The onset of p53-dependent DNA repair or apoptosis is determined by the level of accumulated damaged DNA

Hagai Offer, Neta Erez, Irit Zurer, Xiaohu Tang, Michael Milyavsky, Naomi Goldfinger and Varda Rotter,1

Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel


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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
The p53 tumor suppressor gene plays an important role in both apoptosis and DNA repair pathways that are pivotal for genomic stability. Here we show that the treatment of cells with low doses of {gamma}-irradiation or cisplatin resulted in an immediate enhancement of p53-dependent DNA repair, measured by base excision repair (BER) activity. However, treatment of cells with high doses of DNA damaging agents resulted in a reduction in p53-dependent DNA repair and in the induction of p53-dependent apoptosis. Analysis of p53 upstream molecular events suggested that regulation of p53-associated DNA repair is ATM-dependent. Furthermore, we observed that while dephosphorylation of Ser376 at the C-terminus of the p53 protein was associated with enhancement in DNA repair, phosphorylation at the N-terminal Ser15 resulted in the reduction in DNA repair. The latter is also in correlation with an enhancement in the specific DNA binding activity and in the induction of apoptosis. Treatment of cells with a caspase inhibitor, prior to the damaging agent-blocked apoptosis, had no effect on the DNA repair pattern. Taken together, this suggests that the decision of cells to induce a p53-dependent DNA repair or apoptosis is most probably controlled by the level of genotoxic agent introduced to cells.

Abbreviations: AT, ataxia telangiectasia; BER, base excision repair; NER, nucleotide excision repair


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 Introduction
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The maintenance of genomic stability depends on the ability of cells to sense and recognize damaged DNA and then to either repair or induce an exit from the cell cycle, through apoptosis or cell differentiation (1).

A number of independent studies have suggested a pivotal role for the p53 tumor suppressor gene in several DNA damage and growth control pathways (2–5). By and large, it is suggested that p53, that is activated following genotoxic stress, may trigger the onset of DNA repair, leading to the completion of the cell cycle. Alternatively, p53 may induce apoptosis or terminal differentiation leading to exit from the cell cycle (1,6). The idea that p53 induces apoptosis is unequivocally accepted (6). However, less is known about the molecular mechanism(s) that underlies the role of p53 in the DNA repair machinery. Wild-type p53 was shown to actively participate in various processes of DNA repair and DNA recombination via its ability to interact with components of the repair and recombination machinery and by its various biochemical activities (7). The notion that p53 plays a role in DNA repair pathways in vivo is supported by the observation that p53 knockout mice exhibit an increase in chromosomal abnormalities and a deficiency in global genomic repair (1,8). Moreover, p53 was also suggested to play a direct role in nucleotide excision repair (NER) DNA repair pathway (9). We have shown that wild-type p53 protein directly enhanced base excision repair (BER) activity measured both in vitro and in vivo (10). Furthermore, we found that genotoxic stress induced a p53-dependent modulation in BER activity throughout the cell cycle (11). The idea that p53 is directly involved in BER is also supported by a recent study demonstrating that the stimulation of BER by p53 is correlated with its ability to interact directly both with AP endonuclease and with DNA Polymerase ß (12). Others have reported that p53 is directly involved in rejoining of double-strand breaks following {gamma}-irradiation in mouse embryonic fibroblasts (MEF) (13) and in human lymphoblastic cell lines (14).

Genotoxic stress was shown to stabilize and activate p53 (15–17). It was found that these processes entail phosphorylation of the protein both at the N'- and C'-termini of the molecule. For example, the phosphorylation on Ser15 is believed to induce resistance to MDM-2-dependent degradation of p53 (18). The activated p53 was shown to take part in DNA repair in a transcription-independent manner (19).

Another key cellular protein known to be important for the optimal response of cells to {gamma}-irradiation and for its interaction with p53 is the ATM protein kinase (20–22). ATM is the gene mutated in the genetic disorder ataxia telangiectasia (AT), the symptoms of which include sensitivity to radiation and an increased risk of cancer (23). ATM was shown to be activated by double-strand breaks caused by {gamma}-irradiation and radiomimetic drugs and to phosphorylate several cell-cycle checkpoints and DNA repair-related proteins such as BRCA1, NBS1, CHK2 and p53 (24). ATM was shown to stimulate both Ser15 phosphorylation and the dephosphorylation of Ser376 of the p53 protein in response to {gamma}-irradiation (20–22). Recently, it was suggested that ATM also regulates the stabilization and activation of p53 following DNA damage through MDM-2 phosphorylation, thus preventing MDM-2-dependent p53 degradation (25).

