Analysis of DNA hydroxymethylation in uveal melanoma

Abstract

Purpose: Epigenetic regulation of cancer involves posttranslational modifications of histones and DNA methylation as a means of controlling gene expression without modifying the DNA sequence. These dynamic mechanisms allow tumor cells to adapt faster to their changing microenvironment. The enzymatic conversion of cytosine (C) to 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) silences the methylation and reactivates gene transcription. Hypoxia plays a role in the etiology of uveal melanoma (UM) according to increased expression of HIF1A by tissue microarray immunostaining. Our general hypothesis is that hypoxia modulates DNA methylation in UM. The objectives of our study are to compare the overall level of hydroxymethylation between choroidal melanocytes and UM cells, as well as to evaluate the influence of hypoxia on this epigenetic mark.

Methods: Skin (N=1) and eye (N=6) melanoma tissue sections were used for immunofluorescence analyses using 5-mC and 5-hmC antibodies to study the overall levels of methylation and hydroxymethylation in situ. Melanocytes from the choroid (N=3) and UM cells (N=6) were then exposed to oxygen levels of 21% and 1%, and DNA was isolated to perform immunofluorescence analyses and slot blot using the same antibodies. Levels of 5-mC and 5-hmC were quantified by measuring the densitometric mean (normalization with DAPI or an anti-ssDNA antibody; Mann-Whitney test).

Results: In situ, there was no staining for 5-hmC in UM tumors while neural cells of the retina were positive. The 5-mC staining was fainter or absent in tumor cells compared to retina cells. These results were similar to the positive control (skin). In vitro, both methylation and hydroxymethylation levels were decreased in UM cells compared to normal melanocytes (P < 0.0001). Hypoxic conditions also impaired the overall levels of 5-mC and 5-hmC in all cell types (P < 0.0005 and P < 0.05, respectively).

Conclusions: Overall levels of methylation and hydroxymethylation were reduced in the UM genome, and hypoxia accentuated the loss of these epigenetic marks. Unlike genetic mutations, alterations of DNA methylation are reversible, and represent potential therapeutic targets for restoring the epigenetic balance in tumor cells.