ACCELERATED PUBLICATION
Direct Interactions between HIF-1alpha and Mdm2 Modulate p53 Function*

Delin Chen, Muyang Li, Jianyuan Luo, and Wei GuDagger

From the Institute for Cancer Genetics, and Department of Pathology, College of Physicians & Surgeons, Columbia University, New York, New York 10032

Received for publication, December 16, 2002, and in revised form, February 25, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The p53 tumor suppressor is maintained at low levels in normal cells by Mdm2-mediated degradation and strongly stabilized in response to various types of stress including hypoxia. Although hypoxia-inducible factor 1alpha (HIF-1alpha ) has been implicated to be involved in p53 stabilization, the precise mechanism by which HIF-1alpha regulates p53-mediated function remains unknown. Here, we found that HIF-1alpha directly binds Mdm2 both in vitro and in vivo; in contrast, p53 fails to directly interact with HIF-1alpha in vitro. Interestingly, Mdm2 expression can significantly enhance the in vivo association between p53 and HIF-1alpha , indicating that Mdm2 may act as a bridge and mediate the indirect interaction between HIF-1alpha and p53 in cells. Furthermore, HIF-1alpha protects p53 degradation mediated by Mdm2, and leads to activation of p53-mediated transcription in cells. To elucidate the mechanism of HIF-1alpha -mediated effect, we also found that HIF-1alpha can significantly suppress Mdm2-mediated p53 ubiquitination in vitro and blocks Mdm2-mediated nuclear export of p53. These results have significant implications regarding the molecular mechanism by which p53 is activated by HIF-1alpha in response to hypoxia.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cellular hypoxia is an important phenomenon in developmental biology, normal physiology, and many pathological conditions, including cancer. Hypoxia triggers a multifaceted adaptive response that is primarily mediated by the heterodimeric transcription factor hypoxia-inducible factor (HIF).1 HIF-1 is a heterodimer composed of two subunits, the rate-limiting factor HIF-1alpha and the constitutive expressed HIF-1beta (1, 2). HIF-1beta has been characterized as an aryl hydrocarbon receptor nuclear translocator, and this family of proteins has previously been shown to heterodimerize with the aryl hydrocarbon receptor (3). On the other hand, HIF-1alpha specifically mediates hypoxic responses. In normoxia, HIF-1alpha is maintained at low and often undetectable levels. HIF-1alpha is targeted for degradation by the ubiquitination-proteasome pathway through directly binding to the von Hippel-Lindau tumor suppressor gene (pVHL), which forms the recognition component of an E3 ubiquitin-protein ligase leading to ubiquitination of HIF-1alpha (4-7). Recent reports demonstrate that HIF-1alpha undergoes an iron- and oxygen-dependent modification before it can interact with pVHL. This modification is catalyzed by a specific family of enzymes termed HIF-1alpha -proline hydroxylases (8-10). During hypoxia, this specific hydroxylase is inactive since it requires dioxygen for its activity. As a result, HIF-1alpha accumulates due to the failure of pVHL to recognize its non-hydroxylated form. HIF-1alpha then translocates to the nucleus and dimerizes with the constitutively present HIF-1beta (11).

The importance of the HIF-1alpha response pathway in human tumorigenesis is underscored by the finding that HIF-1alpha is overexpressed in multiple human cancers, because tumor cells, unlike normal cells from the same tissue, are often chronically hypoxic (12). The tumor suppressor protein p53 integrates numerous signals that control cell life and death (13). Wild-type p53 is expressed at low levels in most cells because of its short half-life under normal conditions. In contrast, the p53 protein is stabilized, and its level increases in response to various stresses such as DNA damage, hypoxia, and inappropriate oncogene signaling (14, 15). In its active form, p53 can bind DNA in a sequence-specific manner and activate transcription of target genes. p53 levels are regulated in large part by Mdm2, the product of a p53-inducible gene. Mdm2 can interact with the N terminus of p53, which also contains the major acidic transcriptional activation domain. The interaction between Mdm2 and p53 can inhibit p53 transcriptional activity by interfering with the ability of p53 to contact transcriptional coactivators such as p300/CBP (16). Importantly, Mdm2 binding also promotes the ubiquitination of p53 and its export from the nucleus to the cytoplasm, where p53 is then degraded by cytoplasmic proteasomes (17).

