Automated quantification of DNA demethylation effects in cells via 3D mapping of nuclear signatures and population homogeneity assessment.
Automated quantification of DNA demethylation effects in cells via 3D mapping of nuclear signatures and population homogeneity assessment.
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DOI：
10.1002/cyto.a.20740
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发表时间：
2009-07
期刊：
影响因子：
3.7
通讯作者：
Tajbakhsh, Jian
中科院分区：
文献类型：
--
作者：
Gertych, Arkadiusz;Wawrowsky, Kolja A.;Lindsley, Erik;Vishnevsky, Eugene;Farkas, Daniel L.;Tajbakhsh, Jian
关键词：
当今先进的显微成像技术应用于药物发现的临床前阶段，采用高通量和高含量的细胞三维（3D）分析来更有效地筛选候选化合物。药物疗效可以通过测量细胞群内治疗反应的均匀性来评估。在这项研究中，甲基化胞嘧啶和整体核DNA的拓扑量化核模式被用作细胞对去甲基化抗癌药物：5-氮杂胞苷（5-AZA）和奥曲肽（OCT）治疗的细胞反应的标志。用5-AZA和OCT处理48小时的小鼠垂体卵泡状TtT-GF细胞，以及未处理的群体，用免疫荧光法研究了针对5-甲基胞嘧啶（MeC）和4,6-二氨基-2-苯基吲哚（DAPI）的特异性抗体，以描绘细胞核中甲基化位点和全局DNA （n=163）。细胞图像使用自动3D分析软件进行处理，该软件通过结合种子分水岭分割提取核壳和测量Kullback-Leibler （K-L）散度来分析MeC与DAPI染色位点相对核分布模式的细胞群体均匀性。每个细胞被分配到四个类别中的一个：相似、可能相似、不太相似和不相似。对不同细胞组的评估显示，在未处理的细胞（~100%）和5- aza处理的细胞（90%）中，MeC/DAPI模式相似或可能相似的细胞数量显著增加，而在oct处理的细胞群体中，相同类型的细胞数量较低（64%）。后一组含有不太可能相似或不相似的细胞（28%）（7%）。我们的方法成功地评估了与应用药物的生物学影响相关的细胞行为，即通过去甲基化重组MeC/DAPI分布。在与其他指标的比较中，K-L分化已被证明是一种更有价值和更强大的工具，用于群体内单个细胞的分类，在表观遗传药物筛选中具有潜在的应用。
Today’s advanced microscopic imaging applies to the preclinical stages of drug discovery that employ high-throughput and high-content three-dimensional (3D) analysis of cells to more efficiently screen candidate compounds. Drug efficacy can be assessed by measuring response homogeneity to treatment within a cell population. In this study topologically quantified nuclear patterns of methylated cytosine and global nuclear DNA are utilized as signatures of cellular response to the treatment of cultured cells with the demethylating anti-cancer agents: 5-azacytidine (5-AZA) and octreotide (OCT). Mouse pituitary folliculostellate TtT-GF cells treated with 5-AZA and OCT for 48 hours, and untreated populations, were studied by immunofluorescence with a specific antibody against 5-methylcytosine (MeC), and 4,6-diamidino-2-phenylindole (DAPI) for delineation of methylated sites and global DNA in nuclei (n=163). Cell images were processed utilizing an automated 3D analysis software that we developed by combining seeded watershed segmentation to extract nuclear shells with measurements of Kullback-Leibler’s (K-L) divergence to analyze cell population homogeneity in the relative nuclear distribution patterns of MeC versus DAPI stained sites. Each cell was assigned to one of the four classes: similar, likely similar, unlikely similar and dissimilar. Evaluation of the different cell groups revealed a significantly higher number of cells with similar or likely similar MeC/DAPI patterns among untreated cells (~100%), 5-AZA-treated cells (90%), and a lower degree of same type of cells (64%) in the OCT-treated population. The latter group contained (28%) of unlikely similar or dissimilar (7%) cells. Our approach was successful in the assessment of cellular behavior relevant to the biological impact of the applied drugs, i.e. the reorganization of MeC/DAPI distribution by demethylation. In a comparison with other metrics, K-L divergence has proven to be a more valuable and robust tool for categorization of individual cells within a population, with potential applications in epigenetic drug screening.
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影响因子：
6
作者：
Bastian, BC;Olshen, AB;Pinkel, D
通讯作者：
Pinkel, D
影响因子：
2.6
作者：
deCapoa, A;Menendez, F;Niveleau, A
通讯作者：
Niveleau, A
DOI：
10.1073/pnas.80.16.4919
发表时间：
1983-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
GRUENBAUM, Y;SZYF, M;RAZIN, A
通讯作者：
RAZIN, A
影响因子：
3.7
作者：
Gudla, Prabhakar R.;Nandy, K.;Lockett, S. J.
通讯作者：
Lockett, S. J.
影响因子：
3.7
作者：
Gué, M;Messaoudi, C;Boudier, T
通讯作者：
Boudier, T