Reduced ATM kinase activity and an attenuated p53 response to DNA damage in carcinogen-induced preneoplastic hepatic lesions in the rat
Ilona Silins,
Niklas Finnberg,
Annika Ståhl,
Johan Högberg and
Ulla Stenius,1
Occupational Toxicology Group, Institute of Environmental Medicine, Karolinska Institutet, S-171 77 Stockholm and National Institute for Working Life, S-17184 Solna, Sweden
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Abstract
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In previous studies we have demonstrated that the p53 response to DNA damage in preneoplastic liver lesions, referred to as enzyme-altered foci (EAF), is attenuated. In the present investigation comparative quantitative RTPCR revealed no major difference in the p53 mRNA levels in EAF and non-EAF tissue. When CoCl2 was employed to induce hypoxia-inducible factor (HIF-1
), both non-EAF and EAF hepatocytes readily accumulated p53, whereas EAF hepatocytes did not accumulate p53 upon treatment with diethylnitrosamine (DEN). The p53 response was also induced in EAF hepatocytes by the inhibitor of nuclear export, leptomycin B. An inhibitor of DNA-dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated (ATM), wortmannin, blocked the DEN-induced p53 response in non-EAF hepatocytes. Assay of kinase activity in immunoprecipitated material from EAF and non-EAF tissue revealed attenuated ATM activity in EAF. Immunohistological and western blot analysis of the level of ATM protein was in agreement with the activity measurements and no phosphorylation of Ser15 in p53 was detected in EAF tissue 24 h after a challenging dose of DEN. Taken together with previously published data, these data indicate selective attenuation of the DNA damage pathway in EAF hepatocytes. Down-regulation of DNA damage-induced and ATM-mediated phosphorylation of p53 may confer a growth advantage on EAF hepatocytes.
Abbreviations: ATM, ataxia telangiectasia mutated;; ATR, ATM and Rad3-related; DEN, diethylnitrosamine; DNA-PK, DNA-dependent protein kinase; EAF, enzyme-altered foci; GST-P, glutathione S-transferase placental form; HIF-1
, hypoxia-inducible factor; LI, labelling index; LMB, leptomycin B; MDM2, murine double minute; PB, phenobarbital
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Introduction
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Expression of p53 can be induced by various forms of cellular stress. Genetically distinct pathways for activation of p53 have been elucidated, all of which lead to accumulation of this protein in the nucleus. One such pathway involves kinases which respond to DNA damage by phosphorylating p53 protein (13) and which may induce cell cycle arrest or apoptosis (4). Phosphorylation at N-terminal sites induced by DNA damage are believed to hinder interaction between p53 and MDM2, thereby preventing degradation of p53 (5). Ataxia telangiectasia mutated (ATM) is one of the p53 kinases shown to be activated by different types of DNA damage (1,6).
Hypoxia is another type of stress that may induce accumulation of p53 in the nucleus. In this case p53 is stabilized by physical interaction with hypoxia-inducible factor (HIF-1
), a regulator of hypoxic gene expression (7,8). The hypoxic state can be mimicked by exposure to cobalt chloride (CoCl2), which can activate HIF-1
and stimulate p53 accumulation and the transcription of several genes associated with hypoxia (9,10). Another signal transduction pathway, which probably responds to activated oncogenes and rapid cell proliferation, involves a different set of nuclear proteins, including E2F1, the product of the gene locus CDKN2 and the p19ARF protein (11,12). p19ARF may block interaction between p53 and MDM2, thereby preventing p53 degradation.
In the present investigation we have examined p53 stabilization in mixed primary cultures of hepatocytes containing both glutathione S-transferase placental form (GST-P)-positive and GST-P-negative hepatocytes. These cells were isolated from rats treated with repeated doses of the genotoxic carcinogen diethylnitrosamine (DEN), which gives rise to numerous precarcinogenic lesions in their livers, referred to as enzyme-altered foci (EAF) (13). GST-P-positive hepatocytes are derived from EAF and GST-P-negative hepatocytes are derived from surrounding hepatic tissue (14).
We have reported previously that GST-P-positive hepatocytes isolated from DEN-treated rats do not readily accumulate p53. Upon being challenged with different genotoxic compounds, even at doses that were clearly cytotoxic, or with X-radiation, these cells did not accumulate p53 (1517). Furthermore, the p53 response of EAF in situ to DNA damage was found to be attenuated (18,19). We postulated that this attenuated p53 response cannot be the result of mutations, but must rather be explained by adaptive epigenetic changes (15,17,19).
Several lines of evidence also indicate that the attenuated p53 response cannot be adequately explained by altered xenobiotic metabolism (1519). Different or additional factors may thus modify the p53 response in EAF hepatocytes and in an effort to identify such factors, we have studied signal transduction pathways which might be involved in stabilizing p53 in EAF. We demonstrate that both CoCl2 and the inhibitor of nuclear export, leptomycin B (LMB), readily induce p53 in GST-P-positive cells. We have also found that ATM kinase expression and activity is lower in EAF than in surrounding tissue and that p53 phosphorylated at Ser15 does not accumulate in EAF in response to DEN.
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Materials and methods
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Cell culture and antisense experiments
Female rats (SpragueDawley) were injected intraperitoneally with DEN (Sigma, St Louis) (0.3 mmol/kg body weight) in saline approximately 24 h after birth. At 3 weeks of age, these rats were weaned and once each week thereafter injected with this same dose of DEN (15). One week following the last of 711 such injections, the rats were used for hepatocyte isolation.