The well established role of p53 in the induction of apoptosis following DNA damage on the one hand, and the accumulation of data suggesting that p53 plays a role in DNA repair on the other, prompted us to investigate the possibility that p53 acts as a key regulator in the selection between these pathways.

In the present study we examined in several cell systems the role of p53 in the cellular decision point in response to genotoxic stress. We found that the cellular response depends on the dose of genotoxic agent introduced to the cells. Lower levels of genotoxic agent resulted in the enhancement of BER activity whereas higher levels induced the immediate inhibition of BER activity and instead provoked an apoptotic response. Both pathways were found to be dependent on wild-type p53 and ATM expression.


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Cell lines
The following cell lines were used in this present study. The M1/2 cell line, a p53 non-producer myeloid cell line and derived clones expressing the murine p53 135val ts mutant (26), established previously (27), were used in our experiments. The murine pre-B-cell line 70Z/3 (28) and its stable subclone, 70Z/3-M8, derived by transfection of pSVLM8p53, a p53 mutant cDNA with an alternatively spliced C-terminus and the drug resistant gpt gene (29). Cells at a density of 2.5x108/ml were irradiated by a {gamma}-beam model 150 (Nordion, Canada) fitted with a 60Co source at a dose rate of 90 Rads/min. Nuclear extracts were prepared by a modified protocol as described previously (30). AT lymphoblastoid cell strain (AT 59) and its normal counterpart (NL 5J3) were kindly provided by Dr Y.Shilo (Tel-Aviv University).

MEF preparation
p53+/– and p53–/– mice were crossed and mouse embryo fibroblasts (MEFs) were prepared from 14.5-day-old embryos. Head and organs enriched in blood vessels were removed and tissue was minced and treated with 0.25% trypsin solution for 10 min. Trypsin was inactivated by addition of FCS. Cells from single embryos were plated and genotyped by allele-specific PCR to determine their p53 status. MEFs were propagated in DMEM supplemented with 10% FCS and 2 mM Glutamin. Experiments were performed with third passage of primary MEFs.

Analysis of cell cycle and apoptosis by FACS
Cells were fixed with 70% methanol (Biolab, UK) at room temperature and stained with 50 µg/ml propidium iodide (PI staining) (Sigma). The cells were analyzed by a FACScan flow cytometer (Beckton-Dickinson, USA) using the CellQuest (Beckton-Dickinson) software.

BER assay
BER assay was performed as described (10) previously. Briefly, the assay was carried out in 25 µl containing 40 mM Tris pH 7.6, 12 mM MgCI2, 1 mM DTT, 0.1 mM each of dTTP, dATP, dCTP, 0.01 mM dGTP, 3% PEG, 0.3 µg depurinated pSP65 plasmid, 0.3 µg non-treated plasmid, 0.25 µl [{alpha}-32P]dGTP, 30 mM KCl and 0.5–1.5 µg of nuclear extracts. Samples were incubated at 37°C for 15 min. A 5 µl sample of stop buffer (120 mM EDTA, 1.2% SDS) was added to each sample and incubated for 10 min at 60°C. Proteinase K (20 µg) was added and incubated at 37°C for 1 h, then 170 µl of TE was added. Samples were phenol–chloroform extracted and ethanol precipitated. DNA was linearized with BamHI and fractionated through a 0.7% agarose gel in TBE. Gels were UV photographed, dried and analyzed by phosphoimaging. DNA repair synthesis is presented by PLS (31), calculated by dividing net counts obtained from the phosphoimager (taken as PSL-background: AP– in each individual lane) by the DNA content assessed by UV absorption. Standard deviations are calculated in all experiments.

Determination of p53 protein levels
For western blot analysis, 106 cells were lyzed in sample buffer (140 mM Tris pH 6.8, 22.4% glycerol, 6% SDS, 10% ß-mercaptoethanol and 0.02% Bromophenol Blue) boiled and loaded on 10% polyacrylamide gels containing SDS. Proteins were transferred to nitrocellulose membranes. The p53 protein was detected using PAb-421, PAb-248, PAb-1801 monoclonal antibodies and anti p53 Ser15 antibody (New England Biolabs, USA).

