Detailed assessment of gene activation levels by multiple hypoxia-responsive elements under various hypoxic conditions

Abstract

Objective: HIF-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.

Methods: Human 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.

Conclusion: The 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.

Fig. 1

Relative luciferase activity of various numbers of HREs under various durations of hypoxia (1 % O₂). Left HRE in sense orientation with respect to the Luc2 gene (nHRE+); right HRE (nHRE−) in antisense orientation with respect to the Luc2 gene. A schema of the constructs is attached on top

Fig. 2

Relative luciferase activity of various numbers of HREs under various concentrations of DFO stress. Left HRE in sense orientation with respect to the Luc2 gene (nHRE+); right HRE in antisense orientation with respect to the Luc2 gene (nHRE−). A schema of the constructs is attached on top

Fig. 3

Relative luciferase activity of various numbers of HREs under various hypoxic O₂ concentrations. Left HRE in sense orientation with respect to the Luc2 gene (nHRE+); right HRE in antisense orientation with respect to the Luc2 gene (nHRE−). A schema of the constructs is attached on top

Fig. 4

Ratio of bidirectional activity of various numbers of HRE under various hypoxic conditions. Left DFO stress; Right hypoxic gas stress. Dotted lines indicate the regression line for data of 12HRE; y = 0.0004x + 1.06 (left), y = 0.0125x + 0.744 (right). A schema of the constructs is attached on top

Fig. 5

Results of in vitro ^99mTcO₄ ⁻ uptake assay (top right) and representative in vivo ^99mTcO₄ ⁻ SPECT images (bottom). In the bar graph, the numbers indicate the mean ^99mTcO₄ ⁻ uptake levels, and the error bars represent the standard deviations. In the bottom figure, fusion (coronal) and SPECT (MIP) views in each image set were made from the same mouse. A schema of the constructs is attached on the top left

Fig. 6

Representative staining images of xenograft. Left Hematoxylin–eosin staining; center immunohistochemical staining of pimonidazole; right immunohistochemical staining of NIS. Bars indicate 1 mm