Mutagenesis Advance Access originally published online on June 13, 2007
Mutagenesis 2007 22(5):329-334; doi:10.1093/mutage/gem021
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Wild-type p53 reduces radiation hypermutability in p53-mutated human lymphoblast cells
Department of Environmental and Radiological Health Sciences 1Cell and Molecular Biology Graduate Program, Colorado State University, Fort Collins, CO 80523, USA
Many studies have shown that an alteration of p53 affects various cellular responses to DNA damage after treatment with ionizing radiation. The human lymphoblast cell WTK1, which contains a mutant p53 (ile237), is 10-fold hypermutable at the thymidine kinase (tk) locus compared with TK6 cells, which are from the same donor but contain wild-type p53. These results implied that the specific p53 mutation found in WTK1 may actively contribute to mutagenesis in a gain of function manner. To further investigate this, the present experiments involved transfecting WTK1 cells with a wild-type p53 vector; this restored p53 activity in WTK1 cells, as evidenced by radiation-induced expression of p21. We compared radiosensitivity, as measured both by clonogenic survival and the induction of apoptosis, as well as mutant fractions (MFs) at the tk locus. WTK1 cells expressing wild-type p53 were more sensitive to 
-ray-induced toxicity as measured by either clonogenic survival or apoptosis. The mutation assays revealed that both the spontaneous and -ray-induced MFs were significantly decreased in WTK1 cells expressing wild-type p53; the MFs were similar to those observed in p53-null NH32 cells, also derived from the same donor. These results indicate that wild-type p53 can reduce the apparent gain-of-function hypermutable effects of a particular p53 gene mutation and thereby help maintain genomic stability.
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Introduction |
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p53 is the most commonly mutated gene in human cancer (1
–3). Perhaps one reason for this is that mutations of p53 lead to genomic instability (4,5), and this allows the cancer cell to evolve into ever more malignant forms. How do mutations of p53 produce genomic instability? Despite years of intensive research, we believe that this is still an open question.
It is well known that p53 is involved in the cellular response to DNA damage and can activate cell cycle checkpoints and apoptotic pathways. Problems with checkpoint control can easily lead to instability, as replication and/or mitosis proceeding before DNA repair is completed can have serious consequences (4). A second possibility stems from evidence showing that there is reduced apoptotic death in mutant p53 (mp53) human cells after ionizing radiation (IR); this has led to the suggestion that lack of apoptosis may contribute to the accumulation of autosomal gene mutations (6,7). Finally, there is evidence that p53 plays a direct role in DNA repair pathways (8–20); deficient or altered DNA repair is a third possible explanation for hypermutability in p53 mutant cells.
The human B lymphoblast cell lines used in our work were derived from a single line, WIL2 (21). They all have been rendered heterozygous for the autosomal thymidine kinase (tk) locus (22–24). We and others have shown that WTK1 (25,26) and its progenitor, WI-L2-NS (27), over-express a mutant form of p53 (methionine to isoleucine substitution at codon 237), and no wild-type p53 protein, while TK6 is wild-type for p53. We also generated NH32, a p53-null line, directly from TK6 (23).
These lines respond quite differently to IR. TK6 and NH32 are about equally sensitive to radiation-induced cell killing, but WTK1 are more resistant. Interestingly, radiation induces apoptosis in all three lines. The levels are virtually identical in TK6 and NH32, although the appearance of apoptotic cells is delayed by 
24 h in the p53 null; there is significantly less apoptosis in WTK1, and the kinetics are very similar to those in NH32. Mutagenesis at tk is also different; relative to TK6, background MF or MF induced by 1.5 Gy of X-ray or -ray is elevated by 2- to 3-fold in NH32 (28) and 10-fold in WTK1 (28,29). [It is important to note that in the manuscript describing the isolation of NH32, the class of slow growth mutants was not determined accurately. Therefore, at the time we thought that TK6 and NH32 were equally mutable.]
