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Mayer Cancer Biology Research Laboratory, Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California 94305-5468 [R. A., C. K., A. J. G.], and Harvard School of Public Health, Department of Cancer Cell Biology, Boston, Massachusetts 02115 [R. K. G., C. G. M.]
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ABSTRACT |
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Introduction |
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In addition to protein accumulation, p53 is also posttranslationally modified and associates with accessory proteins to modulate critical downstream target gene expression. Of the many proposed biological activities attributed to p53, its roles in cell cycle regulation and apoptosis have been well established (5) . For example, in response to DNA damage, p53 activation leads to a G1-phase cell cycle arrest that is mediated by the transcriptional up-regulation of the cyclin-dependent kinase inhibitor, p21 (6, 7, 8) . p53 and p21 have also been implicated in maintaining a G2-phase checkpoint in human cells (9) . In some cell types, transcriptional targets of p53 that include bax, Fas, insulin-like growth factor binding protein-3, and p53 induced genes lead to apoptosis, but the exact function of these proteins in mediating p53-dependent apoptosis is not clear (1 , 10 , 11) . Another p53 transcriptional target is the Mdm2 (mouse double minute 2) oncogene, which through a feedback mechanism regulates p53 activity (12) .
The finding that reinforces the importance of the loss of the tumor suppressor activity of p53 in malignant progression is that over half of all human cancers have been found to harbor mutations in the human p53 gene (13 , 14) . Most but not all changes in p53 arise from a missense mutation in one allele that is then followed by a reduction to homozygosity. Experimentally, transgenic mice hemizygous for p53 were found to have a higher risk of tumorigenesis than their wt counterparts (15) . Interestingly, the tumor suppression activity of p53 can also be overcome by its interaction with the MDM2 protein. This interaction between p53 and MDM2 leads to inactivation of p53 as a transcriptional activator (16) or targets p53 for ub-mediated degradation (17 , 18) . Furthermore, induction of Mdm2 gene expression by p53 in response to DNA-damaging agents, in some cell types, suggests that MDM2 may function in mediating p53 degradation during recovery from stress. Thus, MDM2 and p53 form an autoregulatory loop where p53 can control both its levels and activity through the modulation of its negative regulator, MDM2 (19) .
The "high risk" HPVs are a large group of DNA tumor viruses that infect epithelial tissue and are strongly associated with the development of cervical and anogenital cancer (20) . The HPV E6 and E7 genes are expressed in cells of HPV-associated cancers and cooperate in cell transformation (21 , 22) . Their function in transformation, at least in part, depends on their ability to inactivate the p53 and pRb tumor suppressors, respectively. E7 binds and inhibits pRb (23 , 24) , whereas E6, in a similar manner to MDM2, interacts with and targets p53 for degradation via the ub/proteasome degradation pathway (25) . Ubiquitination of p53 requires the formation of a ternary complex of p53, E6, and the endogenous cellular factor, E6AP (26) . This association ultimately leads to the accelerated degradation of p53, thus reducing the physical and functional presence of p53 within a cell (27) .
In cervical carcinoma, recent studies have shown that hypoxia is an independent prognostic factor of survival in patients, irrespective of treatment modality (28) . Tumor hypoxia occurs in most solid tumors from abnormal vasculature development. As a result, blood flow is highly irregular and torpid (29) . Both local tumor control and overall survival were reduced in patients with hypoxic tumors as compared with those with well-oxygenated tumors. Hypoxia is a physiological modulator of p53 and uses p53 to mediate apoptosis (30) . Increased apoptosis in response to hypoxia has been demonstrated to lead to the selection of a clonal population of cells with inactive or mutant p53 protein (31 , 32) .
Previous studies have indicated that hypoxia induces p53 accumulation in human colorectal carcinoma cell lines that express HPV E6 (30) . Here we investigate the mechanism of p53 induction by hypoxia. We provide evidence indicating that hypoxia down-regulates MDM2 protein levels and thereby reduces p53 nuclear export, which leads to its degradation. Additionally, we demonstrate that hypoxia does not alter the protein levels of E6 or E6AP in E6-expressing cells but rather disassociates their interaction with p53, allowing for its nuclear accumulation. Finally, we show that hypoxic activation of p53 does not require the function of the p14ARF protein, indicating that oncogene-induced stabilization of p53 occurs through a separate pathway than that of hypoxia. Taken together, these results suggest a novel means of hypoxic induction of p53 protein that involves disruption of MDM2-mediated nuclear export and the concurrent inhibition of E6/E6AP-mediated degradation.
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Materials and Methods |
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Immunofluorescent Staining.
