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Cancer Research 67, 450-454, January 15, 2007. doi: 10.1158/0008-5472.CAN-06-2710
© 2007 American Association for Cancer Research
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Nutlin3 Blocks Vascular Endothelial Growth Factor Induction by Preventing the Interaction between Hypoxia Inducible Factor 1{alpha} and Hdm2

Gretchen A. LaRusch1, Mark W. Jackson2, James D. Dunbar3, Robert S. Warren4, David B. Donner4 and Lindsey D. Mayo1

1 Departments of Radiation Oncology and Pharmacology, Case Comprehensive Cancer Center, Case Western Reserve University, 2 Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic Foundation, and Case Comprehensive Cancer Center, Cleveland, Ohio; 3 Department of Microbiology and Immunology, Indiana University School of Medicine, and the Walther Cancer Institute, Indianapolis, Indiana; and 4 Department of Surgery, University of California San Francisco, San Francisco, California

Requests for reprints: Lindsey D. Mayo, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202. Phone: 317-278-3173; Fax: 317-278-5413.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Hdm2 is elevated in numerous types of malignancies and is thought to impede the function of wild-type p53. Reactivation of p53 by disrupting the association with Hdm2 was the impetus for the development of Nutlin3. Although regulation of p53 has been the central focus of Hdm2 activity, it also binds other proteins through its p53-binding domain. Here, we show that hypoxia-inducible factor 1{alpha} (HIF1{alpha}) binds to Hdm2 in the domain designated to bind p53. HIF1{alpha} and p53 share a conserved motif that is required to bind Hdm2. Distinct complexes form between Hdm2-HIF1{alpha} and Hdm2-p53 as determined by immunoprecipitation of nuclear extracts and in vitro. The Hdm2 antagonist Nutlin3 prevents the association between Hdm2 and HIF1{alpha}. The vascular endothelial growth factor (VEGF) gene is a transcriptional target of HIF1{alpha}, and under normoxic or hypoxic conditions, Hdm2 increases HIF1{alpha} activity to induce VEGF production. Blocking the association of Hdm2 and HIF1{alpha} by Nutlin3, or ablating Hdm2 expression, diminished the level of VEGF under conditions of normoxia or hypoxia. Our findings establish a unique role for Nutlin3 in attenuating VEGF induction by preventing the association of Hdm2 with HIF1{alpha}. [Cancer Res 2007;67(2):450–4]


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
The p53 tumor suppressor protein is a transcription factor that regulates numerous genes, the gene products of which govern DNA repair, cell cycle arrest, and apoptosis. Loss of p53 activity by point mutation or loss of heterozygosity is evident in a majority of human cancers supporting the notion that loss of function results in tumor progression. The tumors that retain wild-type p53 gene have circumvented normal mechanisms to activate p53. One mechanism of biochemically inactivating p53 involves p53 inducing the Mdm2 gene. Elevated Mdm2 protein binds to p53 to inactivate its transcriptional activity and facilitate the destabilization of p53. This interplay of p53 and Mdm2 has been characterized as the p53-Mdm2 autoregulatory feedback loop (1). Posttranslational modifications of Mdm2 play a dynamic role in regulating the ability of Mdm2 to destabilize p53 (2–4). Since the identification of the major role of Mdm2 in the regulation of the p53 function and considering that Mdm2 may be elevated by other factors in the tumor to inactivate p53 (5, 6), intense investigation has been undertaken to identify molecules that protect p53 from Mdm2. Several compounds have been identified that prevent the association between p53 and Mdm2 (Nutlin3 and Rita; refs. 7–9). In addition, compounds that target the ubiquitin ligase domain of Mdm2 can also protect or engage p53 activity (10).

Mdm2 will interact with numerous proteins, yet a select few interact within the p53-binding domain. Hypoxia-inducible factor 1{alpha} (HIF1{alpha}) has been shown to form a complex with Mdm2 or its human homologue Hdm2 (11, 12). In one report, increased association of HIF1{alpha}-Hdm2 protected p53 from Hdm2-mediated degradation (13). HIF1{alpha} is a transcription factor that regulates genes, the products of which are involved in metabolism and angiogenesis (11, 12). Hdm2-HIF1{alpha} association increases the induction of the vascular endothelial growth factor (VEGF).