To prove that particular p53 mutations may be associated with the observed mutator and hypermutable phenotypes, we transfected the known dominant-negative ala-143 and also the ile-237 p53 cDNAs into TK6 (p53+/+) and thereby obtained isogenic cells that varied only in the status of the p53 gene. We demonstrated that the alteration of p53 status directly led to increases in spontaneous and X-ray-induced mutation frequencies (30). This experiment also showed that the ile-237 mutation found in WTK1 could override the wild-type allele, thus behaving in what we interpreted at the time to be a dominant-negative fashion. The p53 mutant cells showed both a mutator and hypermutable phenotype. However, when we found that these effects were significantly greater in WTK1 than in NH32 (28), this suggested that some forms of mutated p53 may behave in a positive fashion (i.e. a gain-of-function) to affect both spontaneous and radiation-induced mutagenesis.
The experiments described in this paper use the opposite approach of introducing a wtp53 to WTK1 cells. We compared spontaneous and IR-induced mutagenesis at the tk locus, as well as induction of apoptosis and cell killing. We found that expression of wtp53 could reduce some of the effects of mp53.
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Materials and methods |
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Cell lines, plasmids' constructs and transfection conditions
The cell lines TK6 (wtp53), NH32 (double p53 knockout derived from TK6) (23
), Ne72 (p53+/- heterozygote from TK6 with a one allele knockout) and WTK1 (mp53) are originally from the same parental human lymphoblastoid cell WIL2. They all are tk heterozygotes, and all four lines have the active and inactive tk alleles on the same chromosome 17 homologues. The inactive alleles are present and contain single frameshift mutations. Cells were maintained in suspension culture at densities of 5 x 105 to 10 x 105 cells/ml at 37°C in a humidified environment with 5% CO2 using RPMI 1640 growth medium supplemented with 10% heat-inactivated horse serum (Sigma, St Louis, MO, USA). Plasmid pCEP4-wtp53 was generated by insertion of a full-length wtp53 cDNA derived from plasmid pC53-SN3 (wtp53 cDNA, 1.8 kb), under the control of a CMV promoter; it was generously provided by Dr B. Vogelstein (Johns Hopkins Oncology Center, Baltimore, MD, USA). As a negative control, pCEP4 vector without any insert was used. DNA sequencing was used to confirm the direction and sequence accuracy of the insertion after plasmid construction.
Fifteen micrograms of plasmid was introduced into 1 x 107 WTK1 cells by electroporation in a total volume of 0.6 ml using 250 V at 960 µF with a Bio-Rad Gene Pulser. Two days after electroporation, cells were seeded at 1000 cells per well in 96-well microtitre dishes in medium containing 200 µg/ml hygromycin B. Hygromycin-resistant colonies were picked after 2 weeks and expanded. The cell transfected with plasmid pCEP4-wtp53 is designated as WTK1-wt, and that transfected with vector pCEP4 only as WTK1-PC.
p21 induction and western blot analysis
In order to test wtp53 expression and its function, WTK1-PC, WTK1-wt and TK6 (as a positive control) were treated with 1.5 and 3 Gy -irradiation, and harvested for protein extraction 4 h after treatment. Protein lysates were prepared by solubilizing 5 x 106 cells in Laemmli sample buffer (Bio-Rad). Thirty micrograms of total protein per lane was separated on sodium dodecyl sulphate–10% polyacrylamide gels, and transferred to nitrocellulose membranes. The p53 and p21 proteins were detected, respectively, by using the following primary antibodies: mouse monoclonal antibody against p53 (Ab-6; Oncogene Research Products, Cambridge, MA, USA), mouse monoclonal antibody against p21WAF1 (Oncogene Research Products). Secondary antibodies used were horseradish peroxidase-linked whole antibody raised in rabbit against mouse IgG (Amersham Pharmacia, Piscataway, NJ) and were detected by an ECLTM kit (Amersham Pharmacia). After proper development of the membranes, the images were scanned by a Storm scanner and the p53 and p21 expressions were quantitated by the specific software (ImageQuant 5.1, Amersham Biosciences). The p21 protein expression levels were normalized to the loading control ß-actin and standardized to the same background.