Hypoxic induction of p53 requires concomitant induction of HIF-1alpha , whereby HIF-1alpha can then bind to and stabilize p53 (18-21). However, the molecular mechanism by which HIF-1alpha stabilizes p53 remains unknown. Moreover, it is not clear whether HIF-1alpha interacts with p53 directly despite previous indications that p53 associated with HIF-1alpha in cells. Nevertheless, an array of immobilized peptide assay showed that the core domain of p53 has an affinity for the oxygen-dependent degradation domain of HIF-1alpha (22). As described in this report, we have found a strong interaction between HIF-1alpha and Mdm2, while we failed to detect any direct interaction of p53 with HIF-1alpha . Our data demonstrate that HIF-1alpha regulates p53 activity including stability and nuclear export through interactions with Mdm2. These results provide a potential mechanism for p53 stabilization by HIF-1alpha in response to hypoxia.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Transfection-- p53-null H1299 lung carcinoma cells and mouse embryonic fibroblast cells (MEFs) were cultured in Dulbecco's modified Eagle's medium (Mediatech, Richmond, VA) supplemented with penicillin/streptomycin and 10% fetal bovine serum (Mediatech, VA).

1 × 106 or 2 × 105 cells were plated in 10-cm or six-well plates, respectively, and 24 h later transfections were done by calcium phosphate precipitation procedures. After a 24-h incubation with 20% O2, the cells were harvested.

Plasmids-- Full-length HIF-1alpha DNA was generated by PCR using hemagglutinin-tagged HIF-1alpha from David Livingston (Dana Farber Cancer Institute) and was subcloned into pcDNA3.1/v5-His-Topo (Invitrogen) or p3×FLAG-CMV-14 (Sigma). Green fluorescence protein (GFP)-tagged wild-type p53 was obtained from Yanping Zhang (M. D. Anderson Cancer Center). Plasmid DNA for transfections was isolated using Qiagen plasmid maxi kit (Qiagen).

Recombinant Protein Preparation and Glutathione S-Transferase (GST) Pull-down Assay-- GST fusions of p53 and Mdm2 were expressed in Escherichia coli BL-21 (DE3) (Promega) and induced with 0.1 mM isopropyl-1-thio-beta -D-galactopyranoside. Bacterial pellets were lysed in BC500 (25 mM Tris, pH 7.8, 500 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, 0.2% Nonidet P-40, fresh 1 mM PMSF) with sonication. Levels of expressed GST fusion proteins were estimated by incubation with glutathione-Sepharose beads, washing, and quantification by SDS-PAGE followed by staining with Coomassie Brilliant Blue R-250. Known amounts of bovine serum albumin were used as standards.

35S-Labeled in vitro translated HIF-1alpha was prepared by using the TNT system (Promega). Equal amounts (1 µg) of GST, GST-p53, and GST-Mdm2 immobilized on glutathione-Sepharose beads were incubated with in vitro translated HIF-1alpha in BC200 for 3 h at 4 °C. After washing, the bound proteins were eluted with SDS sample buffer and were separated by SDS-PAGE, followed by autoradiography.

Immunoblotting and Co-immunoprecipitation-- Cells were lysed in FLAG lysis buffer (50 mM Tris, 137 mM NaCl, 10 mM NaF, 1 mM EDTA, 1% Triton X-100, 0.2% Sarkosyl, 1 mM DTT, 10% glycerol, pH 7.8) with fresh protease inhibitors (1 mM PMSF, protease inhibitor mixture (Sigma)). Aliquots (30 µg) of cell extracts were resolved in SDS, 8% polyacrymide gels and then transferred to nitrocellulose membranes in 20 mM Tris-HCl, pH 8.0, 150 mM glycine, 20%(v/v) methanol. Membranes were blocked with 5% (v/v) nonfat dry milk, TBST (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20), incubated with alpha -p53 (DO-1) antibody (Santa Cruz), or anti-GFP (Clontech), and detected with ECL reagents (Amersham Biosciences).

