Chemical genomic screening for methylation‐silenced genes in gastric cancer cell lines using 5‐aza‐2′‐deoxycytidine treatment and oligonucleotide microarray

Abstract

To identify novel methylation-silenced genes in gastric cancers, we carried out a chemical genomic screening, a genome-wide search for genes upregulated by treatment with a demethylating agent, 5-aza-2′ ′ ′ ′-deoxycytidine (5-aza-dC). After 5-aza-dC treatment of a gastric cancer cell line (AGS) 579 genes were upregulated 16-fold or more, using an oligonucleotide microarray with 39 000 genes. From these genes, we selected 44 known genes on autosomes whose silencing in gastric cancer has not been reported. Thirty-two of these had CpG islands (CGI) in their putative promoter regions, and all of the CGI were methylated in AGS, giving an estimated number of 421 ± ± ± ± 75 (95% confidence interval) methylation-silenced genes. Additionally, we analyzed the methylation status of 16 potential tumor-related genes with promoter CGI that were upregulated four-fold or more, and 14 of these were methylated in AGS. Methylation status of the 32 randomly selected and 16 potential tumor-related genes was analyzed in 10 primary gastric cancers, and 42 genes (ABHD9 , ADFP, ALDH1A3, ANXA5, AREG, BDNF, BMP7, CAV1, CDH2, CLDN3, CTSL, EEF1A2, F2R, FADS1, FSD1, FST, FYN, GPR54, GREM1, IGFBP3, IGFBP7, IRS2, KISS1, MARK1, MLF1, MSX1, MTSS1, NT5E, PAX6, PLAGL1, PLAU, PPIC, RBP4, RORA, SCRN1, TBX3, TFAP2C, TNFSF9, (1) In various human cancers, silencing of tumorsuppressor genes, such as CDKN2A (p16), CDH1 (Ecadherin) and MLH1, is known to be one of the major mechanisms for their inactivation, along with mutations and LOH. To identify genes silenced by promoter methylation by genome-wide screenings, various techniques have been developed.(2) Most techniques are based on the methylation status of genomic DNA, including MS-RDA and restriction landmark genomic scanning. In contrast, Suzuki et al. developed a technique that screens genes re-expressed after treatment with a demethylating agent, 5-aza-dC, using a microarray.(3) The chemical genomic screening technique is simple and is effective in identifying genes silenced in cell lines. It has been applied to colon, bladder, esophageal, pancreatic and prostate cancers.Gastric cancer is the second most common cause of cancer death in the world.(8) As its molecular basis, deep involvement of aberrant DNA methylation has been indicated by the higher incidences of aberrant DNA methylation of known tumorsuppressor genes than of mutations.(9) We previously searched for genes silenced in MKN28 and MKN74 cell lines using MS-RDA, (10) and identified lysyl oxidase as a novel tumorsuppressor gene.(11) However, the entire picture of methylation-silenced genes in gastric cancers is still unclear, and further searches for methylation-silenced genes are necessary.In the present study, we carried out a chemical genomic screening of methylation-silenced genes in the human gastric cancer cell line AGS.

KISS1 Methylation and Expression as Tumor Stratification Biomarkers and Clinical Outcome Prognosticators for Bladder Cancer Patients

“…12,22 In an attempt to uncover the mechanisms by which KISS1 is lost in bladder cancer, we tested the hypothesis of epigenetic silencing after identifying an enriched 5=-CpG island around the promoter region of KISS1, supporting its susceptibility to be epigenetically modified by hypermethylation. Although KISS1 was identified among the genes restoring their transcript expression after azacytidine treatment using oligonucleotide microarrays in gastric cancer cells, 26 to our knowledge KISS1 has not been reported to be epigenetically altered in bladder cancer. In this report, the effect of KISS1 methylation on its expression and the clinical relevance were evaluated in bladder cancer.…”

The T-Box transcription factor 3 in development and cancer

“…Subsequent studies have also shown that TBX3 levels are epigenetically silenced by methylation in glioblastoma, gastric, bladder and prostate cancer (7). Yamashita et al examined methylation-silenced genes in gastric cancer and revealed that the TBX3 promoter is methylated in at least one primary gastric cancer, but not normal gastric mucosa (75). TBX3 methylation was subsequently revealed to be associated with a significantly lowered survival rate in a cohort of glioblastoma patients (76).…”

DNA methylation profiles of gastric carcinoma characterized by quantitative DNA methylation analysis

“…7 To date, about 87 genes have been characterized to be inactivated by hypermethylation of their promoter CpG islands in GC. 8 Considering the fact that more than 40% of human genes contain promoters within CpG islands [9][10][11] and the number of the genes characterized as hypermethylated in GCs is very limited, the vast majority of genes with CpG islands remain to be tested as to their DNA methylation status in GC.…”