For E6GFP detection, 10 µg of plasmid DNA were electroporated into exponentially growing RKO cells at 0.25 kV and 960 mF using a Bio-Rad Gene Pulser (Bio-Rad, Hercules, CA) and plated on Lab-Tek slides (Nunc, Naperville, Ill.). Cells were treated and fixed as described (30)
, and fluorescence was observed 24 h after transfection by fluorescence microscopy. All hypoxia treatments were performed in a hypoxic chamber (<0.2% O2; Sheldon Corp., Cornelius, OR) or in specially designed aluminum hypoxia chambers.
Immunoblotting.
After treatment, cell extracts were prepared in 200 µl of lysis buffer [10 mM Tris (pH 7.5), 1 mM EDTA, 400 mM NaCl, 10% glycerol, 0.5% NP40, 5 mM NaF, 0.5 mM sodium orthovanadate, 1 mM DTT, and 0.1 mM PMSF]. Protein concentrations for each sample were determined using the D/C protein assay kit (Bio-Rad, Hercules, CA) according to the manufacturer’s specifications. Equivalent amounts of protein were analyzed on NUPAGE 10% or 4–12% SDS-polyacrylamide gels (Novex, San Diego, CA). Primary antibodies used for immunoblotting were: DO-1 mouse monoclonal for human p53, SMP-14 mouse monoclonal antibody for human MDM-2 (Oncogene Science); and the M-19 rabbit polyclonal for human p21 (Santa Cruz Biotechnology, Santa Cruz, CA). The rabbit anti-E6AP polyclonal antisera for human E6AP was kindly provided by Dr. Peter Howley (Harvard Medical School, Boston, MA; Ref. 36
). The rabbit anti-ARF and anti-ub antibodies were obtained from Dr. Yue Xiong (University of North Carolina at Chapel Hill, NC; Ref. 37
) and Dr. Arthur L. Haas (Medical College of Wisconsin, Milwaukee, WI; Ref. 38
), respectively. Blots were probed with either an antirabbit or antimouse IgG-horseradish peroxidase-conjugated (Dako, Carpenteria, CA) secondary antibody. Proteins were then detected using the ECL-PLUS Enhanced Chemiluminescence detection system according to manufacturer’s protocol (Amersham Life Science, Arlington Heights, IL).
Immunoprecipitation.
For immunoprecipitation, cell lysates from above were mixed with an equal volume of TEG buffer [10 mM Tris (pH 7.5), 1 mM EDTA, 20% glycerol, 5 mM NaF, 0.5 mM sodium orthovanadate, 1 mM DTT, and 0.1 mM PMSF], incubated overnight at 4°C while rocking with a anti-p53 primary antibody. The anti-p53 mouse monoclonal pAb1801 (Oncogene Science) was used for coimmunoprecipitation of p53 protein with E6AP, whereas the anti-p53 mouse monoclonal pAb421 (Oncogene Science) was used for coimmunoprecipitation of p53 protein with MDM2. Antibody-protein complexes were then captured by the addition of 40 µl of protein A/G-Plus beads (Santa Cruz Biotechnology) for 2 h at 4°C. The immunoprecipitates were then washed four times with 10X buffer [50 mM HEPES (pH 7.9), 250 mM KCl, 0.2% NP40, 0.1% Triton X-100, 0.01% SDS, and 1 mM DTT], resuspended in 30 µl of SDS gel-loading buffer [0.5 M Tris (pH 6.8), 2 M 
-mercaptoethanol, 12% SDS, 30% glycerol, and 0.1% bromophenol blue], and analyzed on 4–12% NUPAGE SDS-polyacrylamide gels as above. For p53, MDM2, and E6AP detection, blots were probed as above.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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. Hypoxia induced p53 protein accumulation in the RKO.E6 cell line, indicating that E6-mediated degradation of p53 in RKO.E6 cells was somehow attenuated under low oxygen conditions. In contrast, treatment with 4 Gy of IR did not affect the ability of E6 to mediate the degradation of p53. Similarly, UV irradiation failed to induce p53 protein accumulation in RKO.E6 cells, also suggesting that targeted degradation of p53 was unaffected by this form of damage. Although hypoxia induces p53 protein accumulation in a variety of cell lines, p53 protein is transcriptionally latent but functional in inducing apoptosis.4
Because most established human cell lines exhibit low levels of apoptosis, we did not detect a significant increase in apoptosis in the RKO or RKO.E6 cell lines under the stress-inducing conditions used in this study. To determine whether the p53 protein induced by hypoxia in the RKO.E6 cell line was functional, we irradiated cells under a low oxygen environment with 4 Gy of ionizing radiation, continued to incubate the cells for an additional 4 h under low oxygen conditions, and then probed for p21 induction (Fig. 1A)
. p21 protein was detected in cells irradiated under hypoxia or after treatment with the proteasome inhibitor but not in cells treated with hypoxia or UV alone. As hypothesized, the p53-dependent induction of p21 in response to IR was intact in the RKO cell line; however, only a minimal induction of p21 occurred in RKO.E6 cells. The increase in p21 protein induced by MG132 in RKO cells did not require functional p53 (39)
. These results demonstrate that hypoxia can induce p53 protein accumulation that can be transcriptionally activated by DNA damage in an E6-expressing cell.
