Hypoxia induces transcription of a range of physiologically important genes including erythropoietin and vascular endothelial growth factor. The transcriptional activation is mediated by the hypoxia-inducible factor-1 (HIF-1), a heterodimeric member of the basic helix ± loop ± helix PAS family, composed of a and b subunits. HIF-1a shares 48 per cent identity with the recently identi®ed HIF-2a protein that is also stimulated by hypoxia. In a previous study of hemangioblastomas, the most frequent manifestation of hereditary von HippelLindau disease (VHL), we found elevated levels of vascular endothelial growth factor and HIF-2a mRNA in stromal cells of the tumors. Mutations of the VHL tumor suppressor gene are associated with a variety of tumors such as renal clear cell carcinomas (RCC). In this study, we analysed the expression of the hypoxiainducible factors HIF-1a and HIF-2a in a range of VHL wildtype and VHL de®cient RCC cell lines. In the presence of functional VHL protein, HIF-1a mRNA levels are elevated, whereas HIF-2a mRNA expression is increased only in cells lacking a functional VHL gene product. On the protein levels, however, in VHL de®cient cell lines, both HIF-a subunits are constitutively expressed, whereas re-introduction of a functional VHL gene restores the instability of HIF-1a and HIF-2a proteins under normoxic conditions. Moreover, immunohistochemical analyses of RCCs and hemangioblastomas demonstrate up-regulation of HIF-1a and HIF-2a in the tumor cells. The data presented here provide evidence for a role of the VHL protein in regulation of angiogenesis and erythropoiesis mediated by the HIF-1a and HIF-2a proteins. Oncogene (2000) 19, 5435 ± 5443.

Gene licS of Bacillus subtilis encodes an excreted ␤-1,3-1,4-endoglucanase necessary for lichenan utilization. Upstream of licS we found a gene (termed licT) together with its promoter which encodes a transcriptional antiterminator of the BglG family. Genes licT and licS are separated by a palindromic sequence (lic-t) reminiscent of transcriptional terminators recognized by the antiterminator proteins of the BglG family. The LicT protein can prevent termination at terminator lic-t and also at terminator t2 of the Escherichia coli bgl operon and BglG prevents termination at lic-t. The role of LicT in licS regulation by preventing termination at its terminator lic-t appears to be limited since expression of licS is inducible only two-to threefold. This limited regulation is mainly due to a high basal level of licS expression which can in part be attributed to the presence of a second promoter preceding licS and located downstream of lic-t. However, disruption of gene licT leads not only to loss of inducibility of licS but also to loss of growth on lichenan or on its degradation products, indicating its stringent role in ␤-glucan utilization.Evolutionarily conserved mechanisms of gene regulation by means of transcriptional antitermination have recently been described for sucrose metabolism in Bacillus subtilis and for ␤-glucoside utilization in Escherichia coli. The ␤-glucoside (bgl) operon of E. coli consists of three genes (33) which are preceded by a catabolite gene activator protein-cyclic AMPdependent promoter (24). Interestingly, in wild-type strains, the bgl promoter is kept silent in vivo (28) and utilizes insertion sequences as transcriptional enhancers to gain full activity (24,25,31). The first gene of the operon, bglG, encodes the antiterminator protein. It is flanked by two transcriptional terminators (t1 and t2) at which the BglG protein acts to alleviate termination (20,29). Terminators t1 and t2 share a highly conserved sequence motif proximal to and extending into their stem-loop structures (33). It has been shown for terminator t1 that BglG specifically binds to this sequence motif (now termed RAT [3]) at the mRNA level (16), suggesting that binding prevents formation of the terminator structure, thereby allowing transcription to proceed.The second gene of the operon, bglF, encodes the ␤-glucoside-specific transport protein, enzyme II Bgl , which is part of the phosphoenolpyruvate sugar-phosphotransferase system (PTS) and phosphorylates its substrates concomitantly with their transport (6,32,33). Enzyme II Bgl additionally functions as negative regulator of the operon: in the absence of ␤-glucosidic substrates, it phosphorylates the BglG protein, thereby inhibiting its antiterminator activity. This phosphorylation is reversible, allowing induction of the operon upon addition of sugar substrate (1, 2, 29, 30). The third gene, bglB, codes for the hydrolyzing enzyme, a phospho-␤-glucosidase.In B. subtilis, two systems involved in sucrose metabolism are likewise controlled by transcriptional antitermi...

