Cell Division and Cytoskeleton, Danish Cancer Institute, Copenhagen, Denmark
Cell Division and Cytoskeleton, Danish Cancer Institute, Copenhagen, Denmark.
Life Sci Alliance. 2023 Sep 19;6(12). doi: 10.26508/lsa.202302260. Print 2023 Dec.
SiR-DNA/SiR-Hoechst is a far-red fluorescent DNA probe that is routinely used for live-cell imaging of cell nuclei in interphase and chromosomes during mitosis. Despite being reported to induce DNA damage, SiR-DNA has been used in more than 300 research articles, covering topics like mitosis, chromatin biology, cancer research, cytoskeletal research, and DNA damage response. Here, we used live-cell imaging to perform a comprehensive analysis of the effects of SiR-DNA on mitosis of four human cell lines (RPE-1, DLD-1, HeLa, and U2OS). We report a dose-, time-, and light-dependent effect of SiR-DNA on chromosome segregation. We found that, upon the exposure to light during imaging, nanomolar concentrations of SiR-DNA induce non-centromeric chromosome entanglement that severely impairs sister chromatid segregation and spindle elongation during anaphase. This causes DNA damage that is passed forward to the following cell cycle, thereby having a detrimental effect on genome integrity. Our findings highlight the drawbacks in using SiR-DNA for investigation of late mitotic events and DNA damage-related topics and urge the use of alternative labeling strategies to study these processes.
SiR-DNA/SiR-Hoechst 是一种远红色荧光 DNA 探针，常用于间期细胞和有丝分裂中期染色体的活细胞成像。尽管有报道称 SiR-DNA 会诱导 DNA 损伤，但它已被用于 300 多篇研究论文中，涵盖有丝分裂、染色质生物学、癌症研究、细胞骨架研究和 DNA 损伤反应等多个主题。在这里，我们使用活细胞成像技术对 SiR-DNA 对四种人类细胞系（RPE-1、DLD-1、HeLa 和 U2OS）有丝分裂的影响进行了全面分析。我们报告了 SiR-DNA 对染色体分离的剂量、时间和光依赖性影响。我们发现，在成像过程中暴露于光线下时，纳摩尔浓度的 SiR-DNA 会诱导非着丝粒染色体缠结，严重损害后期姐妹染色单体分离和纺锤体伸长，从而导致 DNA 损伤，并传递到下一个细胞周期，从而对基因组完整性产生不利影响。我们的研究结果强调了在使用 SiR-DNA 研究后期有丝分裂事件和与 DNA 损伤相关的课题时存在的缺陷，并敦促使用替代标记策略来研究这些过程。
