Journal Article

Hypermethylation of <i>metallothionein-3</i> CpG island in gastric carcinoma

Dajun Deng, Wa'el El-Rifai, Jiafu Ji, Budong Zhu, Paul Trampont, Jiyou Li, Michael F. Smith and Steven M. Powel

in Carcinogenesis

Volume 24, issue 1, pages 25-29
Published in print January 2003 | ISSN: 0143-3334
Published online January 2003 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/24.1.25
Hypermethylation of metallothionein-3 CpG island in gastric carcinoma

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The expression of metallothionein (MT)-3 is often markedly reduced in gastric carcinoma (GC). The molecular mechanism of this MT-3 downregulation is unknown. Transcriptional silencing of MT-3 by methylation of CpG island was investigated by nucleotide sequencing and denaturing high performance liquid chromatography (DHPLC) analyses. We found that normal brain tissue and a xenografted GC that expressed MT-3 mRNA had unmethylated 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 compared 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 hypermethylated in many gastric carcinomas and may account for the downregulation of MT-3 in gastric carcinogenesis.

Keywords: DHPLC, denaturing high performance liquid chromatography; GC, gastric carcinoma; MT, metallothionein; RMF, relative methylation factor

Journal Article.  4426 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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