Hepatocytes were isolated employing the collagenase perfusion technique and seeded on collagen-coated plates (2x105 cells/35 mm plate) (Sarstedt, Landskrona, Sweden). The cells were then cultured in complete medium for 1.5 h and subsequently in serum-free RPMI 1640 medium containing the various test substances (14). CoCl2, wortmannin and caffeine were all from Sigma. Wortmannin was dissolved in DMSO and added to give a final concentration of 0.10.2 µM. Leptomycin B (kindly provided by Prof. Minoru Yoshida, University of Tokyo) was dissolved in ethanol and added to obtain a final concentration of 5 ng/ml.
In certain experiments, [methyl-3H]thymidine (0.5 µCi/ml) was present in the culture medium. Twenty-four hours after seeding, the cells were fixed in 3.7% formaldehyde for 1.5 h (15). Phosphorothioated p53 antisense oligonucleotides [5'-TCC GAC TGT GAA TCC TCC AT-3' (20); 2 µM] were added to the culture medium 1.5 h before the addition of treatment substances. In ATM antisense experiments, phosphorothioated ATM antisense oligonucleotides (5'-ATC ATT GAG TGC TAG ACT-3' (21); 3 µM) were added to the culture medium 3 h before addition of test substance. All tissue culture media and sera were obtained from Life Technologies (Paisley, UK).
Immunocytochemistry and immunohistochemistry
The fixed cells were stained with mouse monoclonal anti-p53 antibodies (Ab-3; Oncogene, Cambridge, MA) or double-stained with these antibodies and polyclonal rabbit antibodies directed towards GST-P (Biotrin, Ireland). After incubation with these primary antibodies, secondary anti-mouse or anti-rabbit antibodies were applied. Finally, alkaline phosphatasemouse anti-alkaline phosphatase and peroxidaserabbit anti-peroxidase complexes, respectively, were added. Alkaline phosphatase activity was visualized employing New Fuchsin and peroxidase activity with 3,3-diaminobenzidine tetrahydrochloride (DAB) as substrate (15). All secondary antibodies and substrates were purchased from DAKO (Glostrup, Denmark). For performance of autoradiography, the plates were dipped in Kodak NTB-2 emulsion.
Cells were also stained with mouse monoclonal anti-MDM2 antibodies (SMP14; Santa Cruz Biotechnology, Santa Cruz, CA) overnight after which mouse EnVision+TM peroxidase was added and peroxidase activity visualized utilizing DAB as substrate. The percentage of cells that were marker positive was determined by counting at least 500 cells per plate, located in several randomly selected regions of each plate. All experiments were repeated at least three times with different batches of cells. The data were analysed statistically employing a one-way ANOVA test and a value of P < 0.05 was considered statistically significant.
Livers from rats receiving the initial dose and thereafter eight additional doses of DEN (0.3 mmol/kg body weight) were used for ATM immunohistochemistry. One week after the last treatment the livers were perfused in situ with 3.7% buffered formaldehyde at 37°C for 1.5 h and thereafter placed in formaldehyde for 24 h (22). Prior to staining, the slides were placed in citrate buffer and treated in a microwave oven (5x5 min, with a cooling period of 5 min after each treatment). Sections from DEN-rats were stained by overnight incubation with polyclonal antibodies for ATM (H-248; Santa Cruz Biotechnology). ATM was visualized using the rabbit EnVision+TM peroxidase kit with DAB as substrate. The slides were counter-stained with haematoxylin.
Western blotting
Livers from rats receiving the initial dose of DEN and thereafter either 10 additional doses of DEN (0.3 mmol/kg body weight) once weekly (DEN-rats) or phenobarbital (500 p.p.m. in their drinking water) for ~15 months (PB-rats) were studied. Both DEN- and PB-rats were administered a challenging dose of DEN (1.5 and 1.2 mmol/kg body weight, respectively) 24 h prior to killing. Visible hepatic lesions (EAF tissue) and hepatic tissue free from such lesions (non-EAF tissue) were dissected out separately and homogenized in ice-cold 0.25 M sucrose containing 1 mM PMSF and 0.1 mg/ml trypsin inhibitor II-T, after which nuclear fractions were prepared. Pellets and supernatants were suspended in IPB-7 (20 mM TEA, 0.7 M NaCl, 0.5% NP-40, 0.2% DOC, 1 mM PMSF and 0.1 mg/ml trypsin inhibitor II-T, pH 7.8). Livers from rats treated only once with DEN (1.2 mmol/kg body weight) 24 h prior to killing were also treated in a similar manner.
Cell cultures (6x106 cells/10 cm plate) were established in the same manner as that performed for immunocytochemistry. These cultures were washed with PBS and 0.4 ml 0.25 M sucrose containing 0.1 mg/ml trypsin inhibitor II-T and 1 mM PMSF was subsequently added to the plates. After scraping the cells off the plates, the suspension was fractionated by centrifugation. Both tissue and cell samples were immunoprecipitated using monoclonal anti-p53 antibodies (pAb 122; Boehringer Mannheim, Germany) and Protein ASepharose (Pharmacia Biotech Products, Uppsala, Sweden). The precipitates were subjected to SDSPAGE and thereafter blotted onto a PVDF membrane (Bio-Rad, Hercules, CA). The protein bands were subsequently probed using polyclonal antibodies towards p53 (CM-1; Novocastra, Newcastle-upon-Tyne, UK) or towards p53 phosphorylated at residue Ser15 (New England Biolabs, Beverly, MA), followed by incubation with HRP-conjugated secondary antibodies. Visualization was achieved employing the ECL procedure (Amersham, Bucks).
HIF-1
was detected using a mixture of two monoclonal antibodies (OZ12 and OZ15; Neomarkers, Freemont, CA). Rabbit anti-mouse HRP-labelled secondary antibodies were subsequently applied and immunodetection achieved with the ECL procedure.