We show that the p53-binding domain of Hdm2 associates in the NH2 terminus of HIF1{alpha},which shares a highly conserved motif with p53. HIF1{alpha} and Hdm2 form a distinct nuclear complex independent of p53. Furthermore, the Hdm2 antagonist Nutlin3 prevents the association of Hdm2 and HIF1{alpha} and inhibits the induction of VEGF. This work shows that Nutlin3 regulates the activities of Hdm2 involved in inducing the proangiogenic factor VEGF.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Cell culture. Cells were cultured in DMEM supplemented with 10% fetal bovine serum at 37°C under 5% CO2. For growth under hypoxia, the media were changed to DMEM buffered with HEPES to maintain pH in the absence of CO2, and oxygen was displaced with N2 using a pro-oxy 100 regulator. Cells were cultured under 1% oxygen for 24 h.

Immunoprecipitation and Western blotting. Antibodies to HIF1{alpha} (Novus Biologicals, Littleton, CO), Cdc2, p53 (Santa Cruz Biotechnology, Santa Cruz, CA), and Hdm2 (2A10 and IF2, Calbiochem, La Jolla, CA), were used for immunoprecipitations from 100 µg of nuclear extract isolated using the per-NUC/Cyto kit (Pierce, Rockford, IL). Briefly, 100 µg of nuclear extract was added to the antibody preadsorbed to protein A/G agarose, and the volume of the incubate was adjusted to 1 mL using the NP40 lysis buffer [50 mmol/L Tris (pH 7.6), 150 mmol/L NaCl, 0.5% NP40, 10 mmol/L sodium PPi, 10 mmol/L sodium fluoride, 1 mmol/L sodium orthovanadate, supplemented with 10 µg/mL aprotinin, leupeptin, and pepstatin A and 2 mmol/L phenylmethylsulfonyl fluoride]. After 1 h of incubation at 4°C, the incubates were centrifuged, the supernatant was siphoned, and 1 mL NP40 lysis buffer was added to the pellet, which was rotated at 4°C. This wash procedure was repeated thrice, and then Western blots were prepared. Blots were probed with a cocktail of 2A10, SMP14, and IF-2 for Hdm2, or with VEGF or glyceraldehyde-3-phosphate dehydrogenase antibodies, followed by secondary antibody coupled to horseradish peroxidase and detected by chemiluminescence reagent.

Recombinant proteins. HIF1{alpha} and Hdm2 (V5 tagged) were produced using a rabbit reticulocyte lysate system (Promega, Madison, WI), except that the amino acid mixes were combined (cysteine and methonine) such that the reactions produced nonradiolabeled products. The proteins were mixed, and Hdm2 was purified by immunoprecipitation using a V5 antibody and washed with the buffers described above. HIF1{alpha} cDNA was digested with Sma1-Sal1 restriction enzymes. The Sma1-Sal1 fragment was ligated in frame into the pGEX4t-1 glutathione S-transferase (GST) expression vector. The Quik change site-directed mutagenesis kit (Stratagene, La Jolla, CA) mutated phenylalanine 37 to tyrosine in HIF1{alpha}. Mutagenesis was confirmed by sequencing. GST, GST-HIF1{alpha}, or GST-F37Y proteins were produced in bacteria and purified with glutathione beads and then incubated with recombinant human Hdm2, produced as previously described (14), and washed with PBS, and Western blot analysis was conducted.

