Mutagenesis Advance Access published online on November 13, 2007
Mutagenesis, doi:10.1093/mutage/gem041
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The role of p53 in DNA damage-mediated cytotoxicity overrides its ability to regulate nucleotide excision repair in human fibroblasts
Department of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK
The p53 tumour suppressor protein plays a pivotal role in the response of mammalian cells to DNA damage. In addition to its regulatory role in cell cycle progression, p53 regulates apoptosis and can therefore influence cellular survival in response to DNA damage. More recent work has revealed that p53 is also involved in the nucleotide excision repair (NER) of structurally diverse types of DNA damage. The relative influence of p53 on NER and cellular sensitivity to DNA damage was investigated in this study using cells that differ in p53 status. Two cell models were selected: 041 TR fibroblasts in which the expression of p53 is regulated by a tetracycline-inducible promoter, and WI38 primary lung fibroblasts together with their isogenic derivative VA13, in which p53 is abrogated post-translationally by SV40 transformation. Cells were exposed to the clinically and environmentally relevant DNA-damaging agents cisplatin (0–5 µM, 2 h), (±)-anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (0–0.5 µM, 30 min) and UV-C (0–5 J/m2), each of which induce structurally distinct types of DNA damage known to be subject to p53-dependent NER. Sensitivity of the p53-proficient and p53-deficient cells to this DNA damage was then compared at each dose of DNA-damaging agent using the clonogenic survival assay and the colorimetric MTT assay. p53-proficient cells were more sensitive than p53-deficient cells to cisplatin, (±)-anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide and UV-C; these differences in cellular sensitivity were more apparent in the 041 TR cells (up to 3.6-, 5.8- and 1.9-fold, respectively) than the WI38/VA13 cells (up to 2.3-, 1.4- and 1.4-fold, respectively). Thus, despite the well-documented persistence of DNA damage in p53-deficient fibroblasts due to impaired NER, loss of p53 results in reduced DNA damage-mediated cytotoxicity.
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Introduction |
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The biological response to DNA damage is a critical element in the maintenance of genomic integrity [reviewed in (1
)]. A number of defences have evolved to minimize the deleterious effects of DNA damage, including arrest of the cell cycle, apoptosis and complex excision repair mechanisms that remove DNA lesions. The p53 tumour suppressor protein is pivotal in regulating the biological response to DNA damage. It is well established that p53 regulates progression through the cell cycle (2) and apoptotic mechanisms (3). Both ensure that a damaged DNA template is not replicated, either by preventing entry into the DNA synthesis phase of the cell cycle and allowing time for DNA repair to take place or by directing extensively damaged cells to the programmed cell death pathway, thus ensuring that cells with damaged genomes do not divide.
More recently, it has been found that p53 also regulates DNA repair processes. p53 was first shown to be required for nucleotide excision repair (NER) in a study that revealed that p53 enhanced the removal from the genome of UV-induced cyclobutane pyrimidine dimers (CPDs) in human cells (3). Subsequent studies, however, demonstrated that UV-induced 6-4 photoproducts are not subject to p53-dependent NER since their removal is approximately equal in p53-proficient and p53-deficient cells (4–6). Adducts formed by diol epoxide metabolites of the potent chemical carcinogens benzo(a)pyrene and benzo(g)chrysene have been found to be removed in a p53-dependent manner, although this appears to be particularly evident when those adducts are present at low, biologically significant levels (7,8). p53 has also been shown to regulate base excision repair (9,10), but is not required for transcription-coupled NER of CPDs, i.e. the ability of cells to remove these lesions more rapidly from transcribed strands of expressed genes (4–6). Thus, the extent to which p53 is involved in DNA repair is highly dependent upon a combination of factors, including the mechanism required to repair the DNA damage, the structural nature of the DNA damage, the quantity of DNA damage and its location within the human genome.
