Wakuri S, Yamakage K, Kazuki Y, Kazuki K, Oshimura M, Aburatani S, Yasunaga M, Nakajima Y
Hatano Research Institute, Food and Drug Safety Center, Hadano, Kanagawa 257-8523, Japan.
Chromosome Engineering Research Center, Tottori University, Yonago, Tottori 683-8503, Japan; Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Tottori 683-8503, Japan.
Anal Biochem. 2017 Apr 1;522:18-29. doi: 10.1016/j.ab.2017.01.015. Epub 2017 Jan 20.
The luciferase reporter assay has become one of the conventional methods for cytotoxicity evaluation. Typically, the decrease of luminescence expressed by a constitutive promoter is used as an index of cytotoxicity. However, to our knowledge, there have been no reports of the correlation between cytotoxicity and luminescence intensity. In this study, to accurately verify the correlation between them, beetle luciferase was stably expressed in human hepatoma HepG2 cells harboring the multi-integrase mouse artificial chromosome vector. We showed that the cytotoxicity assay using luciferase does not depend on the stability of luciferase protein and the kind of constitutive promoter. Next, HepG2 cells in which green-emitting beetle luciferase was expressed under the control of CAG promoter were exposed to 58 compounds. The luminescence intensity and cytotoxicity curves of cells exposed to 48 compounds showed similar tendencies, whereas those of cells exposed to 10 compounds did not do so, although the curves gradually approached each other with increasing exposure time. Finally, we demonstrated that luciferase expressed under the control of a constitutive promoter can be utilized both as an internal control reporter for normalizing a test reporter and for monitoring cytotoxicity when two kinds of luciferases are simultaneously used in the cytotoxicity assay.
荧光素酶报告基因检测已成为细胞毒性评估的传统方法之一。通常，组成型启动子表达的发光减少被用作细胞毒性指标。然而，据我们所知，尚无关于细胞毒性与发光强度之间相关性的报道。在本研究中，为了准确验证它们之间的相关性，将甲虫荧光素酶稳定表达于携带多整合酶小鼠人工染色体载体的人肝癌HepG2细胞中。我们发现，使用荧光素酶的细胞毒性检测不依赖于荧光素酶蛋白的稳定性和组成型启动子的种类。接下来，将在CAG启动子控制下表达绿色发光甲虫荧光素酶的HepG2细胞暴露于58种化合物。暴露于48种化合物的细胞的发光强度和细胞毒性曲线显示出相似的趋势，而暴露于10种化合物的细胞则不然，尽管随着暴露时间的增加，曲线逐渐接近。最后，我们证明，当在细胞毒性检测中同时使用两种荧光素酶时，组成型启动子控制下表达的荧光素酶既可以用作标准化测试报告基因的内部对照报告基因，也可以用于监测细胞毒性。