Gene reporter assays. LipofectAMINE was used to transfect cells with Rous sarcoma virus ß-galactosidase construct and vegf or epo promoters upstream of the luciferase gene, generous gifts from Amato Giaccia (Stanford University, Stanford, CA) and Gregg Semenza (Johns Hopkins University, Baltimore, MD), respectively. Thirty-six hours after transfection, the cells were harvested, and luciferase and ß-galactosidase were assayed in a luminometer. The ratio of luciferase to ß-galactosidase activity was calculated, and fold induction was determined relative to control. Each data point represents the mean of results from three independent transfections.

shRNA to Hdm2 in U87 cells. The virus was produced as previously described (15). U87 cells were infected with three rounds of virus encoding scrambled (shScr) or two sequences directed to Hdm2 (ShA/C; plasmids encoding the shRNA were a generous gift from Richard Iggo, Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland) and selected with puromycin. The population of puromycin-resistant cells was used to conduct experiments.


   Results and Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Hdm2 forms distinct complexes with p53 and HIF1{alpha}. Several groups have reported the association of Hdm2 and HIF1{alpha}; however, the domains or motifs required for the association were not determined. To determine the domain required for the association of HIF1{alpha} and Hdm2, carboxyl-terminal truncations of Hdm2 ({Delta}Ring finger, {Delta}Zinc finger), and the 110 amino acid p53 interaction domain of Hdm2 were produced in a rabbit reticulocyte lysate system (Fig. 1A ). The data in Fig. 1A show that HIF1{alpha} binds to the to p53-binding domain of Hdm2 (Fig. 1A).


Figure 1
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  		Figure 1. Mapping the HIF1{alpha}-Hdm2 interaction. A, rabbit reticulocyte system produced full-length and deletion mutants of Hdm2 and HIF1{alpha}. HIF1{alpha} was mixed with either Hdm2 or the deletion mutants, and an Hdm2 antibody was used to immunopurify Hdm2. Row 1, input HIF1{alpha}; rows 2–5, the immunoprecipitated Hdm2 (V5 epitope). Western blot analysis detected HIF1{alpha}(top) Hdm2 or the deletion mutants of Hdm2 (bottom). The schematic of Hdm2 showing the ring finger domain, zinc finger domain, the nuclear import and export sequences (NLS-NES), and the p53-binding domain and carboxyl-terminal deletions of Hdm2. B, sequence alignment of E2F, p73, p53, and HIF1{alpha}. Bold letters, conserved amino acids. The schematic of the first 245 amino acids of HIF1{alpha} shows the DNA binding domain (DBD), the helix-loop-helix domain (HLH), and Per-ARNT-Sim (PAS) domains I and II. GST, GST-HIF1{alpha}, and F37Y HIF1{alpha} bound to glutathione beads were incubated with recombinant human Hdm2, washed, and processed for Western blot analysis.

 
Because Hdm2-p53 interaction requires a defined amino acid sequence in p53, we compared the amino acid sequences of HIF1{alpha} and p53. p53 and other proteins (p73 and E2F) that bind to the NH2 terminus of Hdm2 share a conserved motif (Fig. 1B; ref. 16). Upon examination of the HIF1{alpha} amino acid sequence, we identified the conserved motif in the NH2 terminus (Fig. 1B). For the p53-Hdm2 interaction, phenylalanine 19 of p53 inserts into the hydrophobic pocket of Hdm2 and makes van der Waals contacts with isoleucine 61 and glycine 58 (16). Because phenylalanine is important for the p53-Hdm2 association, we generated a HIF1{alpha} mutant whereby phenylalanine 37 was changed to a tyrosine (F37Y). The first 245 amino acids of HIF1{alpha} and F37Y were produced as GST fusion proteins and incubated with recombinant Hdm2. Western blot analysis showed that Hdm2 bound GST-HIF-1{alpha}, but not GST or GST-F37Y (Fig. 2B ), demonstrating the dependence of the conserved motif in HIF1{alpha}.


