Carcinogenesis vol.24 no.1 pp.25–29, 2003 Hypermethylation of metallothionein-3 CpG island in gastric carcinoma

The expression of metallothionein (MT)-3 is often markedly reduced in gastric carcinoma (GC). The mole-cular mechanism of this MT-3 downregulation is unknown. Transcriptional silencing of MT-3 by methylation of CpG island was investigated by nucleotide sequencing and dena-turing high performance liquid chromatography (DHPLC) analyses. We found that normal brain tissue and a xenografted GC that expressed MT-3 mRNA had un-methylated regions of the CpG island in intron1 of this gene. On the other hand, gastric cancer cell lines AGS and MKN445, a xenografted GC, and a representative primary gastric cancer that had no expression of MT-3 mRNA demonstrated hypermethylation of the MT-3 intron1 CpG island. Treatment of the gastric cancer cell lines with 5-azacytidine resulted in new expression of MT-3 mRNA in these cells. A quantifying DHPLC assay was developed to determine the methylation status of this specific region of the MT-3 gene. Fifty-eight primary GC and their corresponding normal gastric epithelial tissues, and 34 normal gastric mucosa were analyzed for MT-3 methylation by DHPLC in the region of methylation abnormalities initially identified. Our DHPLC analyses of the methylated MT-3 product demonstrated that the primary gastric cancers have an average methylation percentage of 6.3 % per tumor com-pared with 2.4 % in normal gastric tissues (P < 0.05). The MT-3 was not methylated in all of eight P53-positive GCs and hypermethylated in eight of 13 P53-negative cases by immunohistochemistry staining (P 0.007). In conclusion, the CpG island in the MT-3 intron1 are abnormally hyper-methylated in many gastric carcinomas and may account for the downregulation of MT-3 in gastric carcinogenesis