Induction of HLA-G expression in a melanoma cell line OCM-1A following the treatment with 5-aza-2′-deoxycytidine

Abstract

The non-classical HLA class I antigen HLA-G is an immune modulator which inhibits the functions of T cells, NK cells, and the Dendritic cells (DC). As a result, HLA-G expression in malignant cells may provide them with a mechanism to escape the immune surveillance. In melanoma, HLA-G antigen expression has been found in 30% of surgically removed lesions but in less than 1% of established cell lines. One possible mechanism underlying the differential HLA-G expression in vivo and in vitro is that the HLA-G gene is epigenetically repressed in melanoma cells in vitro. To test this hypothesis, we treated the HLA-G negative melanoma cell line OCM-1A with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-AC) and analyzed whether HLA-G expression can be restored. Our data strongly suggest that HLA-G is silenced as a result of CpG hypermethylation within a 5' regulatory region encompassing 220 bp upstream of the start codon. After treatment, HLA-G mRNA expression was dramatically increased. Western blot and flow cytometry showed that HLA-G protein was induced. Interestingly, HLA-G cell surface expression on the 5-AC treated OCM-1A cells is much less than that on the HLA-G positive JEG-3 cells while a similar amount of total HLA-G was observed. Possible mechanisms for the difference were analyzed in the study such as cell cold-treatment, peptide loading and antigen processing machinery components (APM) as well as β 2 microglobulin (β 2 -m) expression. Data revealed that the APM component calreticulin might be involved in the lower HLA-G surface expression on OCM-1A cells. Taken together, our results indicated that DNA methylation is an important epigenetic mechanism by which HLA-G antigen expression is modulated in melanoma cells in vitro. Furthermore, to the first time, we hypothesized that the deficiency of calreticulin might be involved in the low HLA-G surface expression on the 5-AC treated OCM-1A cells.

HLA-G expression is induced in Epstein-Barr virus–transformed B-cell lines by culture conditions

“…The mechanism of turning on/off the transcription is not known. Based on the induction of expression of HLA-G using 5-aza2=deoxycytidine it has been suggested that in melanoma cell line methylation of the promoter is one of the major factors [21]. Deacetylation of the histone was also suggested as a possible cause [20].…”

Hypoxia Modulates HLA-G Gene Expression in Tumor Cells

“…Nevertheless, only a few transcription factors and regulatory sequences have been identified until now. Regulation may also act on HLA-G protein expression through the efficiency of translation and the modulation of antigen processing machinery component expression [4,5]. Moreover, in vivo stimulators are likely to be key factors controlling one or several levels of regulation.…”

Effect of 5-Aza-2'-deoxycytidine on SLC22A18 in glioma U251 cells

“…5-Aza-2'-deoxycytidine (5-Aza-CdR) is thought to act by inhibiting DNA methyltransferase that methylates cytosine residues in eukaryotic DNA (8). Several researchers have used it as an experimental tool for demethylation (9,10). This drug appears to be incorporated into the cellular DNA where it acts as a non-competitive inhibitor of the maintenance methylase (9,11).…”

“…Several researchers have used it as an experimental tool for demethylation (9,10). This drug appears to be incorporated into the cellular DNA where it acts as a non-competitive inhibitor of the maintenance methylase (9,11). Methyl moieties are thus passively removed when the DNA undergoes replication and, indeed, numerous inactive cellular and viral genes are activated following exposure to 5-Aza-CdR (12).…”