Cellular effects of a demethylating agent on normal human corneal endothelial cells

Abstract

Purpose : There is increasing evidence that differential DNA methylation plays a significant role in ophthalmic diseases. We previously identified DNA methylation changes in the corneal endothelial tissue of patients with Fuchs endothelial corneal dystrophy (FECD), in particular the hypermethylation of genes related to FECD. 5-Aza-2-deoxycytidine (5-Aza-CdR) is a demethylating agent used to treat human cancers with aberrant DNA methylation. The purpose of this study is to better understand the cellular effects of 5-Aza-CdR on normal human corneal endothelial cells (CEnC).

Methods : Primary CEnC were cultured from a non-FECD cornea obtained from an eye bank and transformed CEnC (HCEnC-21T) were cultured as previously published. Cell cultures were treated with 0.3 mM 5-Aza-CdR or PBS control for 24 hours and assayed for cell viability using the Cell Counting Kit-8 (Dojindo). The Illumina Infinium HM450 BeadChip assay was used to perform global DNA methylation analysis of over 485,000 methylation sites. Statistical and array analyses were performed using R and p-values less than 0.05 were considered statistically significant.

Results : The global DNA methylation profiles of primary and immortalized (HCEnC-21T) normal human CEnC were compared using the Illumina Infinium HM450 BeadChip assay and similar global DNA methylation patterns were observed. HCEnC-21T cells were then treated with either 0.3 µM 5-Aza-CdR or PBS (control) for 24 hours and cell viability, cell growth rate, and global DNA methylation status were measured. No significant difference in cell viability was found between 5-Aza-CdR and PBS-treated cells. At 10 days post-treatment, the cell doubling time peaked at 33 hours for 5-Aza-CdR-treated cells compared with 24 hours in PBS-treated cells. Global demethylation of methylated probes (β > 0.6) was not observed 5 days post-treatment (mean decrease in β value of 0.007), despite an effect on cell growth rate at this time-point. Baseline global DNA methylation levels displayed minimal change following 5-Aza-CdR treatment.

Conclusions : These findings show that 5-Aza-CdR does not induce significant cell toxicity or demethylation changes in normal human corneal endothelial cells. Further analyses of the demethylating effect of 5-Aza-CdR on CEnC from FECD patients with aberrant hypermethylation changes are needed to test its potential as an epigenetic therapy for FECD.

This is a 2021 ARVO Annual Meeting abstract.