Analysis of Chromosomal DNA Fragmentation in Apoptosis by Pulsed-Field Gel Electrophoresis.
Analysis of Chromosomal DNA Fragmentation in Apoptosis by Pulsed-Field Gel Electrophoresis.
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DOI：
10.1007/978-1-0716-0323-9_8
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发表时间：
2020
影响因子：
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通讯作者：
T. Terabayashi;Asako Tokumaru;T. Ishizaki;K. Hanada
中科院分区：
文献类型：
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作者：
T. Terabayashi;Asako Tokumaru;T. Ishizaki;K. Hanada
双链DNA断裂（DSB）形成是细胞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染色体DNA片段化之前引入DNA损伤诱导的DSB。为了分析这些不同的断裂，我们已经开发了一种方法，使用脉冲场凝胶电泳（PFGE）与旋转凝胶电泳系统（RGE），使我们能够区分凋亡DSB和DNA损伤剂诱导的DSB的基础上，他们在电泳凝胶中的流动性。凋亡DSB表现为模糊的低分子量条带（小于500 kb），而损伤诱导的DSB导致紧凑的单一条带（大于500 kb）。此外，使用半胱天冬酶抑制剂Z-VAD-FMK，我们可以确认是否产生断裂的DNA片段作为凋亡反应的一部分。总的来说，我们成功地表征了两种单独的凋亡诱导剂，并显示了这些化合物对DNA断裂诱导的不同影响。
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.