For detection of ATM, EAF and non-EAF tissue from rats administered 11 doses of DEN (0.3 mmol/kg body weight) were dissected out 2 weeks after the final injection and homogenized in lysis buffer (50 mM TrisHCl, 150 mM NaCl, 1% Tween 20, 0.2% NP-40, 1 mM NaF, 1 mM NaVO4, 50 mM glycerophosphate, 10% glycerol, 1 mM PMSF, 2 µg/ml pepstatin A, 5 µg/ml leupeptin, 10 µg/ml aprotinin and 1 mM dithiothreitol, pH 7.5) (23). These homogenates were subjected to immunoprecipitation utilizing polyclonal anti-ATM (H-248; Santa Cruz Biotechnology) and Protein ASepharose. The immunoprecipitates were then separated by gel electrophoresis using the NuPAGE® system (Invitrogen, Groningen, Netherlands) on a 37% polyacrylamide gel and the proteins thereafter blotted onto a PVDF membrane (Bio-Rad) at 30 V for 12 h. Finally, ATM was detected using the polyclonal anti-ATM antibody followed by incubation with HRP-conjugated secondary antibodies and visualization by ECL procedure.
Ku-86 was detected using a goat polyclonal antibody (M-20; Santa Cruz Biotechnology) and anti-goat HRP-conjugated secondary antibodies were subsequently applied. For detection of Cdk2 and P70 S6 kinase, membranes were probed with polyclonal anti-rabbit antibodies (M-2 and C-18, respectively, both from Santa Cruz Biotechnology), followed by incubation with HRP-conjugated secondary antibodies. Immunodetection was achieved using the ECL procedure.
RTPCR
Livers from DEN-rats were immersed in RNAlaterTM (Ambion, Austin, TX). EAF and non-EAF tissue were dissected out from the livers of rats receiving 13 or 14 doses of DEN (0.3 mmol/kg body weight) and total RNA subsequently isolated using the TotallyTM RNA kit (Ambion). Quantitation of total RNA was performed spectrophotometrically, while its quality was assured by agarose gel electrophoresis.
Different quantities of total RNA were amplified employing multiplex comparative RTPCR analysis. This procedure involved the QuantumRNA kit, alternate 18S Internal Standard (Ambion) in combination with the Access RTPCR kit (Promega, Madison, WI) and was performed according to the manufacturers' instructions. The optimal 18S primer/18S competimer ratio was determined empirically to be 1.5/8.5. The primers for rat p53 mRNA were as follows: forward, 5'-TCC TCC CCA ACA TCT TAT CC-3' and reverse, 5'-GCA CAA ACA CGA ACC TCA AA-3' (24). Controls, in which AMV transcriptase was omitted, were run in parallel in order to detect contaminating DNA. Reverse transcription was carried out at 48°C for 45 min, with termination at 94°C for 2 min. The cycling parameters utilized were 1 min at 55°C, 1 min at 72°C and 1.5 min at 94°C, for 40 cycles. This cycling was followed by elongation at 72°C for 7 min. The RTPCR products obtained were analysed using a 2% agarose gel with ethidium bromide, on which the amplicons were separated as 324 (18 S) and 261 base pairs (bp) (p53) bands. The mean band densities were determined using NIH Image 1.62 Software and the ratio of the mean densities of the bands for p53 and 18 S calculated for samples lying within the linear interval of amplification.
Assay of the kinase activity of immunocomplexes
EAF separated into smaller lesions 13 mm in diameter and larger lesions 35 mm in size and non-EAF tissue from rats injected with 11 doses of DEN (0.3 mmol/kg body weight) were dissected 2 weeks after the final dose and homogenized in lysis buffer as described above. Immunoprecipitation was performed on ice utilizing polyclonal anti-ATM and Protein ASepharose. The immunoprecipitates obtained were washed twice in lysis buffer and thereafter once in a high-salt buffer (0.1 M TrisHCl, 0.6 M NaCl, pH 7.4) and once with basic kinase buffer (10 mM HEPES, 50 mM NaCl and 10 mM MgCl2, pH 7.4). Kinase reaction mixture was then added to each sample in order to obtain the following final concentrations: 10 mM HEPES, 50 mM NaCl, 10 mM MgCl2, 10 mM MnCl2, 1 mM DTT, 10 µM ATP, 0.2 µl [
-32P]ATP (3000 Ci/mmol; Amersham) and 1µg PHAS-1 (Stratagene, La Jolla, CA), pH 7.4 in a total volume of 40 µl. These samples were incubated at 30°C for 20 min (25) and the reaction terminated by addition of 40 µl 30% acetic acid. These conditions were demonstrated to give phosphorylation of PHAS-1 by ATM, which was linear with time and protein. The mixtures were thereafter spotted onto P-81 phosphocellulose paper (Whatman, Maidstone, UK), which was then washed five times in 200 ml 10 mM sodium pyrophosphate containing 1% phosphoric acid. After drying overnight, the samples were analysed by liquid scintillation counting.
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Results
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The western blot analysis performed here was in agreement with our immunohistological findings published earlier (19,20). Using the CM-1 antibody, p53 was readily detected in the material immunoprecipitated from the nuclear fraction prepared from the liver of control rats administered only a challenging dose of DEN 24 h prior to killing (Figure 1A
). p53 could also be detected in the nuclear fraction from the lesion-free liver of DEN-rats after a challenging dose of DEN 24 h prior to killing. In contrast, no p53 was detected in samples from several macroscopic EAF (Figure 1A
). Similar results were obtained using the antibodies specific for Ser15 phosphorylated p53. This antibody detected a single band, suggesting that one of the two bands binding the CM-1 antibody contains phosphorylated and the other unphosphorylated p53. p53 was not detected in tissue samples from the livers of untreated rats (data not shown). When the p53 mRNA levels (measured by RTPCR and related to 18 S as an internal standard) were compared, these levels were found to be similar in EAF and non-EAF tissue from DEN-rats (Figure 1B
).

