UP-REGULATION OF VASCULAR ENDOTHELIAL GROWTH-FACTOR EXPRESSION IN A RAT GLIOMA IS CONFERRED BY 2 DISTINCT HYPOXIA-DRIVEN MECHANISMS

Up-regulation of vascular endothelial growth meter (VEGF) expression i s a major event leading to neovascularization in malignant gliomas. Hy poxia is believed to be the crucial environmental stimulus for this up -regulation. To critically assess this hypothesis, we asked whether th e mechanisms defined previously for hypoxia-induced VEGF expression in vitro are similarly involved and sufficient for up-regulation of VEGF gene expression in vivo, using a lacZ reporter gene under the control of VEGF regulatory sequences in an experimental glioma model. Inclusi on of the binding site for hypoxia-inducible factor 1 (HIF 1) in the 5 ' regulatory sequences used in the hybrid gene produced weak beta-gala ctosidase staining in a special tumor cell subtype, the so-called peri necrotic palisading (PNP) cells that flank necrotic regions within the tumor. Deletion of the HIF 1 binding site abolished reporter gene exp ression in the PNP cells, indicating that transcriptional activation o f VEGF expression in gliomas is mediated by HIF 1. Inclusion of 3' unt ranslated sequences from the VEGF gene in the reporter constructs resu lted in an increased beta-galactosidase staining in the PNP cells, sug gesting that mRNA stabilization also contributes to VEGF up-regulation in glioblastoma cells growing as solid tumors. Combination of the 5' flanking region including the HIF 1 site along with 3' untranslated se quences produced increased levels of beta-galactosidase expression in PNP cells. EF 5 immunostaining for regions of low oxygen partial press ure covered the same PNP cells that were stained for beta-galactosidas e. Collectively, the data provide experimental evidence that VEGF gene expression is activated in a distinct tumor cell subpopulation, the p erinecrotic palisading cells of gliomas, by two distinct hypoxia-drive n regulatory mechanisms.