Terabayashi Takeshi, Tokumaru Asako, Ishizaki Toshimasa, Hanada Katsuhiro
Department of Pharmacology, Faculty of Medicine, Oita University, Yufu, Oita, Japan.
Clinical Engineering Research Center, Faculty of Medicine, Oita University, Yufu, Oita, Japan.
Methods Mol Biol. 2020;2119:89-99. doi: 10.1007/978-1-0716-0323-9_8.
Double-strand DNA break (DSB) formation is a key feature of apoptosis called chromosomal DNA fragmentation. However, some apoptosis inducers introduce DNA damage-induced DSBs prior to induction of apoptotic chromosomal DNA fragmentation. To analyze these distinct breaks, we have developed a method using pulsed-field gel electrophoresis (PFGE) with a rotating gel electrophoresis system (RGE) that enables us to distinguish between apoptotic DSBs and DNA damaging agent-induced DSBs based on their mobility in the electrophoresis gel. Apoptotic DSBs appear as smeared low-molecular weight bands (less than 500 kb), while damage-induced DSBs result in a compact single band (more than 500 kb). Furthermore, using a caspase inhibitor, Z-VAD-FMK, we can confirm whether broken DNA fragments are produced as part of an apoptotic response. Overall, we succeeded in characterizing two individual apoptosis inducers and showed the different effects of those compounds on the induction of DNA breaks.
双链DNA断裂（DSB）的形成是凋亡的一个关键特征，称为染色体DNA片段化。然而，一些凋亡诱导剂在诱导凋亡性染色体DNA片段化之前会引发DNA损伤诱导的DSB。为了分析这些不同的断裂，我们开发了一种方法，使用带有旋转凝胶电泳系统（RGE）的脉冲场凝胶电泳（PFGE），这使我们能够根据凋亡DSB和DNA损伤剂诱导的DSB在电泳凝胶中的迁移率来区分它们。凋亡DSB表现为涂抹状的低分子量条带（小于500 kb），而损伤诱导的DSB则产生紧密的单一条带（大于500 kb）。此外，使用半胱天冬酶抑制剂Z-VAD-FMK，我们可以确认断裂的DNA片段是否作为凋亡反应的一部分产生。总体而言，我们成功地对两种单独的凋亡诱导剂进行了表征，并展示了这些化合物对DNA断裂诱导的不同影响。
