ToxSci Advance Access originally published online on December 20, 2007
Toxicological Sciences 2008 102(2):232-240; doi:10.1093/toxsci/kfm305
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Mdm2 as a Sensitive and Mechanistically Informative Marker for Genotoxicity Induced by Benzo[a]pyrene and Dibenzo[a,l]pyrene
Institute of Environmental Medicine, Karolinska Institutet, S-17177 Stockholm, Sweden
1 To whom correspondence should be addressed at Institute of Environmental Medicine, Karolinska Institutet, Box 210, S-171 77 Stockholm, Sweden. Fax: +46-8-343849. E-mail: ulla.stenius{at}ki.se.
Received October 11, 2007; accepted December 9, 2007
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ABSTRACT |
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Mdm2 is an oncoprotein interacting with p53 and maintaining low p53 levels in unstressed cells. Here we investigated the effect of genotoxic compounds on Mdm2 phosphorylation levels. Employing the Mdm2 2A10 antibody and phosphatase treatment we found that Mdm2 accumulated in HepG2 cells when exposed to low concentrations of genotoxic compounds such as mitomycin C, etoposide, 5-fluorouracil, and benzo[a]pyrene (BP). The low-dose responses were not accompanied by p53 accumulation and the effect of low concentrations of BP on Mdm2 was not affected by small interfering RNA for p53. In human lymphoblasts 10nM BP induced an Mdm2 response. Low concentrations of BP also induced binding of Mdm2 to chromatin in HepG2 cells, but no p53 binding or H2AX phosphorylation. The more mutagenic dibenzo[a,l]pyrene as well as higher BP concentrations instead induced
H2AX and p53 Ser15 association with chromatin. Acrolein potentiated the effect of BP on p53 stabilization and chromatin binding. Taken together, these data suggest that (1) Mdm2 is a sensitive biomarker for certain types of genotoxicity, and (2) that polycyclic aromatic hydrocarbons-induced Mdm2 binding to chromatin reflects repairable damage, whereas chromatin binding of p53 Ser15 and H2AX indicates more persistent DNA damage. The analysis of Mdm2 and related endpoints might be useful for evaluating mutagenic potentials of DNA damages. It is suggested that patterns documented here can be used for separating BP doses that induce readily repaired DNA adducts from doses that overwhelm this capacity. Key Words: Mdm2; p53; benzo[a]pyrene; dibenzo[a,l]pyrene; PAH.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The tumor suppressor p53 is stabilized in response to genotoxicity. It is important for the defense against mutations induced by DNA-damaging agents and plays a crucial role in the prevention of cancer (Bartkova et al., 2005
; Oren, 2003). Wild-type p53 is expressed at low levels in unstressed cells and the protein has a short half-life. p53 levels are kept low due to a constant rapid degradation via the ubiquitin–proteasome pathway. A major regulator of this pathway is Mdm2, which functions as an ubiquitin ligase. In response to genotoxicity, DNA-damage signals can prevent the interaction between Mdm2/p53 (Meulmeester et al., 2005), resulting in p53 stabilization. p53 levels increase in DNA-damaged cells, and when accumulated, p53 can function as a transcription factor and induce genes associated with cell cycle arrest or apoptosis (Meulmeester et al., 2005). Mdm2 is also a transcriptional target for p53, thus forming a negative feedback loop with p53 which downregulates p53 with a delay of several hours (Hamstra et al., 2006).
Phosphorylation of p53 and Mdm2 proteins plays an important role in accumulating and activating p53 in response to DNA damage. Phosphorylation of p53 at Ser15 and at Ser20 inhibits the interaction between Mdm2 and p53 and causes nuclear accumulation of p53 (Meulmeester et al., 2005). Certain early Mdm2 phosphorylations can also lead to p53 stabilization (Shinozaki et al., 2003). These results are in line with a model in which DNA-damage–induced modifications of both p53 and Mdm2 inhibit a direct interaction between the two proteins, thus preventing Mdm2 from ubiquitinating p53 (Meulmeester et al., 2005). Recent studies also implicate the VHL gene in these interactions (Roe et al., 2006).
The DNA-damage–responsive kinase ATM might phosphorylate Mdm2 at Ser395 following exposure to genotoxic stress, although ATR can also be involved (Matsuoka et al., 2007). Nevertheless, the phosphorylation of Mdm2 at Ser395 impairs the degradation of p53 and leads to p53 stabilization (Meulmeester et al., 2005; Stommel and Wahl, 2005). This safe guard mechanism may also involve Mdmx (Meulmeester et al., 2005) and COP1 (Dornan et al., 2006). In these and other previous studies Mdm2 alterations were mainly studied at relatively high doses of DNA-damaging agents.