Coimmunoprecipitation assay was performed essentially as described previously (23). In brief, 50 µl of proteasome inhibitor N-acetyl-leucyl-leucyl-norleucinal was added to the cotransfected culture 6 h before harvest. 24 h after transfection, the cells were lysed in BC100 (25 mM Tris, pH 7.8, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, 0.2% Nonidet P-40, fresh 1 mM PMSF) and incubated with anti-FLAG M2 beads (Sigma) overnight at 4 °C. The beads were washed five times with 1 ml of lysis buffer, after which the associated proteins were eluted with BC100, 0.2% Nonidet P-40 plus 0.2 mg/ml FLAG peptide (Sigma). The eluted proteins were resolved on 8% SDS-PAGE and Western blot with the anti-p53 or anti-Mdm2 (SMP14, Santa Cruz) monoclonal antibody.

Luciferase Assay-- Luciferase activity was determined using a dual luciferase assay system (Promega) following the manufacturer's protocol. Cells in six-well plates were removed by scraping into 100 µl of reporter lysis buffer. Cell lysate was collected by centrifugation for 15 min at 12,000 × g. Luciferase activity was measured using a Lumat LB 9507 luminometer (EG&G Wallac, Gaithersburg, MD).

In Vitro Ubiquitination Assays-- The in vitro ubiquitination assay was performed as previously described (24) with some modifications. For a standard reactions, the purified FLAG-p53 proteins from H1299 cells were mixed with other purified components, including E1, E2 (GST-UbcH5C), E3 (GST-Mdm2), and His-ubiquitin in reaction buffer (40 mM Tris, 5 mM MgCl2, 2 mM ATP, 2 mM DTT, pH 7.6). The reaction was stopped after 60 min at 37 °C by additions of SDS sample buffer and subsequently resolved SDS-PAGE gels for Western blot analysis with alpha -p53 (DO-1).

Nuclear Export Assay for p53-- H1299 cells were plated on six-well-containing glass coverslips, and GFP-p53 was transfected as described. 50 µM proteasome inhibitor N-acetyl-leucyl-leucyl-norleucinal was added for 6 h before fixation. Twenty-four hours after transfection, cells on the coverslips were washed three times with phosphate-buffered saline (PBS) and then fixed in 4% paraformaldehyde for 10 min at room temperature. After fixation, cells were washed in PBS three times and then permeabilized in ice-cold PBS containing 0.2% Triton X-100 for 10 min. Cells were blocked in PBS containing 1% bovine serum albumin and 1 µg of DAPI (Sigma)/ml at room temperature for 30 min. Cells were washed three times with PBS, and the stained cells were mounted with mounting medium (Polysciences, Inc., Warrington, PA) and sealed with nail polish. Immunofluorescence was recorded using an immunofluorescence microscope.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Interaction between HIF-1alpha and Mdm2-- Although earlier studies indicated that p53 can bind HIF-1alpha in cells, we repeatedly failed to detect any direct interaction between these two protein in a GST pull-down assay (data not shown), suggesting that HIF-1alpha may interact with p53 through other factors. To examine the notion that p53 may interact with HIF-1alpha through Mdm2, we tested whether HIF-1alpha directly binds Mdm2 in vitro by GST pull-down assays. Both GST-Mdm2 and GST were expressed in bacteria and purified to near homogeneity. As shown in Fig. 1a, the 35S-labeled in vitro translated HIF-1alpha strongly bound to immobolized GST-Mdm2 (lane 2) but not to immobilized GST (lane 3). However, using the same assay, no significant interaction was detected between GST-p53 and HIF-1alpha . Furthermore, we tested the interaction between HIF-1alpha and Mdm2 in vivo by coimmunoprecipitation assays. FLAG-tagged full-length HIF-1alpha was transiently co-transfected with Mdm2 in H1299 cells. Immunoprecipitations were performed with anti-Flag M2 beads, and the precipitated proteins were analyzed by Western blot with an anti-Mdm2 antibody. As shown in Fig. 1b, the Mdm2-HIF-1alpha complexes were readily detected in cells cotransfected with FLAG-HIF-1alpha and Mdm2 (lane 4) but not by Mdm2 alone (lane 3), indicating that there is a specific in vivo interaction between Mdm2 and HIF-1alpha .


