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Visualization of Hypoxia-Inducible Factor-1 Transcriptional Activation in C6 Glioma Using Luciferase and Sodium Iodide Symporter Genes

¹ Department of Nuclear Medicine, Tumor Immunity Medical Research Center, Laboratory of Molecular Imaging and Therapy of Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea; ² Institute of Radiation Medicine, Medical Research Center, Seoul National University, Seoul, Korea; ³ Laboratory of Nuclear Medicine, Korea Institute of Radiological and Medical Science, Seoul, Korea; and ⁴ Kyungpook National University College of Nature Sciences, Daegu, Korea

View larger version (9K): [in this window] [in a new window]   FIGURE 1.  Diagram of HIF-1 specifically expressed hNIS or Luc reporter gene system. Expression of hNIS (A) or Luc (B) gene is regulated by 5HRE (5xHRE) from human VEGF and SV40 promoter. Under hypoxic conditions, HIF-1β complex binds 5HRE and induces transcriptional expression of reporter genes.

View larger version (31K): [in this window] [in a new window]   FIGURE 2.  In vitro effect of hypoxia on activities of reporter proteins according to radioiodide uptake and luciferase assay. C6-5HRE-NIS or C6-5HRE-Luc cells were seeded in 24-well plates and exposed to normoxia or low atmospheric oxygen (<1% O2) (A and C) or various concentrations (0–400 µM) of DFO, which mimic hypoxia, for various times (4–24 h) (B and D). Effect of hypoxia on hNIS activities was measured using radioiodide uptake assays in C6-5HRE-NIS cells (A and B), and luciferase activity was assessed using luciferase assays in C6-5HRE-Luc cells (C and D). Radioiodine uptake and bioluminescent intensity were normalized by measuring cell viabilities using trypan blue assay. *P < 0.001; **P < 0.0001.

View larger version (42K): [in this window] [in a new window]   FIGURE 3.  Induction of HIF-1 and HIF-1 target genes. (A) RT-PCR analysis of transcripts from HIF-1 target genes, VEGF, and hNIS or Luc reporter genes in normoxic (N) or hypoxic (H) C6 and C6-CMV-NIS or C6-CMV-Luc and C6-5HRE-NIS or C6-5HRE-Luc reporter cells. Cells were incubated under N or H (<1% O2) conditions for 24 h, and then semiquantitative RT-PCR was performed on total RNA. (B) Western blotting of HIF-1 and reporter proteins in C6 and C6-CMV-NIS or C6-CMV-Luc and C6-5HRE-NIS or C6-5HRE-Luc cells. Nuclear protein was used to detect HIF-1, and -tubulin was used as loading control.

View larger version (44K): [in this window] [in a new window]   FIGURE 4.  Bioluminescence imaging of HIF-1 transcriptional activation in tumors of Luc group. (A) Control image (no DFO treatment) was acquired 2 wk after tumor challenge (a = C6, b = C6-CMV-Luc, and c = C6-5HRE-Luc), and then images were sequentially obtained at 4 and 24 h after systemic injection of DFO. (B) ROIs were drawn on bioluminescence images to quantify photon fluxes per second. Statistical significances of ROI results were determined using paired t test. Time-dependent increases in bioluminescent signals were detected. *P < 0.05.

View larger version (28K): [in this window] [in a new window]   FIGURE 5.  Scintigraphic imaging of HIF-1 transcriptional activation in NIS group tumors. (A) Scintigraphic imaging of NIS group 1. In NIS group 1, control scintigraphic image (no DFO treatment) was acquired 2 wk after tumor challenge (a = C6, b = C6-CMV-NIS, c = C6-5HRE-NIS, Thy = thyroid, St = stomach, Bl = bladder), and post-DFO image was observed 24 h after administering DFO intraperitoneally. (B) Scintigraphic imaging of NIS group 2. In another hypoxia-induced NIS group 2, tumors were grown for 3 wk (diameter, 2 cm) to allow hypoxia to occur naturally. (C) ROIs on scintigraphic images of NIS group 2. ROIs of thyroid and tumor were drawn on scintigraphic images from B. Tumor or thyroid ratio was calculated for individual mice (n = 4). Statistical significances of ROI results were determined using paired t test. Scintigraphic signal in 3-wk C6-5HRE-NIS tumors was significantly higher than in 2-wk tumors, whereas no significant change was detected in C6 or C6-CMV-NIS tumors. *P < 0.005. (D) Biodistribution of 99mTc-pertechnetate in mice of NIS group 2. After final imaging, tumors were excised, and %ID/g values for 99mTc-pertechnetate in each tumor were calculated (n = 4). Statistical significances of biodistribution results were determined using 2-sided Student t test with equal variances. Radioactivities in C6-5HRE-NIS tumors were significantly greater than those in C6 and C6-CMV-NIS tumors. Bars indicate mean ± SD. *P < 0.005.

View larger version (104K): [in this window] [in a new window]   FIGURE 6.  Comparison between 99mTc autoradiography and pimonidazole staining of tumors in NIS group 2 and immunohistochemical staining for pimonidazole and luciferase in Luc group. This autoradiograph of C6-5HRE-NIS tumor section (C) has higher 99mTc intensity than C6 (A) or C6-CMV-NIS (B) tumor sections. F shows square area in C at higher magnification. Immunohistochemical staining (x40) pattern observed using antipimonidazole antibody (D) shows similar distribution of 99mTc in 3-wk-grown C6-5HRE-NIS tumor section (F). Colocalization immunohistochemical study (x40) for hypoxic marker, pimonidazole (E), and reporter protein, luciferase (G), was performed using 3-wk-grown C6-5HRE-Luc tumors in Luc group. Hypoxic areas as defined by pimonidazole-positive staining in E were similar to luciferase-positive areas in G. Nx = normoxic region; Hx = hypoxic region. Bars indicate 200 µm.