Phosphorylation at Ser395 of Mdm2 is accompanied by decreased reactivity for the monoclonal antibody 2A10 (Balass et al., 2002) and loss of 2A10 reactivity can be restored by employing phosphatase treatment, which unmasks the phosphorylated epitope (Maya and Oren, 2000). Previously we reported that phosphorylation of Mdm2 at the 2A10 epitope was induced in HepG2 and A549 cells by picomolar concentrations of the carcinogenic diol epoxide metabolite of benzo[a]pyrene (BP), (+)-anti-benzo[a]pyrene diol epoxide (BPDE) (Paajarvi et al., 2004, in press). Based on these data, we suggested that the Mdm2 response may be a sensitive marker for DNA damage induced by certain xenobiotics. In previous in vivo studies, employing low doses of diethylnitrosamine, we also detected nuclear Mdm2 alterations in the liver (Finnberg et al., 2004; Silins et al., 2004). This suggested that immunohistological analysis of phosphorylated Mdm2 may greatly facilitate the identification of cells targeted by chemical carcinogens and for establishing carcinogenic "modes of action" at low doses (Cohen et al., 2003).
New and mechanistically informative methods to biomonitor DNA damages need to be developed (Committee on Carcinogenicity of Chemical in Food, C.P.A.T.E., 2004), and future test systems may rely on understanding how perturbed cellular response pathways induce health effects (Krewski et al., 2007). In the present study, the effect of genotoxic agents on Mdm2 has been evaluated. We find that nanomolar concentrations of BP and other DNA-damaging agents rapidly induce Mdm2 phosphorylations. We also characterize a dose-dependent pattern in binding of phosphorylated Mdm2 to chromatin. These alterations may differentiate easily repaired BP-induced damages from more carcinogenic DNA damages.
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MATERIALS AND METHODS |
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Cell culture.
Human hepatocellular carcinoma cells, HepG2 cells, and human lung carcinoma cells, A549 cells were purchased from the American Type Culture Collection. A human lymphoblast cell line (GM00893) was purchased from Coriell Cell Repositories. HepG2 cells were grown in Minimum Essential Medium with Earles salts and L-glutamine supplemented with 1mM sodium pyruvate, nonessential amino acids, 10% inactivated fetal bovine serum, and penicillin/streptomycin. A549 cells were grown in Dulbecco's modified Eagle's medium with glucose (4500 mg/l) and L-glutamine, supplemented with 1mM sodium pyruvate, 10% inactivated fetal bovine serum, and penicillin/streptomycin. Human lymphoblasts were grown in RPMI 1640 medium supplemented with 15% fetal bovine serum, L-glutamine, and penicillin/streptomycin. Chemicals used are 5-fluorouracil (5-FU), mitomycin C, etoposide, BP, caffeine, acrolein purchased from Sigma-Aldrich (St Louis, MO) and dibenzo[a,l]pyrene (DBP) (AccuStandard, Inc, New Heaven, CT). All chemicals were dissolved in dimethyl sulfoxide (DMSO) except for caffeine, which was dissolved in water. Acrolein was also diluted in water. Final concentration of DMSO added to the cells was < 0.2%. An ultraviolet (UV) lamp with intensity of 486 µJ/(cm2s) was used for UV exposure. When synchronized, HepG2 cells were incubated in the same medium as described above, but supplemented with 0.5% serum for 48 h (G0 phase). Starved cells were restimulated to G1 with medium supplemented with 10% serum 6 h before exposure.
Western blotting.
Cells were washed with PBS and lysed in IPB-7 (triethanolamine—HCl 20mM pH 7.8, NaCl 0.7M, 0.5% nonidet P-40, 0.2%, sodium deoxycholate, with 1mM phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, 1 mg/ml pepstatin, 1mM NaF, 1mM NaVO3, 0.1 mg/ml trypsin inhibitor, and 1 mg/ml aprotinin). The samples were subjected to sodium dodecyl sulfate (SDS-PAGE) and thereafter blotted onto a PVDF membrane (Bio-Rad, Hercules, CA). The protein bands were subsequently probed using polyclonal antibodies toward phospho-p53 (Ser15), phospho-p53 Ser46 (Cell Signaling Technology, Beverly, MA), p53 (DO-1): sc-126, poly (ADP ribose) polymerase (PARP) (H-250): sc-7150, Actin (C-11)-R: sc1615, Cdk2 (M2): sc-163, p21 (F-5): sc-6246, Histone H1 (AE-4): sc-8030 (Santa Cruz Biotechnology, Santa Cruz, CA), p53 CM-1 (Novocastra, Newcastle, UK), MDM2 (Ab-2, against the 2A10 epitope) (Calbiochem, Darmstadt, Germany), phospho-ATM Ser1981, phospho-H2AX (Ser139) (Upstate, Lake Placid, NY), and cyclin D1 (Ab-3) (Oncogene, Boston, MA). Proteins were visualized with enhanced chemiluminescence procedure (Amersham Biosciences). Phosphatase treatment was performed as reported previously (Maya and Oren, 2000). In brief, membranes were incubated with 5 U calf intestine alkaline phosphatase (Pase) (Roche Diagnostics, Mannhein, Germany) per milliliter dephosporylation buffer for 1 h at 37°C. Cdk2 and actin were used as a loading control and gave qualitatively similar results. The results were analyzed with National Institutes of Health Image version 1.62 software.