Figure 2
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  		Figure 2. Characterization of p53, HIF1{alpha}, and Hdm2 complexes in vitro and in vivo. A, recombinant p53 (1 µg, 100 pg, and 10 pg) was preincubated with Hdm2 and then added to GST-HIF1{alpha}. Complexes were purified by glutathione beads and analyzed by Western blot. B, Cdc2, HIF1{alpha}, and Mdm2 were immunoprecipitated from nuclear extracts from p53–/– MEFs. Western blot analysis was done to detect HIF1{alpha} and Mdm2 (left). HEPG2 (middle) nuclear extracts were used to immunoprecipitate and Western blot for Hdm2, HIF1{alpha}, and p53. Nuclear extracts were immunoprecipitated for insulin receptor, HIF1{alpha}, or p53. Nuc is input of 50 µg of nuclear extracts from MCF7 (right). Western blot analysis was used to detect HIF1{alpha}, p53, and Hdm2 immunocomplexes. C, immunopurified HIF1{alpha} isolated from nuclear extracts in p53–/– or wild-type MEFs, H1299 cells, or T47D cells under hypoxic conditions (1% oxygen for 24 h). Western blots were prepared and probed for Hdm2 and HIF1{alpha}.

 
To show that p53 and HIF1{alpha} compete for the same binding site in Hdm2, we did in vitro binding assay. Recombinant Hdm2 was preincubated with 10 pg, 100 pg, or 1 ng of recombinant p53. The Hdm2-p53 reactions were then added to GST-HIF1{alpha}-glutathione beads. The data in Fig. 2A show by Western blot analyses that p53 binding to Hdm2 prevents the association with HIF1{alpha}, further demonstrating the competition of both proteins for binding to Hdm2 in the p53 interaction domain.

To examine the complex formation of HIF1{alpha} bound to Hdm2 in vivo, mouse embryo fibroblasts p53–/– (MEF), HEPG2, and MCF7 cells were grown under normoxia, and nuclear extracts were isolated and used to purify the HIF1{alpha} and Mdm2 by immunoprecipitation. Western blots show that Mdm2 forms a complex with HIF1{alpha} in the absence of p53 (Fig. 2B) in MEFs. Immunoprecipitations of nuclear extracts from HEPG2 and MCF7 cells show that both p53 and HIF1{alpha} complex with Hdm2. However, HIF1{alpha} did not copurify with p53, and p53 did not copurify with HIF1{alpha}. Having shown a p53-independent association of Hdm2 and HIF1{alpha} under normoxia, we tested for the interaction of the proteins isolated from MEFs, p53–/– MEFs, H1299 (p53 null), and T47D cells (mutant p53) grown under hypoxia. The Hdm2/HIF1{alpha} complex was isolated from nuclear fractions of each cell type and Western blotted for HIF1{alpha} and Hdm2/Mdm2 (Fig. 2C). The results in Figs. 1 and 2 show that Mdm2 or Hdm2 (human homologue) complex with HIF1{alpha}, and this complex does not copurify p53, and immunopurified p53 does not copurify HIF1{alpha}. These observations are incongruent with a report that shows a trimeric complex of p53/HIF1{alpha}/Mdm2 isolated from whole cell lysates (11). There could be a simple explanation for the different observation. The authors only used an Mdm2 antibody to show a complex of p53 and HIF1{alpha}, which would pull down both p53 and HIF1{alpha} and would not show the distinct complexes.

VEGF is regulated by loss of Hdm2 or Nutlin3 treatment, which prevents Hdm2-HIF1{alpha} complex formation. Experiments were conducted with mdm2-deficient MEFs to assess the functional aspect of the HIF1{alpha}-Hdm2 complex. As knock-out of mdm2 is embryonically lethal, experiments were conducted with p53–/– and p53–/–mdm2–/– MEF. By comparison with MEFs, HIF1{alpha} activity was greater in p53–/– MEFs grown under normoxia or hypoxia as measured by vegf reporter assay (Fig. 3A ). Transactivation of the vegf reporter was diminished in p53–/–mdm2–/– MEFs (Fig. 3A), suggesting a molecular role for Mdm2 in the production of VEGF (Fig. 2). To show that this observation was not limited to murine cells, U87 cells were generated with a scrambled shRNA (scrRNA) or shRNA directed to Hdm2 (A/C). U87 cells under hypoxic conditions did not induce VEGF when ShRNA decreased Hdm2. U87 cells are wild type for p53, and to eliminate the possibility of p53 regulating HIF1{alpha}, we used H1299 cells that lack p53. Transient overexpression of Hdm2 under condition of normoxia or hypoxia in H1299 cells augmented vegf promoter activity, whereas the expression of Hdm2 in the reverse orientation (2mdH) diminished reporter activity (Fig. 3C and D). Although the Hdm2-HIF1{alpha} complex led to an increase in VEGF production, it is not clear how the complex works (17). Both Hdm2 and HIF1{alpha} bind with p300. Hdm2 may be involved in the formation of a p300-Hdm2-HIF1{alpha} complex that would be responsible for inducing HIF1{alpha} target genes. Further investigation is required to resolve the functional aspect of this complex.


