Visualization of Hypoxia-Inducible Factor-1 Transcriptional Activation in C6 Glioma Using Luciferase and Sodium Iodide Symporter Genes
Chan Joo Yeom 1,
June-Key Chung 1*,
Joo Hyun Kang 2,
Yong Hyun Jeon 1,
Kwang Il Kim 1,
Yong Nan Jin 3,
You Mie Lee 4,
Jae Min Jeong 1,
and
Dong Soo Lee 1
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; Institute of Radiation Medicine, Medical Research Center, Seoul National University, Seoul, Korea
2 Laboratory of Nuclear Medicine, Korea Institute of Radiological and Medical Science, Seoul, Korea
3 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
4 Kyungpook National University College of Nature Sciences, Daegu, Korea
* To whom correspondence should be addressed. E-mail: jkchung{at}plaza.snu.ac.kr.
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Abstract |
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Hypoxia-inducible factor-1 (HIF-1) is a transcription factor of hypoxic response in cancer cells and is associated with tumor progression, angiogenesis, metastasis, and resistance to therapy. We assessed whether the human sodium iodide symporter (NIS) reporter systems can be used to visualize transcriptional activation of HIF-1 in C6 glioma. Methods: Two types of plasmid-expressing human NIS or luciferase (Luc) genes, controlled by 5 copies of hypoxia response element (5HRE), were constructed: p5HRE-NIS or p5HRE-Luc. C6 glioma cells were stably transfected with p5HRE-NIS or p5HRE-Luc plasmids (C6-5HRE-NIS or C6-5HRE-Luc). Hypoxic conditions were modeled by exposing culture medium to desferrioxamine (DFO) or a low oxygen atmosphere (<1% O2) in a hypoxic chamber. HIF-1 transcription activity was assessed by measuring cellular 125I uptake and luminescent intensities. Reverse-transcription polymerase chain reaction and Western blotting were performed to observe the messenger RNA and protein levels of reporter and target genes under hypoxic or normoxic conditions. C6, C6-cytomegalovirus (CMV)-NIS, or C6-CMV-Luc and C6-5HRE-NIS or C6-5HRE-Luc cells were injected subcutaneously into nude mice (the NIS and Luc groups, respectively). Two weeks after tumor challenge, bioluminescence and 99mTc scintigraphic images were acquired before and after intraperitoneal DFO administration. Natural hypoxia in tumors was induced by growing tumors for 3 wk. Ex vivo studies, such as biodistribution, immunohistochemistry, and 99mTc autoradiography, were performed. Results: Time- and concentration-dependent increases of 125I uptake and bioluminescence were observed in hypoxically stressed reporter cells. Also, messenger RNA and protein levels of reporter and target genes increased under hypoxic conditions. 99mTc uptake and bioluminescence signals from C6-5HRE-NIS and C6-5HRE-Luc tumors increased during hypoxia. In the biodistribution study, a larger amount of 99mTc accumulated in C6-5HRE-NIS tumors than in the other tumors not containing 5HRE (P < 0.005). In the Luc group, immunostaining showed similar distribution patterns for luciferase and pimonidazole, and in the NIS group, autoradiography of C6-5HRE-NIS tumors showed a distribution similar to that observed for pimonidazole immunostaining. Conclusion: The transcriptional activation of HIF-1 induced by hypoxia or DFO was visualized by both bioluminescence and scintigraphic reporter gene systems.
Key Words:
hypoxia, hypoxia-inducible factor-1, molecular imaging, sodium iodide symporter, luciferase