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Fig. 1. Lack of p53 and Ser15-phosphorylated p53 response and unaltered mRNA levels in EAF lesions from DEN-treated rats. (A) Western blot analysis of nuclear immunoprecipitated p53 was performed employing either antibodies directed towards p53 itself (CM-1) or towards p53 phosphorylated at Ser15. Several small macroscopic lesions and lesion-free tissue from the livers of DEN- and PB-rats were dissected out and subfractionated. The liver of a control rat was treated in a similar manner. All of the animals received a challenging dose of DEN 24 h prior to killing. (B) Comparative multiplex RTPCR analysis of the relative amounts of p53 mRNA in EAF and non-EAF tissue (negative Polaroid image). 18S rRNA was used as an internal standard.
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In control experiments we analysed hepatic lesions in rats treated with phenobarbital (PB), a non-genotoxic carcinogen. Long-term treatment with PB apparently selects for EAF with other phenotypes, and these lesions exhibited enhanced immunohistological staining for p53 in response to a challenging dose of DEN (19). In this case, the pattern of phosphorylation of p53 was the same in both EAF and non-EAF hepatocytes, i.e. challenge with DEN induced phosphorylation at Ser15 in both of these tissues (Figure 1A
).
In further studies here we employed hepatocytes isolated from DEN-treated rats. We have described several indications that GST-P-positive cells in these cultures originate from EAF, i.e. that these positive cells demonstrate resistance to xenobiotics, a lack of responsiveness to TGFß, an increased LI and other features characteristic of EAF (14,15,20,26). We have also demonstrated that the p53 response to different genotoxic compounds and X-radiation is attenuated (1519). Figure 2
illustrates that in primary culture of hepatocytes isolated from DEN-rats and exposed to CoCl2 during a period of 24 h, p53-positive nuclei were observed in both GST-P-negative and GST-P-positive cells. The staining for p53 was located strictly in the nucleus. Since GST-P staining was present mainly in the cytoplasm, this latter staining did not interfere with the identification of p53-positive and p53-negative hepatocytes.

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Fig. 2. Rat hepatocytes in culture stained for both p53 and glutathione S-transferase placental form (GST-P). Cultures of hepatocytes isolated from EAF-bearing DEN-rats were exposed to 20 µM CoCl2 for 24 h. Three p53-positive cells (red nucleus) can be seen. One of these cells is also GST-P positive (brown cytoplasm), while the other two are GST-P negative.
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In cultures exposed to CoCl2 the frequency of p53-positive cells was counted and Figure 3
depicts the result of one such typical experiment. As shown, after 24 h the highest frequencies of p53-positive, GST-P-negative cells were observed with 1025 µM CoCl2. The use of higher concentrations resulted in cytotoxicity and, consequently, decreasing frequencies of p53 expression in GST-P-negative cells. GST-P-positive cells are more resistant to the cytotoxicity of CoCl2 and continuously increasing frequencies of p53-positive staining in these cells were observed with increasing concentrations of CoCl2 up to 40 µM (the highest concentration tested here). Thus, when the cell cultures were exposed to 40 µM CoCl2, ~15% of the GST-P-positive cells were also p53-positive (Figure 3
).
Compilation of data from such experiments with cells isolated from six different DEN-rats demonstrated that 20.3 ± 4.9% of the GST-P-negative cells and 4.1 ± 1.4% of the GST-P-positive cells were stained positively for p53 after exposure to 10 µM CoCl2 for 24 h. The corresponding values for cells not exposed to CoCl2 were 4.2 ± 2.9 and 0.26 ± 0.15% for GST-P-negative and GST-P-positive cells, respectively. In a previous analogous study in which the hepatocyte cultures were exposed to DEN instead of CoCl2, not more than 12% of the GST-P-positive hepatocytes also stained for p53, even when clearly toxic concentrations were employed (15).
Figure 3
also illustrates the effect of antisense p53 oligonucleotides on the induction of this protein in GST-P-negative and GST-P-positive cells. As expected, antisense oligonucleotides decreased p53 induction by CoCl2 in both cell types. The specificity of this antisense effect has been demonstrated in a previous study (20).
The effect of CoCl2 on DNA synthesis was also examined and Figure 4A
depicts the effect on the labelling index (LI). As shown previously (15), a higher LI is demonstrated by GST-P-positive cells than by GST-P-negative cells. We have also found earlier that the LI of GST-P-positive cells in culture is unaffected by exposure to 0.3 mM DEN (15). In contrast, exposure to 10 µM CoCl2 resulted in >50% inhibition of LI in both GST-P-negative and GST-P-positive cells. At the highest concentration of CoCl2 tested (40 µM) only 2% of the GST-P-positive cells still demonstrated DNA synthesis, compared with ~10% in the absence of CoCl2. This extent of inhibition corresponds roughly to the increase in the fraction of p53-positive GST-P-positive cells (cf. Figure 3
).
MDM2 is one of the genes responsive to p53 (27) and the expression of this protein was thus used as an indicator of p53 function. Exposure to CoCl2 resulted in a concentration-dependent increase in cells staining positively for MDM2 (Figure 4B
). This staining was localized to nuclei and the frequency of MDM2-positive cells was roughly the same as that of p53-positive cells.
The effects of CoCl2 on HIF-1
and p53 expression were then studied further using western blot analysis. It was found that treatment of hepatocyte cultures from DEN-rats with CoCl2 resulted in an increase in the level of HIF-1
, as well as increasing levels of p53 (Figure 5
). As expected, both of these proteins were expressed in the nuclear compartment, but not in the cytoplasm of the hepatocytes.