Indirect immunofluorescence
Cells were harvested, cytocentrifuged onto slides and processed for immunofluorescence using standard protocols. Briefly, the detection of p53 was carried out by using as the primary antibody against p53, mouse monoclonal antibody Ab-6 (Oncogene Research Products). The primary antibodies were detected using tetramethyl rhodamine isothiocyanate-conjugated rabbit anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA). Following incubation with secondary antibodies, the DNA was stained with 0.2 µg of 4'6'-diamidino-2-phenylindole per millilitre (DAPI). In parallel, three groups were set up for each cell line. Stained cells were examined with a fluorescence microscope under UV light. The p53 expression level was classified subjectively into three groups: high, medium and low. For each cell line, at least 100 cells were scored.
Cell growth and cell survival assays
We measured cell proliferation to evaluate the effects of adding wtp53 on growth. Cells were seeded at a density of 4 x 105 cells/ml in T-25 culture flasks. Three flasks of each cell line were seeded initially. Cells were counted each day using a Coulter cell counter (Beckman Coulter, Inc., Fullerton, CA), and the total number of cells was calculated and plotted. Cells remaining in flasks were diluted back to a density of 4 x 105 cells/ml every day. In addition, we measured colony formation both for control WTK1-PC and WTK1-wt cells. For this, cells were seeded into 96-well microtitre dishes at 1 cell per well in hygromycin; after 14 days, colonies were counted with a dissecting microscope and the plating efficiency (PE) was determined by the formula: PE = –ln ((96 – C)/96), where C is the number of colonies in the dish. Standard deviations (SDs) were calculated from three independent experiments.
Cell survival after -irradiation of log-phase cultures was determined using colony formation as the criterion. Cells were untreated or irradiated with 1, 3 or 5 Gy and immediately seeded into 96-well plates at appropriate concentrations. Colonies were counted 14 days later. PE was determined as above, except that the value was divided by the number of cells seeded per well. Data are from three independent experiments.
Flow cytometric analysis for apoptosis
Cells were harvested daily from day 1 to day 6 after 1.5 and 3 Gy irradiation. Cell suspensions were centrifuged in 15-ml tubes and washed twice with phosphate-buffered saline (PBS). The cells were re-suspended in the remaining solution and 4 ml of a 10-fold diluted diethyleneglycol formaldehyde lysing solution (Becton-Dickinson, Basel, Switzerland) was added to the cell suspension in order to permeabilize and fix the cells. After incubation for 10 min at room temperature, the suspension was centrifuged and the supernatants were washed once with PBS, re-suspended in 200 µl of FACS flow solution (Becton-Dickinson) and 5 µl of a 1 mg/ml stock solution of propidium iodide (PI, Sigma, Mannheim, Germany) and 50 µl of a 1 mg/ml stock solution of RNase (Bovine pancreas RNase A, Serva, Heidelberg, Germany) were added to each tube. A 27 µl/ml concentration of H33342
[GenBank]
was employed in order to delay photobleaching during flow cytometric analysis. Cells from each probe were analysed using a flow cytometer (Becton Dickinson, San Jose, CA, USA) employing the CellQuest program. First, a scatter diagram of forward light scatter versus side light scatter was used to exclude debris. Apoptotic cells were then identified using a scatter diagram of PI fluorescence versus forward light scatter. Apoptotic cells were defined as those cells showing a reduced DNA staining (PI staining).
