DNA damage during mitosis in human cells delays the metaphase/anaphase transition via the spindle-assembly checkpoint
DNA damage during mitosis in human cells delays the metaphase/anaphase transition via the spindle-assembly checkpoint
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DOI：
10.1016/s0960-9822(02)01226-5
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发表时间：
2002-10-29
期刊：
影响因子：
9.2
通讯作者：
Rieder, CL
中科院分区：
文献类型：
--
作者：
Mikhailov, A;Cole, RW;Rieder, CL
背景资料：有丝分裂过程中的DNA损伤触发了酵母和苍蝇胚胎中ATM激酶介导的细胞周期检查点途径，从而延迟了分裂的进展。最近的数据表明，哺乳动物也是如此。在这里，我们使用激光显微手术和拓扑异构酶II α的抑制剂来破坏各种哺乳动物细胞在有丝分裂后的DNA。然后，我们遵循这些细胞的命运，并强调有丝分裂的进展，纺锤体结构，和染色体behaviors.Results的时间：我们发现，在后期前期产生的DNA断裂不妨碍进入前中期。如果损伤较小，细胞会按时完成有丝分裂。然而，在许多细胞类型中，更显著的损伤实质上延迟退出有丝分裂。在人类（HeLa，CFPAC-1和hTERT-RPE）细胞中，这种延迟发生在中期，在双极纺锤体形成和细胞周期蛋白A破坏之后，并且它不依赖于功能性p53通路。用ATM激酶抑制剂预处理细胞不能消除由于染色体损伤引起的中期延迟。免疫荧光研究表明，在中期染色体损伤的细胞阻断包含一个或多个Mad 2阳性动粒，并迅速覆盖块时，细胞显微注射显性负结构的Mad 2（Mad 2DeltaC）。结论：我们的结论是，延迟有丝分裂诱导的DNA损伤是不是由于ATM介导的DNA损伤检查点途径。相反，这种损伤导致动粒附着和功能的缺陷，反过来又维持了内在的基于Mad-2的纺锤体组装检查点。
Background: DNA damage during mitosis triggers an ATM kinase-mediated cell cycle checkpoint pathway in yeast and fly embryos that delays progression through division. Recent data suggest that this is also true for mammals. Here we used laser microsurgery and inhibitors of topoisomerase IIalpha to break DNA in various mammalian cells after they became committed to mitosis. We then followed the fate of these cells and emphasized the timing of mitotic progression, spindle structure, and chromosome behavior.Results: We find that DNA breaks generated during late prophase do not impede entry into prometaphase. If the damage is minor, cells complete mitosis on time. However, more significant damage substantially delays exit from mitosis in many cell types. In human (HeLa, CFPAC-1, and hTERT-RPE) cells, this delay occurs during metaphase, after the formation of a bipolar spindle and the destruction of cyclin A, and it is not dependent on a functional p53 pathway. Pretreating cells with ATM kinase inhibitors does not abrogate the metaphase delay due to chromosome damage. Immunofluorescence studies reveal that cells blocked in metaphase by chromosome damage contain one or more Mad2-positive kinetochores, and the block is rapidly overridden when the cells are microinjected with a dominant-negative construct of Mad2 (Mad2DeltaC).Conclusions: We conclude that the delay in mitosis induced by DNA damage is not due to an ATM-mediated DNA damage checkpoint pathway. Rather, the damage leads to defects in kinetochore attachment and function that, in turn, maintain the intrinsic Mad-2-based spindle assembly checkpoint.