Given the significant proportion of human tumours that exhibit mutations in the p53 gene, it is of interest to establish how the loss of p53 affects the cellular responses to DNA damage. In the case of environmentally induced DNA damage such as UV photoproducts and carcinogen–DNA adducts, loss of p53 is deleterious since it affects the removal of DNA damage and the ability to undergo apoptosis, thus increasing the chances of carcinogenesis. It is, however, of particular interest from a therapeutic point of view because many chemotherapeutic agents induce toxic DNA damage as part of their mode of action, and loss of p53 would disrupt the cellular response to such DNA damage and possibly influence therapeutic outcome. Alterations in DNA repair efficiency within tumour cells have been shown to affect cytotoxicity of chemotherapeutic drugs (11,12); therefore, if cytotoxic DNA damage induced by a chemotherapeutic agent is repaired by p53-dependent NER, loss of p53 would be predicted to increase cellular sensitivity. Evidence does suggest that DNA damage induced by the platinum-based chemotherapeutic drug cisplatin is repaired in a p53-dependent manner (13,14); this would indicate that cisplatin-induced DNA damage can persist in tumours in which p53 is absent or mutated and thus has more opportunity to exert cytotoxic effects. However, p53 also regulates cellular toxicity through apoptosis, and while toxic DNA damage may persist in p53-deficient cells due to impaired repair, the loss of p53-dependent apoptosis is also likely to reduce the extent to which this persisting DNA damage is able to initiate cell death.
There is dispute in the literature as to the effect of p53 on the ultimate fate of the cell. Some studies have found that p53 increases the sensitivity of cells to DNA-damaging agents and/or results in increased apoptosis (14–17). However, other studies have found that the loss of p53 and the resulting persistence of certain types of DNA damage increase cellular sensitivity (18–20), possibly due to a reduction in DNA repair capacity. In light of the increased knowledge concerning the role of p53 in NER of certain types of DNA damage, the aim of this study was to ascertain the relative influence of p53 on NER and cytotoxicity. The work described in this paper evaluates the effect of p53 on the cytotoxicity of chemically and physically induced DNA damage from environmental and clinical origins: cisplatin, (±)-anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) and UV-C, each of which induces structurally distinct types of DNA damage that are repaired in a p53-dependent manner. The data from these experiments are compared with existing knowledge of p53-dependent NER to determine the relative effect of loss of p53 function on NER and cytotoxicity.
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Materials
041 TR cells, originally obtained from Dr G. Stark (Cleveland Clinic Foundation, Cleveland, OH, USA), were derived from spontaneously immortalized Li-Fraumeni syndrome skin fibroblasts and stably transfected with a tetracycline-regulated system for the expression of wild-type p53 (21
). WI38 primary human Caucasian foetal lung fibroblasts and their isogenic SV40-transformed derivative VA13 (in which p53 is abrogated post-translationally) were obtained from the European Collection of Animal Cell Cultures (ECACC, 90020107 and 85062512, respectively). Antibodies used in western blotting procedures were from DakoCytomation (Glostrup, Denmark), Santa Cruz Biotechnologies (Santa Cruz, CA, USA) or Sigma Chemical Co. (Poole, Dorset, UK), as indicated below. Tissue culture additives were obtained from Invitrogen/Gibco (Paisley, UK). A BPDE was obtained from the NCRI Chemical Carcinogen Reference Standards Repository (Kansas City, MO, USA). Cisplatin and all other reagents were from Sigma Chemical Co.
Cell culture
041 TR cells were grown as monolayers in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (FBS) and 2 mM L-glutamine in the continuous presence of G418 (600 µg/ml) and hygromycin (50 µg/ml) to maintain selection pressure for transfected cells. Cells were grown in the continuous presence of tetracycline (2 µg/ml) to suppress p53 expression. When p53 expression was required, tetracycline was withdrawn from growth medium 24 h prior to experiments. WI38 and VA13 cells were grown as monolayers in DMEM, supplemented with 10% FBS, 2 mM L-glutamine and 5% non-essential amino acids. All cell cultures were incubated at 37°C in a humidified atmosphere of 5% CO2 and were diluted every 2–3 days to maintain growth and viability. WI38 were grown for no more than 30 passages, as recommended by ECACC.
Treatment of cells with DNA-damaging agents
Exponentially growing cells were incubated, at a density required for subsequent processing, with cisplatin (0–25 µM) in serum-free DMEM for 2 h in a humidified atmosphere of 5% CO2 at 37°C. Cisplatin was diluted from a 2 mM stock in 0.9% saline solution, while BPDE was diluted from a 5 mM stock in anhydrous tetrahydrofuran. Following incubation for 2 h with cisplatin or 30 min with BPDE, media containing DNA-damaging agents was removed, cells were washed twice in phosphate-buffered saline (PBS) and incubated further in serum-containing DMEM. For treatment with UV-C radiation, exponentially growing cells were washed twice with PBS and then exposed to UV radiation (0–5 J/m2) at 254 nm using an ultraviolet lamp (Model UVLS-28, UltraViolet Products, Upland, CA, USA), before being washed once with PBS and incubated further in serum-containing DMEM. After treatment with DNA-damaging agents, cells were subjected to the clonogenic survival assay or the 1-(4,5-demethylthiazoyl-2-yl)-3,5-diphenylformazan (MTT) assay to monitor cellular toxicity by two independent biological endpoints, as described below.
