BackgroundCycling and chronic tumor hypoxia are involved in tumor development and growth. However, the impact of cycling hypoxia and its molecular mechanism on glioblastoma multiforme (GBM) progression remain unclear.MethodologyGlioblastoma cell lines, GBM8401 and U87, and their xenografts were exposed to cycling hypoxic stress in vitro and in vivo. Reactive oxygen species (ROS) production in glioblastoma cells and xenografts was assayed by in vitro ROS analysis and in vivo molecular imaging studies. NADPH oxidase subunit 4 (Nox4) RNAi-knockdown technology was utilized to study the role of Nox4 in cycling hypoxia-mediated ROS production and tumor progression. Furthermore, glioblastoma cells were stably transfected with a retroviral vector bearing a dual reporter gene cassette that allowed for dynamic monitoring of HIF-1 signal transduction and tumor cell growth in vitro and in vivo, using optical and nuclear imaging. Tempol, an antioxidant compound, was used to investigate the impact of ROS on cycling hypoxia-mediated HIF-1 activation and tumor progression.Principal FindingsGlioblastoma cells and xenografts were compared under cycling hypoxic and normoxic conditions; upregulation of NOX4 expression and ROS levels were observed under cycling hypoxia in glioblastoma cells and xenografts, concomitant with increased tumor cell growth in vitro and in vivo. However, knockdown of Nox4 inhibited these effects. Moreover, in vivo molecular imaging studies demonstrated that Tempol is a good antioxidant compound for inhibiting cycling hypoxia-mediated ROS production, HIF-1 activation, and tumor growth. Immunofluorescence imaging and flow cytometric analysis for NOX4, HIF-1 activation, and Hoechst 3342 in glioblastoma also revealed high localized NOX4 expression predominantly in potentially cycling hypoxic areas with HIF-1 activation and blood perfusion within the endogenous solid tumor microenvironment.ConclusionsCycling hypoxia-induced ROS via Nox4 is a critical aspect of cancer biology to consider for therapeutic targeting of cycling hypoxia-promoted HIF-1 activation and tumor progression in GBM.

The herpes simplex virus type 1 thymidine kinase (HSV1-tk)/ green fluorescent protein (TKGFP) dual-reporter gene and a multimodality imaging approach play a critical role in monitoring therapeutic gene expression, immune cell trafficking, and protein-protein interactions in translational molecular-genetic imaging. However, the cytotoxicity and low temporal resolution of TKGFP limits its application in studies that require a rapid turnover of the reporter. The purpose of this study was to construct a novel mutant TKGFP fusion reporter gene with low cytotoxicity and high temporal resolution for use in the real-time monitoring of temporal dynamics and spatial heterogeneity of hypoxia-inducible factor 1 (HIF-1) signal transduction activity mediated by hypoxia and reoxygenation in vitro and in vivo. Methods: Destabilized TKGFP was produced by inserting the nuclear export signal (NES) sequence at the N terminus and fusing the degradation domain of mouse ornithine decarboxylase (dMODC) at the C terminus. The stability of TKGFP in living NG4TL4 cells was determined by Western blot analysis, HSV1-tk enzyme activity assay, and flow cytometric analysis. The suitability of NESTKGFP:dMODC as a transcription reporter was investigated by linking it to a promoter consisting of 8 copies of hypoxiaresponsive elements, whose activities depend on HIF-1. The dynamic transcriptional events mediated by hypoxia and reoxygenation were monitored by NESTKGFP:dMODC or TKGFP and determined by optical imaging and PET. Results: Unlike TKGFP, NESTKGFP:dMODC was unstable in the presence of cycloheximide and showed a short half-life of protein and enzyme activity. Rapid turnover of NESTKGFP:dMODC occurred in a 26S proteasome-dependent manner. Furthermore, NESTKGFP:dMODC showed an upregulated expression and low cytotoxicity in living cells. Studies of hypoxia-responsive TKGFP and NESTKGFP: dMODC expression showed that NESTKGFP:dMODC as a reporter gene had better temporal resolution than did TKGFP for monitoring the dynamic transcriptional events mediated by hypoxia and reoxygenation; the TKGFP expression level was not optimal for the purpose of monitoring. Conclusion: In translational molecular-genetic imaging, NESTKGFP:dMODC as a reporter gene, together with optical imaging and PET, allows the direct monitoring of transcription induction and easy determination of its association with other biochemical changes.Key Words: herpes simplex virus type 1 thymidine kinase/green fluorescent protein (TKGFP); mouse ornithine decarboxylase (MODC); hypoxia; hypoxia-inducible factor 1 (HIF-1); moleculargenetic imaging

In vivo PET study confirmed the modulatory role of EtOH, administered acutely or chronically, in [1- C]-acetate kinetics and metabolism in the rat brain. Acute EtOH intoxication may inhibit the transport and metabolism of acetate in the brain, whereas chronic EtOH exposure may lead to the adaptation of the rat brain to EtOH in acetate utilization. [1- C]-acetate PET imaging is a feasible approach to study the effect of EtOH on acetate metabolism in rat brain.