Chromatin isolation.
Chromatin was isolated essentially as described in Al Rashid et al. (2005) and in references therein. Cells were lysed in IPB-7 (as described above, including protease inhibitors) and vortexed to ensure rupture of both cellular and nuclear membranes (checked by light microscopy). Two fractions were isolated by centrifugation (14,100 x g), one supernatant containing the cytoplasm and the soluble nuclear fraction and one nonsoluble pellet containing chromatin (Al Rashid et al., 2005). The pellet was washed three times with IPB-7, homogenized in IPB-7 by sonication and thereafter subjected to SDS-PAGE and Western blot.
Immunoprecipitation.
Immunoprecipitation was performed as previously (Paajarvi et al., 2005). In brief, diluted protein was incubated together with antibodies against the protein to immunoprecipitate for 1 h. Thereafter Protein A/G PLUS-Agarose sc-2003 (Santa Cruz Biotechnology) was added, and the mix was immunoprecipitated over night. The day after, the samples were centrifuged and the pellet was washed with IPB-7 for 20 min. This procedure was repeated three times. Last time the pellet was washed with PBS (including protease inhibitors). The pellet was then diluted in water and the samples were prepared for Western blot.
Small interfering RNA transfection.
HepG2 cells were transfected the day after plating by using SignalSilence p53 (Cell Signaling Technology, Beverly, MA) and TransIT-TKO Transfection Reagent (Mirus Bio Corporation, Madison, WI). The cells were transfected for 24 h according to manufacturer's protocol.
Statistical analysis.
Statistical analysis was conducted using Student's t-test. The data were presented as means ± SD. All experiments were repeated at least three times with different batches of cells. Results were considered to be statistically significant at p 
0.05.
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RESULTS |
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Mdm2 is a More Sensitive Marker than p53 for BP, Mitomycin C, Etoposide and 5-FU, but not for UV
HepG2 cells were used in most experiments due to their metabolic competence and well-documented ability to activate polycyclic aromatic hydrocarbons (PAHs) and other mutagens (Knasmuller et al., 1998
). We applied the same analytical procedure as in previous studies on PAH metabolites (Paajarvi et al., 2004, in press), that is, samples were treated with alkaline phosphatase and then submitted to Western blot analysis, employing the 2A10 antibody. The ability of the 2A10 antibody to recognize Mdm2 is inhibited by phosphorylations on Ser260 and/or Ser395, but alkaline phophatase treatment restores antibody recognition (Balass et al., 2002). First, we compared the effects of genotoxic agents on p53 and on Mdm2 levels, as seen after phosphatase treatment.
The data presented in Figure 1A show that BP and an alkylating drug, mitomycin C, induced Mdm2 alteration in lower concentrations than they induced p53 alterations. After 24 h treatment, there was a marked effect on Mdm2 levels of 0.01µM of BP and of 0.1µM mitomycin C, but no effect on p53 at any of these concentrations. Mitomycin C has been shown to transcriptionally downregulate Mdm2 (Inoue et al., 2001) and this effect was suggested by lower levels seen at 10µM for both BP and mitomycin C. We also tested BP in A549 cells, which lacks major metabolic capacity, but only very weak responses (not shown) were seen. The topoisomerase II inhibitor, etoposide, was tested in both HepG2 cells and in A549 cells. Low concentrations of etoposide induced a pattern (Fig. 1B) similar to that induced by low concentrations of BP and mitomycin C, that is, an Mdm2 response without a p53 response. The Mdm2 response could also be seen after 6 h (Fig. 1B). Figure 1C shows the effect of an uracil analog, 5-FU in HepG2 cells. Also 5-FU induced an Mdm2 response without affecting p53 levels. In Figures 1A and 1C Mdm2 blots without Pase treatment are shown. Less clear effects are seen in these blots and the increased detectability caused by Pase indicates a 2A10-specific phosphorylation. The response induced by higher concentrations (e.g., 5-FU in Fig. 1C) and detected without Pase treatment suggests a p53-induced accumulation of Mdm2 (Paajarvi et al., in press).
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Taken together, these data indicate that Mdm2 has the potential to be a more sensitive indicator than p53 stabilization for genotoxicity induced by BP, mitomycin C, etoposide, and 5-FU.
