Flowchart: Preparation: HIF-1
 

 

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J Neurooncol. 2006 Apr 13; [Epub ahead of print]

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Inhibition of hypoxia inducible factor-1alpha (HIF-1alpha) decreases vascular endothelial growth factor (VEGF) secretion and tumor growth in malignant gliomas.

Jensen RL, Ragel BT, Whang K, Gillespie D.

Department of Neurosurgery, University of Utah, Salt Lake City, Utah, USA.

INTRODUCTION: Hypoxia inducible factor-1alpha (HIF-1alpha) regulates vascular endothelial growth factor (VEGF), the presumed principal mediator of angiogenesis in malignant gliomas, under normal physiologic conditions. We examined the effect of HIF-1alpha on VEGF secretion, tumor growth, and angiogenesis in malignant gliomas. METHODS: We examined 175 human gliomas for expression of HIF-1alpha and its downstream-regulated proteins. HIF-1alpha expression and VEGF secretion in glioma cell lines under normoxia and hypoxia were examined using ELISA and Western blot. Malignant glioma cell lines were transfected with dominant-negative HIF-1alpha (DN-HIF-1alpha) expression vector or siRNA constructs against the HIF-1alpha gene. Growth studies were conducted on cells with the highest VEGF/HIF-1alpha inhibition isolated from stable transfected cell lines. MIB-1-labeling index and microvascular density (MVD) measurements were performed on the in vivo tumors. RESULTS: HIF-1 expression correlates with malignant glioma phenotype and was not confined to perinecrotic, pseudopalisading cells. VEGF and HIF-1 expression was high in glioma cell lines even under normoxia, and increased after exposure to hypoxia or growth factor stimulation. Cells transfected with DN-HIF-1alpha or HIF-1alpha siRNA demonstrated decreased HIF-1alpha and VEGF secretion. In vivo but not in vitro growth decreased in response to VEGF and HIF-1 inhibition. HIF-1 siRNA studies showed decreased VEGF secretion and in vitro and in vivo growth of glioma cell lines. MVD was unchanged but MIB-1 proliferation index decreased for both types of HIF-1 inhibition. CONCLUSIONS: VEGF and HIF-1alpha are elevated in malignant gliomas. HIF-1alpha inhibition results in VEGF secretion inhibition. HIF-1alpha expression affects glioma tumor growth, suggesting clinical applications for malignant glioma treatment.

PMID: 16612574 [PubMed - as supplied by publisher]

RNA. 2006 Apr 6; [Epub ahead of print]

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Assessing IRES activity in the HIF-1{alpha} and other cellular 5' UTRs.

Bert AG, Grepin R, Vadas MA, Goodall GJ.

1Division of Human Immunology, Hanson Institute, Institute of Medical and Veterinary Science (IMVS), Adelaide, SA 5000, Australia.

Dicistronic reporter plasmids, such as the dual luciferase-containing pR-F plasmid, have been widely used to assay cellular and viral 5' untranslated regions (UTRs) for IRES activity. We found that the pR-F dicistronic reporter containing the 5' UTRs from HIF-1alpha, VEGF, c-myc, XIAP, VEGFR-1, or Egr-1 UTRs all produce the downstream luciferase predominantly as a result of cryptic promoter activity that is activated by the SV40 enhancer elements in the plasmid. RNA transfection experiments using dicistronic or uncapped RNAs, which avoid the complication of cryptic promoter activity, indicate that the HIF-1alpha, VEGF, c-myc, and XIAP UTRs do have some IRES activity, although the activity was much less than that of the viral EMCV IRES. The translation of transfected monocistronic RNAs containing these cellular UTRs was greatly enhanced by the presence of a 5' cap, raising questions as to the strength or mechanism of IRES-mediated translation in these assays.

PMID: 16601206 [PubMed - as supplied by publisher]