Hypoxia induces p53 through a pathway distinct from most DNA-damaging and stress-inducing agents
Alan Renton,
Susana Llanos and
Xin Lu1
Ludwig Institute for Cancer Research, Imperial College of Science, Technology and Medicine at St Mary's Campus, Norfolk Place, London W2 1PG, UK
1 To whom correspondence should be addressed Email: x.lu{at}ic.ac.uk
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Abstract
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The p53 tumour suppressor gene is a transcription factor that can induce cell cycle arrest and apoptosis. In response to various stress-inducing signals, p53 level increases and this is accompanied with increased activities of p53. Interestingly, the methylxanthine caffeine can abrogate the p53 accumulation induced by certain DNA-damaging agents by an unknown mechanism. In an effort to understand how different signals induce p53, human tumour cell lines were treated with combinations of various stress-inducing agents and caffeine. Caffeine inhibited the accumulation of p53 induced by leptomycin B (LMB), an inhibitor of CRM1, but not N-acetyl-leu-leu-norleucinal, a proteasome inhibitor. Furthermore, caffeine also inhibited the accumulation of p53 by a variety of stress-inducing agents in vivo, such as 5-fluorouracil, doxorubicin, mitomycin C, camptothecin and roscovitine. However, caffeine failed to affect the accumulation of p53 in hypoxia (HYP)-treated cells. These results suggested that HYP must use a distinct pathway from most DNA-damaging and stress-inducing agents to induce p53.
Abbreviations: ACD, actinomycin D; ALLN, N-acetyl-leu-leu-norleucinal; ATM, Ataxia telangiectasia-mutated; CAM, camptothecin; DXR, doxorubicin; HYP, hypoxia; LMB, leptomycin B; UV, ultraviolet
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Introduction
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It has been well established that p53 is primarily regulated through control of its protein level. This is mainly mediated by (h)MDM2, which targets p53 for ubiquitin-mediated proteasomal degradation, maintaining it at low levels in unstressed cells (1,2). A multitude of stress signals, such as the DNA-damaging agents ultraviolet-irradiation (UV-irradiation) and
-irradiation, have been shown to stabilize p53 in the nucleus (36). This nuclear accumulation is necessary for p53 to carry out its functions of cell cycle arrest and/or apoptosis. However, very little is known about the mechanisms by which p53 is accumulated in response to different stress signals. The protein product of the CRM1 gene functions as a Ran-GTP-dependent nuclear export receptor in Schizosaccharomyces pombe (fission yeast) (7) and in humans (8). A Streptomyces-derived antibiotic, leptomycin B (LMB), can specifically block CRM1-mediated nuclear export by binding to CRM1 (7,9). LMB treatment of human cells induces nuclear accumulation of p53 (10). Furthermore, other publications also support the hypothesis that nuclear export of p53 is important for its protein stability (11,12). Therefore, it led us to speculate that stress signals that induce the stabilization of p53 must interfere with the processes involved in the nuclear export and/or protein degradation of p53.
Previous studies have shown that caffeine can abrogate the p53 accumulation induced by a variety of DNA-damaging agents, including
-irradiation (13), UVC-irradiation (14) and the topoisomerase inhibitor camptothecin (CAM) (15). Most important here, temperature-sensitive and caffeine-resistant mutants in S.pombe have been shown to contain a mutation at the CRM1 gene locus (16). Therefore, it is possible that caffeine may alter the CRM1 pathway to inhibit the accumulation of p53 in response to DNA damage. However, caffeine is known to have an array of effects upon cellular metabolism and the cell cycle. Hence, caffeine could also inhibit the accumulation of p53 by enhancing its degradation. The understanding of how caffeine abrogates the accumulation of p53 induced by stress signals may help us to elucidate the mechanisms of p53 stabilization elicited by various stress-inducing agents.
