Skip to main content
Article thumbnail
Location of Repository

Real-Time Imaging of HIF-1α Stabilization and Degradation

By Ekaterina Moroz, Sean Carlin, Katerina Dyomina, Sean Burke, Howard T. Thaler, Ronald Blasberg and Inna Serganova

Abstract

HIF-1α is overexpressed in many human cancers compared to normal tissues due to the interaction of a multiplicity of factors and pathways that reflect specific genetic alterations and extracellular stimuli. We developed two HIF-1α chimeric reporter systems, HIF-1α/FLuc and HIF-1α(ΔODDD)/FLuc, to investigate the tightly controlled level of HIF-1α protein in normal (NIH3T3 and HEK293) and glioma (U87) cells. These reporter systems provided an opportunity to investigate the degradation of HIF-1α in different cell lines, both in culture and in xenografts. Using immunofluorescence microscopy, we observed different patterns of subcellular localization of HIF-1α/FLuc fusion protein between normal cells and cancer cells; similar differences were observed for HIF-1α in non-transduced, wild-type cells. A dynamic cytoplasmic-nuclear exchange of the fusion protein and HIF-1α was observed in NIH3T3 and HEK293 cells under different conditions (normoxia, CoCl2 treatment and hypoxia). In contrast, U87 cells showed a more persistent nuclear localization pattern that was less affected by different growing conditions. Employing a kinetic model for protein degradation, we were able to distinguish two components of HIF-1α/FLuc protein degradation and quantify the half-life of HIF-1α fusion proteins. The rapid clearance component (t1/2 ∼4–6 min) was abolished by the hypoxia-mimetic CoCl2, MG132 treatment and deletion of ODD domain, and reflects the oxygen/VHL-dependent degradation pathway. The slow clearance component (t1/2 ∼200 min) is consistent with other unidentified non-oxygen/VHL-dependent degradation pathways. Overall, the continuous bioluminescence readout of HIF-1α/FLuc stabilization in vitro and in vivo will facilitate the development and validation of therapeutics that affect the stability and accumulation of HIF-1α

Topics: Research Article
Publisher: Public Library of Science
OAI identifier: oai:pubmedcentral.nih.gov:2660410
Provided by: PubMed Central
Download PDF:
Sorry, we are unable to provide the full text but you may find it at the following location(s):
  • http://www.pubmedcentral.nih.g... (external link)
  • Suggested articles

