Adaptation to hypoxia is regulated by hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor consisting of an oxygen-regulated alpha subunit and a constitutively expressed beta subunit. Although HIF-1 is regulated mainly by oxygen tension through the oxygen-dependent degradation of its alpha subunit, in vitro it can also be modulated by cytokines, hormones and genetic alterations. To investigate HIF-1 activation in vivo, we determined the spatial and temporal distribution of HIF-1 in healthy mice subjected to varying fractions of inspiratory oxygen. Immunohistochemical examination of brain, kidney, liver, heart, and skeletal muscle revealed that HIF-1alpha is present in mice kept under normoxic conditions and is further increased in response to systemic hypoxia. Moreover, immunoblot analysis showed that the kinetics of HIF-1alpha expression varies among different organs. In liver and kidney, HIF-1alpha reaches maximal levels after 1 h and gradually decreases to baseline levels after 4 h of continuous hypoxia. In the brain, however, HIF-1alpha is maximally expressed after 5 h and declines to basal levels by 12 h. Whereas HIF-1beta is constitutively expressed in brain and kidney nuclear extracts, its hepatic expression increases concomitantly with HIF-1alpha. Overall, HIF-1alpha expression in normoxic mice suggests that HIF-1 has an important role in tissue homeostasis.

Leukocyte infiltration and necrosis are two biological phenomena associated with the development of neovascularization during the malignant progression of human astrocytoma. Here, we demonstrate expression of interleukin (IL)-8, a cytokine with chemotactic and angiogenic properties, and of IL-8–binding receptors in astrocytoma. IL-8 expression is first observed in low grade astrocytoma in perivascular tumor areas expressing inflammatory cytokines. In glioblastoma, it further localizes to oxygen-deprived cells surrounding necrosis. Hypoxic/anoxic insults on glioblastoma cells in vitro using anaerobic chamber systems or within spheroids developing central necrosis induced an increase in IL-8 messenger RNA (mRNA) and protein expression. mRNA for IL-8–binding chemokine receptors CXCR1, CXCR2, and the Duffy antigen receptor for chemokines (DARC) were found in all astrocytoma grades by reverse transcription/PCR analysis. In situ hybridization and immunohistochemistry localized DARC expression on normal brain and tumor microvascular cells and CXCR1 and CXCR2 expression to infiltrating leukocytes. These results support a model where IL-8 expression is initiated early in astrocytoma development through induction by inflammatory stimuli and later in tumor progression increases due to reduced microenvironmental oxygen pressure. Augmented IL-8 would directly and/or indirectly promote angiogenesis by binding to DARC and by inducing leukocyte infiltration and activation by binding to CXCR1 and CXCR2.

Studies in tissue culture cells have implicated p300 and CBP acetyltransferases in myogenic regulatory factor (MRF) mediated transcription and terminal differentiation of skeletal muscle cells. However, in vivo data placing p300 and CBP on myogenic differentiation pathways are not yet available. In this report we provide genetic evidence that p300 but not CBP acetyltransferase (AT) activity is required for myogenesis in the mouse and in embryonic stem (ES) cells. A fraction of embryos carrying a single p300 AT- deficient allele exhibit impaired MRF expression, delayed terminal differentiation and a reduced muscle mass. In mouse embryos lacking p300 protein, Myf-5 induction is severely attenuated. Similarly, ES cells homozygous for a p300 AT or a p300 null mutation fail to activate Myf5 and MyoD transcription efficiently, while Pax3, acting genetically upstream of these MRFs, is expressed. In contrast, ES cells lacking CBP AT activity express MyoD and Myf5 and undergo myogenic differentiation. These data reveal a specific requirement for p300 and its AT activity in the induction of MRF gene expression and myogenic cell fate determination in vivo.

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Expression of the monocyte chemoattractant protein-1 (MCP-1) was examined in human central nervous system tumours (glioblastomas and astrocytomas) and normal human brain. Northern blot analysis demonstrated constitutive expression of MCP-1 mRNA in 6 of 12 glioblastoma cell lines. Expression could be stimulated by interleukin (IL)-1 beta and tumour necrosis factor (TNF)-alpha in all cell lines tested. Immunoprecipitation demonstrated secretion of both isoforms, MCP-1 alpha and -beta, of the MCP-1 protein. Reverse-transcription polymerase chain reaction and Northern blot analysis on tissues demonstrated MCP-1 mRNA expression in 17 of 17 glioblastomas, 3 of 6 anaplastic astrocytomas and 6 of 6 low-grade astrocytomas, as well as in fetal brain but not in normal adult brain. In situ hybridization on 2 glioblastomas and 1 low-grade astrocytoma indicates that neoplastic astrocytes and endothelial cells express MCP-1 mRNA in vivo. Moreover, tumour cyst fluids of glioblastomas and astrocytomas were able to induce monocyte chemoattraction in an in vitro assay. This chemotactic activity was specifically neutralized by anti-MCP-1 antibodies in 9 of 10 samples, further demonstrating the production of bioactive MCP-1 in vivo and supporting an important role for this factor in the infiltration of monocytes/macrophages into tumour tissue.