Electrophoretic mobility shift assays
The DNA mobility shift assay was performed as described (32) previously. Briefly, 10–20 fmol of radio-end-labeled DNA oligonucleotide TCGAGAGGC)ATGTCTAGGCATGTCTC (33), was mixed with 5 µg of nuclear extract, 1 µl of anti-p53 monoclonal antibody PAb-421 (34) ascitic fluid, 2 µg (2 µl) of poly (dI–dC) and 10 µl of buffer (25 mM Tris–HCl, 100 mM KCl, 6.25 mM MgCl2, 0.5 mM EDTA, 1 mM DTT and 10% glycerol). The reactions were incubated for 15 min on ice and another 15 min at room temperature loaded on a 4% polyacrylamide gel and electrophoresed.


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p53 induces DNA repair or apoptosis in M1/2 cells
Previously, we have shown that M1/2 cells undergo p53-dependent apoptosis in response to elevation in the level of wild-type p53 (27,35). We have further found that wild-type p53 expression in these cells facilitated BER activity. Interestingly, expression of the mutant p53 protein interfered with these activities (10,11,35). In order to examine the relationship between p53-dependent repair activity and the induction of p53-dependent apoptosis, we used the M1/2 parental cells and a derived clone expressing the p53Val135 temperature sensitive mutant. This p53 ts mutant was shown to acquire a wild-type conformation at the permissive temperature (32°C) and a mutant conformation at the restrictive temperature (37°C). The M1/2 cells were transferred to 32°C for different time periods, collected and examined for apoptosis or DNA repair activity. As controls, we used stable cell lines generated by infection with the empty retroviral vector, pLXSN.

Figure 1AGo depicts the results of a DNA ladder assay representing typical apoptotic DNA fragmentation in cells containing p53 at its wild-type conformation but not in control cells (PL). As seen, the apoptotic ladder peaked at 36 h following shift to the permissive temperature (32°C). The reduction in the intensity of the ladder at 48 h is probably due to disintegration of cells at this time point.



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Fig. 1. p53 induces DNA repair or apoptosis in M1/2 cells. M1/2 cells infected with either empty retroviral vector (PL) or the temperature sensitive p53 (ts) containing vector were incubated at 32°C for 0, 12, 24, 36 or 48 h, as indicated. (A) Agarose gel showing DNA fragmentation. M, molecular weight DNA marker. (B) FACS analysis of apoptotic cells measured by Annexin V binding. The squares represent cells infected with the empty vector and the diamonds represent cells infected with ts-p53. (C) Duplicate samples of linearized AP+ and AP– plasmids of the various reactions were separated by gel electrophoresis and exposed to UV (upper panel) or analyzed for radioactivity by phosphoimaging (lower panel). Repair synthesis (%) is calculated as described in Materials and methods.

 
Figure 1BGo shows the percentage of apoptosis in these cells measured by Annexin V binding and analysis by FACS. As seen, empty vector containing cells exhibited low levels of apoptosis, whereas cells expressing wild-type p53 exhibited high levels of apoptosis starting at 24 h following the shift to 32°C.

Figure 1CGo shows BER activity measured at several time points following shift cells to 32°C. The in vitro BER assay that we established (10) previously consisted of two different sized plasmids: an AP+ (3 kb) containing apurinic or apyrimidinic sites (AP) and an AP– (4.5 kb) lacking AP sites that were incubated together with a nuclear extract and a radioactively labeled nucleotide ([{alpha}-32P]dGTP). Upon completion of the reaction, the two plasmids were linearized, separated through an agarose gel and the levels of radioactivity, indicating DNA repair synthesis, were measured by phosphoimaging.

As seen in Figure 1CGo, under the conditions used, only the AP+ plasmids showed incorporation of radioactive nucleotides representing DNA repair synthesis. Judged by quantitative analysis of the pattern of DNA repair, it appears that the DNA repair activity remained constant in the control cells (pLXSN), while the cells expressing wild-type p53 protein (p53ts-53) exhibited enhanced BER activity. This enhanced activity remained constant until 24 h following the shift to the permissive temperature and then declined. At 36 h, the time point in which we observed an increase in p53 induced apoptosis (see Figure 1BGo), there was a significant decrease in DNA repair activity.