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Fig. 5. Western blot analysis of p53 and HIF-1 in hepatocytes from DEN-rats exposed to CoCl2. Hepatocyte cultures derived from EAF-bearing DEN-rats were exposed to CoCl2 for 24 h. Cytosolic (cyt) and nuclear (nuc) fractions were prepared from each culture and western blotting performed as described in Materials and methods.
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In additional experiments the combined effects of different types of stimuli on the p53 response were examined. In GST-P-negative cells, both DEN and proteasome inhibitor (PSI) [which stabilizes p53 by inhibiting the ubiquitin pathway for its degradation (28)] produced effects on p53 that were additive to the effect of CoCl2 (Figure 6A
). In the case of GST-P-positive cells, the effect of PSI was also additive to that of CoCl2, whereas DEN did not produce an additive effect (Figure 6B
). The lack of additivity in the p53 responses to CoCl2 and DEN in GST-P-positive hepatocytes indicates that the lack of response of these cells to DEN alone did not reflect any threshold phenomenon in our method of detection.
Leptomycin B is a drug that has been reported to inhibit nuclear export of proteins containing a nuclear export signal (29,30). Such signals are present both in p53 and MDM2 and LMB has been shown to inhibit nuclear export of p53 (31,32). Here, hepatocyte cultures exposed to LMB for 24 h were then double-stained for GST-P and p53. Such treatment with 5 ng/ml LMB medium induced nuclear p53 in 16.2 ± 1.9 and 10.5 ± 1.5% of GST-P-negative and GST-P-positive cells, respectively (Figure 7
). Thus, LMB elicited a similar p53 nuclear response in both these kinds of cells, indicating a basal protein synthesis in both cell types.
Other investigators have demonstrated that the activity of certain kinases, including ATM, which are involved in the p53 response to DNA damage (33) can be inhibited by wortmannin (25) or caffeine (34,35). These kinase inhibitors were subsequently tested in our model system. As shown in Figure 8A
, p53 induction by DEN was inhibited by wortmannin; 150 nM of this inhibitor resulted in an ~50% inhibition. In contrast, wortmannin had no effect on the CoCl2-induced p53 response (data not shown), indicating that these responses of hepatocytes to CoCl2 and DEN do not involve the same pathway. Figure 8B
illustrates that 1 mM caffeine decreased the percentage of hepatocytes demonstrating a p53 response to DEN from 12.5 to 5%. Quantitation of p53 protein by western blot analysis revealed that treatment with either wortmannin or caffeine decreased the level of Ser15-phosphorylated p53 in DEN-treated cells (Figure 8C
), further supporting the conclusion that the DEN-induced p53 response observed here involves the activity of members of phosphatidyl-inosital-3, family of kinases. We also studied the effect of antisense ATM oligonucleotides on the p53 response. As shown in Figure 9
, antisense treatment attenuated the p53 response induced by DEN. Sense oligonucleotides had no effect (data not shown).

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Fig. 8. Inhibition of Ser15-phosphorylation and p53 stabilization induced by DEN using wortmannin and caffeine. Hepatocyte cultures isolated from EAF-bearing DEN-rats were treated with 0.3 mM DEN and (A) wortmannin or (B) caffeine for 24 h, after which the percentages of p53-positive cells on the plates were determined. Mean values ± SD for three plates are shown. *P < 0.05 compared with the corresponding control value. (C) p53 was immunoprecipitated from the nuclear fractions of hepatocyte cultures exposed to DEN (0.3 mM), wortmannin and/or caffeine for 24 h and subsequently analysed by western blotting employing an antibody specific for Ser15-phosphorylated p53.
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Fig. 9. Inhibition of p53 stabilization induced by DEN using antisense ATM oligonucleotides. Hepatocyte cultures derived from EAF-bearing DEN-rats were treated with DEN and antisense ATM oligonucleotides for 22 h as indicated. Thereafter the percentages of p53-positive cells on the plates were determined. Mean values ± SD for three plates are shown. *P < 0.05 compared with DEN only.
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These results prompted us to study the expression of ATM in EAF tissue. An immunohistological study suggested a lower expression of ATM in most EAF, as compared with the surrounding tissue (Figure 10
). It was noted that most nuclei in EAF tissue (in rats treated 710-fold with DEN) exhibited no staining or a lower staining intensity than nuclei in surrounding tissue, which were fairly even stained. A weak cytoplasmic staining, seen in non-EAF tissue, was absent in EAF tissue. The levels of ATM protein were also quantitated by western blot analysis. This protein was readily detected in tissue free from macroscopic lesions, but not detectable in EAF tissue (Figure 11A
). Similar results were obtained using material immunoprecipitated with two different antibodies directed towards ATM. In contrast, Western blot analysis of the cell cycle regulating p70S6 kinase and a subunit of DNA-PK, Ku-86, revealed no major differences in expression between EAF and non-EAF tissues (Figure 11B
). Employing an immunocomplex kinase assay, phosphorylation of the exogenous substrate, PHAS-I, was found to be lower in ATM immunoprecipitated from EAF than in non-EAF tissue (Figure 11C
). Separation of macroscopic EAF according to size revealed that the largest lesions demonstrate the lowest kinase activity.

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Fig. 10. Reduced immunohistochemical staining for ATM in EAF lesions in DEN-treated rats. Liver section stained for ATM and counter-stained with haematoxylin is shown. (A) Part of an EAF (lower left corner) and surrounding tissue can be seen. (B) The same EAF at higher magnification. Note the ATM immunostaining (brown) in surrounding hepatocytes, where nuclei have a more intense brown staining than the cytoplasm. In EAF hepatocytes the haematoxylin staining (grey/blue) predominates.
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Discussion
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The findings presented here suggest that stabilization of p53 in response to DEN-induced damage in rat hepatocytes is at least partially mediated via a signal transduction pathway dependent on ATM kinase. We have also provided evidence that this pathway is attenuated in EAF hepatocytes and that nuclear accumulation of p53 can be induced readily in EAF cells via other pathways and by stimuli other than DNA damage.
