AIM:To study the mechanisms responsible for inactivationof a novel esophageal cancer related gene 4 (ECRG4) inesophageal squamous cell carcinoma (ESCC).METHODS:A pair of primers was designed to amplify a220 bp fragment,which contains 16 CpG sites in the corepromoter region of the ECRG 4 gene.PCR products ofbisulfite-modified CpG islands were analyzed by denaturinghigh-performance liquid chromatography (DHPLC),whichwere confirmed by DNA sequencing.The methylation statusof ECRG 4 promoter in 20 cases of esophageal cancer andthe adjacent normal tissues,5 human tumor cell lines(esophageal cancer cell line-NEC,EC109,EC9706;gastriccancer cell line-GLC;human embryo kidney cell line-Hek293)and 2 normal esophagus tissues were detected.Theexpression level of the ECRG 4 gene in these samples wasexamined by RT-PCR.RESULTS:The expression level of ECRG 4gene was varied.Of 20 esophageal cancer tissues,nine were unexpressed,six were lowly expressed and five were highly expressedcompared with the adjacent tissues and the 2 normalesophageal epithelia.In addition,4 out of the 5 human celllines were also unexpressed.A high frequency of methylationwas revealed in 12 (8 unexpressed and 4 lowly expressed)of the 15 (80%) downregulated cancer tissues and 3 of the4 unexpressed cell lines.No methylation peak was observedin the two highly expressed normal esophageal epitheliaand the methylation frequency was low (3/20) among the20 cases in the highly expressed adjacent tissues.Themethylation status of the samples was consistent with theresult of DNA sequencing.CONCLUSION:These results indicate that the inactivationof ECRG 4gene by hypermethylation is a frequent molecularevent in ESCC and may be involved in the carcinogenesis ofthis cancer. AIM: To study the mechanisms responsible for inactivation of a novel esophageal cancer related gene 4 (ECRG4) inesophageal squamous cell carcinoma (ESCC) .METHODS: A pair of primers were designed to amplify a220 bp fragment which contains 16 CpG sites in the core promoter region of the ECRG 4 gene. PCR products of bisulfite-modified CpG islands were analyzed by denaturing high-performance liquid chromatography (DHPLC), which were confirmed by DNA sequencing. The methylation status of ECRG 4 promoter in 20 cases of esophageal cancer and the adjacent normal tissues, 5 human tumor cells lines (esophageal cancer cell line-NEC, EC109, EC9706; gastric cancer cell line-GLC; human embryo kidney cell line-Hek293) and 2 normal esophagus tissues were detected. The expression level of the ECRG 4 gene in these samples wasexamined by RT -PCR.RESULTS: The expression level of ECRG 4 gene was varied. Of 20 esophageal cancer tissues, nine were unexpressed, six were lowly expressed and five were highly expressedcompared with the adjac ent tissues and the 2 normalesophageal epithelia. In addition, 4 out of the 5 human celllines were also unexpressed. A high frequency of methylation was revealed in 12 (8 unexpressed and 4 lowly expressed) of the 15 (80%) downregulated cancer tissues and 3 of the4 unexpressed cell lines. No methylation peak was observed in the two highly expressed normal esophageal epithelia and the methylation frequency was low (3/20) among the20 cases in the highly expressed adjacent tissues .methylation status of the samples was consistent with theresult of DNA sequencing .CONCLUSION: These results indicate that the inactivation of ECRG 4gene by hypermethylation is a frequent molecularevent in ESCC and may be involved in the carcinogenesis of cancer.