Experimental eye research / short communication format characterization of DNA hydroxymethylation in the ocular choroid.

DNA methylation and hydroxymethylation represent important epigenetic modifications involved in cell differentiation. DNA hydroxymethylation can be used to classify independent biological samples by tissue type. Relatively little is known regarding the genomic abundance and function of 5-hydroxymethylcytosine (5-hmC) in ocular tissues. The choroid supplies oxygen and nutrients to the outer retina through its dense network of blood vessels. This connective tissue is mainly composed of pigmented melanocytes, and stromal fibroblasts. Since DNA hydroxymethylation level is relatively high in cutaneous melanocytes, we investigated the presence of 5-hmC in choroidal melanocytes, as well as the expression of ten-eleven translocation methylcytosine dioxygenases (TETs) and isocitrate dehydrogenases (IDHs) implicated in this DNA demethylation pathway. Immunofluorescence, DNA slot blots and liquid chromatography coupled to tandem mass spectrometry performed with choroidal tissues and melanocytes within these tissues revealed that they have a relatively high level of 5-hmC. We also examined the expression of TET1/2 and IDH1/2 in choroidal melanocytes by gene expression profiling, qPCR and Western blotting. In addition, we detected decreased levels of 5-hmC when choroidal melanocytes were exposed to a lower concentration of oxygen. Our study therefore demonstrates that DNA hydroxymethylation is present in choroidal melanocytes, and that the abundance of this epigenetic mark is impacted by hypoxia.

Differential responses of choroidal melanocytes and uveal melanoma cells to low oxygen conditions.

PURPOSE: Tissue culture is traditionally performed at atmospheric oxygen concentration (21%), which induces hyperoxic stress, as endogenous physiologic oxygen tension found in tissues varies between 2% and 9%. This discrepancy may lead to misinterpretation of results and may explain why effects observed in vitro cannot always be reproduced in vivo and vice versa. Only a few studies have been conducted in low physiologic oxygen conditions to understand the development and differentiation of cells from the eye. METHODS: The aim of this study was to investigate the growth and gene expression profile of melanocytes from the choroid permanently exposed to 21% (hyperoxic) or 3% (physiologic) oxygen with proliferation assays and DNA microarray. The cellular behavior of the melanocytes was then compared to that of cancer cells. RESULTS: The gross morphology and melanin content of choroidal melanocytes changed slightly when they were exposed to 3% O2, and the doubling time was statistically significantly faster. There was an increase in the percentage of choroidal melanocytes in the active phases of the cell cycle as observed by using the proliferation marker Ki67. The caveolin-1 senescence marker was not increased in choroidal melanocytes or uveal melanoma cells grown in hyperoxia. In comparison, the morphology of the uveal melanoma cells was similar between the two oxygen levels, and the doubling time was slower at 3% O2. Surprisingly, gene expression profiling of the choroidal melanocytes did not reveal a large list of transcripts considerably dysregulated between the two oxygen concentrations; only the lactate transporter monocarboxylate transporter (MCT4) was statistically significantly upregulated at 3% O2. CONCLUSIONS: This study showed that the oxygen concentration must be tightly controlled in experimental settings, because it influences the subsequent cellular behavior of human choroidal melanocytes.