Mutation assays
All lymphoblastoid cell lines were maintained in standard culture condition, and pre-treated with CHAT (deoxycytidine, hypoxanthine, aminopterin and thymidine) medium for 2 days to eliminate pre-existing tk- mutants. Following the treatment, the cells were cultured in a density of 4 x 105 cells/ml in 100 ml of THC medium (CHAT without aminopterin), for another 2 days. Then, cells were untreated or -irradiated with 1.5 Gy, and then cultured for 3 days to allow for phenotypic expression. Mutants were scored by seeding 2000 cells per well in 96-well microtitre dishes in complete medium supplemented with 2 µg/ml of trifluorothymidine (TFT). A PE control was also prepared in a separate microtiter dish at a concentration of 1 cell per well in complete medium without TFT. After 10 days, plates were re-fed with fresh TFT and then returned to the incubator. After an additional 11 days, each well was scored for the presence or absence of a viable colony, and mutant fractions (MFs) were calculated as described previously (31).
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Results |
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Restoration of wtp53 function
We introduced the wtp53 gene on an extrachromosomal vector into human WTK1 lymphoblast cells, which express endogenous mutant but not wtp53. As a control, we also transfected WTK1 cells with an empty vector. Populations of cells resistant to hygromycin were isolated and characterized. As can be seen in Figure 1A, the endogenous mp53 in WTK1-PC transfected with the vector only is not induced appreciably by IR; p21 is not induced at all. However, IR did induce a very slight increase in p53 and a marked increase in p21 in WTK1-wt cells (Figure 1B). TK6 cells shown in Figure 1C have wtp53 only, and exhibited a robust induction of p53 and p21. We quantified the radiation-induced increases in p21 expression levels in WTK1-wt cells, and compared them with TK6; as can be seen in Figure 1D, p21 levels in WTK1-wt were
29% of the TK6 response after 1.5 Gy and 59% after 3 Gy. These results suggested that wtp53 gene transfer can have dominant effects over mp53, and that p53 function can be restored.
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To determine how p53 protein was distributed within the population of transfectants, we utilized immunofluorescence staining for p53. As seen in Figure 2A, p53 expression mainly was in the nucleus and the levels of expression seemed more homogenous in WTK1-PC cells than in WTK1-wt cells. This fits with the idea that having only an endogenous gene will produce consistent levels, while having a variable number of transgenes will lead to more heterogeneity. We performed a semi-quantitative analysis of p53 expression level by classifying individual cells as exhibiting high, medium or low levels of fluorescence as determined by microscopy. These data are summarized in Figure 2B. There were more cells with high levels of p53 expression in WTK1-wt cells (25.9 ± 3.5% compared with 6.5 ± 2.4% in WTK1-PC, P < 0.01). Also, cells with low levels of expression were decreased significantly (P < 0.01). In comparison, TK6 and WTK1 showed homogenous staining patterns under these conditions; NH32 remained dark (data not shown).
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Effects of wtp53 on cell growth
We investigated the effects of wtp53 gene transfer on cell growth. As shown in Figure 3, transfection of WTK1 cells with wtp53 had no effect on the population growth rate. In addition, we measured the PEs for the control and wtp53 transfectants; PE of WTK1-wt was slightly lower (50.7 ± 2.8% compared with 60.7 ± 2.9% in the control cells), but the difference was not significant (P = 0.96). Furthermore, there were no apparent differences in the cell cycle distribution patterns, as monitored in the flow cytometry experiments described below (data not shown).
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Increase of radiosensitivity after transfection of wtp53
We next investigated the effects of wtp53 co-expression with mp53 on sensitivity to
-irradiation. We measured both clonogenic survival and apoptosis. These assays were performed in TK6 and p53-null NH32 cells as well. Cell survival as measured by the clonogenic assay further confirmed that wtp53 gene transfer can have dominant effects over mp53. As shown in Figure 4A, addition of the wtp53 increased radiosensitivity (for all three dose points, P values ranged from 0.016 to 0.019), although not to the level of TK6. Interestingly, the dose–response for WTK1-wt was very similar to that of p53-null NH32 cells (Figure 4A). These results supported the idea that p53 mutated cells are generally more resistant to IR than cells with wtp53, and restoration of p53 function leads to increased radiosensitivity.
