title: 3-D DNA methylation phenotypes correlate with cytotoxicity levels in prostate and liver cancer cell models.
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.
authors: Translational Cytomics Group, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.
authors: Arkadiusz
authors: A
authors: Gertych
authors: Jin Ho
authors: JH
authors: Oh
authors: Kolja A
authors: KA
authors: Wawrowsky
authors: Daniel J
authors: DJ
authors: Weisenberger
authors: Jian
authors: J
authors: Tajbakhsh
mesh_terms: D044503:5-Methylcytosine / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001374:Azacitidine / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003562:Cytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D019175:DNA Methylation; D006801:Humans; D008113:Liver Neoplasms; D008297:Male; D010641:Phenotype; D011471:Prostatic Neoplasms
chemical_list: D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D003562:Cytidine; D044503:5-Methylcytosine; C009131:pyrimidin-2-one beta-ribofuranoside; D001374:Azacitidine
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