Lewis Cody W, Golsteyn Roy M
a Cancer Cell Laboratory, Department of Biological Sciences, University of Lethbridge , Lethbridge , AB , Canada.
Cell Cycle. 2016 Nov 16;15(22):3131-3145. doi: 10.1080/15384101.2016.1231287. Epub 2016 Sep 16.
We have examined the relationship between checkpoint adaptation (mitosis with damaged DNA) and micronuclei. Micronuclei in cancer cells are linked to genomic change, and may induce chromothripsis (chromosome shattering). We measured the cytotoxicity of the cancer drug cisplatin in M059K (glioma fibroblasts, IC50 15 μM). Nearly 100% of M059K cells were positive for histone γH2AX staining after 48 h treatment with a cytotoxic concentration of cisplatin. The proportion of micronucleated cells, as confirmed by microscopy using DAPI and lamin A/C staining, increased from 24% to 48%, and the total micronuclei in surviving cells accumulated over time. Promoting entry into mitosis with a checkpoint inhibitor increased the number of micronuclei in cells whereas blocking checkpoint adaptation with a Cdk inhibitor reduced the number of micronuclei. Interestingly, some micronuclei underwent asynchronous DNA replication, relative to the main nuclei, as measured by deoxy-bromo-uracil (BrdU) staining. These micronuclei stained positive for histone γH2AX, which was linked to DNA replication, suggesting that micronuclei arise from checkpoint adaptation and that micronuclei may continue to damage DNA. By contrast the normal cell line WI-38 did not undergo checkpoint adaptation when treated with cisplatin and did not show changes in micronuclei number. These data reveal that the production of micronuclei by checkpoint adaptation is part of a process that contributes to genomic change.
我们研究了关卡适应（DNA受损时的有丝分裂）与微核之间的关系。癌细胞中的微核与基因组变化相关，且可能诱发染色体碎裂（染色体破碎）。我们测定了抗癌药物顺铂对M059K细胞（胶质瘤成纤维细胞，IC50为15 μM）的细胞毒性。用细胞毒性浓度的顺铂处理48小时后，近100%的M059K细胞组蛋白γH2AX染色呈阳性。通过使用DAPI和核纤层蛋白A/C染色的显微镜检查确认，微核化细胞的比例从24%增加到48%，存活细胞中的微核总数随时间累积。用关卡抑制剂促进细胞进入有丝分裂会增加细胞中的微核数量，而用细胞周期蛋白依赖性激酶（Cdk）抑制剂阻断关卡适应则会减少微核数量。有趣的是，通过脱氧溴尿嘧啶（BrdU）染色测量发现，一些微核相对于主核进行异步DNA复制。这些微核组蛋白γH2AX染色呈阳性，这与DNA复制有关，表明微核源自关卡适应，且微核可能会继续损伤DNA。相比之下，正常细胞系WI-38在用顺铂处理时不会发生关卡适应，微核数量也没有变化。这些数据表明，关卡适应导致微核产生是促成基因组变化过程的一部分。