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Materials and methods
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Cell lines and reagents
MCF-7 and U2OS cells were grown in supplemented tissue culture medium (Dulbecco's modified Eagle medium containing 10% fetal calf serum, 100 U/ml penicillin and streptomycin, and 2 mM L-glutamine; all from Gibco BRL, Paisley, UK, except serum, which was from PAA Laboratories, Cambridge, UK) at 37°C, 10% CO2 and subconfluent monolayers were used for experimentation. LMB is a gift from Dr Minoru Yoshida (Department of Biotechnology, Graduate School of Agriculture and Life Sciences, University of Tokyo, Tokyo, Japan). DO1, PC10, SMP14 and SX118 are mouse monoclonal antibodies to human p53, PCNA, mdm2 and p21waf1/cip1. The secondary antibodies used are FITC-conjugated or HRP-conjugated anti-mouse immunoglobulin (Ig) (F3008, Sigma, Poole, UK and P0161 DAKO, Ely, UK, respectively).
Immunofluorescence staining
Cells were treated under the conditions as indicated and then fixed with methanol:acetone 1:1 solution at the indicated time points. For Figure 2B, cells were fixed with 4% paraformaldehyde for 15 min and made permeable with 0.1% Triton X-100 for 4 min. The cells were then stained with DO1 anti-p53 primary antibody and FITC-conjugated anti-mouse Ig secondary antibody. Dishes were mounted with Citifluor shielding agent (Citifluor, London, UK), and immunocomplexes visualized and photographed using a Zeiss Axiophot microscope and camera at x400 magnification. For Figure 2B, the nuclear staining of p53 was visualized and photographed using confocal microscopy.

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Fig. 2. Caffeine partially abrogates LMB-, but not ALLN-induced p53 stabilization in MCF7 and U2OS. (A) Western analysis of the effect of caffeine on p53 stabilization. MCF7 and U2OS cells were treated for 9 h with combinations of 10 ng/ml LMB, 5 mM caffeine, DMSO, 50 µM ALLN, and then harvested. Fifteen micrograms of total protein were separated on 12% gels and subjected to western analysis using DO1 anti-p53 antibody. (B) Immunofluorescence analysis of the effect of caffeine on p53 stabilization. MCF7 cells were treated for 8 h with combinations of ethanol as a negative control, 20 ng/ml LMB, 5 mM caffeine, DMSO (a negative control), and 50 µM ALLN. Fixing and immunofluorescence for p53 were carried out as described in the Materials and methods.
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Immunoblotting
Cells were treated with various agents under indicated conditions and then harvested at the indicated time points. Adhered cells were washed with phosphate-buffered saline, pelleted and protein extracts made in lysis buffer [50 mM Tris pH 7.6, 150 mM NaCl, 5 mM ethylenediaminetetra-acetic acid (EDTA), 1% Nonidet P-40, 5% protease inhibitor cocktail (Roche Diagnostics, Lewes, UK), 10 mM NaF, 1 mM Na3VO4, 10 mM ß-glycerophosphate and 2 mM dithiothreitol (DTT)] on ice. Twenty micrograms total proteins in 5x sample buffer [62.5 mM Tris pH 6.8, 2% sodium dodecyl sulphate (SDS), 10% glycerol, 0.72 M ß-mercaptoethanol and 0.02% bromophenol blue] were separated on a 12% SDS denaturing gel, transferred to nitrocellulose, and subjected to western analysis using antibodies as indicated. HRP-conjugated anti-mouse secondary antibody was used (P0161, DAKO, diluted 1:1000). Detection was carried out using the Enhanced Chemi-Luminenscence (ECL) kit (Amersham Life Science, Little Chalfont, UK).
Treatment of cells with stress-inducing agents and caffeine
MCF7 and U2OS cells were treated for 9 h with combinations of 10 ng/ml LMB, 5 mM caffeine, dimethyl sulphoxide (DMSO) (diluted 1:2000), 50 µM N-acetyl-leu-leu-norleucinal (ALLN), and then harvested. For the other stress-inducing agents, MCF7 cells were treated for 16 h with combinations of 10 µg/ml 5FU, DMSO (diluted 1:86 200), 1 µM doxorubicin (DXR), DMF/HCl (diluted 1:3985), 1 µM actinomycin D (ACD), 5 µg/ml cross-linkers cisplatin (CDDP), 10 µg/ml mitomycin C (MMC), 0.5 µM CAM, 5 µg/ml etoposide (ETOP), 50 µM roscovitine (RSC), 10 J/m2 UVC-irradiation and 5 mM caffeine and then harvested.