Figure 3
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  		Figure 3. Vegf reporter activity. A, parental, p53–/–, and p53–/–mdm2–/– MEFs were transfected with the vegf promoter and ß-gal and then cultured under normoxia or hypoxia. B, U87 scrambled (ShScr) or Hdm2 knockdown (ShA/C) cells were treated under normoxic or hypoxic conditions, and cellular extracts were harvested for Western blot analysis of Hdm2, VEGF, and glyceraldehyde-3-phosphate dehydrogenase. C, normoxia (left), hypoxia (right). H1299 cells were transiently cotransfected with wild-type or mutant (mutant HRE elements) vegf promoter, ß-gal expression vector, and Hdm2 or Hdm2 expressed in the reverse orientation (2mdH). H1299 cells transiently transfected with the vegf reporter and ß-gal were pretreated with 10 µmol/L Nutlin3 for 10 min, then cells were incubated under hypoxia or normoxia for 24 h and harvested for analysis thereafter. D, H1299 cells were transiently transfected with the epo promoter driving luciferase, and ß-gal then 24 h thereafter were treated with 10 µmol/L Nultin3 for 24 h under normoxia or hypoxia and then harvested for analysis. Columns, mean reporter assays from three independent transfections; bars, SD.

 
To test if Nutlin3 would have an effect on vegf production, the vegf luciferase construct was transiently transfected into H1299 cells. Transfected cells were then treated with 10 µmol/L Nutlin3 and incubated under normoxic or hypoxic conditions for 24 h. Nutlin3 effectively prevented the induction of vegf in transient assays comparable to transient transfection of antisense Hdm2 (Fig. 3C). Additionally, we examined another HIF1{alpha}-responsive promoter (erythropoietin) linked to luciferase (epo). Nutlin3 decreased the induction of the epo promoter (Fig. 3D) similar to that of the vegf promoter (Fig. 3C) under conditions of normoxia or hypoxia.

Given that Nutlin3 diminished vegf and epo promoter activity in transient assays, we next examined in vitro how Nutlin3 would affect the formation of the HIF1{alpha}-Hdm2 complex. Nutlin3 (1 nmol/L, 10 nmol/L, and 1 µmol/L) was preincubated with recombinant Hdm2 and incubated with GST-HIF1{alpha} or p53. Complexes were purified with glutathione beads (HIF1{alpha}) or DO-1 agarose beads (p53) and analyzed by Western blot. The data in Fig. 4A show that Nutlin3 prevented the association of p53 or HIF1{alpha} with Hdm2. Next, we examined the ability of Nutlin3 to influence the Hdm2-HIF1{alpha} association by immunoprecipitation of Hdm2 from nuclear extracts isolated from cells under hypoxic conditions in the absence or presence of 10 µmol/L Nutlin3. The data in Fig. 4B show that the nuclear HIF1{alpha}-Hdm2 complex was disrupted by the presence of Nutlin3 in H1299 cells. Similar results were observed in U87 cells (wild-type p53), in that Nutlin3 prevented the complex formation of Hdm2 with HIF1{alpha} when Hdm2 was immunoprecipitated (Fig. 4D). An examination of endogenous VEGF levels in U87 cells shows diminished levels when cells are incubated under normoxic or hypoxic conditions in the presence of Nutlin3 (Fig. 4D). Thus, Nutlin3 is able to block the formation of the complex of Hdm2 and HIF1{alpha}, and this disruption has a dramatic effect on the levels of VEGF in the absence or presence of p53.


