Department of Nuclear Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200025, China.
Nucl Med Biol. 2011 May;38(4):599-604. doi: 10.1016/j.nucmedbio.2010.11.005. Epub 2011 Feb 4.
As intracellular iodine is released rapidly, increased expression of sodium/iodide symporter (NIS) is required for effective radioiodine treatment of tumor. As Egr1 promoter is activated by ¹³¹I and may promote human NIS (hNIS) expression, hNIS also induces ¹³¹I uptake and activates Egr1, so the existence of a positive feedback effect of ¹³¹I-promoted Egr1-hNIS expression is possible. Our purpose was to investigate the possible existence of this positive feedback effect through a series of in vitro pioneer studies.
Recombinant baculovirus (Bac-Egr1-hNIS) encoding the hNIS gene under the control of a radiation-inducible Egrl promoter was constructed. To test ¹³¹I-promoted hNIS expression, human malignant glioma U87 cells were transfected with Bac-Egr1-hNIS, stimulated with or without ¹³¹I; the expression of hNIS protein was detected by immunofluorescence and flow cytometry test. In addition, the uptake and efflux of ¹³¹I were determined after the incubation of Bac-Egr1-hNIS-transfected U87 cells with or without ¹³¹I.
Immunocytochemical staining and flow cytometry test showed a higher hNIS protein expression in Bac-Egr1-hNIS-transfected U87 cells with ¹³¹I stimulation than in cells without stimulation. Bac-Egr1-hNIS-transfected U87 cells accumulated up to about 4.05 times of ¹³¹I after ¹³¹I stimulation. The amount of ¹³¹I uptake in both groups showed a baculovirus dose-dependent manner. However, rapid efflux of radioactivity was observed in both groups, with 50% lost during the first 2 min after the ¹³¹I-containing medium had been replaced by a nonradioactive medium.
Our results indicated that an improved transgene expression of ¹³¹I-stimulated hNIS in U87 cells using a baculovirus vector containing the Egr1 promoter is possible, and the increased expression of hNIS is responsible for a higher ¹³¹I uptake. It might provide a reference for the existence of a positive feedback effect in ¹³¹I-promoted Bac-Egr1-hNIS expression in malignant glioma and is an interesting aspect of NIS-related studies.
由于细胞内碘迅速释放，因此需要增加钠/碘转运体（NIS）的表达，以实现肿瘤的有效放射性碘治疗。由于 Egr1 启动子被 ¹³¹I 激活，并且可能促进人 NIS（hNIS）的表达，hNIS 还诱导 ¹³¹I 摄取并激活 Egr1，因此 ¹³¹I 促进 Egr1-hNIS 表达的正反馈效应可能存在。我们的目的是通过一系列体外探索性研究来研究这种正反馈效应的可能存在。
构建了编码 hNIS 基因的重组杆状病毒（Bac-Egr1-hNIS），该基因受辐射诱导的 Egr1 启动子控制。为了测试 ¹³¹I 促进的 hNIS 表达，用 Bac-Egr1-hNIS 转染人恶性神经胶质瘤 U87 细胞，并用或不用 ¹³¹I 刺激；通过免疫荧光和流式细胞术检测 hNIS 蛋白的表达。此外，在 Bac-Egr1-hNIS 转染的 U87 细胞与或不与 ¹³¹I 孵育后，测定 ¹³¹I 的摄取和外排。
免疫细胞化学染色和流式细胞术检测结果显示，在有 ¹³¹I 刺激的 Bac-Egr1-hNIS 转染的 U87 细胞中，hNIS 蛋白表达高于无刺激组。¹³¹I 刺激后，Bac-Egr1-hNIS 转染的 U87 细胞可积累高达约 4.05 倍的 ¹³¹I。两组的 ¹³¹I 摄取量均呈杆状病毒剂量依赖性方式增加。然而，两组中均观察到放射性物质的快速外排，¹³¹I 含量在非放射性培养基替换含 ¹³¹I 培养基后的前 2 分钟内丢失了 50%。
我们的结果表明，使用含有 Egr1 启动子的杆状病毒载体，可能提高了 ¹³¹I 刺激的 U87 细胞中转基因 hNIS 的表达，并且 hNIS 的高表达导致更高的 ¹³¹I 摄取。这可能为恶性神经胶质瘤中 ¹³¹I 促进的 Bac-Egr1-hNIS 表达中的正反馈效应的存在提供参考，并且是 NIS 相关研究的一个有趣方面。
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