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is a mitogen and chemotactic factor for endothelial cells in vitro and an angiogenesis and vascular permeability factor in vivo. Due to its properties, VEGF is a candidate for both angiogenesis and vascular permeability/oedema induction which typically occur in glioblastomas. In this study we test the hypothesis that the antioedema effect of dexamethasone is mediated by downregulation of VEGF or VEGF receptor expression. VEGF mRNA and protein levels of two rat glioma cells lines, C6 and GS-9L, were determined after incubation with dexamethasone under normoxic and hypoxic conditions. In normoxic C6 and GS9L cells, we observed 50-60% downregulation of VEGF mRNA by dexamethasone (P=0.015 and P=0. 01, respectively). This effect was dependent on glucocorticoid-receptor (GR) function. The inhibitory effect of dexamethasone on VEGF gene expression by tumour cells was markedly reduced by hypoxia which suggests that the upregulation of VEGF driven by hypoxia overcomes the effect of the dexamethasone. Dexamethasone did not alter VEGFR-2 mRNA levels in human umbilical endothelial cells. In a subcutaneous glioma tumour model, we observed only a 15% decrease in VEGF mRNA expression in dexamethasone treated animals (n = 12) compared with controls animals (P = 0.24). We conclude that dexamethasone may decrease brain tumour-associated oedema by reduction of VEGF expression in tumour cells. However, the highly reduced activity on hypoxic tumour cells suggests that dexamethasone efficacy may be limited by hypoxia in rapidly growing tumours.

Hemangioblastomas, the most frequent manifestation of the hereditary von Hippel-Lindau disease (VHL), are highly vascularized tumors of the central nervous system. In previous studies, the endothelial-specific mitogen vascular endothelial growth factor (VEGF) was shown to be up-regulated in the stromal cells, the putative neoplastic cells in hemangioblastomas. Therefore, it was suggested that secretion of VEGF by stromal cells is the pathogenetic cause of the vascular lesions in hemangioblastomas. The novel basic helix loop helix transcription factor HRF/HIF-2alpha is a candidate regulator of VEGF expression during development. We therefore investigated expression of HRF/HIF-2alpha in hemangioblastomas and found the overexpression of VEGF mRNA in stromal cells to be highly correlated with elevated expression levels of HRF/HIF-2alpha mRNA. This finding is suggestive for a role of HRF in VEGF-dependent vascular growth in hemangioblastomas and could provide a link between transcriptional activation of the VEGF gene and loss of function of the VHL gene product.

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The HlpA protein which is encoded by the hlpA gene in the plastid genome of the cryptomonad alga Chryptomonas phi is structurally related to the non-sequence-specific DNA-binding and DNA-bending HU family of chromatin-associated proteins. The expression of the HlpA protein complements the mutant phenotype of Bacillus subtilis cells impaired in the Hbsu protein (B. subtilis HU), as measured by the resistance of the cells to methylmethane sulphonate. To analyse the interactions of HlpA with DNA, we expressed the protein in Escherichia coli and purified it to homogeneity. HlpA interacts preferentially with four-way junction DNA or DNA minicircles, when compared with linear DNA, recognising DNA structure. HlpA and E. coli HU display comparable affinities for all types of DNA tested; however, HlpA exhibits a stronger tendency to self-associate in the presence of DNA. Accordingly, HlpA oligomerises more readily than HU in protein crosslinking experiments. In the presence of topoisomerase I, HlpA constrains negative superhelical turns in closed circular plasmid DNA. The HlpA protein mediates the joining of distant recombination sites into a complex nucleoprotein structure, as judged by beta-mediated site-specific recombination. The results presented provide evidence that HlpA is a functional plastid equivalent of nuclear and mitochondrial HMG1-like proteins and bacterial HU proteins.