to
confirm equal loading. LFS 087, AG02804D, GM637 and all actin blots were
allowed to expose film from between 15 sec to 1 min. p53 blots for GM9503,
GM969, NF, NFE6, SKOV-3 and LFS 041 were allowed to expose film
overnight.
among the human cell lines tested, suggesting that factors other
than p53 expression levels can influence constitutive BER of
the reporter gene in untreated cells. Surprisingly, HCR was
actually greater in some p53-compromised cells compared to
three of the four normal human fibroblasts tested. In particular,
LFS087 and GM637 cells showed greater HCR levels
compared to the normal fibroblasts in untreated cells. One
explanation for this could be the high constitutive levels of p53
protein expression in these cells (Figure 5). It has been reported
that abrogation of p53 by the SV40 large T antigen or
expression of mutant p53 results in accumulation of p53
(44,45), which explains the high constitutive levels. In
addition, it has been shown that SV40-transformed cells still
have some functional p53 that is not bound by the large
T antigen (45), and even the p53 that is bound is capable of
performing some of its transcription factor role (58). p53 has
five functional protein domains [as reviewed in ref. (15)], and it
is therefore possible that a mutated p53 or even large T antigenbound p53 could still posses many of its functions. Therefore,
the accumulated p53 mutated, abrogated or otherwise, could
contribute to a greater constitutive proficiency of BER in
LFS041, LFS087, AG02804D and GM637 cells compared to
three of the four normal fibroblasts tested. Even the p53compromised SKOV-3, which express no p53 protein, did not
show a significant reduction in HCR compared to the normal
fibroblasts suggesting that factors other than p53 expression
contribute to BER in the reporter gene.
Using direct measurement of BER kinetics, Le et al. reported
that low priming doses of ionizing radiation result in a more
rapid removal of thymine glycols in A549 human lung
carcinoma cells when given 4 h before a challenging dose
(23). By examining DNA repair synthesis of plasmid-borne
apurinic sites in cell extracts, Offer et al. (24) have reported
that low doses of ionizing radiation or cisplatin result in
enhancement of BER. Using a rat model of temporary middle
cerebral artery occlusion, Lan et al. reported an inducible BER
activity in the cortex, but not the caudate region of the rat brain
following ischaemia-induced oxidative injury. The enhanced
BER activity was attributed to up-regulation of gene expression
and activation of selective BER enzymes, particularly DNA
Pol-b and OGG1 (25). In contrast, Bercht et al. reported that
the repair rate of oxidative lesions induced by the photosensitizer Ro19-8022 plus light in MEFs, MCF-7 breast cancer
cells and primary human fibroblasts was not increased if
a priming dose of either Ro19-8022 plus light or the alkylating
agent methylmethanesulphonate was applied 6 or 18 h prior to
the challenging dose (27).
We report here that UVC pre-treatment of the normal
fibroblast strains resulted in an increase in the expression of
p53 and a concomitant enhanced HCR of the MB þ VL-treated
reporter gene. GM9503, GM969 and GM38A show a significant enhancement of HCR (50%) following low-dose UVC
pre-treatment to cells and NF fibroblasts show an HCR
enhancement of 25%, which was not significant (Figures 3
and 4 and Table 1, column 3). The lower extent of enhancement
of the NF cells could be attributed to several factors. It has been
shown that the p53 response is attenuated in cells that are grown
to a high density (59) and in cells that are of high passage
number (60). As the NF cells were of considerably higher
passage number than the other normal fibroblast strains used, it
is possible that the UVC-induced p53 response is attenuated in
the NF fibroblasts. The correlation of enhancement of HCR with
up-regulation of wild-type p53 following UVC in all the normal
human fibroblasts is consistent with an involvement of p53 in
the enhancement of BER in normal human fibroblasts.
In contrast to the results for the normal fibroblasts, upregulation of HCR did not correlate with up-regulation of p53
expression for the p53-compromised cells following UVC pretreatment. In particular, the SKOV-3, LFS 087 and NF-E6
showed no up-regulation of p53 expression following UVC,
and yet these cells show significant enhancement of HCR
following UVC pre-treatment. This indicates that BER of the
MB þ VL-treated reporter gene can be induced by UVC pretreatment of cells through both p53-dependent and p53independent mechanisms. Offer et al. have also reported both
p53-dependent and p53-independent up-regulation of BER
following gamma rays or cisplatin (24). It has been suggested
that DNA damage up-regulates Pol-b as well as p53 expression
(51). It is therefore possible that the induced BER of the MB þ
VL-treated reporter gene in UVC-pre-treated cells with compromised p53 results, in part at least, from an increased expression
of Pol-b.
In contrast to the results of constitutive HCR in untreated
cells (Table 1, column 2), HCR values in UVC-pre-treated cells
were generally less in the p53-compromised cells compared to
81
S. N. Kassam and A. J. Rainbow
the normal fibroblasts (Table 1, column 4). In particular, HCR
values for the LFS 041 and LFS 087 cell lines expressing
mutant p53 were significantly less compared to that in
GM9503 fibroblasts for UVC-pre-treated cells, and the LFS
087 line showed a significantly reduced HCR compared to all
the normal strains tested. These results are consistent with
a negative gain of function for the effects of some p53
mutations on BER as suggested previously by Offer et al. (48).
The results of the present report indicate that BER of MB þ
VL-damaged DNA is inducible by UVC pre-treatment in human
cells and that the enhancement in BER results from both p53dependent and p53-independent mechanisms. The major
relevant UV components of sunlight are UVA and UVB.
UVB, like UVC, is directly absorbed by DNA generating CPD
and 6-4PP (9). In contrast, UVA does not directly excite DNA
but rather indirectly damages DNA by a photosensitized reaction
through the formation of ROS and results in the formation of
non-bulky oxidative DNA base modifications including 8-OxoG
(3,4) that are removed by BER. The results of the present work
suggest that exposure of skin cells to the UVB component of
sunlight may result in an up-regulation of BER that enhances the
removal of UVA-induced oxidative DNA damage.
Funding
National Cancer Institute of Canada with funds from the
Canadian Cancer Society (NCIC 016066).
Acknowledgements
Conflict of interest statement: None declared.
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Received on May 26, 2008; accepted on August 27, 2008
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