The findings with RTPCR, the CoCl2-induced responses and the effect of LMB all indicate that basal synthesis of the p53 protein occurs in EAF cells. The fact that p53 antisense treatment inhibited CoCl2-induced p53 accumulation in both GST-P-positive and GST-P-negative cells indicates the involvement of de novo protein synthesis rather than, for example, translocation of some postulated cytoplasmic pool of p53 to the nucleus. The effect of LMB supports this conclusion and indicates that the attenuated p53 response is not due to impaired nuclear import.
Phosphorylation of p53 at residue Ser15 has been shown to occur in response to DNA damage (3638). Such Ser15 phosphorylation is catalysed by multiple protein kinases, including ATM, which may be activated by different types of damages (1,6), including those induced by arsenite (37) and cadmium (39). The results obtained here with kinase inhibitors are consistent with an involvement of ATM in our model system. Thus, we found that 150 nM wortmannin inhibited the DEN-induced p53 response in hepatocytes by 50%, a value in good agreement with EC50 values of 200 nM reported in the literature (25). Reported EC50 values for inhibition of DNA-PK and ATR by wortmannin are, on the other hand, 16 nM and 1.8 µM, respectively, making these kinases less likely candidates for involvement here. Our results with caffeine and antisense oligonucleotides also favour an involvement of ATM.
Phosphorylation at residue Ser15 may hinder interaction between p53 and MDM2 (36), even though it has been suggested that Ser20 may be more important in preventing this interaction (40). Our present data do not exclude involvement of other pathways for DEN-induced p53 phosphorylations. In experiments to be published elsewhere, we have examined the effect of a single exposure to DEN on p53 phosphorylations in naïve rats. In this system increased phosphorylation occurs at Ser15 and Ser390, whereas phosphorylation at Ser20 is unaltered. We also detect phosphorylations of MDM2 which are compatible with an involvement of ATM (41).
The kinase assay on immunoprecipitated material from EAF and non-EAF tissue revealed decreased ATM activity in EAF. Furthermore, western blot analysis and immunohistology showed a lower level of ATM protein in EAF tissue. These data, together with the lack of Ser15 phosphorylation of p53 in EAF and the inhibition studies, indicate inhibition of ATM-mediated phosphorylation of p53 in EAF hepatocytes.
In previous studies we have reported both in vivo and in vitro observations which support the conclusion that the attenuated p53 response in EAF hepatocytes cannot be explained solely on the basis of altered xenobiotic metabolism (1519). While the present study was in progress, other investigators reported that the expression of p53 in EAF in vivo in 2-AAF-treated rats is not induced by radiation (42). This observation is in agreement with our findings, since radiation activates p53 without the involvement of xenobiotic-metabolizing enzymes. Our findings presented here provide additional evidence in line with these data and indicate that the attenuated p53 response in EAF cells can, at least in part, be explained by a lower level of ATM activity.
Data presented here suggest that EAF from DEN-rats constitutively express a lower amount of ATM protein than the surrounding tissue. The mechanism remains to be elucidated, but there are physiological alterations that might lead to down-regulation of ATM. For example it has been reported that ATM kinase activity can be inactivated by caspases during or even prior to apoptosis (43,44). Cleavage of ATM by caspases may generate an enzymatically inactive protein, which may bind to DNA and act in a trans-dominant negative fashion to prevent signalling of DNA damage. ATM can also bind to peroxisomes (45), which might prevent its participation in signalling of nuclear DNA damage. More recently it was reported that EGF, via the Sp1 promoter, can down-regulate ATM transcription (46). Whether this is of importance here is not known, but in a previous study we have found that EAF hepatocytes do not respond to EGF-induced DNA replication (20).
It has been suggested that many EAF develop as adaptive lesions in response to toxic stress (47). It is reasonable to postulate that adaptation to genotoxic liver carcinogens may involve an attenuated response to DNA damage, with attenuated activation of p53. The liver plays a central role in the detoxification of such compounds and DNA damage might be more seriously threatening than other acute effects induced by genotoxic carcinogens. A decrease in liver cell mass may result from an arrest in the cell cycle and apoptosis and, if repeated often enough, effects might eventually compromise the animal's capacity to survive toxic stress. From this perspective, attenuation of the signal transduction pathway for DNA damage in EAF cells would seem to be an adequate response. Selective down-regulation of DNA damage signalling to p53 appears more plausible than, for example, blocked p53 synthesis or increased p53 degradation, which might impair functions of this protein without relevance for genotoxic stress. We thus conclude that selective inhibition of ATM activity in EAF hepatocytes supports the hypothesis that many EAF are adaptive precarcinogenic lesions.
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Notes
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1 Email: ulla.stenius{at}imm.ki.se 
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Acknowledgments
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This work was supported financially by the National Institute for Working Life, Sweden.