For hypoxia (HYP) treatment, cells were exposed to HYP by being sealed in an AnaeroGen 2.5 l jar with an AnaeroGen sachet and an Oxoid anaerobic indicator (Oxoid, Basingstoke, UK), and then incubated at 37°C. Within 2 h the oxygen levels had decreased to <1%, as determined by the indicator.
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Results
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Caffeine inhibits the expression levels of p53 and its target genes induced by LMB, but not by ALLN
As a result of the published link between caffeine-resistance and mutation in the CRM1 gene, we decided to begin by examining what effect caffeine has on LMB-induced p53. Two wild-type p53 expressing cell lines, MCF7 and U2OS, were incubated with 2 ng/ml LMB for durations ranging from 0 to 24 h (Figure 1A and data not shown), as well as with LMB for 24 h at concentrations ranging from 0.2 to 20 ng/ml (Figure 1B and data not shown). In agreement with the results of Freedman and Levine (10) p53 was accumulated in the nucleus of LMB-treated MCF7 and U2OS. Furthermore, the data show that 8 h and 20 ng/ml are the optimal conditions for LMB-induced p53 stabilization, and so these were used for the majority of subsequent experiments. As further supporting evidence, U2OS cells were treated with 20 ng/ml LMB and then harvested for western blot analysis at time points ranging from 0 to 10 h (Figure 1C). In agreement with the above figures, these results show that 8 h treatment with LMB is sufficient to significantly induce the expression of p53.

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Fig. 1. Treatment with LMB induces p53 nuclear accumulation in MCF7 and U2OS. (A) Immunofluorescence time course. MCF7 and U2OS were treated with 220 ng/ml LMB, and then fixed with methanol:acetone at the indicated time points and stained with DO1 anti-p53 primary antibody and visualized with FITC-conjugated anti-mouse Ig secondary antibody (Sigma). (B) Immunofluorescence doseresponse. MCF7 and U2OS cells were treated for 24 h with ETOH as a negative control, or LMB (at the indicated concentrations). Fixing and immunofluorescence for p53 were carried out as described in (A). (C) Western blotting time course. U2OS cells were treated with ethanol, 10 J/m2 UVC-irradiation, or 20 ng/ml LMB, and then harvested at the indicated timepoints. Twenty micrograms of total proteins were separated on a 12% SDS denaturing gel and the presence of p53 was detected by anti-p53 antibody DO.1.
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The proteasome and calpain inhibitor ALLN can induce p53 nuclear accumulation by preventing p53 from degradation (17). As LMB and ALLN stabilize p53 through different pathways, we investigated the ability of caffeine to abrogate p53 accumulation after treatment with LMB or ALLN. MCF7 and U2OS cells were treated with combinations of caffeine and LMB or ALLN, and then harvested for western analysis or fixed and subjected to anti-p53 immunofluorescence. Both LMB and ALLN were able to induce p53 in MCF7 and U2OS cells, and caffeine could only inhibit the accumulation of p53 induced by LMB, but not by ALLN (Figure 2A). Furthermore, caffeine performed this function in a dose-dependent manner: both 2 and 5 mM caffeine could abrogate p53 accumulation, but 5 mM was more effective (data not shown). Thus, 5 mM caffeine was used throughout the study. The immunofluorescence data show that the inhibition of LMB-induced p53 accumulation by caffeine is affected through the reduction of nuclear-accumulated p53 (Figure 2B). Less p53-positive nuclei were seen in those dishes treated with LMB and caffeine, than those treated with LMB alone at the same dose. All these results indicate that caffeine is able to specifically inhibit the accumulation of p53 induced by LMB, but not by ALLN.
