Mutagenesis Advance Access originally published online on October 3, 2008
Mutagenesis 2009 24(1):75-83; doi:10.1093/mutage/gen054
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UV-inducible base excision repair of oxidative damaged DNA in human cells
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 + 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 β-galactosidase (β-gal) reporter gene, to examine the role of the p53 tumor suppressor in constitutive and inducible BER of MB + VL-damaged DNA in human cells. Host cell reactivation (HCR) of β-gal activity for MB + 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 + 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 pre-treatment of the normal fibroblast strains resulted in an enhanced HCR of the MB + 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 pre-treatment. These results indicate that BER of MB + VL-damaged 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.
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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 β (Pol-β) 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 (GG-NER) 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-β, 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 (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 up-regulation 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 + VL is known to create predominantly 8-OxoG in the DNA of cell-free 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 β-galactosidase (β-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 + 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 + VL-induced DNA damage in human cells. We report here that BER of MB + 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.
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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 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
-minimal essential media (-MEM) supplemented with 10% foetal bovine serum and antimycotic–antibiotic 100 µg/ml penicillin, 100 µg/ml streptomycin and 250 ng/ml amphotericin B (Gibco BRL, Grand Island, NY). Media for the E6 transformants were supplemented with 250 µg/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 +72 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 + VL
Preparation of MB was as described previously (36). A total of 80 µl of Ad5HCMVLacZ virus was added to 3.6 ml of phosphate buffered saline (PBS) containing 20 µg/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 µl of irradiated virus was removed, diluted appropriately in unsupplemented -MEM and used to infect the cell monolayers.
HCR experiments
SV40-transformed cells, LFS cells and tumor cells were seeded at 3.8 x 104 cells per well and primary human fibroblasts were seeded at 1.9 x 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 µl of untreated or MB + VL-treated virus for 90 min at a multiplicity of infection of 20 or 40 plaque-forming units per cell, overlaid with 160 µl of complete -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 µl 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 β-gal as described previously (36). Incubation medium was aspirated from each well and 60 µl of chlorophenolred β-D-galactopyranoside (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 β-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 β-gal survival curves, the average background level of β-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 -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 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 µg 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.
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Constitutive HCR of the MB + 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 + VL-induced DNA damage, we examined HCR of an MB+VL-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 β-gal activity for MB + 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 β-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.
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HCR of the MB + VL-treated reporter gene is increased in UVC-pre-treated compared to untreated cells
We also examined the HCR of the MB + 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 β-gal activity for MB + 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 + VL-treated reporter suggesting that BER of MB + 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) also showed an increased HCR in pre-UVC-treated cells, although the enhancement was not significant.
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HCR of the MB + VL-treated reporter gene in UVC-pre-treated cell lines, relative to UVC-pre-treated GM9503 cells
UVC-induced DNA damage is known to activate p53 via post-translational 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 + VL-treated reporter gene in normal fibroblasts with that in the p53-compromised cells with UVC pre-treatment. To do this, we determined the D37 value of each of each cell line for survival of β-gal activity for MB + VL-treated AdCA17 in UVC-pre-treated 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 + 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 the MB + 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 SV40-transformed cell lines, GM637 and AG02804D, also showed high p53 expression levels in both untreated and UVC-pre-treated 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).
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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 + 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 + VL-damaged reporter gene in normal human fibroblasts. In contrast, p53 expression did not correlate with enhanced HCR of the MB + 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).
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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 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-β (50) and may also modulate the activity of Pol-β, 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-β 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 β (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 + VL-treated reporter gene in untreated cells did not correlate with wild-type p53 expression levels 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 antigen-bound 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 p53-compromised 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-β 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 + 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, up-regulation of HCR did not correlate with up-regulation of p53 expression for the p53-compromised cells following UVC pre-treatment. 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 + VL-treated reporter gene can be induced by UVC pre-treatment of cells through both p53-dependent and p53-independent 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-β as well as p53 expression (51). It is therefore possible that the induced BER of the MB + VL-treated reporter gene in UVC-pre-treated cells with compromised p53 results, in part at least, from an increased expression of Pol-β.
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 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 + VL-damaged DNA is inducible by UVC pre-treatment in human cells and that the enhancement in BER results from both p53-dependent 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.
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National Cancer Institute of Canada with funds from the Canadian Cancer Society (NCIC 016066).
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Conflict of interest statement: None declared.
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* To whom correspondence should be addressed. Tel: +1 905 525 9140; Fax: +1 905 522 6066; Email: rainbow{at}mcmaster.ca
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Received on May 26, 2008; accepted on August 27, 2008.
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