Thus, it seems that both DNA repair and apoptosis are p53-dependent but occur at different time patterns following expression of wild-type p53 as a result of a shift to the permissive temperature.

DNA damaging dose regulates p53-associated DNA repair activity
Next we examined whether p53-dependent BER activity correlates with the amount of genotoxic agent introduced to cells. To that end, we used two cell types, the M1/2 expressing a ts p53Val135 and MEFs, expressing endogenous wild-type p53. The two were exposed to increasing levels of {gamma}-irradiation or to cisplatin, respectively. M1/2 cells, containing the ts p53Val135 (p53ts-53) or the empty vector (pLXSN), grown at 37°C, were irradiated with increasing doses of {gamma}-irradiation (25–200 R), incubated for 2 h at 37°C and then collected. The nuclear extracts obtained were subjected to the DNA repair assay either at 37 or 32°C. This protocol agrees with our previous observation that the ts-p53 protein from cells grown at 37°C acquires the wild-type conformation upon incubation at 32°C in vitro.

Figure 2AGo shows an increase in p53-dependent BER activity with a peak at 50 R, followed by a significant decrease. p53-independent BER activity measured in M1/2 cell under the same conditions exhibited an increase in BER activity as a function of increased dose of {gamma}-irradiation. p53-independent BER measured in M1/2 cells expressing mutant p53 at 37°C exhibited the same pattern as M1/2 null cells (Figure 2BGo). The pattern exhibits a {gamma}-irradiation-dependent increase in BER activity that reaches a peak at 150 R. However, in agreement with our previous findings a slight reduction in the levels of BER, following expression of mutant p53 was evident, indicating a possible p53 mutant gain of function that is interfering with the repair process.



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Fig. 2. (AC) DNA repair activity induced by {gamma}-irradiation or cisplatin is dose dependent. M1/2 cells infected with either empty retroviral vector (squares) or the temperature sensitive p53 (diamonds) were treated with different doses of {gamma}-irradiation, or with 5 µg/ml cisplatin for different time periods. (D) p53 +/– or p53–/– MEFs were treated with 5 µg/ml cisplatin for increasing time points, as indicated. (A) Repair synthesis of cells irradiated with increasing amounts of {gamma}-irradiation measured at 32°C. (B) Repair synthesis of {gamma}-irradiated cells (as in A) measured at 37°C. (C) Repair synthesis of cells treated with 5 µg/ml cisplatin for different time periods, measured at 32°C. (D) Western blot analysis of p53 protein level in 53+/– MEFs treated with 5 µg/ml cisplatin for indicated times as measured by PAb-248 and repair synthesis of cells treated with 5 µg/ml cisplatin for different time periods [p53+/– (diamonds) or p53–/– (squares) cells].

 
To further establish these observations we used cisplatin, which has a time course accumulative DNA damage effect as an additional DNA damaging agent. M1/2 cells containing ts-p53 or the empty vector were treated with 5 µg/ml cisplatin for the indicated time periods (Figure 2CGo) at 37°C. Nuclear extracts were tested for BER synthesis at 32°C. As seen in Figure 2CGo, p53-dependent BER activity measured in cells expressing wild-type p53 increased up to 3 h following cisplatin treatment and then decreased. On the other hand, control cells exhibited an increase in p53-independent BER activity that correlated with the duration of the cisplatin treatment. Interestingly, the basal level of BER activity in wild-type p53 producer cells (Figure 2A and BGo) was higher than that measured in p53 null cells.

To further establish p53 dependency of these phenomena, we treated MEFs, derived from p53–/– mice or their p53+/– littermates with 5 µg/ml cisplatin. As seen in Figure 2DGo, an increase in p53 protein levels, measured by western blot analysis was evident in p53 producer cells following cisplatin treatment. Figure 2DGo shows that the BER synthesis in the p53+/– cells at the initial time point (0 h) was significantly higher than that observed in the p53–/– cells. In the p53–/– cells, BER activity was increased slightly as a response to cisplatin treatment and was rising moderately throughout the experiment. However, the repair activity in the p53+/– cells increased after 1 h of treatment with cisplatin and decreased as cells were treated with drug for a longer time. It should be noted that our stock of p53–/– cells was limited and thus we were forced to examine less time points.