The specific effect of caffeine on inhibiting the accumulation of p53 by LMB but not by ALLN is also reflected in the expression of p53 target genes. As a transcription factor, p53 induces the expression of hMDM2 and p21WAF1. Furthermore, LMB is known to stabilize transactivationally active p53 (10). Figure 3A shows that LMB-induced p53 was able to upregulate the expression of p21waf1/cip1, a known transcriptional target of p53, in MCF7 and U2OS. Caffeine reduced this transcriptional upregulation. In addition, caffeine attenuated the level of hMDM2 induced by LMB, but had no effect on that induced by ALLN (Figure 3B). The data are similar to those shown above for p53 stabilization. Interestingly, there is a slight difference in sensitivity to abrogation by caffeine displayed by the MCF7 and U2OS cell lines with regard to p53 induction. The abrogation was more pronounced in MCF7 than in U2OS cells, correlating with the differences in inhibition of p53 levels between these cell lines. A similar pattern was also seen in terms of hMDM2: caffeine seems to have a greater abrogational effect in MCF7 than in U2OS cells. The reason for such difference in response is not known.

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Fig. 3. Caffeine inhibits the expression levels of p53 target genes p21waf1 and mdm2 induced by LMB but not ALLN. (A) Western analysis of the effect of caffeine on p53 and p21 expression. MCF7 and U2OS cells were treated for 9 h with ethanol (diluted 1:500) or combinations of 20 ng/ml LMB and 5 mM caffeine, and then harvested. Twenty micrograms of total protein were separated on a 12% gel and subjected to western analysis using both anti-p53 antibody DO1 and anti-p21 antibody SX118. (B) Western analysis of the effect of caffeine on p53 and hMDM2 expression. MCF7 and U2OS cells were treated for 8 h with combinations of ethanol, 20 ng/ml LMB, 5 mM caffeine, DMSO, 50 µM ALLN and 10 J/m2 UVC-irradiation, and then harvested. Thirty micrograms of total protein were separated on 12% gels and subjected to western analysis using anti-p53 antibody DO1, as well as on 10% gels using anti-hMDM2 antibody SMP14. All blots were then re-probed with anti-PCNA antibody PC10 to check that loading was even.
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Caffeine can inhibit p53 accumulation induced by a variety of DNA-damaging and stress-inducing agents, except HYP
Caffeine has been shown to override the accumulation of p53 induced by certain agents, such as UVC-irradiation (14) and CAM (15), and the ability of caffeine to partially abrogate UVC-induced p53 stabilization in the cell lines used here is shown in Figure 3B. We have already demonstrated that caffeine is able to specifically inhibit the accumulation of p53 induced by LMB but not ALLN, suggesting that stress signals such as UVC and CAM may stabilize p53 by inhibiting its nuclear export through the CRM1 pathway, rather than by inhibiting its degradation in the proteasome. However, these observations could also be explained by the alteration of a process upstream of both nuclear export and proteasomal destruction. To further understand these phenomena, we treated cells with combinations of caffeine and a variety of stress-inducing agents that have been shown to stabilize p53. It is well documented that DNA-damaging agents such as 5-fluorouracil (5FU), DXR and ACD that ultimately lead to the inhibition of DNA and RNA synthesis are able to induce p53 accumulation via ill-defined mechanisms (6). The same can be said for the alkylating agents and CDDP and MMC, as well as agents that do not bind DNA directly or affect nucleotide synthesis, such as the topoisomerase II inhibitor ETOP (6), the topoisomerase I inhibitor CAM (15), and the cyclin-dependent kinase 1 and 2 inhibitor RSC (18).
Therefore, MCF7 cells were treated with combinations of caffeine and either DXR, ACD, 5FU, CDDP, MMC, CAM, ETOP, RSC or UVC-irradiation as a control. As can be seen in Figure 4A, caffeine was able to inhibit the p53 accumulation induced by these agents in MCF7 cells. In terms of UVC- and CAM-stabilized p53, the data agree with those presented by Fallis et al. (14) and Nelson and Kastan (15), respectively. Exposing cells to hypoxic conditions is also known to stabilize p53. Thus, MCF7 cells were treated with combinations of caffeine and HYP or UVC-irradiation as a control. The results (see Figure 4B) show that caffeine had little effect upon HYP-stabilized p53 in MCF7 cells, whereas it inhibited UVC-stabilized p53 under the same conditions. The data suggest that p53 can be stabilized by two types of stress signals. Most stress signals appear to induce p53 accumulation through a pathway sensitive to the inhibitory effect of caffeine. However, HYP is able to stabilize p53 in a caffeine-resistant manner.