In the experiment shown in Figure 2, stabilization of p53 was monitored 6 h after 5–240 s exposure to UV radiation (486 µJ/(cm2s)). It can be seen that 20 s UV exposure induced p53 accumulation and a weak alteration in Mdm2 levels. Longer exposure times (40–240 s) decreased Mdm2 levels, which represents an alternative mechanism for p53 accumulation induced by certain DNA-damaging agents (Inoue et al., 2001). We also found that UV induced an ATM phosphorylation at Ser1981 at the dose which downregulated Mdm2 and stabilized p53 (Fig. 2). These data indicate that p53 is a more sensitive marker of UV-induced DNA damage than Mdm2.
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Characterization of Mdm2 Phosphorylation Induced by BP
A concentration of 0.01µM BP induced an Mdm2 response (Fig. 1A), and this is the lowest BP concentration that induced unscheduled DNA synthesis (UDS) in HepG2 cells (Valentin-Severin et al., 2004
). In order to characterize functional aspects of Mdm2 alterations we now focused BP-induced effects. In Figure 3A the increased detectability of Mdm2 2A10 antibody achieved by phosphatase treatment after BP exposure is documented. The difference between phosphatase and no phosphatase treatment reflects the amount of Mdm2 2A10-specific phosphorylation, as shown in the graph (Fig. 3A). A peak of 2A10-specific phosphorylation was seen at 3µM BP.
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In an effort to exclude a role for p53 in the Mdm2 response to BP in low doses we used small interfering RNA (siRNA) for p53 (Fig. 3B). It was found that the Mdm2 response induced by 0.1µM BP was not affected by siRNA for p53, although siRNA decreased p53 levels. As a control we tested siRNA p53 on the effect of mitomycin C on p21 induction, and as expected this response was inhibited.
In additional experiments with BP we also tested the effect of wortmannin, an inhibitor of ATM and other kinases (not shown). As previously (Paajarvi et al., in press), we found that wortmannin in low concentrations abolished the Mdm2 response. p53/Mdm2 levels and responses can be influenced by the cell cycle. In Figure 3C it is shown that the Mdm2 response studied here was not markedly affected by cell cycle phase synchronization (described in "Materials and Methods"). The p53 response was also similar in growing HepG2 cells, in G0 arrested cells as well as in cells in G1.
Mdm2 is a More Sensitive Marker for BP than for DBP
We continued by comparing the effects of BP with the effects induced by DBP (Fig. 4A). DNA adducts formed by metabolites of this latter PAH are less efficiently repaired (Dreij et al., 2005; Yoon et al., 2004), and DBP is 100–200 times more carcinogenic than BP (Courter et al., 2007; Yu et al., 2006). We used a 24-h incubation period and found that DBP-induced p53 Ser15 and p53 Ser46 in lower concentrations (0.1µM) than BP (10µM). Both concentrations coincided with threshold concentrations inducing apoptosis in HepG2 cells (Staal et al., 2007). They also correlated to the induction of ATM phosphorylation. DBP induced an increase of Mdm2 phosphorylated at the 2A10 epitope, but only in concentrations that also affected p53 (0.1µM). As previously, 0.01µM of BP induced Mdm2 phosphorylation at the 2A10 epitope but no p53 alterations (Fig. 4A). Thus BP, but not DBP, induced an isolated Mdm2 response at low concentrations. The highest concentration (10µM) of both BP (as also shown in Fig. 1A) and DBP decreased Mdm2 levels, as previously discussed for the metabolites (Paajarvi et al., 2004).
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In order to extend the idea that Mdm2 can be used as a biomarker we studied human lymphoblasts. It was found that 0.01 and 0.1µM BP induced a clear response as detected by the 2A10 antibody and by employing phosphatase treatment, but no p53 response (Fig. 4B). The phosphatase treatment markedly improved the detectability of Mdm2, indicating that the increase seen was related to a phosphorylation.
Low Doses of BP, but not of DBP, Induces Chromatin Binding of Mdm2
Both Mdm2 and p53 can associate with chromatin (White et al., 2006) and binding of p53 phosphorylated at Ser15 to chromatin has been suggested to indicate persistent DNA damage (Al Rashid et al., 2005). Furthermore, it has been suggested that Mdm2 takes part in DNA repair by colocalizing with the Mre11-Nbs1-Rad50 complex to double-strand DNA breaks (Alt et al., 2005) or by interacting with DNA polymerase 
(Alt et al., 2005; Ganguli and Wasylyk, 2003). In an effort to study chromatin binding at low doses, we measured the amount of Mdm2 and p53 associated with a chromatin-enriched fraction. The fractions were checked by Western blot analysis. Phosphorylated Akt Ser473 is a soluble protein found in both the cytoplasm and in the nucleus in stressed cells (Roudier et al., 2006), and we did not detect it in the chromatin-enriched fraction. Histone H1 was also analyzed and was enriched about 10-fold (densitometric analysis) in the chromatin fraction (Fig. 5C).
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Low concentrations (0.01–1.0µM) of BP readily induced Mdm2 in the chromatin-enriched fraction after 3 h (Fig. 5A) or 6 h (not shown), but 10µM BP did not (Fig. 5A).