Western blotting
Total cellular protein was isolated by lysing cells in 100 µl of a stock buffer consisting of Triton X-100 (1%), Tris–HCl (50 mM, pH 7.4), sodium chloride (150 mM), ethylenediaminetetraacetic acid (5 mM), together with Protease Inhibitor Cocktail (Sigma, Poole, UK) used according to the manufacturer's instructions. Whole-cell extracts were prepared, protein concentrations were determined by the Bradford method and equal amounts of protein (10 µg) were resolved by NuPAGE 4–12% Bis–Tris pre-cast polyacrylamide gels (Invitrogen, Paisley, UK) and electroblotted to a nitrocellulose membrane. Membranes were then subjected to immunoblotting with mouse monoclonal antibodies to human p53 (DO-7, DakoCytomation), rabbit polyclonal antibodies to human p21 (C19, Santa Cruz Biotechnology) and mouse monoclonal antibodies to actin (AC-15, Sigma) diluted 1:1000, 1:1000 and 1:5000, respectively, in 1% non-fat milk in PBS. This was followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies (goat anti-mouse from DakoCytomation, goat anti-rabbit from Santa Cruz Biotechnology, both diluted 1:5000 in 1% non-fat milk and PBS). Proteins were detected by enhanced chemiluminescence (Supersignal chemiluminescence detection, Pierce, Rockford, IL) and transparency film (Hyperfilm ECL, Amersham, Biosciences, Little Chalfont, UK).
Clonogenic survival assay
Cells were seeded into 10 cm diameter cell culture plates at a density of 104 cells per dish and left to settle for 24 h before treatment with either DNA-damaging agents or appropriate solvent vehicles, as described above. Cells were then allowed to recover in normal growth medium and left for between 7 and 14 days to allow colony formation. Cells were washed with PBS and fixed with 70% (v/v) methanol prior to staining with 5% methylene blue. Established colonies were counted by image analysis. Images were photographed using a Zeiss MR GrabV.10 camera, AxiCam type MR TV lens (25 mm) with associated light panels (Medalight®, LP-100 cold cathode lamp) and KsRun 3.0 software on an imaging computer system (Image Associates Ltd, Bicester, UK). Once images had been captured, ImageJ 1.29 software was used to count colonies. Survival was calculated as a percentage of the number of colonies observed in control experiments.
MTT assay
The MTT assay is a colorimetric assay and a rapid measure of short-term cell viability. MTT is reduced by mitochondrial enzymes in viable cells to form insoluble purple formazan crystals; this conversion is directly proportional to the number of viable cells and can be monitored easily by spectrophotometry. Cells were seeded into 24-well plates at a density of 105 cells per well and left to settle for 24 h. Cells were then treated with DNA-damaging agents as described above before being washed twice in PBS and incubated in growth media for 18 h. MTT, diluted from a 5 mg/ml stock in PBS, was added to each well to give a final concentration of 0.5 mg/ml and cells were incubated for a further 5 h. After this, media was removed, 500 µl DMSO was added to the cells and 100 µl of the resulting mixture was transferred to a 96-well plate to be subjected to analysis on a microplate reader (Multiskan Ascent, Thermo Electron Corporation, Vantaa, Finland) at 595 nm.
Statistical analysis
Cellular sensitivity of each cell type was expressed as a percentage of the survival of control cells. The difference between the sensitivity of p53-proficient and p53-deficient cells at each dose of DNA-damaging agent was expressed as a ‘toxicity ratio’, which was calculated by dividing the mean percentage survival of p53-deficient cells by the mean percentage survival of p53-proficient cells. The Student's t-test was then used to evaluate the statistical significance of the difference between the sensitivity of p53-proficient and p53-deficient cells at each dose of DNA-damaging agent, based on three independent experiments each performed in triplicate.
