Abstract
ObjectiveHIF-1/HRE pathway is a promising target for the imaging and the treatment of intractable malignancy (HIF-1; hypoxia-inducible factor 1, HRE; hypoxia-responsive element). The purposes of our study are: (1) to assess the gene activation levels resulting from various numbers of HREs under various hypoxic conditions, (2) to evaluate the bidirectional activity of multiple HREs, and (3) to confirm whether multiple HREs can induce gene expression in vivo.MethodsHuman colon carcinoma HCT116 cells were transiently transfected by the constructs containing a firefly luciferase reporter gene and various numbers (2, 4, 6, 8, 10, and 12) of HREs (nHRE+, nHRE−). The relative luciferase activities were measured under various durations of hypoxia (6, 12, 18, and 24 h), O2 concentrations (1, 2, 4, 8, and 16 %), and various concentrations of deferoxamine mesylate (20, 40, 80, 160, and 320 µg/mL growth medium). The bidirectional gene activation levels by HREs were examined in the constructs (dual-luc-nHREs) containing firefly and Renilla luciferase reporter genes at each side of nHREs. Finally, to test whether the construct containing 12HRE and the NIS reporter gene (12HRE-NIS) can induce gene expression in vivo, SPECT imaging was performed in a mouse xenograft model.Results(1) gene activation levels by HREs tended to increase with increasing HRE copy number, but a saturation effect was observed in constructs with more than 6 or 8 copies of an HRE, (2) gene activation levels by HREs increased remarkably during 6–12 h of hypoxia, but not beyond 12 h, (3) gene activation levels by HREs decreased with increasing O2 concentrations, but could be detected even under mild hypoxia at 16 % O2, (4) the bidirectionally proportional activity of the HRE was confirmed regardless of the hypoxic severity, and (5) NIS expression driven by 12 tandem copies of an HRE in response to hypoxia could be visualized on in vivo SPECT imaging.ConclusionThe results of this study will help in the understanding and assessment of the activity of multiple HREs under hypoxia and become the basis for hypoxia-targeted imaging and therapy in the future.
Highlights
In tumor cells at low oxygen levels, genetic response to hypoxia is initiated by binding of hypoxia-inducible factor1 (HIF-1), which is a major transcriptional activator of hypoxia-regulated genes, to hypoxia-responsive elements (HREs), which are transcription enhancers located in the
Gene activation levels by various numbers of HREs under various durations of hypoxia The expression of the firefly luciferase reporter gene induced by various numbers (2, 4, 6, 8, 10, and 12) of HREs was assessed after hypoxic stress at 1 % O2 during four different test periods (6, 12, 18, and 24 h; Fig. 1)
The findings in this study were as follows: (1) gene activation levels tended to increase with increasing HRE copy number, but saturation was observed in constructs with more than 6 or 8 copies of the HRE, (2) gene activation levels by the HRE increased remarkably during 6–12 h of hypoxia, but not beyond 12 h, (3) gene activation levels by the HRE decreased with increasing O2 concentrations, but could be detected even under mild hypoxia in 16 % O2, (4) the bidirectionally proportional activity of the HRE was confirmed regardless of the hypoxic conditions, and (5) NIS expression upregulated by 12 tandem copies of HREs (12HRE) in response to tumor hypoxia could be visualized in in vivo SPECT imaging
Summary
In tumor cells at low oxygen levels, genetic response to hypoxia is initiated by binding of hypoxia-inducible factor (HIF-1), which is a major transcriptional activator of hypoxia-regulated genes, to hypoxia-responsive elements (HREs), which are transcription enhancers located in theAnn Nucl Med (2014) 28:1011–1019 First author HRE originHRE copy number Direction of HREO2 concentration (%) Hypoxia duration (h)Dachs [4] Shibata [12] Ruan [13] Post [14] Current study mPGK hVEGF hEPO hVEGF, hEPO hVEGFSense Sense Sense Bidirectional Sense, antisense, bidirectionalHRE hypoxia-responsive element, mPGK mouse phosphoglycerate kinase-1, hVEGF human vascular endothelial growth factor, hEPO human erythropoietin promoter regions of hypoxia-regulated genes such as erythropoietin (Epo) and vascular endothelial growth factor (VEGF) genes [1, 2]. Multiple copies of HREs have been synthesized and applied in in vivo experiments for imaging reporter gene expression in response to hypoxia [6,7,8] and for hypoxiatargeted gene therapy [9,10,11]. The correlation between hypoxia and gene activation levels by such multiple HREs has to rely upon in vitro experiments of cell cultures, because there is no appropriate method to evaluate hypoxia in essentially inhomogeneous tumor masses. Shibata et al [12] have suggested that the saturated hypoxia responsiveness of ten copies of an HRE is similar to that of five copies, but Ruan et al [13] reported that hypoxic induction of gene expression by nine copies was approximately three times higher than from six copies. Post et al [14] investigated the bidirectional function of the HRE only as a classical enhancer element; their results seemed to be erroneous based on the wide variation in HRE background activity observed under normoxia
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