H2AX was not detected at these time points (not shown). Instead, this marker was seen after 24 h but only at the higher concentrations of BP, that is, 7 and 10µM (Figs. 5B and 5C). This is in line with previous data on BP and H2AX (Toyooka and Ibuki, 2005). DBP, on the other hand, did not induce Mdm2 at 3 h (Fig. 5A), 6 h (not shown), or 24 h (Fig. 5B) but induced H2AX at 1µM. We also analyzed p53 binding to chromatin at 24 h. It was found that 7 and 10µM of BP, but not 1 and 3µM, induced chromatin association of p53 Ser15 (Fig. 5C). There was no effect of nanomolar concentrations of DBP on p53 Ser15 associated to chromatin although 1µM DBP induced a clear response (Figs. 5B and 5C). This is in line with previously published data (Al Rashid et al., 2005). Thus, DBP for 24 h induced both p53 and H2AX in the chromatin fraction whereas Mdm2 was not seen. Interestingly, in these experiments with BP and DBP, H2AX and p53 in the chromatin-enriched fraction was only observed in the absence of Mdm2 and vice versa.
The BP-induced Mdm2 response in the chromatin fraction was paralleled by the response in the soluble fraction (cf. Figs. 1A, 3A, and 5A
). However, cells exposed to low concentrations (0.1µM) of chemotherapeutic drugs for 24 h did not induce changes in the chromatin fraction. High concentrations of mitomycin C, etoposide, and 5-FU, all induced chromatin binding of Mdm2 as well as p53 (Fig. 6), suggesting that only high concentrations of these compounds induced chromatin binding of Mdm2. It was thus observed that the Mdm2 response to these drugs in the chromatin fraction differed from that induced by BP.
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Acrolein Potentiates the Effect of BP on p53
Recently, it was shown that acrolein inhibits repair of BPDE induced DNA adducts (Feng et al., 2006
). We investigated the combined effect of acrolein and BP and found that this combination induced p53 stabilization and phosphorylation of p53 at Ser15 in the soluble fraction (Fig. 7A). Neither BP (1µM) nor acrolein alone induced these p53 responses. An interacting effect between acrolein and BP was also documented in the chromatin fraction where the combination induced p53 binding to chromatin (Fig. 7B). This is in line with the suggestion that p53 binding to chromatin indicates persistent DNA damage (Al Rashid et al., 2005). No significant effect of the combination of acrolein and BP was seen on Mdm2 (Fig. 7C)
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Caffeine Inhibits Chromatin Binding of Mdm2
We analyzed the effect of caffeine, an inhibitor of ATM. As shown above (Fig. 5A), 0.1µM BP induced Mdm2 binding to chromatin within 3 h. This response was inhibited by 1mM caffeine (Fig. 8A). This is compatible with an involvement of ATM because millimolar concentrations of caffeine inhibits ATM although other kinases might also be inhibited (Sarkaria et al., 1999
). We also found that ATM associated with H2AX under conditions, which lead to H2AX phosphorylation. Thus, a binding was induced by 1µM DBP, but not by 1µM BP (Fig. 8B). This is in agreement with data presented in Figures 4A and 5B.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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In this study we show that Mdm2 phosphorylation induced by low doses of BP and other DNA-damaging chemicals can be detected in cell lysates by employing Western blot analysis combined with alkaline phosphatase treatment. We show that Mdm2 phosphorylation was induced by concentrations of BP that did not induce detectable p53 accumulation. We also documented dose-dependent alterations in the chromatin-enriched fraction. Thus, low concentrations of BP induced a rapid Mdm2 chromatin binding but no H2AX phosphorylation or p53 binding, and a reversed pattern was induced by high BP concentrations and by DBP. By combining BP with an inhibitor of DNA repair, acrolein (Feng et al., 2006
), p53 binding was potentiated. The lowest concentrations of BP that induced Mdm2 phosphorylation at the 2A10 epitope coincided with lowest concentrations that induce UDS (Valentin-Severin et al., 2004) or that induce cell cycle disturbances but only minimal apoptosis (Staal et al., 2007).
It has been indicated that p53 Ser15 will not be phosphorylated in response to nongenotoxic agents, and suggested that a cluster of phosphorylation sites associated with Ser15 (Ser9, Thr18, and Ser20) may be a marker for detecting DNA damage (Saito et al., 2003). We show here that Mdm2 is a more sensitive marker and that environmentally relevant concentrations of BP-induced Mdm2 phosphorylation in concentrations that did not induce p53 phosphorylation. This was also shown for BP metabolites (Paajarvi et al., 2004). However, we have not shown that the Mdm2 response alone is selective for genotoxic compounds, so supported by the data on lymphoblasts we conclude that phosphorylation of the 2A10 epitope in Mdm2 can be a sensitive marker for genotoxicity when used in combination with p53 phosphorylations.