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Results |
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Characterization of p53 status in 041 TR, WI38 and VA13 cells
The levels of p53 protein, and its downstream transactivation target p21, were assessed in whole extracts of 041 TR, WI38 and VA13 cells by western blotting over a 24-h period following induction of DNA damage (Figure 1, summarized in Table I). This was to establish whether the 041 TR, WI38 and VA13 cells were suitable models to assess the role of p53 in the cellular response to different DNA-damaging agents. In the absence of tetracycline, 041 TR cell extracts showed increasing p53 expression up to 24 h after treatment with 20 µM cisplatin, with p21 levels showing a similar expression profile (absence of p21 was noted at 2 h at all exposures and is likely to be the result of poor transfer to the nitrocellulose membrane in this region of the gel). However, treatment of 041 TR cells, grown in the continuous presence of tetracycline, with 20 µM cisplatin showed that no detectable p53 or p21 was expressed at any time up to 24 h after treatment (Figure 1). This confirmed that expression of transcriptionally active p53 in 041 TR cells was efficiently suppressed by tetracycline. p53-proficient WI38 cells treated with 20 µM cisplatin showed induction of p53 to a maximum at 24 h after treatment (Figure 1); a similar expression profile for p21 demonstrated that this p53 was transcriptionally active. In the isogenic SV40-transformed VA13 cells, in which p53 function is abrogated, treatment with 20 µM cisplatin resulted in constitutively high levels of p53 up to 24 h after treatment (Figure 1). This is expected since the SV40 large T-antigen binds to p53, inhibiting p53-dependent transcription by forming a complex in which p53 is transcriptionally inactive (22
–24) and therefore inhibiting its normal function, including the auto-regulatory mechanism with mdm2. Indeed, the reduced function of the p53 protein in VA13 cells was demonstrated by the very low levels of p21 expression. In keeping with previously published results (4,7), similar profiles of expression were observed when cells were treated with 0.5 µM BPDE and 5 J/m2 UV-C (data not shown). These experiments confirmed that 041 TR, WI38 and VA13 cells were suitable models with which to investigate the role of p53 in DNA damage-dependent cytotoxicity.
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Cellular sensitivity to cisplatin
Exponentially growing cells were exposed to increasing concentrations of cisplatin as described in the Materials and Methods. Toxicity ratios were established by comparing the toxicity of equimolar concentrations of cisplatin in p53-proficient and p53-deficient backgrounds; this gave a useful measure of the relative toxicity of each DNA-damaging agent in the presence or absence of p53 (Table II). In addition, the Student's t-test was used at each dose of DNA-damaging agent to establish whether the difference between survival in p53-proficient and p53-deficient cells was statistically significant (P < 0.05). At all concentrations of cisplatin, p53-proficient 041 TR cells (i.e. cultured in the absence of tetracycline) were more sensitive to the cytotoxic effects of cisplatin when compared to p53-deficient 041 TR cells (i.e. cultured in the presence of tetracycline) (Figure 2A). Calculation of toxicity ratios revealed that p53-deficient 041 TR cells were up to 3.6-fold more resistant to the cytotoxic effects of cisplatin than isogenic p53-proficient cells (P < 0.01, Table II). These results were mirrored in experiments using WI38 and VA13 cells, which revealed that p53-proficient WI38 cells were more sensitive than their SV40-transformed, p53-deficient derivative VA13. Toxicity ratios revealed that WI38 cells were up to 2.3-fold more sensitive to cisplatin than VA13 cells; less than the ratio between p53-proficient and p53-deficient 041 TR cells but still statistically significant at all cisplatin concentrations (P < 0.01, Table II). The MTT assay revealed a similar relationship between p53 status and sensitivity to cisplatin. In keeping with data obtained from clonogenic analysis, the MTT assay also indicated that p53-proficient 041 TR cells were more sensitive to the cytotoxic effects of cisplatin when compared with p53-deficient 041 TR cells (Figure 2B). Enhanced toxicity was also observed in the p53-proficient WI38 cell line compared with the p53-deficient VA13 cells, albeit to a lesser extent than the 041 TR cell line. Toxicity ratios measured by the MTT assay were observed to be less than for comparable clonogenic assay experiments, but revealed that the 1.6-fold difference in toxicity p53-proficient and p53-deficient 041 TR was greater than the 1.2-fold toxicity ratio for WI38/VA13 cells (Table II). Indeed, the differences between survival in p53-proficient and -deficient cells were only statistically significant at 10–25 µM cisplatin (P < 0.05) in 041 TR cells and at 5 µM for WI38/VA13 (P < 0.05); a reflection of the comparatively small toxicity ratio and the greater calculated experimental error observed in the MTT assay. Furthermore, cellular survival was noticeably higher as monitored by the MTT assay when compared with data obtained using the clonogenic survival assay (Figure 2B).
