A computational platform for robotized fluorescence microscopy (II): DNA damage, replication, checkpoint activation, and cell cycle progression by high-content high-resolution multiparameter image-cytometry
A computational platform for robotized fluorescence microscopy (II): DNA damage, replication, checkpoint activation, and cell cycle progression by high-content high-resolution multiparameter image-cytometry
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DOI：
10.1002/cyto.a.22265
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发表时间：
2013-04-01
期刊：
影响因子：
3.7
通讯作者：
Faretta, Mario
中科院分区：
文献类型：
--
作者：
Furia, Laura;Pelicci, Pier Giuseppe;Faretta, Mario
在稀有细胞群体中的复杂分子网络的解剖受到当前技术的限制，这些技术不允许同时定量，高分辨率定位和多个参数的统计学稳健分析。我们已经开发了一种新的计算平台（自动显微镜图像细胞仪，A.M.I.CO）的定量图像分析数据的共聚焦或宽视场自动化显微镜。我们已经将这种图像细胞术技术应用于非转化乳腺细胞中自发DNA损伤时检查点激活的研究。细胞周期谱和活跃的DNA复制与（i）Ki 67相关，以监测增殖;（ii）磷酸化组蛋白H2 AX（H2 AX）和53 BP 1，作为DNA损伤反应（DDR）的标志物;和（iii）p53和p21，作为检查点激活标志物。我们的数据表明，细胞周期调节机制的存在，涉及不同的功能H2 AX和53 BP 1在DDR，和p53和p21在检查点激活和静止调节细胞周期。定量分析，事件选择，和物理重新定位，然后已被用于相关的蛋白质表达在人口水平与分子之间的相互作用，测量与邻近连接分析，具有前所未有的统计相关性。(c)2012年国际细胞计数促进学会
Dissection of complex molecular-networks in rare cell populations is limited by current technologies that do not allow simultaneous quantification, high-resolution localization, and statistically robust analysis of multiple parameters. We have developed a novel computational platform (Automated Microscopy for Image CytOmetry, A.M.I.CO) for quantitative image-analysis of data from confocal or widefield robotized microscopes. We have applied this image-cytometry technology to the study of checkpoint activation in response to spontaneous DNA damage in nontransformed mammary cells. Cell-cycle profile and active DNA-replication were correlated to (i) Ki67, to monitor proliferation; (ii) phosphorylated histone H2AX (H2AX) and 53BP1, as markers of DNA-damage response (DDR); and (iii) p53 and p21, as checkpoint-activation markers. Our data suggest the existence of cell-cycle modulated mechanisms involving different functions of H2AX and 53BP1 in DDR, and of p53 and p21 in checkpoint activation and quiescence regulation during the cell-cycle. Quantitative analysis, event selection, and physical relocalization have been then employed to correlate protein expression at the population level with interactions between molecules, measured with Proximity Ligation Analysis, with unprecedented statistical relevance. (c) 2012 International Society for Advancement of Cytometry