Our data show that low concentrations of BP (0.01–3µM) induced Mdm2 binding in the chromatin-enriched fraction, but no p53 Ser15 binding or H2AX phosphorylation. Higher concentrations of BP (7 and 10µM) induced p53 Ser15 binding and H2AX, but did not induce Mdm2 binding. Thus, the Mdm2 response versus the p53 and H2AX responses in the chromatin fraction were mutually exclusive. This response switch correlates to recently published toxicity data, which show that BP concentrations up to 3µM induce cell cycle disturbances, whereas higher concentrations induced extensive apoptosis (Staal et al., 2007). The two responses may thus reflect critical differences in DNA-damage severity. This is supported by the observation that DBP, which may cause less profound and more slowly repaired conformational changes in DNA (Dreij et al., 2005; Yoon et al., 2004), induced p53 Ser15 binding to chromatin and H2AX. Furthermore, H2AX (Rebbaa et al., 2006) and p53 Ser46 (Mayo et al., 2005) have been associated with apoptosis and binding of p53 Ser15 (Al Rashid et al., 2005) to chromatin with persistent DNA damage. It may thus be suggested that the isolated Mdm2 response to low BP doses reflects readily repaired DNA damages. The p53 and H2AX responses, on the other hand, reflect damages associated with apoptosis and a perturbed DNA repair system.
Our interpretation given above is corroborated by the finding that acrolein, recently shown to inhibit repair of BP adducts (Feng et al., 2006), potentiated BP-induced p53 binding to chromatin. In fact, the combined exposure to BP and acrolein produced a response similar to that induced by the chemotherapeutic drugs. These chemicals have been selected for clinical use and for their efficacy as anticancer agents and for their capacity to induce apoptosis, and it can be surmised that this characteristic was reflected in the response seen in the chromatin fraction.
Our data suggest that analyzing DNA-damage signaling pathways might be more informative than measuring, for example number of DNA adducts. Recently published data show that BP (3µM) induces about 115 adducts per 108 nucleotides, whereas equitoxic concentrations of DBP (0.1µM) induces 10 adducts per 108 nucleotides (Staal et al., 2007). This suggests that DBP is only three times more potent inducer of DNA adducts than BP. As DBP is 100–200 times more potent inducer of carcinogenesis (Courter et al., 2007) these data challenge the notion that PAH adduct levels reflect tumor potency (Ross et al., 1995). Adduct measurements may include adducts in nondividing cells or adducts occupying regions of DNA that are not repaired (Jenkins et al., 2005). For example, adducts may include unrepaired DNA damages in senescent cells, permanently arrested by p53 or other tumor suppressors (Krishnamurthy et al., 2004). In line with this reasoning, it has been observed that mutations and tumors may exhibit sublinear dose–response curves even when adducts exhibit linear dose effect curves (Hoshi et al., 2004).
In summary, the present study indicate that Mdm2 alterations can be associated with readily repaired DNA adducts induced by low doses of BP. Further studies may confirm if these Mdm2 alterations also can be used as a mechanistically informative biomarker for genotoxic agents. The analysis of Mdm2 signaling may lead to the design of cellular models which can define threshold doses that overwhelm the DNA repair pathways and that may reflect breakpoints in the dose–response curve for environmental carcinogens.
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FUNDING |
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The Swedish Board for Laboratory Animals; and Integrated Assessment of Health Risks of Environmental Stressors in Europe funded under the EU Sixth Framework Program.
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REFERENCES |
|---|
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Al Rashid ST, Dellaire G, Cuddihy A, Jalali F, Vaid M, Coackley C, Folkard M, Xu Y, Chen BP, Chen DJ, et al. Evidence for the direct binding of phosphorylated p53 to sites of DNA breaks in vivo. Cancer Res. (2005) 65:10810–10821.
Alt JR, Bouska A, Fernandez MR, Cerny RL, Xiao H, Eischen CM. Mdm2 binds to Nbs1 at sites of DNA damage and regulates double strand break repair. J. Biol. Chem. (2005) 280:18771–18781.
Balass M, Kalef E, Maya R, Wilder S, Oren M, Katchalski-Katzir E. Characterization of two peptide epitopes on Mdm2 oncoprotein that affect p53 degradation. Peptides (2002) 23:1719–1725.[CrossRef][Web of Science][Medline]
Bartkova J, Horejsi Z, Koed K, Kramer A, Tort F, Zieger K, Guldberg P, Sehested M, Nesland JM, Lukas C, et al. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature (2005) 434:864–870.[CrossRef][Medline]
Cohen SM, Meek ME, Klaunig JE, Patton DE, Fenner-Crisp PA. The human relevance of information on carcinogenic modes of action: Overview. Crit. Rev. Toxicol. (2003) 33:581–589.[Web of Science][Medline]
Committee on Carcinogenicity of Chemical in Food, C.P.A.T.E. Guidance on a Strategy for the Risk Assessment of Chemical Carcinogens (2004) London, UK: Committee on Carcinogenicity.