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Cellular sensitivity to BPDE
p53-deficient 041 TR cells were more resistant to the cytotoxic effects of BPDE than isogenic p53-proficient cells (Figure 3A), with a toxicity ratio of up to 5.8 and a high degree of statistical significance (P < 0.01 at all concentrations, Table II). Similar results were obtained in WI38/VA13 cells, with p53-proficient WI38 cells more sensitive to BPDE than their p53-abrogated derivative at all concentrations assayed (Figure 3B). In keeping with the experiments undertaken with cisplatin, the difference between toxicity in p53-proficient WI38 cells and p53-deficient VA13 cells was smaller than was observed with the 041 TR cells; toxicity ratios increased to a maximum of 1.4 and statistical significance was observed at higher concentrations (P < 0.01, Table II).
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The MTT assay revealed that 041 TR p53-deficient cells were more resistant to the cytotoxic effects of BPDE when compared to 041 TR p53-proficient cells (Figure 3B, Table II), with toxicity ratios increasing with BPDE concentration up to a maximum of 1.4 (Table II). Differences between survival in p53-proficient and -deficient cells were statistically significant at all concentrations (P < 0.05, Table II). Similarly, p53-proficient WI38 cells were more sensitive than p53-deficient derivatives VA13, with comparable toxicity ratios ranging from 1.2 to 1.4 (Table II); once again, these differences in sensitivity were significant at all concentrations (P < 0.05, Table II). In keeping with similar experiments with cisplatin, overall toxicity as indicated by the MTT assay was less than that observed in clonogenic assay experiments undertaken with identical conditions (Figure 3), suggesting that the clonogenic assay is a more stringent measure of the cytotoxicity of DNA-damaging agents.
Cellular sensitivity to UV-C radiation
Both p53-proficient and p53-deficient 041 TR cells showed a large decrease in survival in the clonogenic survival assay at UV doses above 1 J/m2 (Figure 4A). 041 TR cells grown in the absence of tetracycline were between 1.0- and 1.9-fold more sensitive to the cytotoxic effects of UV-C radiation (Table II), demonstrating that p53 sensitizes 041 TR cells to the cytotoxic effects of UV-C, albeit to a lesser extent than was observed with cisplatin or BPDE and with less statistical significance across the range of doses (P < 0.001 for 1 and 3 J/m2 only, Table II). This was supported by similar results in the WI38/VA13 pair, with comparable toxicity ratios (1.0–1.4) and a similar degree of statistical significance (P < 0.001 at 3 J/m2 and P < 0.05 at 0.05 J/m2, Table II). The MTT assay revealed that 041 TR p53-proficient cells were only slightly more sensitive to the cytotoxic effects of UV when compared to 041 TR p53-deficient cells (toxicity ratios 1.0–1.3, Figure 4B), and even less difference was observed between the UV sensitivities of the WI38 and VA13 cells (toxicity ratio 1.0–1.2, Table II, Figure 4B). In both cases, statistically significant differences were observed at higher doses (P < 0.05 at 3 J/m2 and above, Table II). In keeping with previous experiments, the overall cytotoxicity measured by the MTT assay was significantly less than observed using the clonogenic survival assay.
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Discussion |
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The results from this study indicate that the presence or absence of functional p53 in human cells has a profound effect on the cytotoxicity of a range of DNA-damaging agents. This has been established for three structurally distinct types of DNA damage in two independent cellular backgrounds. They demonstrate that the presence of functional p53 increases cellular sensitivity to cisplatin, BPDE and UV-C radiation, in two matched pairs of cell lines that differ in p53 status. Both the clonogenic survival assay and the MTT assay demonstrated that p53-deficient cells treated with cisplatin, BPDE and UV were more resistant to the cytotoxic effects of these DNA-damaging agents than p53-proficient cells.