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Fig. 1.   HIF-1alpha interacts with Mdm2 in vitro and in vivo. a, the in vitro interaction of Mdm2 and HIF-1alpha . The GST (lane 3) and GST-Mdm2 (lane 2) fusion protein were used in a GST pull-down assay with in vitro translated 35S-labeled full-length HIF-1alpha . b, Mdm2 interacts with HIF-1alpha in vivo. Western blot analysis of whole cell extracts (lanes 1 and 2) or immunoprecipiates with the anti-FLAG M2 beads (IP/M2) (lanes 3 and 4) from cells cotransfected with FLAG-HIF-1alpha (10 µg) and Mdm2 (10 µg) (lanes 2 and 4) or Mdm2 alone (lane 1 and 3) by anti-Mdm2 antibody.

Mdm2 Enhances the in Vivo Binding between p53 and HIF-1alpha -- To examine the possibility that the HIF-1alpha -p53 interaction is mediated by Mdm2, we tested whether Mdm2 expression is required for the HIF-1alpha -p53 interaction in cells. p53 and FLAG-HIF-1alpha were transiently transfected with or without Mdm2 in H1299 cells. Immunoprecipitations were carried out from cell extracts using anti-Flag M2 beads. As shown in Fig. 2, p53 was barely detectable in the HIF-1alpha -associated immunocomplexes in the absence of Mdm2 expression (lane 5), further confirming the idea that p53 cannot directly bind HIF-1alpha in vivo. In contrast, when Mdm2 was expressed in the cells, p53 was efficiently coprecipitated with HIF-1alpha by the same method (lane 6). These results indicate that Mdm2 acts as a bridge mediating the binding between HIF-1alpha and p53.


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Fig. 2.   Mdm2 enhances the binding between p53 and HIF-1alpha . Western blot analysis of whole cell extracts (lanes 1-3) or immunoprecipites with the anti-FLAG M2 beads (IP/M2) (lanes 4-6) from cells cotransfected with FLAG-HIF-1alpha (10 µg) and p53 (5 µg) (lanes 2 and 5), plus Mdm2 (5 µg) (lanes 3 and 6) or p53 alone (lanes 1 and 4) by anti-p53 monoclonal antibody (DO-1).

HIF-1alpha Abrogates Mdm2-mediated p53 Degradation and Transcription Repression-- Mdm2 is a key regulator of p53 and can both inhibit p53 transcriptional activity and target it for degradation (24). We tested the possibility whether HIF-1alpha could protect p53 from degradation mediated by Mdm2. The p53-null H1299 cells were transfected with CMV-p53, CMV-Mdm2, CMV-GFP, and pTOPO-HIF-1alpha . 24 h after transfection the cells were lysed in a FLAG lysis buffer and analyzed by Western blot. As shown in Fig. 3a, HIF-1alpha effectively rescues p53 from Mdm2-mediated degradation. Overexpression of Mdm2 significantly induced p53 degradation (lane 2 versus lane 1), whereas this degradation was inhibited in a dose-dependent manner upon overexpression of HIF-1alpha (lanes 3 and 4 versus lane 2). Interestingly, HPV E6 protein can induce p53 degradation through the E6/E6 AP ubiquitin ligase complex. However, overexpression of HIF-1alpha cannot protect p53 degradation mediated by E6 (Fig. 3b). Taken together, these results demonstrated an effect of HIF-1alpha directly on Mdm2-mediated degradation of p53. Furthermore, we used p53 transcriptional activity assay to support the notion. To this end, we cotransfected MEFs (p53-/-) with vectors expressing p53, Mdm2, and HIF-1alpha , along with a reporter construct containing the minimal p21 promoter (p21min-luc). As indicated in Fig. 3c, cotransfection of Mdm2 with p53 strongly repressed p21 luciferase activity due to p53 degradation. However, HIF-1alpha significantly abrogated the inhibitory effect of Mdm2 in a dose-dependent manner. The above data demonstrate that HIF-1alpha can strongly stabilize p53 by abrogating Mdm2-mediated effects, which leads to activation of p53-mediated transcription.