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We used flow cytometry to measure apoptosis after an IR dose of 3 Gy. The apoptotic cells were identified as those displaying a reduced DNA staining (PI staining for sub-G1). A small increase in apoptosis was observed in the wtp53 WTK1 transfectants over the time course measured. As shown in Figure 4B, apoptosis levels in wtp53 WTK1 transfectant ranged from 6.5 to 17.6%, as compared with 2.9 to 15.3% seen in WTK1-PC cells. The differences were significant at all time points, with P < 0.01 on the second and third days, and P < 0.05 on the fourth and fifth days); however, the introduction of the wtp53 had only a small effect on apoptosis.
wtp53 reduces the hypermutability observed in WTK1 cells
We measured mutagenesis at the heterozygous tk locus. The background mutant frequencies (BMFs) are shown in Figure 5A. The BMF in WTK1-wt was 75 ± 18 x 10–6, and this is significantly decreased (P = 0.008) from the value of 169 ± 44 x 10–6 seen in the control WTK1-PC line; however, it was significantly higher than the backgrounds of 6.8 ± 1.4 x 10–6 in TK6 (P = 0.0003), 7.4 ± 4.8 x 10–6 seen in the p53+/– heterozygote Ne72 (P = 0.0004) and 18.3 ± 3.1 x 10–6 seen in the p53-null NH32 (P = 0.00009). Induced mutant frequencies are shown in Figure 5B. MFs induced by 1.5 Gy -ray were lower in the WTK1-wt (321 ± 69 x 10–6; P = 0.0005) than in the WTK1 control cell (1847 ± 450 x 10–6). The mutant frequency induced by IR in WTK1-wt cells was close to that seen in the p53-null NH32 line (170 ± 80 x 10–6, P = 0.03), but it is obviously higher than those seen in both Ne72 (67.6 ± 23.3 x 10–6; P = 0.0004) and TK6 (37 ± 4 x 10–6, P = 0.0002). These results indicated that there is a significant relationship between mutagenesis at an autosomal locus and p53 status in human lymphoblast cells.
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Discussion |
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This study shows that p53 function can be partially restored in the human lymphoblast cell line WTK1, which contains an mp53. We found that the exogenous wtp53 was able to function in the presence of the particular ile237 mp53 and regulate the p21WAF1 downstream factor. p53 restoration had clear phenotypic consequences as well. There was a modest increase in sensitivity to
-radiation as measured by clonogenic survival; however, apoptosis was increased only slightly, and likely was not responsible for the altered radiosensitivity. The most profound effect was that spontaneous and radiation-induced mutageneses were significantly decreased. The fact that introduction of wtp53 had only a very small effect on apoptosis but a large effect on mutagenesis leads to the interesting conclusion that mutant p53-mediated hypermutability was not caused by suppression of apoptosis. Our results demonstrate that wild-type p53 can reduce the apparent hypermutable effects of the particular p53 gene mutation on mutagenesis at the autosomal tk locus. As shown in Figure 5, both the background and induced mutation frequencies were significantly decreased in the wild-type p53 transfectants; the background mutation frequency was significantly higher than that in both p53 wild-type TK6 and p53-null NH32 cells. The induced mutation frequency in WTK1-wt cells was close to that seen in the NH32 line. Therefore, our results indicate that the restoration of the wild-type p53 activity in cells with mutant p53 can assist to maintain genomic stability.
So we return to the question of how mutations of p53 produce genomic instability. The first hypothesis was that p53 induces a G1 block after DNA damage. This allows the cell time to repair before the sensitive S-phase. Otherwise, replication of damaged DNA could lead to genetic changes. Although this is often proposed in the human lymphoblast cell systems we are using, it has been documented that there is no p53-mediated G1 block (25). Therefore, it is not a viable explanation for the hypermutability observed in our experiments.