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References
|
---|
-
Banin,S., Moyal,L., Shieh,S.-Y., Taya,Y., Anderson,C.W., Chessa,L., Smorodinsky,N.I., Prives,C., Reiss,Y., Shiloh,Y. and Ziv,Y. (1998) Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science, 281, 16741679.[Abstract/Free Full Text]
-
Woo,R.A., McLure,K.G., Lees-Miller,S.P., Rancourt,D.E. and Lee,P.W.K. (1998) DNA-dependent protein kinase acts upstream of p53 in response to DNA damage. Nature, 394, 700704.[ISI][Medline]
-
Prives,C. and Hall,P.A. (1999) The p53 pathway. J. Pathol., 187, 112126.[ISI][Medline]
-
El-Deiry,W.S., Harper,W.J., O'Connor,P.M. et al. (1994) WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res., 54, 11691174.[Abstract]
-
Vogelstein,B., Lane,D. and Levine,A.J. (2000) Surfing the p53 network. Nature, 408, 307310.[ISI][Medline]
-
Canman,C.E. and Lim,D.-S. (1998) The role of ATM in DNA damage responses and cancer. Oncogene, 17, 33013308.[ISI][Medline]
-
An,W.G., Kanekal,M., Simon,M.C., Maltepe,E., Blagosklonny,M.V. and Neckers,L.M. (1998) Stabilization of wild-type p53 by hypoxia-inducible factor 1
. Nature, 392, 405408.[ISI][Medline]
-
Goldberg,M.A., Dunning,S.P. and Bunn,H.F. (1988) Regulation of the erythropoietin gene: evidence that the oxygen sensor is a heme protein. Science, 242, 14121415.[ISI][Medline]
-
Wang,G.L. and Semenza,G.L. (1993) Characterization of hypoxia-inducible factor 1 and regulation of DNA binding activity by hypoxia. J. Biol. Chem., 268, 2151321518.[Abstract/Free Full Text]
-
Minchenko,A., Salceda,S., Bauer,T. and Caro,J. (1994) Hypoxia regulatory elements of the human vascular endothelial growth factor gene.Cell. Mol. Biol. Res., 40, 3539.[ISI][Medline]
-
Kamijo,T., Zindy,F., Roussel,M.F., Quelle,D.E., Downing,J.R., Ashmun,R.A., Grosveld,G. and Sherr,C.J. (1997) Tumor supression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF. Cell, 91, 649659.[ISI][Medline]
-
Pomerantz,J., Schreiber-Agus,N., Liégeois,N.J. et al. (1998) The INK4a tumor supressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2's inhibition of p53. Cell, 92, 713723.[ISI][Medline]
-
Pitot,H.C. (1990) Altered hepatic foci: their role in murine hepatocarcinogenesis. Annu. Rev. Pharmacol. Toxicol., 30, 465500.[ISI][Medline]
-
Stenius,U., Warholm,M., Martens,U. and Högberg,J. (1994) Isolation of glutathione S-transferase P-positive hepatocytes from carcinogen-treated rats by use of ethacrynic acid as selecting agent.Carcinogenesis, 15, 15611566.[Abstract]
-
Stenius,U. and Högberg,J. (1995) GST-P-positive hepatocytes isolated from rats bearing enzyme-altered foci show no signs of p53 protein induction and replicate even when their DNA contains strand breaks. Carcinogenesis, 16, 16831686.[Abstract]
-
Lennartsson,P., Stenius,U. and Högberg,J. (1998) Attenuated p53 expression and lack of effect on TGF
on cell replication in enzyme altered foci. Toxicol. In Vitro, 12, 607610.[ISI]
-
van Gijssel,H.E., Stenius,U., Mulder,G.J. and Meerman,J.H.N. (2000) Lack of p53 protein expression in preneoplastic rat hepatocytes in vitro after exposure to N-acetoxy-acetylaminofluorene, X-rays or a proteasome inhibitor. Eur. J. Cancer, 36, 106112.[ISI]
-
Lennartsson,P., Högberg,J. and Stenius,U. (1998) Wild-type p53 expression in liver tissue and in enzyme-altered foci: an in vivo investigation on diethylnitrosamine-treated rats.Carcinogenesis, 19, 12311237.[Abstract]
-
Finnberg,N., Stenius,U. and Högberg,J. (2000) Xenobiotics modulate the p53 response to DNA damage in preneoplastic enzyme-altered foci in rat liver; effects of diethylnitrosamine and phenobarbital. Toxicol. Sci., 54, 95103.[Abstract/Free Full Text]
-
Lennartsson,P., Stenius,U. and Högberg,J. (1999) p53 expression and TGF-
-induced replication of hepatocytes isolated from rats exposed to the carcinogen diethylnitrosamine.Cell. Biol. Toxicol., 15, 3138.[ISI][Medline]
-
Daniel,R., Katz,R.A., Merkel,G., Hittle,J.C., Yen,T.J. and Skalka,A.M. (2001) Wortmannin potentiates integrase-mediated killing of lymphocytes and reduces the efficiency of stable transduction by retroviruses. Mol. Cell. Biol., 21, 11641172.[Abstract/Free Full Text]
-
Martens,U. and Stenius,U. (1999) Immunohistochemical detection of induced expression of wild-type p53 tumor supressor protein in the livers of rats treated with diethylnitrosamine. Histochem. J., 31, 7578.[ISI][Medline]
-
Pandita,T.K., Lieberman,H.B., Lim,D.-S., Dhar,S., Zheng,W., Taya,Y. and Kastan,M.B. (2000) Ionizing radiation activates the ATM kinase throughout the cell cycle. Oncogene, 19, 13861391.[ISI][Medline]
-
Higami,Y., Shimokawa,I., Ando,K., Tanaka,K. and Tsuchiya,T. (2000) Dietary restriction reduces hepatocyte proliferation and enhances p53 expression but does not increase apoptosis in normal rats during development. Cell Tissue Res., 299, 363369.[ISI][Medline]
