Quantitative assessment of chromosome instability induced through chemical disruption of mitotic progression.
Quantitative assessment of chromosome instability induced through chemical disruption of mitotic progression.
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DOI：
10.1080/15384101.2016.1175796
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发表时间：
2016-07-02
期刊：
影响因子：
--
通讯作者：
Dasso M
中科院分区：
文献类型：
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作者：
Markossian S;Arnaoutov A;Saba NS;Larionov V;Dasso M
大多数实体瘤是非整倍体，携带异常数量的染色体，并且它们经常在染色体不稳定（CIN）现象中错误分离整个染色体。虽然CIN可以通过破坏许多有丝分裂途径而引起，但尚不清楚这些机制中哪一个是最关键的，或者其他机制是否也可以在体内发挥重要作用。确定候选CIN调节因子的相对重要性的一个困难是缺乏对活细胞中CIN的直接定量分析：虽然肉眼可以检测到严重的有丝分裂异常，但中等水平的CIN可能不明显，因此测量起来有问题。为了解决这个问题，我们开发了第一个基于人类人工染色体（HAC）的定量活细胞检测，用于人类细胞中有丝分裂染色体分离。我们生产了U2OS-Phoenix细胞，携带融合到后期促进复合体/环体（APC/C）底物hSecurin破坏盒（DB）的alphoidtetO-HAC编码eGFP拷贝和融合到mCherry的四环素抑制因子序列（tetri -mCherry）。在HAC错分离时，没有获得HAC拷贝的子细胞在随后的间期呈GFP阴性。HAC也可以根据rt - mcherry信号进行实时监控。u20s - phoenix细胞显示出较低的固有CIN水平，可以通过不同机制靶向有丝分裂进程的药物增强CIN水平。该检测允许直接检测临床重要药物诱导的CIN，而没有明显的有丝分裂缺陷，使我们能够对低于可识别形态破坏所需阈值的CIN水平进行评分。
Most solid tumors are aneuploid, carrying an abnormal number of chromosomes, and they frequently missegregate whole chromosomes in a phenomenon termed chromosome instability (CIN). While CIN can be provoked through disruption of numerous mitotic pathways, it is not clear which of these mechanisms are most critical, or whether alternative mechanisms could also contribute significantly in vivo. One difficulty in determining the relative importance of candidate CIN regulators has been the lack of a straightforward, quantitative assay for CIN in live human cells: While gross mitotic abnormalities can be detected visually, moderate levels of CIN may not be obvious, and are thus problematic to measure. To address this issue, we have developed the first Human Artificial Chromosome (HAC)-based quantitative live-cell assay for mitotic chromosome segregation in human cells. We have produced U2OS-Phoenix cells carrying the alphoidtetO-HAC encoding copies of eGFP fused to the destruction box (DB) of anaphase promoting complex/cyclosome (APC/C) substrate hSecurin and sequences encoding the tetracycline repressor fused to mCherry (TetR-mCherry). Upon HAC missegregation, daughter cells that do not obtain a copy of the HAC are GFP negative in the subsequent interphase. The HAC can also be monitored live following the TetR-mCherry signal. U2OS-Phoenix cells show low inherent levels of CIN, which can be enhanced by agents that target mitotic progression through distinct mechanisms. This assay allows direct detection of CIN induced by clinically important agents without conspicuous mitotic defects, allowing us to score increased levels of CIN that fall below the threshold required for discernable morphological disruption.