Translational Cytomics Group, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.
BMC Pharmacol Toxicol. 2013 Feb 11;14:11. doi: 10.1186/2050-6511-14-11.
The spatial organization of the genome is being evaluated as a novel indicator of toxicity in conjunction with drug-induced global DNA hypomethylation and concurrent chromatin reorganization. 3D quantitative DNA methylation imaging (3D-qDMI) was applied as a cell-by-cell high-throughput approach to investigate this matter by assessing genome topology through represented immunofluorescent nuclear distribution patterns of 5-methylcytosine (MeC) and global DNA (4,6-diamidino-2-phenylindole = DAPI) in labeled nuclei.
Differential progression of global DNA hypomethylation was studied by comparatively dosing zebularine (ZEB) and 5-azacytidine (AZA). Treated and untreated (control) human prostate and liver cancer cells were subjected to confocal scanning microscopy and dedicated 3D image analysis for the following features: differential nuclear MeC/DAPI load and codistribution patterns, cell similarity based on these patterns, and corresponding differences in the topology of low-intensity MeC (LIM) and low in intensity DAPI (LID) sites.
Both agents generated a high fraction of similar MeC phenotypes across applied concentrations. ZEB exerted similar effects at 10-100-fold higher drug concentrations than its AZA analogue: concentration-dependent progression of global cytosine demethylation, validated by measuring differential MeC levels in repeat sequences using MethyLight, and the concurrent increase in nuclear LIM densities correlated with cellular growth reduction and cytotoxicity.
3D-qDMI demonstrated the capability of quantitating dose-dependent drug-induced spatial progression of DNA demethylation in cell nuclei, independent from interphase cell-cycle stages and in conjunction with cytotoxicity. The results support the notion of DNA methylation topology being considered as a potential indicator of causal impacts on chromatin distribution with a conceivable application in epigenetic drug toxicology.
基因组的空间组织正被评估为一种新的毒性指标，与药物诱导的全球 DNA 低甲基化和伴随的染色质重排一起使用。3D 定量 DNA 甲基化成像（3D-qDMI）作为一种高通量的逐细胞方法，通过评估代表 5-甲基胞嘧啶（MeC）和全局 DNA（4,6-二脒基-2-苯基吲哚=DAPI）核分布模式的免疫荧光核分布模式来评估基因组拓扑结构，从而研究这一问题在标记的核中。
通过比较齐扎滨（ZEB）和 5-氮杂胞苷（AZA）的剂量，研究了全球 DNA 低甲基化的差异进展。用人前列腺癌和肝癌细胞进行共聚焦扫描显微镜检查和专门的 3D 图像分析，以获得以下特征：核 MeC/DAPI 负载和共分布模式的差异，基于这些模式的细胞相似性，以及低强度 MeC（LIM）和低强度 DAPI（LID）位点拓扑的相应差异。
两种药物在应用浓度下都产生了大量相似的 MeC 表型。ZEB 在比其 AZA 类似物高 10-100 倍的药物浓度下产生相似的作用：通过使用 MethyLight 测量重复序列中的差异 MeC 水平来验证的全胞嘧啶去甲基化的浓度依赖性进展，以及核 LIM 密度的同时增加与细胞生长减少和细胞毒性相关。
3D-qDMI 证明了定量测量细胞核中药物诱导的 DNA 去甲基化的剂量依赖性空间进展的能力，该方法独立于细胞间周期阶段，并与细胞毒性相关。这些结果支持了 DNA 甲基化拓扑结构被认为是对染色质分布产生因果影响的潜在指标的观点，并且在表观遗传药物毒理学中具有应用前景。
