Abstract
BackgroundThe 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.MethodsDifferential 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.ResultsBoth 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.Conclusions3D-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.
Highlights
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
Zebularine exerts a comparably lower degree of cytotoxicity than 5-azacytidine We evaluated cultured DU145 prostate cancer cells and Huh-7 hepatocellular carcinoma cell lines for imagingbased DNA methylation analysis using the 3D-qDMI system to determine DNA methylation phenotypes of cells after 5-zacytidine and zebularine administration
It is conceivable that a reduction in H3K9me3 could lead to local DNA decondensation as extensively reported elsewhere [10,68]. These findings support our topologic approach in using DAPI signals as a convenient way of reporting changes in heterochromatin organization and distribution, extensively discussed in previous works [43,44]: as we found that DAPI staining is compatible with the hydrochloric acid-treatment conditions of fixed cells we applied for MeC-signal retrieval without any detectable obscuring of both signals [64]
Summary
DNA methylation occurs at the drug discovery field has so far been mostly focusing on screening the effect of candidate agents on the levels of molecular cell signaling and metabolism. In recent years of the post-genomic era, chromatin conformation and the higher–order genome organization, which set the framework for the global orchestration as well the locus-specific regulation of gene expression in the human cell nucleus [15,16,17,18], are gaining more attention in therapy; the reason being that these functional structures can become affected as a consequence of epigenetic interference by chromatin-modifying agents such as inhibitors of DNA methylation [19]. Zebularine (ZEB) or 1-β-D-ribofuranosyl-2(1H)-pyrimidone has recently emerged as a new DNMT inhibitor (DNMTi), with properties that makes it a potential drug candidate for oral administration: (i) stability at pH ranges between 1.0 and 7.0 in aqueous solutions, (ii) far less toxicity than AZA and decitabine to cultured cells, and (iii) no detectable toxicity in a T-cell lymphoma mouse model [23,24,25,26,27]
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