Yeom Chan Joo, Chung June-Key, Kang Joo Hyun, Jeon Yong Hyun, Kim Kwang Il, Jin Yong Nan, Lee You Mie, Jeong Jae Min, Lee Dong Soo
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.
J Nucl Med. 2008 Sep;49(9):1489-97. doi: 10.2967/jnumed.107.044461. Epub 2008 Aug 14.
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.
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% O(2)) in a hypoxic chamber. HIF-1 transcription activity was assessed by measuring cellular (125)I 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 (99m)Tc 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 (99m)Tc autoradiography, were performed.
Time- and concentration-dependent increases of (125)I 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. (99m)Tc uptake and bioluminescence signals from C6-5HRE-NIS and C6-5HRE-Luc tumors increased during hypoxia. In the biodistribution study, a larger amount of (99m)Tc 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.
The transcriptional activation of HIF-1 induced by hypoxia or DFO was visualized by both bioluminescence and scintigraphic reporter gene systems.
缺氧诱导因子-1（HIF-1）是癌细胞缺氧反应的转录因子，与肿瘤进展、血管生成、转移及治疗耐药性相关。我们评估了人钠碘同向转运体（NIS）报告系统是否可用于可视化C6胶质瘤中HIF-1的转录激活。
构建了两种由5个缺氧反应元件（5HRE）控制的表达人NIS或荧光素酶（Luc）基因的质粒：p5HRE-NIS或p5HRE-Luc。用p5HRE-NIS或p5HRE-Luc质粒（C6-5HRE-NIS或C6-5HRE-Luc）稳定转染C6胶质瘤细胞。通过将培养基暴露于去铁胺（DFO）或缺氧箱中的低氧气氛（<1% O₂）来模拟缺氧条件。通过测量细胞¹²⁵I摄取和发光强度来评估HIF-1转录活性。进行逆转录聚合酶链反应和蛋白质印迹以观察缺氧或常氧条件下报告基因和靶基因的信使核糖核酸及蛋白质水平。将C6、C6-巨细胞病毒（CMV）-NIS或C6-CMV-Luc以及C6-5HRE-NIS或C6-5HRE-Luc细胞分别皮下注射到裸鼠体内（分别为NIS组和Luc组）。肿瘤接种两周后，在腹腔注射DFO前后采集生物发光和⁹⁹ᵐTc闪烁显像图像。通过使肿瘤生长3周诱导肿瘤自然缺氧。进行了诸如生物分布、免疫组织化学和⁹⁹ᵐTc放射自显影等离体研究。
在缺氧应激的报告细胞中观察到¹²⁵I摄取和生物发光随时间和浓度的增加。此外，在缺氧条件下报告基因和靶基因的信使核糖核酸及蛋白质水平增加。缺氧期间，来自C6-5HRE-NIS和C6-5HRE-Luc肿瘤的⁹⁹ᵐTc摄取和生物发光信号增加。在生物分布研究中，与其他不含5HRE的肿瘤相比，更多的⁹⁹ᵐTc积聚在C6-5HRE-NIS肿瘤中（P<0.005）。在Luc组中，免疫染色显示荧光素酶和匹莫硝唑的分布模式相似，在NIS组中，C6-5HRE-NIS肿瘤的放射自显影显示出与匹莫硝唑免疫染色相似的分布。
通过生物发光和闪烁显像报告基因系统均可可视化缺氧或DFO诱导的HIF-1转录激活。