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Fig. 4. Caffeine can partially abrogate p53 stabilization induced by a variety of DNA-damaging and stress-inducing agents, except HYP. (A) MCF7 cells were treated for 16 h with combinations of 10 µg/ml 5FU, 1 µM DXR, 1 µM ACD, 5 µg/ml CDDP, 10 µg/ml MMC, 0.5 µM CAM, 5 µg/ml ETOP, 50 µM RSC, 10 J/m2 UVC-irradiation and 5 mM caffeine, and then harvested. The relevant controls for the solvent were included and labelled as CONTROL. (B) MCF7 cells were treated with combinations of 10 J/m2 UVC-irradiation, HYP, and 5 mM caffeine, and then harvested at the indicated time points. For both (A) and (B), 10 µg total protein was separated on 10% gels and subjected to western analysis using anti-p53 antibody DO1.
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Discussion
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The similarity between the effects of caffeine upon LMB-induced p53 and p53 induced by the majority of the stress-inducing agents tested, together with the finding by Kumada et al. (16) that caffeine-resistant S.pombe mutants contain mutations in the yeast crm1 locus, indicated that inhibition of CRM1-dependent nuclear export may be part of the regulatory mechanism that stabilizes p53 response to stress. However, several recent publications have shown the hMDM2-mediated degradation of p53 can occur in both cellular compartments (20,21). Therefore, caffeine may in some way upregulate the degradation of p53, even p53 located in the nucleus. This is consistent with our observation that caffeine inhibited LMB-, but not ALLN-stabilized p53, implying that caffeine induces the degradation of p53 trapped in the nucleus, and that it acts upstream of the inhibitory target of ALLN: calpain and the proteasome. It also agrees with the recent finding that caffeine can inhibit the kinases Ataxia telangiectasia-mutated (ATM) and ATM-related (ATR), both of which phosphorylate the p53 N-terminus in response to DNA damage, contributing to its accumulation (22,23). In addition, caffeine is known to have multiple functions. Therefore, it is possible that caffeine can inhibit the accumulation of p53 by many different pathways.
We do not yet know the precise mechanisms through which caffeine prevents the accumulation of p53 in response to various stress signals. Moreover, the ability of caffeine to inhibit p53 accumulation in response to different DNA-damaging agents differs slightly suggesting that the pathways used by these DNA-damaging agents are not identical. For example, caffeine is more effective to prevent the accumulation of p53 induced by ACD than that induced by camptothecin (CAM). However, the ability of caffeine to inhibit the p53 induction by most of the DNA-damaging and stress-inducing agents tested suggested that a common pathway must be involved. Most interestingly, this caffeine-sensitive pathway was not used by HYP as caffeine failed to inhibit the accumulation of p53 in HYP-treated cells. This observation suggested that under the conditions used in this study, HYP induced p53 through an ATM- and ATR-independent pathway as caffeine is a known inhibitor of ATM and ATR. One possible explanation is that HYP-mediated p53 stabilization is elicited through the accumulation of HYP-inducible factor 1
(HIF1
), which binds p53 and prevents its degradation (24). Taken together, the data presented here provide an interesting model from which to further enhance understanding of the vital processes involved in the accumulation of p53 after exposure to stressful conditions. This understanding is likely to improve our ability to utilize specific molecular therapy to specifically activate p53 in the clinical cancer therapy.
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Acknowledgments
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We thank Dr Minoru Yoshida (Department of Biotechnology, Graduate School of Agriculture and Life Sciences, University of Tokyo, Tokyo, Japan) for kindly donating the LMB (which was dissolved in ethanol and stored at -20°C), and Dr Damian Yap for his help in preparing the manuscript. This work was funded by the Ludwig Institute for Cancer Research.
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Received June 17, 2002;
revised December 23, 2002;
accepted March 3, 2003.