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Updated: Jul 17, 2026
Induction and Testing of Hypoxia in Cell Culture
Published on: August 12, 2011
Nutlin3 blocks vascular endothelial growth factor induction by preventing the interaction between hypoxia inducible
Gretchen A LaRusch1, Mark W Jackson, James D Dunbar
1Department of Radiation Oncology, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, Ohio, USA.
Abstract:
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 1alpha (HIF1alpha) binds to Hdm2 in the domain designated to bind p53. HIF1alpha and p53 share a conserved motif that is required to bind Hdm2. Distinct complexes form between Hdm2-HIF1alpha and Hdm2-p53 as determined by immunoprecipitation of nuclear extracts and in vitro. The Hdm2 antagonist Nutlin3 prevents the association between Hdm2 and HIF1alpha. The vascular endothelial growth factor (VEGF) gene is a transcriptional target of HIF1alpha, and under normoxic or hypoxic conditions, Hdm2 increases HIF1alpha activity to induce VEGF production. Blocking the association of Hdm2 and HIF1alpha 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 HIF1alpha.
Insights
The Hdm2 protein interacts with hypoxia-inducible factor 1-alpha (HIF1alpha), promoting VEGF production. Nutlin3 disrupts this Hdm2-HIF1alpha interaction, reducing VEGF levels in cancer cells.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Hdm2 protein is frequently overexpressed in various cancers, inhibiting wild-type p53 tumor suppressor activity.
- Nutlin3, an Hdm2 antagonist, was developed to reactivate p53 by disrupting the Hdm2-p53 interaction.
- Hdm2's p53-binding domain also interacts with other proteins, suggesting broader regulatory roles.
Purpose of the Study:
- To investigate the interaction between Hdm2 and hypoxia-inducible factor 1-alpha (HIF1alpha).
- To determine if Nutlin3 affects the Hdm2-HIF1alpha complex formation.
- To elucidate the role of the Hdm2-HIF1alpha interaction in vascular endothelial growth factor (VEGF) production.
Main Methods:
- Immunoprecipitation of nuclear extracts and in vitro binding assays to detect Hdm2-HIF1alpha and Hdm2-p53 complexes.
- Treatment with Nutlin3 to assess its effect on Hdm2-HIF1alpha association.
- Analysis of VEGF gene expression under normoxic and hypoxic conditions following Hdm2 modulation or Nutlin3 treatment.
Main Results:
- HIF1alpha binds to Hdm2 within the p53-binding domain, sharing a conserved motif with p53.
- Distinct Hdm2-HIF1alpha and Hdm2-p53 complexes were identified.
- Nutlin3 inhibited the Hdm2-HIF1alpha association and decreased VEGF production induced by HIF1alpha under both normoxic and hypoxic conditions.
- Ablating Hdm2 expression also diminished VEGF levels.
Conclusions:
- HIF1alpha interacts with Hdm2 via a conserved motif in the p53-binding domain.
- Hdm2 positively regulates HIF1alpha-mediated VEGF production.
- Nutlin3 attenuates VEGF induction by disrupting the Hdm2-HIF1alpha interaction, revealing a novel therapeutic mechanism beyond p53 reactivation.
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