p53 is a pivotal protein in eliciting cellular toxicity in response to DNA damage, being involved in both the intrinsic and extrinsic mechanisms of apoptosis. Several studies have highlighted the importance of p53 in sensitizing a range of human cell types to DNA-damaging agents. For example, tetracycline-regulated p53 expressed in human osteosarcoma cells increased the sensitivity of these cells to cisplatin (14), and human glioblastoma cells expressing wild-type p53 were more sensitive to cisplatin than those expressing mutant p53 (16). Another study indicated that mutation in the C-terminal region of p53 decreased sensitivity of head and neck squamous cell carcinoma cell lines to cisplatin (17). These studies of cellular sensitivity are supported by large-scale gene expression analysis; testicular germ cell tumours treated with cisplatin led predominantly to the increase of p53-regulated pathways involved in activating cell death (25), and in silico research predicts that functional p53 correlates with cisplatin sensitivity (26). p53 has also been shown to sensitize cells to other DNA-damaging agents, including UV radiation (3) and 4-nitroquinoline oxide (27). However, others have found that p53-deficient and p53-mutated cells were more sensitive to the cytotoxic effects of DNA-damaging agents. Mouse testicular teratocarcinoma cells with mutant p53 were observed to be more sensitive to cisplatin than those retaining wild-type activity (28). Similarly, loss or abrogation of p53 function was observed to increase apoptosis and/or decrease survival of human cells in response to cisplatin (29), BPDE (18) and UV (19,29). In light of these conflicting theories and an increased understanding of the role of p53 in NER, the aim of this study was to undertake a systematic investigation of how the loss of p53 affects the balance between altering cellular sensitivity and the ability to repair DNA damage by NER.
Experiments were performed in two isogenic matched pairs of human cell lines. The 041 TR cells, in particular, have been used extensively to demonstrate p53-dependent NER of UV-induced DNA damage and carcinogen–DNA adducts (4,7,8). Similar results were obtained in both matched pairs of cell lines, with both demonstrating that the presence of p53 increased cellular sensitivity to all three DNA-damaging agents used. However, the difference in sensitivity between p53-proficient and p53-deficient cells was generally more pronounced in the 041 TR cell system compared with WI38/VA13, as demonstrated in Figures 2–4. The results from the Student's t-test (Table II) also indicate a greater and more frequent degree of statistical significance between cellular sensitivity of p53-proficient and p53-deficient cells in the 041 TR system (P < 0.05 in 21 of 28 possible incidences) than in WI38/VA13 (17 of 28 incidences) and is likely to be due to the different mechanisms by which the activity of p53 is experimentally controlled in these cells. Similarly, two independent techniques were used for the analysis of cellular sensitivity in this study. Given that the aim of this study was to investigate the ultimate effect on the cell of the presence of absence of p53, monitoring cell death was more appropriate than monitoring apoptosis, as several studies have revealed that apoptosis in response to DNA damage does not correlate with ultimate cell killing (28,30–32). The results indicate that the clonogenic assay is a more sensitive and stringent measure of cellular survival than the MTT assay; the clonogenic assay (P < 0.05 in 20 of 28 possible incidences) also generated more statistically significant results than the MTT assay (18 of 28 incidences) according to the Student's t-test, a finding which is consistent with other studies in our laboratory with a range of DNA-damaging agents. Brown and Wouters (33) concur, in a review of the literature, that the clonogenic assay is likely to be the most reliable indicator of cell death in response to genotoxic insult. They also note that short-term toxicity assays such as the MTT assay and detection of apoptotic markers might be less suitable for measuring differences in toxicity between p53 wild-type and p53-mutant cell lines because these assays do not allow enough time for an accurate assessment of total cell death, focusing instead on very early induction of cell death by apoptosis (33).
The DNA-damaging agents used in this study, which span environmental and clinical significance, were selected for investigation because the damage they induce is known to be subject to p53-dependent NER. p53 is required for the repair of the major BPDE–dG adduct, which accounts for 
90% of the DNA damage induced by benzo(a)pyrene (7,34); the requirement for p53 was identified as being particularly important at low levels of DNA damage (7). UV-C radiation induces CPDs and 6-4 photoproducts; the former, accounting for 60% of DNA damage induced by UV-C, requires p53 for efficient NER, while the 6-4 photoproducts that account for the majority of the remaining DNA damage do not (5). Studies indicate that p53 increases the efficiency of the repair of cisplatin-induced DNA damage (13,14); furthermore, we have recently found, using 32P-post-labelling analysis, that p53 is required for the NER of cisplatin-induced intrastrand cross-links, the most clinically relevant lesions formed by this chemotherapeutic agent which comprise 90% of total DNA damage (S. Bhana, A. Hewer, D. H. Phillips, D. R. Lloyd, unpublished data). While these previous studies have shown that p53 is necessary for the removal of DNA damage induced by these agents and that the DNA damage persists in p53-deficient cells, the results from the present study demonstrate that it is the p53-proficient cells that are more sensitive to the cytotoxic effects of the DNA-damaging agents. Thus, the results demonstrate that the role of p53 in cytotoxicity is more consequential to the ultimate fate of the fibroblasts than its role in NER.
