A B S T R A C TNeuroglobin is a recently identified globin molecule that is expressed predominantly in the vertebrate brain. Neuroglobin expression increases in oxygen-deprived neurons, suggesting it protects neurons from ischemic cell death. We report that neuroglobin transcript and protein are expressed in human glioblastoma cells, and that this expression increases in hypoxia in vitro. We also show that neuroglobin is up-regulated in hypoxic microregions of glioblastoma tumor xenografts. Our finding of hypoxic up-regulation of neuroglobin in human glioblastoma cells may provide insight into how tumor cells adapt to and survive in hypoxic microenvironments. Published by Elsevier B.V. All rights reserved. IntroductionNeuroglobin (Ngb), a recently discovered member of the vertebrate globin family, is a small monomeric heme protein with oxygen binding properties (Burmester et al., 2000;Giuffre et al., 2008). Although Ngb's amino acid sequence shows little similarity to that of hemoglobin or myoglobin, amino acids that confer hemoglobin and myoglobin function are conserved as are all characteristics of the globin fold (reviewed in Brunori and Vallone, 2007). Insertion/deletion events are rare, and approximately half of the positions are strictly conserved . As its name suggests, Ngb is expressed primarily in neuronal tissues, with highest concentrations (w100 mM) found in the retina, localized near mitochondria (Geuens et al., 2003;Pesce et al., 2002). High Ngb levels are also found in the brain in regions that show greatest oxygen consumption . Lower levels of Ngb are reported in pancreas, adrenal gland and testes, but Ngb is absent from liver, heart, striated muscle, lung, small bowel, kidney and vasculature Mammen et al., 2002).Although 8 years have passed since the discovery of Ngb, its physiological function remains elusive . Ngb binds oxygen, however, its concentration in tissues other than retina is too low to suggest it might function as an oxygen delivery molecule (Schmidt-Kastner et al., 2006). Based on its regional (greatest in brain areas with high oxygen consumption) and subcellular (near mitochondria) distributions, Ngb may play an important role in scavenging reactive oxygen or nitrosative species, or by activation of yet unknown protective mechanisms (Brunori and Vallone, 2007;Fordel et al., 2006Fordel et al., , 2007bGreenberg et al., Abbreviations: Ngb, neuroglobin; GBM, glioblastoma multiforme; RT, reverse transcription; qRT-PCR, quantitative real-time polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; HRP, horseradish peroxidase; DAB, 3,3 0 diamino benzidine tetrahydrochloride; GFAP, glial fibrillary acidic protein; HIF-1, hypoxia-inducible factor-1; VEGFR2, vascular endothelial growth factor receptor 2.

Background: Hypoxia-tolerant human glioma cells reduce oxygen consumption rate in response to oxygen deficit, a defense mechanism that contributes to survival under moderately hypoxic conditions. In contrast, hypoxia-sensitive cells lack this ability. As it has been previously shown that hypoxia-tolerant (M006x, M006xLo, M059K) and -sensitive (M010b) glioma cells express differences in mitochondrial function, we investigated whether mitochondrial DNA-encoded mutations are associated with differences in the initial response to oxygen deficit.