J Neurooncol. 2006 Apr 13; [Epub
ahead of print] RNA.
2006 Apr 6; [Epub ahead of print]
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,
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]
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,
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]