Courter LA, Musafia-Jeknic T, Fischer K, Bildfell R, Giovanini J, Pereira C, Baird WM. Urban dust particulate matter alters PAH-induced carcinogenesis by inhibition of CYP1A1 and CYP1B1. Toxicol. Sci. (2007) 95:63–73.
Dornan D, Shimizu H, Mah A, Dudhela T, Eby M, O'Rourke K, Seshagiri S, Dixit VM. ATM engages autodegradation of the E3 ubiquitin ligase COP1 after DNA damage. Science (2006) 313:1122–1126.
Dreij K, Seidel A, Jernstrom B. Differential removal of DNA adducts derived from anti-diol epoxides of dibenzo[a,l]pyrene and benzo[a]pyrene in human cells. Chem. Res. Toxicol. (2005) 18:655–664.[CrossRef][Web of Science][Medline]
Feng Z, Hu W, Hu Y, Tang MS. Acrolein is a major cigarette-related lung cancer agent: Preferential binding at p53 mutational hotspots and inhibition of DNA repair. Proc. Natl. Acad. Sci. U.S.A. (2006) 103:15404–15409.
Finnberg N, Silins I, Stenius U, Hogberg J. Characterizing the role of MDM2 in diethylnitrosamine induced acute liver damage and development of pre-neoplastic lesions. Carcinogenesis (2004) 25:113–122.
Ganguli G, Wasylyk B. p53-independent functions of MDM2. Mol. Cancer Res. (2003) 1:1027–1035.
Hamstra DA, Bhojani MS, Griffin LB, Laxman B, Ross BD, Rehemtulla A. Real-time evaluation of p53 oscillatory behavior in vivo using bioluminescent imaging. Cancer Res. (2006) 66:7482–7489.
Hoshi M, Morimura K, Wanibuchi H, Wei M, Okochi E, Ushijima T, Takaoka K, Fukushima S. No-observed effect levels for carcinogenicity and for in vivo mutagenicity of a genotoxic carcinogen. Toxicol. Sci. (2004) 81:273–279.
Inoue T, Geyer RK, Yu ZK, Maki CG. Downregulation of MDM2 stabilizes p53 by inhibiting p53 ubiquitination in response to specific alkylating agents. FEBS Lett. (2001) 490:196–201.[CrossRef][Web of Science][Medline]
Jenkins GJ, Doak SH, Johnson GE, Quick E, Waters EM, Parry JM. Do dose response thresholds exist for genotoxic alkylating agents? Mutagenesis (2005) 20:389–398.
Knasmuller S, Parzefall W, Sanyal R, Ecker S, Schwab C, Uhl M, Mersch-Sundermann V, Williamson G, Hietsch G, et al. Use of metabolically competent human hepatoma cells for the detection of mutagens and antimutagens. Mutat. Res. (1998) 402:185–202.[Web of Science][Medline]
Krewski D, Acosta D, Andersen M, Anderson H, Bailar J III, Boekelheide K, Brent R, Charnley G, Cheung V, Green S, et al. Toxicity testing in the twenty-first century: A vision and a strategy. Report in brief. The National Academies of Sciences (2007).
Krishnamurthy J, Torrice C, Ramsey MR, Kovalev GI, Al-Regaiey K, Su L, Sharpless NE. Ink4a/Arf expression is a biomarker of aging. J. Clin. Invest. (2004) 114:1299–1307.[CrossRef][Web of Science][Medline]
Matsuoka S, Ballif BA, Smogorzewska A, McDonald ER 3rd, Hurov KE, Luo J, Bakalarski CE, Zhao Z, Solimini N, Lerenthal Y, et al. ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. Science (2007) 316:1160–1166.
Maya R, Oren M. Unmasking of phosphorylation-sensitive epitopes on p53 and Mdm2 by a simple Western-phosphatase procedure. Oncogene (2000) 19:3213–3215.[CrossRef][Web of Science][Medline]
Mayo LD, Seo YR, Jackson MW, Smith ML, Rivera Guzman J, Korgaonkar CK, Donner DB. Phosphorylation of human p53 at serine 46 determines promoter selection and whether apoptosis is attenuated or amplified. J. Biol. Chem. (2005) 280:25953–25959.
