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Expression of theXAGE-1antigen is restricted to germ cells of the testis and a variety of neoplastic tissues. To date, the molecular mechanism for regulating expression of this cancer/testis antigen gene has been unknown. To evaluate methylation as a potential mechanism for regulating expression of this gene, we first correlated gene methylation status (measured by sequencing of bisulfide-modified DNA and COBRA) to expression of XAGE-1 mRNA in normal and cancerous cells. This analysis revealed dense methylation of the CpG island in theXAGE-1gene promoter for the normal and cancerous cells that do not express this gene but loss of this methylation in normal testis, cancer cell lines and the primary gastric cancers where the gene is highly expressed. Further supporting the role of methylation in regulating expression ofXAGE-1were observations that treatment of 2 heavily methylated cell lines, SNU620 and HT29, with 5′-aza-deoxycytidine resulted in demethylation ofXAGE-1promoter and corresponding expression of this gene. Finally, we cloned various segments of the CpG-richXAGE-1gene promoter linked to a luciferase reporter construct and transiently transfected this construct into HCT116 cells. These experiments confirmed transcriptional regulatory activity for the promoter region that incorporates the CpG island and demonstrated thatin vitromethylation of this island results in loss of promoter activity. Collectively, these studies indicate thatXAGE-1expression in normal and cancerous tissues is regulated by methylation of the CpG island in the gene promoter.