Based on these two experimental cell models it appears that the expression of wild-type p53 modulates the pattern of BER. While exposure of cells to low levels of genotoxic agent enhanced DNA repair activity, high levels reduced it. This biphasic response was not evident in the p53-independent BER activity measured in the p53 null or in cells expressing mutant p53.

Reduction in BER activity in cells exposed to high levels of damaged DNA is independent of the induction of apoptosis
In order to further establish the observation that p53-dependent BER activity correlated with levels of genotoxic agent introduced to cells we have focused on another cellular experimental model.

70Z/3, a pre-B-cell line expressing endogenous wild-type p53 and a 70Z/3-M8-derived cell line, in which the wild-type p53 protein was shown to be inactivated by overexpression of mutant p53 (36) were analyzed. Both cell lines were irradiated with increasing doses of {gamma}-irradiation (Figure 3Go). Two hours later, cells were collected; nuclear extracts were prepared and assayed for BER activity. As seen in Figure 3AGo, there was an increase in BER activity in 70Z/3 that peaked at 100 R. At higher doses of radiation a significant decrease below the basal level of repair activity was noticed. On the other hand, the repair activity of 70Z/3-M8 cells was not increased significantly above the basal level.



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Fig. 3. (A and B) Modulation of wild-type p53 associated DNA repair pattern following genotoxic stress does not depend on the induction of apoptosis. 70Z/3 (diamonds) or 70Z/M8 cells (squares) were irradiated with increasing doses of {gamma}-irradiation, or treated with Z-VAD (50 µM) 30 min before the irradiation (70Z/3 only). (A) Repair synthesis of cells irradiated with increasing amounts of {gamma}-irradiation. (B) Twenty-four hours later, apoptosis was measured by FACS analysis of PI stained cells. (C) Repair synthesis in 70Z/3 treated with Z-VAD (circles) or with solvent (diamond). (D) Twenty-four hours later, apoptosis in Z-VAD treated (circles) or solvent treated (diamond) 70Z/3 cells was measured by FACS analysis.

 
To evaluate apoptosis cells were {gamma}-irradiated; 24 h later cells were labeled with PI and analyzed by FACS. As seen in Figure 3BGo, wild-type p53 containing cells (70Z/3) showed an increase in apoptosis, whereas cells expressing mutant p53 did not exhibit any significant changes in cell viability as a function of {gamma}-irradiation.

It should be noted that the initial increase in BER correlated with the resistance to the induction of apoptosis at the range of up to 100 R. However, at higher {gamma}-irradiation doses, there was an immediate decrease in BER activity. To further resolve the correlation between BER activity and apoptosis we have treated cells with a caspase inhibitor. This was aimed to examine the possibility that apoptosis executors, such as caspases may abrogate the function of proteins that are essential for BER (37).

Figure 3CGo shows that the pattern of BER synthesis was unchanged following pre-treatment of cells with a broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), 30 min prior to {gamma}-irradiation. As expected, apoptosis measured by PI incorporation into cells treated with zVAD-fmk was inhibited (Figure 3DGo). Therefore, we conclude that although BER activity and apoptosis are both wild-type p53-dependent, the decision to turn off repair activity following treatment with higher doses of genotoxic agent is independent of the induction of apoptosis. Reduction in BER activity is not regulated by the mere onset of the apoptotic pathway that occurs in cells that are exposed to high levels of genotoxic agents.

Low levels of {gamma}-irradiation induce protein stabilization, but not p53-specific DNA binding
Activation and stabilization of p53 were shown to be mediated by post-translational modifications that may occur at different domains of the molecule (38). In the following experiments we examined whether the onset of BER or apoptosis are associated with differential post-translational modifications of p53. To that end, we exposed 70Z/3 cells to increasing doses of {gamma}-irradiation and analyzed the phosphorylation status of p53 and its capacity to specifically bind to DNA. As seen in Figure 4AGo, protein stabilization measured by immunoblotting with PAb-248, was already evident following exposure of cells to 50 R. This was further augmented as a function of the increase in the dose of {gamma}-irradiation. Next, we examined the status of phosphorylation of the C-terminus of p53. Dephosphorylation of Ser376 at the C-terminus was shown to expose the PAb-421 epitope (39). This binding occurred only following the exposure of cells to 100 R. No further increase was evident as the dose of {gamma}-irradiation was increased. Furthermore, phosphorylation of Ser15 at the N-terminus was evident only following exposure of cells to 150 R and higher. Figure 4BGo represents in a quantitative way the relative levels of specific p53 protein forms expressed. Data were obtained by scanning the individual bands and calculating the ratios.



