Open Access Research article

3-D DNA methylation phenotypes correlate with cytotoxicity levels in prostate and liver cancer cell models

Arkadiusz Gertych13, Jin Ho Oh12, Kolja A Wawrowsky14, Daniel J Weisenberger5 and Jian Tajbakhsh12*

Author Affiliations

1 Translational Cytomics Group, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA

2 Chromatin Biology Laboratory, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA

3 Bioinformatics Laboratory, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA

4 Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA

5 USC Epigenome Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, 90089, USA

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BMC Pharmacology and Toxicology 2013, 14:11  doi:10.1186/2050-6511-14-11

Published: 11 February 2013

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.

Keywords:
DNA methylation phenotype; Chromatin distribution; High-throughput cell assay; 3D image analysis; MethyLight; Repetitive element; Epigenetic drug