Detailed assessment of gene activation levels by multiple hypoxia responsive elements under various hypoxic conditions [an abstract of entire text]

hypoxia responsive element (HRE)

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Introduction: HIF-1/HRE pathway, which regulates various hypoxia-targeted genes, is a promising target for the imaging and the treatment of intractable malignancy (HIF-1; hypoxia inducible factor 1, HRE; hypoxia responsive elements). The purposes of our study are: (1) to assess the gene activation levels resulting from various numbers of HREs under various durations of hypoxia and hypoxic conditions, (2) to evaluate the bidirectional activity of multiple HREs in tandem, and (3) to confirm whether multiple HREs can induce gene expression in vivo in response to tumor hypoxia. Methods: We designed twelve constructs containing a firefly luciferase reporter gene and various numbers (2, 4, 6, 8, 10, and 12) of HREs(nHRE+, nHRE－). Human colon carcinoma HCT116 cells were transiently transfected and measured the relative luciferase activities by HREs under various durations of hypoxia (6, 12, 18, and 24 h), hypoxic O2 concentrations(1,2, 4, 8, and 16%), and hypoxia-like chemical stress using various concentration of deferoxamine mesylate (20, 40, 80, 160, and 320μg/mL growth medium). The bidirectional gene activation levels by HREs also examined in the constructs (dual-luc-nHREs) containing firefly and Renilla luciferase reporter genes at each side of various numbers (2, 4, 6, 8, 10, and 12). Finally, HCT116 cells were stably transfected with the reporter construct containing 12HRE and the NIS reporter gene (pGL4-12HRE-NIS). To test whether multiple HREs can induce gene expression in vivo in response to tumor hypoxia, SPECT imaging of tumor hypoxia was performed in a mouse subcutaneous xenograft model. Results: The new findings from this study are as follows: (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 in vivo SPECT imaging. Conclusion: The results of this study will help understanding and assessment of the activity of multiple HREs under hypoxia and become the basis for hypoxia-targeted imaging and therapy in the future.