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Fig. 4. Analysis of p53 protein level, DNA binding activity and post-translational modifications following increasing amounts of DNA damage. 70Z/3 cells were exposed to increasing amounts of {gamma}-irradiation. (A) Western blot analysis of p53 protein determined by PAb-248, PAb-421 and P-Ser15. (B) Graphic representation of the western blot analysis, emphasizing the percentage of modified p53 out of the total p53 protein measured by binding PAb-248 (gray bars). Dephosphorylation of Ser376 (PAb421/PAb248) (open bars) and Ser15 phosphorylation (Ser15/PAb248)(dark bars), respectively. (C) Nuclear extracts obtained from {gamma}-irradiated as well as from non-treated cells were analyzed for specific DNA binding activity of p53 using EMSA, as described in Materials and methods. The arrow points to the p53-DNA–PAb-421 complex.

 
In order for p53 to be active as a transcription factor it needs to bind to specific sequences in DNA. This activity was found to be essential for p53-dependent apoptosis but to be non-essential for p53-dependent BER (12,19). Therefore, we next measured the ability of p53 to bind to its consensus sequence following increasing doses of {gamma}-irradiation. As seen in Figure 4CGo, the specific DNA binding activity of p53 appears in nuclear extracts exposed to 150 R and higher. Thus, it seems that increasing doses of {gamma}-irradiation results in different post-translational modifications of p53 affecting its specific DNA binding activity and its mode of action.

AT cells are impaired in their response to {gamma}-irradiation induced BER
ATM, the gene defective in the genetic disorder AT, is known to be an upstream regulator of p53 in response to {gamma}-irradiation (40), and cells derived from AT patients are hypersensitive to {gamma}-irradiation (41). ATM was shown to phosphorylate p53 at the Ser15 residue and to be associated with dephosphorylation of p53 at residue Ser376, following exposure to {gamma}-irradiation (39). Next we examined BER activity in cells derived from AT patients in response to {gamma}-irradiation. As seen in Figure 5AGo, exposure of AT cells to increasing levels of {gamma}-irradiation did not alter significantly the pattern of BER activity measured. However, a significant increase in DNA repair synthesis following as low as 50 R of {gamma}-irradiation was detected in nuclear extracts obtained from normal counter part cells. As observed above, higher doses of {gamma}-irradiation resulted in suppression of BER activity bellow the basal level in these controls. These results suggest that while control cells exhibit a p53-dependent BER activity controlled by levels of {gamma}-irradiation introduced to cells, AT cells exhibit a p53-independent BER activity that is not affected by exposure of cells to increasing levels of {gamma}-irradiation.



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Fig. 5. Analysis of DNA repair pattern and p53 protein post-translational modifications following {gamma}-irradiation of AT cells. (A) DNA repair synthesis in AT derived (squares) cells versus their normal (diamonds) counterparts irradiated with increasing amounts of {gamma}-irradiation. (B) Western blot analysis of p53 protein in the AT cells and in their normal counterparts (NL) as determined by PAb-1801, PAb-421and P-Ser15.

 
In order to examine this hypothesis, we measured the p53 protein levels and its phosphorylation state in both cell types. As seen in Figure 5BGo, the total amount of p53 protein in normal cells, estimated by PAb-1801, was gradually increased and correlated with {gamma}-irradiation dose, reaching maximum expression at 400 R. The pattern of p53 dephosphorylation at the Ser376 residue in control cells, as detected by PAb-421, showed an initial increase at 50 R and remained unchanged following an increase in the irradiation dose. Moreover, phosphorylation at the Ser15 residue was evident only following exposure of these cells to 100 R (Figure 5BGo). This is in agreement with our above observation with the 70Z/3 cells (see Figure 4AGo). However, in AT cells, there was no detectable p53 protein accumulation or dephosphorylation at Ser376 following 50 or 100 R of {gamma}-irradiation. Exposure of AT cells to 400 R induced detectable levels of p53, as well as phosphorylation at Ser15. This is in agreement with previous reports (20,39) and may represent modification of p53 by a protein kinase other than ATM, e.g. ATR. Thus, it seems that p53-dependent BER depends on functional ATM in cells and involves dephosphorylation of p53 at residue Ser376.