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Fig. 3.   a, protection of p53 from Mdm2-mediated degradation by HIF-1alpha . Western blot analysis of extracts from cells transfected with p53 (lane 1) or cotransfected with p53 (1 µg) and Mdm2 (2 µg) (lane 2) or in combination with different amounts of HIF-1alpha (lanes 3 and 4), by the anti-p53 monoclonal antibody (DO-1). The CMV-GFP expression vector was included in each transfection as a transfection efficiency control, and the levels of GFP were detected with the anti-GFP monoclonal antibody (JL-8, Clontech). b, failure to protect E6-mediated p53 degradation. Western blot analysis of extracts from cells transfected with p53 (lane 1) or cotransfected with p53 (1 µg) and E6 (0.5 µg) (lane 2) or in combination with different amounts of HIF-1alpha (lanes 3 and 4). c, abrogation of Mdm2-mediated repression of p53-dependent transcription activation. MEF (p53-/-) cells were transfected with p53 alone (0.25 µg) or cotransfected with p53 (0.25 µg) and Mdm2 (0.5 µg) or in combination with indicated amount of HIF-1alpha expression vector (µg) together with the p21-Luc reporter construct (2 µg). d, HIF-1alpha suppresses Mdm2-mediated p53 ubiquitination. The ubiquitination reactions were performed in the absence of Mdm2 as control (lane 1), with Mdm2 (lane 2), and Mdm2 plus HIF-1alpha (lane 3).

HIF-1alpha Suppresses p53 Ubiquitination Mediated by Mdm2-- To investigate whether HIF-1alpha can suppress p53 ubiquitination mediated by Mdm2, we carried out an in vitro ubiquitination assay. As shown in Fig. 3d, ubiquitinated p53 was easily detected at the reaction with Mdm2 but no HIF-1alpha (lane 2 versus lane 1). However, in the presence of HIF-1alpha , the levels of ubiquitinated p53 decreased dramatically (lane 3 versus lane 2). This result demonstrates that HIF-1alpha can significantly suppress Mdm2-mediated p53 ubiquitination.

HIF-1alpha Blocks Nuclear Export Mediated by Mdm2-- Current models indicate that Mdm2 can also induce nuclear export of p53 (25, 26). To test the possibility that HIF-1alpha can block this export, we transfected H1299 cells with vectors expressing GFP-p53, Mdm2 or HIF-1alpha and examined them by immunofluorescence microscope (Fig. 4a). The distribution of GFP-p53 was almost entirely nuclear when expressed alone, but it localized to the cytoplasm to varying extents when coexpressed with Mdm2. We scored the extent by which p53 localized to the cytoplasm when expressed alone or when expressed together with Mdm2 (Fig. 4b). Cells cotransfected with p53 and Mdm2 had a 76% cytoplasmic expression versus 24% when p53 was transfected alone, indicating that nuclear Mdm2 can promote the cytoplasmic localization of p53 (25, 26). Interestingly, only 26% of cytoplasmic expression occurred in cells cotransfected with HIF-1alpha in addition to p53 and Mdm2 (Fig. 4, a and b). These results indicate that HIF-1alpha can strongly block Mdm2-mediated p53 nuclear export.


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Fig. 4.   HIF-1alpha blocks p53 nuclear export mediated by Mdm2. a, subcellular localization of GFP-p53 in H1299 cells transfected with GFP-p53 alone (top row), GFP-p53 + Mdm2 (middle row), and GFP-p53 + Mdm2 + HIF-1alpha (bottom row). GFP-p53 images are shown in the left column, costaining with DAPI in the middle column, and merge of GFP-p53 and DAPI in right column. b, the proportion of cells expressing cytoplasmic p53 following transfection of GFP-p53, Mdm2, or HIF-1alpha in H1299 cells. 150 cells were scored, and data from three independent experiments were averaged.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

