[show abstract][hide abstract] ABSTRACT: BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
[show abstract][hide abstract] ABSTRACT: Targeting chromatin and its basic components through epigenetic drug therapy has become an increased focus in the treatment of complex diseases. This boost calls for the implementation of high-throughput cell-based assays that exploit the increasing knowledge about epigenetic mechanisms and their interventions for genotoxicity testing of epigenetic drugs. 3D quantitative DNA methylation imaging is a novel approach for detecting drug-induced DNA demethylation and concurrent heterochromatin decondensation/reorganization in cells through the analysis of differential nuclear distribution patterns of methylcytosine and gDNA visualized by fluorescence and processed by machine-learning algorithms. Utilizing 3D DNA methylation patterns is a powerful precursor to a series of fully automatable assays that employ chromatin structure and higher organization as novel pharmacodynamic biomarkers for various epigenetic drug actions.
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