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. The half-life of p53 in nonirradiated RKO cells was between 30 and 60 min. In RKO.E6 cells, p53 protein was undetectable because of E6-mediated degradation (Fig. 1C)
. In contrast, hypoxia increased the half-life of p53 up to 120 min in both RKO and RKO.E6 cells, demonstrating that hypoxia extends the half-life of p53 in both an E6-positive and -negative background. We only observed an increase in p53 half-life in RKO cells in response to IR, whereas levels remained undetectable in RKO.E6-treated cells.
Hypoxia Down-Regulates ARF and MDM2 Protein Levels.
Recent studies have proposed that the regulation of p53 protein stability can be mediated by the p14ARF protein and through an autoregulatory loop with the MDM2 protein (37
, 41)
. To determine a role for ARF or MDM2 in regulating p53 levels in E6-expressing cells, we immunoblotted for each protein in cells treated with hypoxia for 0, 6, 12, or 24 h (Fig. 2A)
. In RKO cells, we observed p53 protein accumulation by 6 h, being maximal at 24 h. Under these same conditions, we found that hypoxia induced a rapid decrease of both ARF and MDM2 proteins. Similarly, ARF and MDM2 also decreased within 6 h in RKO.E6 cells. However, an increase in p53 protein was only detected after 12 h, confirming the observation that basal p53 levels in E6 cells are normally lower than those seen in non-HPV-infected cells. In addition, hypoxia does not affect the expression of ub.
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. E6AP protein levels were detectable and remained unchanged in both cell lines, even after 24 h of treatment. Because reliable E6 antibodies that would detect endogenous E6 protein in RKO cells do not exist, we fused the E6 gene to the NH2-terminus of the GFP and measured GFP expression in transiently transfected RKO cells (Fig. 2, B and C)
. In this system, we observed no change in the expression of GFP fluorescence or protein in aerated or hypoxia-treated cells.
Previous studies have established that neither E6 nor E6AP by itself stably associates with p53 and that E6AP can form a stable complex with E6 in the absence of p53 (27
, 42)
, indicating that only a ternary complex is stable. Because hypoxia did not affect E6GFP or E6AP levels, we determined whether endogenous E6AP could still interact with p53 under hypoxia. We found that E6AP would only coprecipitate with p53 under aerobic growth conditions and after proteasome inhibitor treatment. We observed a substantially reduced interaction of E6AP with p53 under hypoxia (Fig. 2C)
. Taken together, these results suggest that the pathway leading to stabilization of p53 protein by hypoxia in E6-expressing cells does not require the down-regulation of E6 or E6AP protein levels but the inhibition of the formation of E6/E6AP/p53 complexes.
Hypoxia Inhibits MDM2-mediated Degradation of p53.
Recent studies have described a novel nuclear export signal domain in MDM2 that allows it to be exported from the nucleus, suggesting that its ability to shuttle from the nucleus to the cytoplasm allows it to efficiently target p53 for degradation (43
, 44)
. Given this finding and our observation that hypoxia down-regulates MDM2 expression, we investigated whether ectopic expression of hMDM2 and wt p53 in p53/MDM2 null MEFs would still lead to accelerated p53 degradation under hypoxia (Fig. 3A)
. Cotransfection of hMDM2 and p53 lead to a decrease in the amount of p53 detected in these cells under both aerobic and hypoxic conditions. Cotransfection of p53 with E6GFP also led to degradation under both aerobic and hypoxic conditions. Thus, transient overexpression of MDM2 or E6 in cells was sufficient to mediate the degradation of p53 under hypoxia. In contrast, a MDM2 nuclear shuttling mutant, MDM2NES, was unable to degrade p53 under both aerobic or hypoxic conditions, suggesting the ability of MDM2 to export p53 from the nucleus was required for p53 destabilization. As a negative control, we did not observe a decrease in p53 protein levels in cells cotransfected with an MDM2 nuclear localization mutant or a MDM2 mutant incapable of p53 binding. These observations also suggest that the enforced expression of E6 in a cell can overcome hypoxia-mediated uncoupling of p53 from E6/E6AP complexes. We suggest that hypoxia does not alter the ability of the ub/proteasome degradation machinery responsible for the destruction of p53 within a cell, but that hypoxia-induced p53 protein can be attributed in large part to MDM2 down-regulation or destabilization of p53/E6AP complexes.