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The involvement of p53 in the maintenance of genomic stability was shown to be associated with several pathways. While in some cases p53 induces an exit from the cell cycle by growth arrest, apoptosis or cell differentiation, in other events, p53 was shown to induce DNA repair, which may permit eventually the continuation of the cell cycle (1,8). However, it is still unclear as to the specific factors that will determine which of the pathways will be induced by activated p53.

Our present study supports the notion that levels of genotoxic agent introduced to cells may determine in a p53-dependent manner whether cells will undergo apoptosis or induce DNA repair. We report here that low doses of damaging agent significantly up regulate p53-dependent BER, while high doses suppress it but rather induces apoptosis. p53-dependent BER activity, measured in either primary embryonic fibroblasts, pre-B cells that express endogenous wild-type p53 or in myeloid cells expressing the wild-type p53 of the ts mutant, was enhanced at low levels of genotoxic agent and reduced in the presence of high levels of genotoxic agent, either {gamma}-irradiation or cisplatin. On the other hand, p53-independent BER activity measured in p53 null primary embryonic cells or in cells in which p53 was inactivated by overexpression of mutant p53 did not show this biphasic pattern. Our results also suggest that turning off BER is not dependent on the onset of apoptosis. Indeed, blockage of apoptosis by a specific caspase inhibitor did not change at all the pattern of BER in the cells.

In all, this may indicate that increasing doses of genotoxic agents cause the accumulation of activated p53 that determines the onset of BER or apoptosis.

The conclusion that levels of accumulated p53 in cells may determine the onset of a specific p53 pathway is in agreement with our previous studies. We observed that while high levels of p53 induce HL-60 cells to undergo apoptosis, low levels of p53 induced, in the same cells, the onset of cell differentiation (42). Likewise, induction of p53 expression in both SaOS2 and H1299 p53 null cells, induced growth arrest at low p53 levels and apoptosis at high p53 levels (43).

In our previous studies we observed that p53 facilitates BER activity both in vitro and in vivo (10,11,19). We found that the involvement of p53 in BER was transcriptionally independent (19). However, another study suggested that transcription-deficient p53 protein was inefficient in BER activity (12). Modulation in the repair machinery components following DNA damage is a relatively unclear phenomenon, but it is presumed to involve transcriptional induction of the repair factors or post-translational modifications of existing repair proteins (44). Furthermore, changes in repair activity could be an indirect consequence of the changes in cellular metabolism and/or induction of programmed cell death, following high doses of DNA damage (45). Caspase-dependent cleavage of PARP (46) and ATM (47) proteins, which participate in DNA repair and DNA damage signaling, could provide an example of such repair/signaling factors that are inactivated during apoptosis induction. Our study clearly distinguishes between the suppression of BER due to degradation of the repair factors occurring in the course of apoptosis progression and the possibility that down regulation of repair is an early and autonomous checkpoint regulated event, independent of the cell death execution machinery.

Recently, it was also shown that the stimulation of BER by p53 is correlated with its ability to interact directly both with AP endonuclease (APE) and with DNA Polymerase ß. Moreover, p53 was shown to stabilize the interaction between DNA Pol ß and abasic sites (12).

Our present findings provide further important clues concerning the physical association of wild-type p53 with the BER machinery and modulations of this repair activity by DNA damage through signaling to p53. The observation that treatment of 70Z/3 cells with the broad-spectrum caspase inhibitor, zVAD-fmk, did not abolish the decrease in BER activity, following higher doses of {gamma}-irradiation, suggests that the p53-dependent decision to turn off this DNA repair machinery is independent of apoptosis activation.