While these results are consistent with those described in other studies using cells from a variety of sources, it must be acknowledged that the p53 response may vary between cells that originate from different tissues and species and which may exhibit different p53 mutations. Furthermore, the extent to which p53-independent cellular toxicity occurs in different cell types, and in response to different DNA-damaging agents, may collectively account for inconsistencies in the literature. Several studies have indicated that DNA damage can elicit apoptosis or cytotoxicity via p53-independent mechanisms (18,20,35). For instance, in observing increased sensitivity of p53-deficient fibroblasts to UV, Wani et al. (18) proposed that these cells undergo apoptosis through a p53-independent mechanism triggered by the persisting DNA damage that eludes repair. In a related study, El-Mahdy et al. (19) found that virally mediated degradation of p53 in mammary epithelial cells made them more sensitive to UV-induced DNA damage as measured by clonogenic and MTT analysis, which was attributed to unrepaired DNA damage in the non-transcribed strand of expressed genes stimulating p53-independent cytotoxic mechanisms; McKay et al. (29) further support the notion that transcription-blocking DNA damage can stimulate p53-independent apoptosis. p53-independent apoptosis in a testicular germ cell tumour cell line was found in another study to be activated by cisplatin through the mitogen-activated protein kinase—extracellular signal-regulated kinase-signalling pathway (36). The p53-related proteins p63 and p73 have also been implicated in DNA damage-dependent apoptosis (37), mediated by diverse pathways that are not necessarily dependent upon p53 (38,39). In the presence of robust and sensitive p53-independent mechanisms of cytotoxicity, unrepaired DNA damage may well promote cytotoxic pathways in p53-deficient cells under certain experimental conditions. The extent to which these p53-independent mechanisms take place is likely to vary among different cell types and the DNA-damaging agent. While we found p53-proficient cells to be consistently more sensitive than p53-deficient cells, the extent of cell death in p53-deficient cells treated with UV-C and cisplatin suggested that there was a significant degree of p53-independent cell death. Indeed, the difference in cellular sensitivity to UV-C radiation between p53-proficient cells and p53-deficient cells was relatively small in comparison to similar experiments with other DNA-damaging agents; this may reflect a high degree of p53-independent cell death in response to UV-induced DNA damage, and the fact that CPDs, which account for only 60% UV-C-induced DNA damage, are the only UV-C-induced DNA lesions subject to p53-dependent NER.
In summary, the results presented here demonstrate that the presence of functional p53 renders human fibroblasts more sensitive to the cytotoxic effects of cisplatin, BPDE and UV, even though the loss of p53 has been shown previously to lead to persisting DNA damage. Given the 50% frequency rate of p53 mutations in human tumours, these results are a particularly important consideration in terms of cisplatin-based cancer chemotherapies; the data suggest that p53-proficient tumours would be more sensitive to the cytotoxic effects of cisplatin, while tumours in which p53 function has been lost would be more resistant. The fact that very few testicular germ cell tumours exhibit mutations in p53 (40) may explain why cisplatin is so effective in the treatment of testicular cancer but much less effective in the treatment of other solid tumours; with p53 retained, testicular tumours will remain exquisitely sensitive. The nature and location of the p53 mutation may, however, have a more profound influence on some mechanisms than others which may in turn affect therapy; studies to investigate the impact of such mutations on the cellular response to DNA damage are currently in progress.
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Biotechnology and Biological Sciences Research Council studentship to S.B.; EB Hutchinson Trust.
Conflict of interest statement: None declared.
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* To whom correspondence should be addressed. Tel: 01227 827357; Fax: 01227 763912; Email: D.Lloyd{at}kent.ac.uk
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Received on April 26, 2007; revised on August 20, 2007; accepted on September 19, 2007.
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