Figure 4
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  		Figure 4. Nutlin3 prevents Hdm2-HIF1{alpha} association. A, recombinant Hdm2 was preincubated with Nutlin3 (1 nmol/L, 10 nmol/L, or 1 µmol/L), and GST or GST-HIF1{alpha} was added. Western blot analysis was done for Hdm2 using 2A10 antibody (left). Experiments were done as with HIF1{alpha}, except that recombinant p53 was added and DO-1 (p53) agarose beads were used to purify complexes. 2A10 detected Hdm2, and rabbit polyclonal antibody detected p53 (right). B, H1299 cells were treated with either vehicle or 10 µmol/L Nutlin3 and incubated under hypoxia or normoxia for 24 h. Hdm2 or control immunoglobulin G was used to immunopurify Hdm2 from protein extracts. Western blot detected Hdm2 and HIF1{alpha}. C, Hdm2 was immunopurified from cellular extracts of U87 cells pretreated with Nutlin3 (10 µmol/L) under conditions of normoxia or hypoxia, and HIF1{alpha} or Hdm2 was detected by Western blot (left) or cellular extracts (right). D, whole cell lysates were prepared for Western blot analysis for the detection of VEGF and glyceraldehyde-3-phosphate dehydrogenase.

 
Great interest has been generated recently in developing compounds that can reestablish p53 activity. Numerous approaches have been used to free Hdm2 from p53, such as altering the levels of Hdm2 by antisense oligonucleotides and using small disruptive peptides or small molecules that target the p53-binding domain of Hdm2. Several lead compounds have proven to be successful in preventing such interaction. Nutlin3 was successful in preventing the association of p53 and Hdm2 and, in xenograph models, decreased tumor volume similar to doxorubicin treatment. We previously showed that the Nutlin3 compound was specific to Hdm2 and not to its family member Hdmx (Mdm4) in preventing the association with p53 (18). This shows that Nutlin3 effects are specific to Hdm2 and the utility of this small molecule inhibitor not only to protect p53 from Hdm2 but also to prevent Hdm2 from activating HIF1{alpha}and mediating VEGF induction.

Hdm2 plays a role in tumorigenesis that can be independent of p53. Indeed, hemangiosarcoma, a highly vascularized tumor, was observed with high frequency in Mdm2 transgenic mice. Moreover, tumor development in these mice was independent of p53 (19). About 70% of patients with hemangiosarcoma have high levels of VEGF (20). These two studies and our work presented herein support a role of Hdm2 in promoting the angiogenic factor VEGF. The oncogenic activity of Hdm2 may manifest itself by playing a dual role in blocking p53 activity while also enhancing HIF1{alpha} activity. However, targeting Hdm2 using Nutlin3 or Nutlin3-like molecules in the treatment of patients may target the tumor by reactivating p53 to bring about a decrease in tumor volume, which has been shown in xenograph models, and by inhibiting HIF1{alpha}-mediated angiogenesis.