It is known that once checkpoint proteins are activated, they transduce the appropriate signal to their downstream target genes. These, in turn, mediate the specific cellular response that is controlled by the level and type of DNA lesion (48). The ability of ATM to phosphorylate p53 on Ser15 directly and MDM-2 at several serine residues (25), is at least in part responsible for the proper activation of the p53-dependent DNA damage checkpoint. We present here the first evidence that both p53 induction and suppression of BER following {gamma}-irradiation is deficient in AT cells. The defective BER induction observed in AT cells could be an additional factor contributing to the IR hypersensitivity and genomic instability characterizing the AT phenotype.

Post-translational modifications of p53, induced by DNA damage, are critical for eliciting its biological activities (38,49). However, the significance of particular post-translational modification on p53-mediated cellular activities are still unclear and controversial. This is mainly because of the use of experimental systems based on p53 overexpression. We demonstrated here that dephosphorylation of Ser376, which exposes the PAb-421 epitope, is an early event, activated by low doses of genotoxic agents. Furthermore, this modification correlated with enhanced p53-associated BER activity. It was shown before that Ser376 dephosphorylation is associated with ATM-dependent activation of p53 following DNA damage leading to association of p53 with the 14-3-3-protein (39). However, in our hands only a fraction of the stabilized p53 becomes PAb-421 positive. An additional significance of this modification is supported by our finding that cells derived from AT patients, carrying wild-type p53 are both deficient in Ser376 dephosphorylation and in the induction of BER in response to {gamma}-irradiation. It is worth mentioning that the observed p53-associated BER depends on the integrity of the p53 C-terminus (19). Although the molecular mechanism responsible for enhanced p53-associated BER as a function of the C-terminus phosphorylation state remains to be defined, we can propose several models. The PAb-421-positive fraction of p53 can facilitate BER through enhanced interaction with Ref-1 and DNA Pol ß or other repair factors such as 14-3-3, via changes in the oligomerization state of p53 and by modulating its DNA binding properties.

Our present findings point to the possibility that BER activity induction was invariantly correlated with low levels of p53 protein, while BER suppression was observed in nuclear extracts containing high levels of p53. These quantitative changes in p53 protein level were associated with qualitative changes in p53 phosphorylation status. In addition to Ser376 dephosphorylation observed following low doses of IR, Ser15 phosphorylation was evident only following high doses of IR. The reduction in p53-associated BER following high doses of irradiation correlated with Ser15 phosphorylation. Others have shown that Ser15 phosphorylation is associated with enhanced expression of p53 target genes and apoptotic response (50). Here we observed a correlation between Ser15 phosphorylation on the one hand and enhanced specific DNA binding activity of p53 and apoptosis on the other hand. This Ser15 phosphorylation following {gamma}-irradiation was ATM dependent (20–22). Obviously, we cannot exclude a role for other post-translational modifications occurring in the p53 protein, which may differentially be induced by various doses of genotoxic agents.

Accordingly, we propose a model (Figure 6Go) for the selection of the differential pathways by p53-dependent checkpoint activation. When DNA damage occurs, the DNA damage sensing machinery estimates the level of damage. Then p53 is being post-translationally modified leading to the activation of a DNA damage checkpoint. The DNA damage checkpoint is activated according to the DNA damage level by tightly regulated p53 post-translational modifications. ATM kinase activity mediates, at least in part, this p53 activation (20,21). In response to low and moderate levels of DNA damage, p53 protein activates the DNA repair machinery represented by BER in our present studies. This is probably by direct participation of p53 in the repair complex. On the other hand, high levels of DNA damage may induce different p53 post-translational modifications that down regulate BER pathway and in turn induce an apoptotic response. The role of p53 in both the induction and suppression of DNA repair, which may strongly affect cell survival, has to be taken into consideration when DNA damage inducing agents are used in cancer treatments.



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Fig. 6. A proposed model for the selection of the induction of different pathways following p53-dependent checkpoint activation.

 


    Notes
 
1 To whom correspondence should be addressed Email: varda.rotter{at}weizmann.ac.il Back


    Acknowledgments
 
This work was supported in part by grants from the Israel-USA Binational Science Foundation (BSF) and the DIP (Deutsch-Israelische Projektkooperation) and the Kadoori Foundation. V.R. is the incumbent of the Norman and Helen Asher Professorial Chair in Cancer Research at the Weizmann Institute.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received October 30, 2001; revised February 19, 2002; accepted March 1, 2002.