    Citations

    1. (2003). 2ME2 inhibits tumor growth and angiogenesis by disrupting microtubules and dysregulating HIF.
    2. (2001). A conserved family of prolyl-4-hydroxylases that modify HIF.
    3. (2004). A Fenton reaction at the endoplasmic reticulum is involved in the redox control of hypoxia-inducible gene expression.
    4. (2005). A hypoxia-independent hypoxia-inducible factor-1 activation pathway induced by phosphatidylinositol-3 kinase/Akt in HER2 overexpressing cells.
    5. (1992). A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation.
    6. (2005). Accumulation of hypoxia-inducible factor-1alpha is limited by transcription-dependent depletion.
    7. (2008). Akt/mTOR pathway assures the synthesis of HIF-1alpha protein in a glucoseand reoxygenation-dependent manner in irradiated tumors.
    8. (2006). Akt1 activation can augment hypoxia-inducible factor-1alpha expression by increasing protein translation through a mammalian target of rapamycin-independent pathway.
    9. (2005). Both microtubule-stabilizing and microtubule-destabilizing drugs inhibit hypoxia-inducible factor-1alpha accumulation and activity by disrupting microtubule function.
    10. (2007). Cell type-specific, topoisomerase II-dependent inhibition of hypoxia-inducible factor-1alpha protein accumulation by NSC
    11. (2006). Cell-type-specific regulation of degradation of hypoxia-inducible factor 1 alpha: role of subcellular compartmentalization.
    12. (2007). Constitutive/ Hypoxic Degradation of HIF-{alpha} Proteins by the Proteasome Is Independent of von Hippel Lindau Protein Ubiquitylation and the Transactivation Activity of the Protein.
    13. (2003). Cytoplasmically retargeted HSV1-tk/GFP reporter gene mutants for optimization of non-invasive molecular-genetic imaging.
    14. (2001). Design of the linkers which effectively separate domains of a bifunctional fusion protein.
    15. (2006). Development of novel therapeutic strategies that target HIF-1.
    16. (2005). Echinomycin, a small-molecule inhibitor of hypoxia-inducible factor-1 DNAbinding activity.
    17. (2002). Geldanamycin induces degradation of hypoxia-inducible factor 1alpha protein via the proteosome pathway in prostate cancer cells.
    18. (2003). Heat shock protein 90.
    19. (2001). HER2 (neu) signaling increases the rate of hypoxia-inducible factor 1alpha (HIF-1alpha) synthesis: novel mechanism for HIF-1-mediated vascular endothelial growth factor expression.
    20. (2003). HIF-1 as a target for drug development.
    21. (2005). hNISIRES-eGFP dual reporter gene imaging.
    22. (2002). How oxygen makes its presence felt.
    23. (2005). HSP90 and the chaperoning of cancer.
    24. (2002). Hsp90 regulates a von Hippel Lindau-independent hypoxia-inducible factor-1 alphadegradative pathway.
    25. (2005). Hypoxia inducible factor-1: a novel target for cancer therapy.
    26. (2004). Hypoxia inducible factor-1alpha as a cancer drug target.
    27. (2002). Hypoxia-inducible erythropoietin gene expression in human neuroblastoma cells.
    28. (1997). Hypoxia-inducible factor 1alpha (HIF-1alpha) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes.
    29. (2001). Hypoxia-inducible factor-1 alpha (HIF-1 alpha) escapes O(2)-driven proteasomal degradation irrespective of its subcellular localization: nucleus or cytoplasm.
    30. (2005). Hypoxia-inducible factor-1alpha and the glycolytic phenotype in tumors.
    31. (2005). Identification of a novel small-molecule inhibitor of the hypoxia-inducible factor 1 pathway.
    32. (2006). Identification of MAPK phosphorylation sites and their role in the localization and activity of hypoxia-inducible factor-1alpha.
    33. (2002). Identification of small molecule inhibitors of hypoxia-inducible factor 1 transcriptional activation pathway.
    34. (2007). Imaging of hypoxia-driven gene expression in an orthotopic liver tumor model.
    35. (2008). In vivo bioluminescence imaging monitoring of hypoxia-inducible factor 1alpha, a promoter that protects cells, in response to chemotherapy.
    36. (2002). Insulin-like growth factor 1 induces hypoxia-inducible factor 1-mediated vascular endothelial growth factor expression, which is dependent on MAP kinase and phosphatidylinositol 3-kinase signaling in colon cancer cells.
    37. (2004). Interaction of the PAS B domain with HSP90 accelerates hypoxia-inducible factor-1alpha stabilization.
    38. (2000). Loss of PTEN facilitates HIF-1-mediated gene expression.
    39. (2001). Maity A
    40. (2004). Molecular imaging of temporal dynamics and spatial heterogeneity of hypoxia-inducible factor-1 signal transduction activity in tumors in living mice.
    41. (2005). Molecularly targeted therapy for gastrointestinal cancer.
    42. (2006). Mouse model for noninvasive imaging of HIF prolyl hydroxylase activity: assessment of an oral agent that stimulates erythropoietin production.
    43. (2004). mTOR inhibition reverses Akt-dependent prostate intraepithelial neoplasia through regulation of apoptotic and HIF-1-dependent pathways.
    44. (2007). Nuclear expression of hypoxia-inducible factor-1alpha in clear cell renal cell carcinoma is involved in tumor progression.
    45. (2005). Optical imaging of tumor hypoxia and evaluation of efficacy of a hypoxia-targeting drug in living animals.
    46. (1999). Overexpression of hypoxia-inducible factor 1alpha in common human cancers and their metastases.
    47. (2002). Oxygen-dependent ubiquitination and degradation of hypoxia-inducible factor requires nuclear-cytoplasmic trafficking of the von Hippel-Lindau tumor suppressor protein.
    48. (1997). PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer.
    49. (2007). RACK1 competes with HSP90 for binding to HIF-1alpha and is required for O(2)-independent and HSP90 inhibitor-induced degradation of HIF-1alpha.
    50. (1999). Reciprocal positive regulation of hypoxia-inducible factor 1alpha and insulin-like growth factor 2.
    51. (2002). Regulation and destabilization of HIF-1alpha by ARD1-mediated acetylation.
    52. (2003). Regulation of angiogenesis by hypoxia: role of the HIF system.
    53. (2003). Regulation of colon carcinoma cell invasion by hypoxia-inducible factor 1.
    54. (2006). Regulation of gene expression by HIF-1. Novartis Found Symp 272: 2–8; discussion 8–14,
    55. (2002). Regulation of hypoxia-inducible factor 1alpha expression and function by the mammalian target of rapamycin.
    56. (1998). Regulation of hypoxia-inducible factor 1alpha is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway.
    57. (2004). Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex.
    58. (1999). Regulation of the hypoxia-inducible transcription factor 1alpha by the ubiquitin-proteasome pathway.
    59. (2006). Role of carbonic anhydrase IX expression in prediction of the efficacy and outcome of primary epirubicin/tamoxifen therapy for breast cancer.
    60. (2008). Short hairpin RNA interference therapy for ischemic heart disease.
    61. (1998). Signal transduction in hypoxic cells: inducible nuclear translocation and recruitment of the CBP/p300 coactivator by the hypoxia-inducible factor-1alpha.
    62. (2003). Targeting HIF-1 for cancer therapy.
    63. (2001). Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation.
    64. (2007). The combination of hypoxia-response enhancers and an oxygen-dependent proteolytic motif enables real-time imaging of absolute HIF-1 activity in tumor xenografts.
    65. (2000). The expression and distribution of the hypoxia-inducible factors HIF-1alpha and HIF-2alpha in normal human tissues, cancers, and tumor-associated macrophages.
    66. (2005). The insulin-PI3K/TOR pathway induces a HIF-dependent transcriptional response in Drosophila by promoting nuclear localization of HIF-alpha/Sima.
    67. (2005). The proinflammatory cytokine interleukin 1beta and hypoxia cooperatively induce the expression of adrenomedullin in ovarian carcinoma cells through hypoxia inducible factor 1 activation.
    68. (2001). Transcription factor HIF-1 is a necessary mediator of the pasteur effect in mammalian cells.
    69. (1999). Transcription-dependent nuclear-cytoplasmic trafficking is required for the function of the von Hippel-Lindau tumor suppressor protein.
    70. (2008). Visualization of hypoxia-inducible factor-1 transcriptional activation in C6 glioma using luciferase and sodium iodide symporter genes.
    71. (2003). YC-1: a potential anticancer drug targeting hypoxia-inducible factor 1.

    To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.