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. After hypoxia treatment, we did not observe a decline in the extent of p53-ub conjugates, most likely resulting from undetectable MDM2 activity, but there was a substantial induction in the amount of p53-ub conjugates after reoxygenation. At the same time, p53 levels decreased while MDM2 protein returned to normal levels. Thus, upon reoxygenation MDM2 expression returned with a concurrent increase in p53-ub conjugates and a decrease in total p53 levels. As expected, p53-ub conjugates were present after IR but not after UV treatment, although p53 protein levels increased in both lanes whereas MDM2 levels only increased after IR (3)
. These results confirm our observation that simply inhibiting the expression of MDM2 could result in p53 accumulation, whereas increasing the levels of MDM2 results in accelerated p53 degradation. |
Discussion |
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. Because IR-induced p53 is also ARF independent, we propose that the induction of p53 by hypoxia is separable from that of DNA damage agents and mitogenic oncogenes such as E1A or c-Myc (46
, 47)
. Nevertheless, ARF down-regulation is an intriguing observation, and it is conceivable that hypoxia may regulate tumor progression by both down-regulating the ARF tumor suppressor pathway and by selecting for apoptosis-resistant mutants. We have found that hypoxia is a potent inducer of Ser-15 phosphorylation in both E6-positive and -negative cell lines.5 Recent studies have suggested that this modification disrupts the p53/MDM2 interaction, thus leading to p53 stabilization after IR (48) . The decrease in MDM2 levels after hypoxic treatment indicates that phosphorylation of Ser-15 may not be necessary for hypoxia-induced p53 accumulation. However, we did detect a low level of p53-ub conjugate formation, even after 24 h of hypoxia, suggesting that there remains residual MDM2 activity capable of mediating the ubiquitination of p53. Thus, p53 phosphorylation may inhibit the efficient ubiquitination of p53 by residual MDM2 protein.
Ectopic expression of MDM2 under both aerated or hypoxic conditions was sufficient to mediate the degradation of p53 in p53/MDM2 null MEFs (Fig. 3A)
, indicating that the cellular machinery necessary for p53 degradation is still intact under hypoxic conditions. Because the overexpression of a MDM2 export mutant failed to degrade p53, degradation of p53 under hypoxic conditions requires nuclear export. Recently, it has been suggested that E6-mediated degradation of p53 required the nuclear export function of MDM2 (49)
. However, we found that enforced expression of an E6GFP fusion protein was sufficient to mediate the degradation of p53 in cells lacking MDM2. Because the E6GFP fusion protein was both nuclear and cytoplasmic in these overexpression studies and to date no export function has been attributed to E6 or GFP, we believe that E6-mediated degradation of p53 can precede nuclear export of p53. That is, E6 may first interact with E6AP and while in the cytoplasm; before p53 has translocated into the nucleus, E6 may direct E6AP-dependent ubiquitination and degradation of p53. Thus, under normal physiological conditions were MDM2 and E6 protein are functional, hypoxia is able to uncouple p53 from E6-dependent degradation in the cytoplasm and inhibit MDM2 mediated export of p53, which results in p53 accumulation. On the basis of these studies, we propose that the mechanism by which hypoxia induces p53 protein levels involves both the disruption of MDM2-mediated nuclear export and the concurrent inhibition of E6/E6AP-mediated degradation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by NIH Grants RO1 CA64489 and PO1 CA67166. 
2 To whom requests for reprints should be addressed, at Stanford University School of Medicine, Department of Radiation Oncology, CBRL GK220A, Stanford, CA 94350-5468. Phone: (650) 723-7366; Fax: (650) 723-7382; E-mail: giaccia{at}leland.stanford.edu
3 The abbreviations used are: wt, wild type; HPV, human papillomavirus; E6AP, E6-associated protein; MEF, mouse embryonic fibroblast; GFP, green fluorescent protein; ARF, alternative reading frame; Mdm2, mouse double minute 2; hMDM2, human version of Mdm2; ub, ubiquitin; PMSF, phenylmethylsulfonyl fluoride.
4 C. Koumenis et al., submitted for publication.
5 C. Koumenis and R. M. Alarcón, submitted for publication.
Received 10/ 1/99. Accepted 10/29/99.
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150, or 5 µg of E6GFP. Cells were treated with hypoxia for 24 h, and lysates were made in RIPA buffer. Antibody DO-1 was used for immunodetection of p53 protein. B, hypoxia does not alter p53 basal ubiquitination, but reoxygenation induces MDM2 protein and p53-ub conjugate formation. Cell extracts were prepared in RIPA buffer [2 mM Tris (pH 7.5), 5 mM EDTA, 150 mM NaCl, 1.0% NP40, 1.0% deoxycholate, 0.025% SDS, and 1 mM PMSF] and were immunoprecipitated with p53 antibody DO-1. p53-ub conjugates were detected as described