Up-Regulation of Vascular Endothelial Growth Factor Expression in a Rat Glioma Is Conferred by Two Distinct Hypoxia-driven Mechanisms'

expression in glioma tumors growing in animals, which is restricted to the distinctive PNP cells, we used the transplantable GS9L glioma cell line. When injected s.c. into syngeneic rats, GS9L cells form tumors with distinctive necrotic foci that are histologically similar to human gliomas. In situ hybridization revealed high level expression of VEGF mRNA in PNP cells of these experimental tumors (14). To study the mech anisms regulating this expression, we stably transfected GS9L cells with mouse VEGF-lacZ reporter gene constructs and injected the resultant cell clones into syngeneic rats. The data reveal that although GS9L cells clonally selected to contain the reporter gene were used to produce the transplanted tumors, such that all GS9L cells in the tumor should contain the VEGF reporter gene, only the PNP cells up-regulated its expression, confirming that the special microenvironment of the PNP cells confers a distinct regulation onto the VEGF gene. Two elements previously implicated by in vitro studies demonstrably contribute to this cell type-specific expression in the solid tumors: the HIF 1 binding site; and 3' untranslated sequences that affect mRNA stability. EF 5 immuno staining for regions of low oxygen partial pressure overlapped with expression of a-gal driven by mouse VEGF regulatory sequences. Thus, we could demonstrate that hypoxia is indeed the microen ABSTRACT Up-regulation of vascular endothelial growth factor (VEGF) expression is a major event leading to neovascularization in malignant gliomas. Hypoxia is believed to be the crucial environmental stimulus for this up-regulation. To critically assess this hypothesis, we asked whether the 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. Inclusion of the binding site for hypoxia-inducible factor 1 (HIF 1) in the 5' regulatory sequences used in the hybrid gene produced weak @3-galactosidase staining in a special tumor cell subtype, the so-called perinecrotic palisading (PNP) cells that flank necrotic regions within the tumor. Deletion of the HIF 1 bindingsite abolished reporter gene expressionin the PNP cells, indicating that transcriptional activation of VEGFexpression in gliomas is mediated by HIF 1. Inclusion of 3' untranslated sequences from the VEGF gene in the reporter constructs resulted in an increased @3-galactosidasestaining in the PNP cells, suggesting that mRNA stabilization also contributes to VEGF up-regulation in glioblastoma ceUsgrowing as solid tumors. Corn bination of the 5' flanking region including the HIF 1 site along with 3' untranslated sequences produced increased leveis of @3-galactosidaseex pression in PNP cells. EF 5 immunostaining for regions of low oxygen PNP @1-ga experimental evidence that VEGFgene expression is activated in a distinct tumor cell subpopulation, the perinecroticpalisading cells ofgliornas, by two distinct hypoxia-driven regulatory mechanisms.