-
Sarkaria,J.N., Tibbetts,R.S., Busby,E.C., Kennedy,A.P., Hill,D.E. and Abraham,R.T. (1998) Inhibition of phosphoinositide 3-kinase related kinases by the radiosensitizing agent wortmannin. Cancer Res., 58, 43754382.[Abstract]
-
Stenius,U. (1993) Different inhibition of DNA synthesis by transforming growth factor ß and phenobarbital on GST-P-positive and GST-P-negative hepatocytes. Carcinogenesis, 14, 159161.[Abstract]
-
Momand,J., Zambetti,G.P., Olson,D.C., George,D. and Levine,A.J. (1992) The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell, 69, 12371245.[ISI][Medline]
-
Lopes,U.G., Erhardt,P., Yao,R. and Cooper,G.M. (1997) p53-dependent induction of apoptosis by proteasome inhibitors. J. Biol. Chem., 272, 1289312896.[Abstract/Free Full Text]
-
Fornerod,M., Ohno,M., Yoshida,M. and Mattaj,I.W. (1997) CRM1 is an export receptor for leucine-rich nuclear export signals. Cell, 90, 10511060.[ISI][Medline]
-
Wolff,B., Sanglier,J.-J. and Wang,Y. (1997) Leptomycin B is an inhibitor of nuclear export: inhibition of nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 (HIV-1) Rev protein and Rev-dependent mRNA. Chem. Biol., 4, 139147.[ISI][Medline]
-
Roth,J., Dobbelstein,M., Freedman,D.A., Shenk,T. and Levine,A.J. (1998) Nucleo-cytoplasmic shuttling of the hdm2 oncoprotein regulates the levels of the p53 protein via a pathway used by the human immunodeficiency virus rev protein. EMBO J., 17, 554564.[Abstract/Free Full Text]
-
Stommel,J.M., Marchenko,N.D., Jimenez,G.S., Moll,U.M., Hope,T.J. and Wahl,G.M. (1999) A leucine-rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking. EMBO J., 18, 16601672.[Abstract/Free Full Text]
-
Rotman,G. and Shiloh,Y. (1999) ATM: A mediator of multiple responses to genotoxic stress. Oncogene, 18, 61356144.[ISI][Medline]
-
Blasina,A., Price,B.D., Turenne,G.A. and McGowan,C.H. (1999) Caffeine inhibits the checkpoint kinase ATM. Curr. Biol., 9, 11351138.[ISI][Medline]
-
Sarkaria,J.N., Busby,E.C., Tibbetts,R.S., Roos,P., Taya,Y., Karnitz,L.M. and Abraham,R.T. (1999) Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine. Cancer Res., 59, 43754382.[Abstract/Free Full Text]
-
Shieh,S.-Y., Ikeda,M., Taya,Y. and Prives,C. (1997) DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2. Cell, 91, 325334.[ISI][Medline]
-
Yih,L.-H. and Lee,T.-C. (2000) Arsenite induces p53 accumulation through an ATM-dependent pathway in human fibroblasts. Cancer Res., 60, 63466352.[Abstract/Free Full Text]
-
Tibbetts,R.S., Brumbaugh,K.M., Williams,J.M., Sarkaria,J.N., Cliby,W.A., Shieh,S., Taya,Y., Prives,C. and Abraham,R.T. (1999) A role for ATR in the DNA damage-induced phosphorylation of p53. Genes Dev., 13, 152157.[Abstract/Free Full Text]
-
Matsuoka,M. and Igisu,H. (2001) Cadmium induces phosphorylation of p53 at serine 15 in MCF-7 cells. Biochem. Biophys. Res. Commun., 282, 11201125.[ISI][Medline]
-
Hirao,A., Kong,Y.-Y., Matsuoka,S., Wakeham,A., Ruland,J., Yoshida,H., Liu,D., Elledge,S.J. and Mak,T.W. (2000) DNA damage-induced activation of p53 by the checkpoint kinase Chk2. Science, 287, 18241827.[Abstract/Free Full Text]
-
Maya,R., Balass,M., Kim,S.-T., Shkedy,D., Leal,J.-F.M., Shifman,O., Moas,M., Buschman,T., Ronai,Z., Shiloh,Y., Kastan,M.B., Katzir,E. and Oren,M. (2001) ATM-dependent phosphorylation of Mdm2 on serine 395: role in p53 activation by DNA damage. Genes Dev., 15, 10671077.[Abstract/Free Full Text]
-
Van Gijssel,H.E., Ohlson,L.C.E., Torndahl,U.-B., Mulder,G.J., Eriksson,L.C., Porsch-Hällström,I. and Meerman,J.H.N. (2000) Loss of nuclear p53 protein in preneoplastic rat hepatocytes is accompanied by Mdm2 and Bcl-2 overexpression and by defective response to DNA damage in vivo. Hepatology, 32, 701710.[ISI][Medline]
-
Smith,G.C.M., Dádda di Fagagna,F., Lakin,N.D. and Jackson,S.P. (1999) Cleavage and inactivation of ATM during apoptosis. Mol. Cell. Biol., 19, 60766084.[Abstract/Free Full Text]
-
Hotti,A., Järvinen,K., Siivola,P. and Hölttä,E. (2000) Caspases and mitochondria in c-Myc-induced apoptosis: identification of ATM as a new target of caspases. Oncogene, 19, 23542362.[ISI][Medline]
-
Watters,D., Kedar,P., Spring,K., Bjorkman,J., Chen,P., Gatei,M., Birrell,G., Garrone,B., Srinivasa,P., Crane,D.I. and Lavin,M.F. (1999) Localization of a portion of extranuclear ATM to peroxisomes. J. Biol. Chem., 274, 3427734282.[Abstract/Free Full Text]
-
Gueven,N., Keating,K.E., Chen,P., Fukao,T., Khanna,K.K., Watters,D., Rodemann,P.H. and Lavin,M.F. (2001) Epidermal growth factor sensitizes cells to ionizing radiation by down-regulating protein mutated in ataxia-telangiectasia.J. Biol. Chem., 276, 88848891.[Abstract/Free Full Text]
-
Farber,E. (1990) Clonal adaptation during carcinogenesis. Biochem. Pharmacol., 39, 18371846.[ISI][Medline]
Received March 22, 2001;
revised July 9, 2001;
accepted August 24, 2001.