Meulmeester E, Pereg Y, Shiloh Y, Jochemsen AG. ATM-mediated phosphorylations inhibit Mdmx/Mdm2 stabilization by HAUSP in favor of p53 activation. Cell Cycle (2005) 4:1166–1170.[Web of Science][Medline]
Oren M. Decision making by p53: Life, death and cancer. Cell Death Differ. (2003) 10:431–442.[CrossRef][Web of Science][Medline]
Paajarvi G, Jernström B, Seidel A, Stenius U. Exposure of mammalian cells to diol epoxides from benzo[a]pyrene and dibenzo[al]pyrene and effects on Mdm2 and p53. Polycyclic Aromatic Compounds (2004) 24:537–548.[CrossRef][Web of Science]
Paajarvi G, Jernstrom B, Seidel A, Stenius U. Anti-diol epoxide of benzo[a]pyrene induces transient Mdm2 and p53 Ser15 phosphorylation, while anti-diol epoxide of dibenzo[a,l]pyrene induces a nontransient p53 Ser15 phosphorylation. Mol. Carcinog. (in press).
Paajarvi G, Roudier E, Crisby M, Hogberg J, Stenius U. HMG-CoA reductase inhibitors, statins, induce phosphorylation of Mdm2 and attenuate the p53 response to DNA damage. FASEB J. (2005) 19:476–478.
Rebbaa A, Zheng X, Chu F, Mirkin BL. The role of histone acetylation versus DNA damage in drug-induced senescence and apoptosis. Cell Death Differ. (2006) 13:1960–1967.[CrossRef][Web of Science][Medline]
Roe JS, Kim H, Lee SM, Kim ST, Cho EJ, Youn HD. p53 stabilization and transactivation by a von Hippel-Lindau protein. Mol. Cell (2006) 22:395–405.[CrossRef][Web of Science][Medline]
Ross JA, Nelson GB, Wilson KH, Rabinowitz JR, Galati A, Stoner GD, Nesnow S, Mass MJ. Adenomas induced by polycyclic aromatic hydrocarbons in strain A/J mouse lung correlate with time-integrated DNA adduct levels. Cancer Res. (1995) 55:1039–1044.
Roudier E, Mistafa O, Stenius U. Statins induce mammalian target of rapamycin (mTOR)-mediated inhibition of Akt signaling and sensitize p53-deficient cells to cytostatic drugs. Mol. Cancer Ther. (2006) 5:2706–2715.
Saito S, Yamaguchi H, Higashimoto Y, Chao C, Xu Y, Fornace AJ Jr, Appella E, Anderson CW. Phosphorylation site interdependence of human p53 post-translational modifications in response to stress. J. Biol. Chem. (2003) 278:37536–37544.
Sarkaria JN, Busby EC, Tibbetts RS, Roos P, Taya Y, Karnitz LM, Abraham RT. Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine. Cancer Res. (1999) 59:4375–4382.
Shinozaki T, Nota A, Taya Y, Okamoto K. Functional role of Mdm2 phosphorylation by ATR in attenuation of p53 nuclear export. Oncogene (2003) 22:8870–8880.[CrossRef][Web of Science][Medline]
Silins I, Stenius U, Hogberg J. Induction of preneoplastic rat liver lesions with an attenuated p53 response by low doses of diethylnitrosamine. Arch. Toxicol. (2004) 78:540–548.[Web of Science][Medline]
Staal YC, Hebels DG, van Herwijnen MH, Gottschalk RW, van Schooten FJ, van Delft JH. Binary PAH-mixtures cause additive or antagonistic effects on gene expression but synergistic effects on DNA adduct formation. Carcinogenesis (2007) 28:2632–2640.
Stommel JM, Wahl GM. A new twist in the feedback loop: Stress-activated MDM2 destabilization is required for p53 activation. Cell Cycle (2005) 4:411–417.[Web of Science][Medline]
Toyooka T, Ibuki Y. Co-exposure to benzo[a]pyrene and UVA induces phosphorylation of histone H2AX. FEBS Lett. (2005) 579:6338–6342.[CrossRef][Web of Science][Medline]
Valentin-Severin I, Thybaud V, Le Bon AM, Lhuguenot JC, Chagnon MC. The autoradiographic test for unscheduled DNA synthesis: A sensitive assay for the detection of DNA repair in the HepG2 cell line. Mutat. Res. (2004) 559:211–217.[Web of Science][Medline]
White DE, Talbott KE, Arva NC, Bargonetti J. Mouse double minute 2 associates with chromatin in the presence of p53 and is released to facilitate activation of transcription. Cancer Res. (2006) 66:3463–3470.
Yoon JH, Besaratinia A, Feng Z, Tang MS, Amin S, Luch A, Pfeifer GP. DNA damage, repair, and mutation induction by (+)-Syn and (-)-anti-dibenzo[a,l]pyrene-11,12-diol-13,14-epoxides in mouse cells. Cancer Res. (2004) 64:7321–7328.
Yu Z, Loehr CV, Fischer KA, Louderback MA, Krueger SK, Dashwood RH, Kerkvliet NI, Pereira CB, Jennings-Gee JE, Dance ST, et al. In utero exposure of mice to dibenzo[a,l]pyrene produces lymphoma in the offspring: Role of the aryl hydrocarbon receptor. Cancer Res. (2006) 66:755–762.
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