How cells sense changes in ambient oxygen is a central question in biology. In mammalian cells, lack of oxygen, or hypoxia, leads to stabilization of a sequence-specific DNA binding transcriptional factor called HIF. Downstream genes of HIF are linked to processes such as angiogenesis and glucose metabolism (20). On the other hand, hypoxia induces accumulation of the tumor suppressor p53. Earlier studies indicated that HIF-1alpha interacts with p53 in vivo (18); however, the nature of this interaction has not been elucidated. In this study, we demonstrate for the first time that HIF-1alpha directly binds to Mdm2 both in vitro and in vivo. In the absence of Mdm2, the binding between HIF-1alpha and p53 is almost undetectable. Furthermore, Mdm2 expression significantly induces the indirect interaction between p53 and HIF-1alpha in cells, indicating that Mdm2 may act as a bridge mediating the p53-HIF-1alpha interaction. In this regard, we failed to detect any significant interaction between p53 and HIF-1alpha in MEF Mdm2-/- cells, and stabilization of p53 induced by HIF-1alpha expression was also severely abrogated (data not shown). Our findings seem at odds with the recent report of tight binding between the ODD domain of HIF-1alpha and the core domain of p53 by an array of immobilized peptide assay (22). It is likely that the experiment was based on p53 pepetide fragments, which may not be equivalent to the native, folded protein. However, the existence of Mdm2 somehow might cause the conformational change of p53 native protein, thereby favoring the exposure of binding sites of p53 to HIF-1alpha .

In addition, our results demonstrate that HIF-1alpha protects p53 from degradation mediated by Mdm2 and can abrogate p53 transcriptional repression by Mdm2. Considering that p53 degradation is mainly induced by Mdm2 in normal cells, we also found that Mdm2-mediated ubiqutination of p53 is significantly inhibited by HIF-1alpha . Since Mdm2-mediated p53 ubiquitination promotes its nuclear export (25, 26), we further demonstrate that HIF-1alpha expression can block Mdm-2-mediated nuclear export of p53. Thus, these results have significant implications regarding the molecular mechanism by which HIF-1alpha modulates p53 function in vivo.

Our results are also consistent with published results indicating HIF-1alpha interacts with the wild-type p53 protein but not the tumor-derived p53 mutant form in cells (18). Since wild-type p53 protein is capable of inducing Mdm2 expression in cells, the observed interaction between p53 and HIF-1alpha is most likely mediated by endogenous Mdm2. In contrast, because the tumor-derived p53 mutant is completely inactive in transcriptional activation of endogenous Mdm2, HIF-1alpha failed to interact with p53 since there is no (or very low levels) Mdm2 in cells expressing mutated p53. Recent studies also indicate that Mdm2 is involved in modulating HIF-1alpha stability under hypoxic conditions (21), further supporting the notion that HIF-1alpha directly interacts with Mdm2 but not p53. Mdm2 is a potent E3 ubiquitin ligase and induces both p53 degradation and nuclear export of p53 through ubiquitination. Therefore, it is possible that Mdm2 also directly mediates degradation and nuclear export of HIF-1alpha . Our study, together with several other studies (15, 18, 21), strongly implicates the important role of HIF-1alpha in the regulation of the p53-Mdm2 pathway in response to hypoxia.

    ACKNOWLEDGEMENTS

We thank C. Brooks for carefully reading the manuscript and other members of W. Gu laboratory for sharing unpublished data and critical comments. We thank David Livingston and Yanping Zhang for providing plasmids.

    FOOTNOTES

* This work was supported in part by grants from the Leukemia & Lymphoma Society, Avon Foundation, the Stewart Trust, the Irma T. Hirschl Trust, and National Institutes of Health/NCI (to W. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Leukemia & Lymphoma Society Scholar. To whom correspondence should be addressed: Berrie Research Pavilion, Rm. 412C, Inst. for Cancer Genetics, Columbia University, 1150 St. Nicholas Ave., New York, NY 10032. Tel.: 212-851-5282 (office) or 212-851-5285/5286 (laboratory); Fax: 212-851-5284; E-mail: wg8@columbia.edu.

Published, JBC Papers in Press, February 26, 2003, DOI 1074/jbc.C200694200

    ABBREVIATIONS

The abbreviations used are: HIF, hypoxia-inducible factor; E3, ubiquitin-protein isopeptide ligase; MEF, mouse embryonic fibroblast cell; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline; DAPI, 4',6-diamidino-2-phenylindole; CMV, cytomegalovirus.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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