   Acknowledgments
 
Grant support: National Cancer Institute grant CA109262, American Cancer Society grant IRG 91-022-09, General Motors Cancer Research Fund (L.D. Mayo). NIH grant F32HL072661 (M.W. Jackson).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7/21/06. Revised 10/19/06. Accepted 11/13/06.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. Wu X, Bayle JH, Olson D, Levine AJ. The p53-mdm-2 autoregulatory feedback loop. Genes Dev 1993;7:1126–32.[Abstract/Free Full Text]
  2. Mayo LD, Donner DB. A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus. Proc Natl Acad Sci U S A 2001;98:11598–603.[Abstract/Free Full Text]
  3. Khosravi R, Maya R, Gottlieb T, Oren M, Shiloh Y, Shkedy D. Rapid ATM-dependent phosphorylation of MDM2 precedes p53 accumulation in response to DNA damage. Proc Natl Acad Sci U S A 1999;96:14973–7.[Abstract/Free Full Text]
  4. Goldberg Z, Vogt Sionov R, Berger M, et al. Tyrosine phosphorylation of Mdm2 by c-Abl: implications for p53 regulation. EMBO J 2002;21:3715–27.[CrossRef][Medline]
  5. Ries S, Biederer C, Woods D, et al. Opposing effects of Ras on p53: transcriptional activation of mdm2 and induction of p19ARF. Cell 2000;103:321–30.[CrossRef][Medline]
  6. Kinyamu HK, Archer TK. Estrogen receptor-dependent proteasomal degradation of the glucocorticoid receptor is coupled to an increase in mdm2 protein expression. Mol Cell Biol 2003;23:5867–81.[Abstract/Free Full Text]
  7. Vassilev LT, Vu BT, Graves B, et al. In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 2004;303:844–8.[Abstract/Free Full Text]
  8. Doggrell SA. RITA—a small-molecule anticancer drug that targets p53. Expert Opin Investig Drugs 2005;14:739–42.[CrossRef][Medline]
  9. Koblish HK, Zhao S, Franks CF, et al. Benzodiazepinedione inhibitors of the Hdm2:p53 complex suppress human tumor cell proliferation in vitro and sensitize tumors to doxorubicin in vivo. Mol Cancer Ther 2006;5:160–9.[Abstract/Free Full Text]
  10. Yang Y, Ludwig RL, Jensen JP, et al. Small molecule inhibitors of HDM2 ubiquitin ligase activity stabilize and activate p53 in cells. Cancer Cell 2005;7:547–59.[CrossRef][Medline]
  11. Ravi R, Mookerjee B, Bhujwalla ZM, et al. Regulation of tumor angiogenesis by p53-induced degradation of hypoxia-inducible factor 1{alpha}. Genes Dev 2000;14:34–44.[Abstract/Free Full Text]
  12. Bardos JI, Chau NM, Ashcroft M. Growth factor-mediated induction of HDM2 positively regulates hypoxia-inducible factor 1{alpha} expression. Mol Cell Biol 2004;24:2905–14.[Abstract/Free Full Text]
  13. Chen D, Li M, Luo J, Gu W. Direct interactions between HIF-1{alpha} and Mdm2 modulate p53 function. J Biol Chem 2003;278:13595–8.[Abstract/Free Full Text]
  14. Mayo LD, Turchi JJ, Berberich SJ. Mdm-2 phosphorylation by DNA-dependent protein kinase prevents interaction with p53. Cancer Res 1997;57:5013–6.[Abstract/Free Full Text]
  15. Kaeser MD, Pebernard S, Iggo RD. Regulation of p53 stability and function in HCT116 colon cancer cells. J Biol Chem 2004;279:7598–605.[Abstract/Free Full Text]
  16. Kussie PH, Gorina S, Marechal V, et al. Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain. Science 1996;274:948–53.[Abstract/Free Full Text]
  17. Nieminen AL, Qanungo S, Schneider EA, Jiang BH, Agani FH. Mdm2 and HIF-1{alpha} interaction in tumor cells during hypoxia. J Cell Physiol 2005;204:364–9.[CrossRef][Medline]
  18. Patton JT, Mayo LD, Singhi AD, Gudkov AV, Stark GR, Jackson MW. Levels of HdmX expression dictate the sensitivity of normal and transformed cells to Nutlin-3. Cancer Res 2006;66:3169–76.[Abstract/Free Full Text]
  19. Jones SN, Hancock AR, Vogel H, Donehower LA, Bradley A. Overexpression of Mdm2 in mice reveals a p53-independent role for Mdm2 in tumorigenesis. Proc Natl Acad Sci U S A 1998;95:15608–12.[Abstract/Free Full Text]
  20. Zietz C, Rossle M, Haas C, et al. MDM-2 oncoprotein overexpression, p53 gene mutation, and VEGF up-regulation in angiosarcomas. Am J Pathol 1998;153:1425–33.[Abstract/Free Full Text]



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