The second hypothesis is that p53-mediated apoptosis eliminates cells with pre-mutagenic lesions (defined as DNA damage with the potential to be processed into a mutation). If that occurs, a mutagenic mechanism would not have a chance to function and produce a viable, mutated cell. There are many studies showing reduced apoptosis in p53 mutant cells, mainly after IR treatment (32–36). One of the most compelling experiments that supports this hypothesis is from the Kronenberg laboratory. They transfected p53 wild-type TK6 lymphoblastoid cells with a vector containing bcl-2 and showed that radiation mutagenesis was significantly increased (6). Yet, there are reasons to question reduced apoptosis as the primary mechanism. The first is a logical one. We believe that a double-strand break (DSB) is a pre-mutagenic lesion. It is well known that at the typical IR doses used for mutagenesis studies, there are a significant number of DSBs induced (e.g. 80 DSBs per cell after 2 Gy). Yet, this dose only induces apoptosis in 30–40% of the cells. Thus, many cells with lots of DSBs/pre-mutagenic lesions do not undergo apoptosis. Second, the experiments shown in Figures 4 and 5 provide evidence against the hypothesis; adding a vector with wild-type p53 to cells with a mutated form led to a substantial reduction in radiation mutagenesis, but had little or no effect on apoptosis.
The third hypothesis is that p53 participates directly in DNA repair, and its absence results in a more error-prone repair. Furthermore, the presence of a mutated form of p53 can lead to even greater hypermutability. The combination of the quantitative dose–response and the mutational spectra data has led us to believe that p53 effects on DSB repair are responsible for its effects on mutagenesis. This could be mediated through effects on homologous recombination (HR), non-homologous end joining (NHEJ) or both, as there is evidence linking p53 with both processes.
In one of our previous studies, we found that recombination between two transfected plasmids was significantly higher in WTK1 than in TK6 (37). We found that plasmid integration was higher as well. At that time we thought that was also attributable to elevated recombination, but now we consider it possible that elevated NHEJ activity could also be involved. A number of other studies have concluded that the presence of wild-type p53 reduces the levels of spontaneous inter- and intra-molecular HR (8,9,12,14,19,38). The mechanisms by which this occurs have not been elucidated. However, other studies have indicated that reduced HR is not associated with p53 function at the G1/S checkpoint (16) or with its transactivation function (12). Therefore, these findings are consistent with the notion of a direct effect, and p53 has been shown to bind directly to proteins involved in HR including replication protein A (15), Rad51 (17) and the WRN helicase mutated in Werner's syndrome (11,39,40). WRN appears to be involved in the initiation of HR (41,42).
There is limited evidence that p53 also may be involved directly with NHEJ. Tang et al. (18) showed that p53 could enhance the rejoining of cohesive ends after irradiation, and that this was independent of the effects on the G1/S checkpoint and transactivation; they postulated that it occurred via the single-strand annealing function of the C-terminal end. Bill et al. (10) showed that end joining in vitro of linearized plasmid DNA was more efficient in extracts of WTK1 than of TK6 lymphoblasts; this experiment would be consistent with Tang et al. if one postulated that the mutant form of the protein in WTK1 was still able to promote end joining, and that the presence of higher levels of p53 protein in WTK1 afforded it higher levels of NHEJ.
In summary, our study indicated that the wild-type p53 might reduce the excessive mutagenesis seen at an autosomal locus that is driven by the endogenous mutant p53, and perform the normal process of maintaining genomic integrity.
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Acknowledgments |
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We are grateful to Xiaofan Cao for some of the statistical analysis of the data. This work was supported by Grant CA49696 from National Institutes of Health (H.L.L.).
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Notes |
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* To whom correspondence should be addressed: Qinming Zhang M.D. M.Sc., Department of Environmental and Radiological Health Sciences, Colorado State University, 1618 Campus Delivery, Fort Collins, CO 80523, USA. Tel: +970 491 2688; Fax: +970 491 0623; Email: zhangqm{at}colostate.edu
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References |
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Received on February 8, 2007; revised on April 12, 2007; accepted on April 20, 2007.
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