Hypermethylation of metallothionein-3 CpG island in gastric carcinoma.
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.
Deng
Dajun
D
Peking University Health Science Center and Beijing Institute for Cancer Research, Beijing, 100034, China. dengdajun@sina.com
El-Rifai
Wa'el
W
Ji
Jiafu
J
Zhu
Budong
B
Trampont
Paul
P
Li
Jiyou
J
Smith
Michael F
MF
Powel
Steven M
SM
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D001921:Brain / Q000378:metabolism; D018899:CpG Islands / Q000235:genetics*; D019175:DNA Methylation; D005260:Female; D006801:Humans; D007438:Introns / Q000235:genetics; D008297:Male; D000090023:Metallothionein 3; D051379:Mice; D008875:Middle Aged; D009419:Nerve Tissue Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000235:genetics*; D014407:Tumor Cells, Cultured
D000090023:Metallothionein 3; C000715430:Mt3 protein, mouse; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger