DIPG-67. Hypoxia-Inducible Factors Regulate Diffuse Intrinsic Pontine Glioma Growth in Normoxic Culture

Diffuse intrinsic pontine glioma (DIPG) are incurable tumors and the leading cause of pediatric brain tumor deaths. They exhibit low blood perfusion and regions of necrosis, indicative of a low-oxygen environment that supports activation of hypoxia-inducible factors (HIF) that are associated with increased proliferation, invasion, and therapy resistance. However, previous reports suggest that HIF2-alpha slows growth in some glioma models. We therefore sought to test the hypothesis that HIFs regulate DIPG growth. We cultured the human DIPG tumors SU-DIPG-IV, VUMC-DIPG-X, and SU-DIPG-XIII at ambient oxygen tension and 5% carbon dioxide. We measured protein expression by Western blot and growth by trypan blue exclusion or tetrazolium reduction following exposure to the hypoxia-mimetic (HM) compounds, cobalt (II) chloride or deferoxamine, or selective HIF inhibitors. All three DIPG cultures retained stable expression of HIF1-alpha and HIF2-alpha protein at ambient oxygen tension, unchanged by HM treatment. Selective inhibition of HIF2-alpha by TC-S 7009 increased apparent growth, whereas selective inhibition of HIF1-alpha by CAY10585 did not. We conclude that hypoxia-independent HIF expression unchanged by either HM treatment or HIF inhibition suggests impaired HIF degradation, in which hypoxia-induced activation of HIF target genes more likely depends on transcriptional co-activators rather than blocked proteasomal degradation. In both ambient and hypoxic conditions, HIF2-alpha activity may oppose DIPG growth. Future experiments will investigate whether the effects of HIF2-alpha inhibition on tumor growth can be explained by enhanced HIF1-alpha activity through desequestration of common binding partners, or through direct action of HIF2-alpha on previously reported apoptotic pathways

DIPG-67. Hypoxia-Inducible Factors Regulate Diffuse Intrinsic Pontine Glioma Growth in Normoxic Culture

Diffuse intrinsic pontine glioma (DIPG) are incurable tumors and the leading cause of pediatric brain tumor deaths. They exhibit low blood perfusion and regions of necrosis, indicative of a low-oxygen environment that supports activation of hypoxia-inducible factors (HIF) that are associated with increased proliferation, invasion, and therapy resistance. However, previous reports suggest that HIF2-alpha slows growth in some glioma models. We therefore sought to test the hypothesis that HIFs regulate DIPG growth. We cultured the human DIPG tumors SU-DIPG-IV, VUMC-DIPG-X, and SU-DIPG-XIII at ambient oxygen tension and 5% carbon dioxide. We measured protein expression by Western blot and growth by trypan blue exclusion or tetrazolium reduction following exposure to the hypoxia-mimetic (HM) compounds, cobalt (II) chloride or deferoxamine, or selective HIF inhibitors. All three DIPG cultures retained stable expression of HIF1-alpha and HIF2-alpha protein at ambient oxygen tension, unchanged by HM treatment. Selective inhibition of HIF2-alpha by TC-S 7009 increased apparent growth, whereas selective inhibition of HIF1-alpha by CAY10585 did not. We conclude that hypoxia-independent HIF expression unchanged by either HM treatment or HIF inhibition suggests impaired HIF degradation, in which hypoxia-induced activation of HIF target genes more likely depends on transcriptional co-activators rather than blocked proteasomal degradation. In both ambient and hypoxic conditions, HIF2-alpha activity may oppose DIPG growth. Future experiments will investigate whether the effects of HIF2-alpha inhibition on tumor growth can be explained by enhanced HIF1-alpha activity through desequestration of common binding partners, or through direct action of HIF2-alpha on previously reported apoptotic pathways

DIPG-67. Hypoxia-Inducible Factors Regulate Diffuse Intrinsic Pontine Glioma Growth in Normoxic Culture

Diffuse intrinsic pontine glioma (DIPG) are incurable tumors and the leading cause of pediatric brain tumor deaths. They exhibit low blood perfusion and regions of necrosis, indicative of a low-oxygen environment that supports activation of hypoxia-inducible factors (HIF) that are associated with increased proliferation, invasion, and therapy resistance. However, previous reports suggest that HIF2-alpha slows growth in some glioma models. We therefore sought to test the hypothesis that HIFs regulate DIPG growth. We cultured the human DIPG tumors SU-DIPG-IV, VUMC-DIPG-X, and SU-DIPG-XIII at ambient oxygen tension and 5% carbon dioxide. We measured protein expression by Western blot and growth by trypan blue exclusion or tetrazolium reduction following exposure to the hypoxia-mimetic (HM) compounds, cobalt (II) chloride or deferoxamine, or selective HIF inhibitors. All three DIPG cultures retained stable expression of HIF1-alpha and HIF2-alpha protein at ambient oxygen tension, unchanged by HM treatment. Selective inhibition of HIF2-alpha by TC-S 7009 increased apparent growth, whereas selective inhibition of HIF1-alpha by CAY10585 did not. We conclude that hypoxia-independent HIF expression unchanged by either HM treatment or HIF inhibition suggests impaired HIF degradation, in which hypoxia-induced activation of HIF target genes more likely depends on transcriptional co-activators rather than blocked proteasomal degradation. In both ambient and hypoxic conditions, HIF2-alpha activity may oppose DIPG growth. Future experiments will investigate whether the effects of HIF2-alpha inhibition on tumor growth can be explained by enhanced HIF1-alpha activity through desequestration of common binding partners, or through direct action of HIF2-alpha on previously reported apoptotic pathways

(14) N solid-state NMR spectroscopy of amino acids

(14) N ultra-wideline solid-state NMR (SSNMR) spectra were obtained for 16 naturally occurring amino acids and four related derivatives by using the WURST-CPMG (wideband, uniform rate, and smooth truncation Carr-Purcell-Meiboom-Gill) pulse sequence and frequency-stepped techniques. The (14) N quadrupolar parameters were measured for the sp(3) nitrogen moieties (quadrupolar coupling constant, CQ , values ranged from 0.8 to 1.5 MHz). With the aid of plane-wave DFT calculations of the (14) N electric-field gradient tensor parameters and orientations, the moieties were grouped into three categories according to the values of the quadrupolar asymmetry parameter, ηQ : low (≤0.3), intermediate (0.31-0.7), and high (≥0.71). For RNH3(+) moieties, greater variation in N-H bond lengths was observed for systems with intermediate ηQ values than for those with low ηQ values (this variation arose from different intermolecular hydrogen-bonding arrangements). Strategies for increasing the efficiency of (14) N SSNMR spectroscopy experiments were discussed, including the use of sample deuteration, high-power (1) H decoupling, processing strategies, high magnetic fields, and broadband cross-polarization (BRAIN-CP). The temperature-dependent rotations of the NH3 groups and their influence on (14) N transverse relaxation rates were examined. Finally, (14) N SSNMR spectroscopy was used to differentiate two polymorphs of l-histidine through their quadrupolar parameters and transverse relaxation time constants. The strategies outlined herein permitted the rapid acquisition of directly detected (14) N SSNMR spectra that to date was not matched by other proposed methods