FREQUENT EPIGENETIC INACTIVATION OF RIZ1 BY PROMOTER HYPERMETHYLATION IN HUMAN GASTRIC CARCINOMA Yasuhiro OSHIMO 1,2 , Naohide OUE 1 , Yoshitsugu MITANI 1 , Hirofumi NAKAYAMA 1 , Yasuhiko KITADAI 2 , Kazuhiro YOSHIDA 3 , Kazuaki CHAYAMA 2 and Wataru YASUI 1 * 1 Department of Molecular Pathology, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima, Japan 2 Department of Medicine and Molecular Science, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima, Japan 3 Department of Surgical Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan The retinoblastoma protein-interacting zinc finger gene, RIZ1 (GenBank accession number U17838), is involved in chromatin-mediated gene expression and is also a target for frameshift mutation in microsatellite-unstable cancers. Methylation of the RIZ1 promoter CpG island has been shown to be a common mechanism in inactivating the RIZ1 gene in human liver and breast cancers. We investigated levels of RIZ1 mRNA in 45 gastric carcinoma tissues by quan- titative RT-PCR and in gastric carcinoma cell lines by RT- PCR. In addition, we examined CpG island methylator phe- notype (CIMP) status, p53 mutation status, and the correlation between promoter methylation status and RIZ1 mRNA expression. CIMP status was investigated by examin- ing the methylation status of MINT1, MINT2, MINT12, MINT25 and MINT31. p53 mutation status was examined by PCR-single strand conformation polymorphism and pro- moter methylation status was examined by methylation-spe- cific PCR. Promoter hypermethylation of the RIZ1 gene was found in 31 (69%) of 45 gastric carcinoma tissues and in 3 (21%) of 14 corresponding non-neoplastic mucosae, the inci- dence being significantly different (p  0.002). None of the 12 normal gastric tissues from young non-cancer individuals showed hypermethylation. Promoter hypermethylation was associated with reduced RIZ1 expression in gastric carcinoma tissues (p  0.029). Promoter hypermethylation of the RIZ1 gene was significantly associated with CIMP (p  0.002). Mutation status of the p53 gene was not associated with methylation status or RIZ1 expression in gastric carcinoma. In gastric carcinoma cell lines MKN-28 and KATO-III, the RIZ1 promoter was hypermethylated and RIZ1 transcription was inactive. Treatment of these cells with demethylating agent 5-aza-2-deoxycytidine restored RIZ1 transcription. Our results suggest that transcriptional inactivation of the RIZ1 gene by promoter hypermethylation may participate in stomach carcinogenesis. © 2004 Wiley-Liss, Inc. Key words: DNA methylation; gastric carcinoma; RIZ1; CIMP; p53 Epigenetic mechanisms, including DNA methylation and alter- ation of chromatin structure, are important ways to silence many genes, especially defective tumor suppressor genes, involved in human cancers. 1,2 Recent studies have shown that promoter hy- permethylation is a crucial mechanism in transcriptional silencing of tumor suppressor genes in gastric cancer. 3–13 We also showed that DNA methylation occurs for MGMT, 14 p16 INK4a , RAR-beta, CDH1, 15 TSP1, 16 HLTF, 17 and cyclin D2 18 in gastric carcinomas. The retinoblastoma protein-interacting zinc finger gene RIZ was isolated with a functional screen for Rb-binding protein. 19 Domain analysis suggests that RIZ1 is a putative methyltransferase. The PR (PRDI-BF1 and RIZ)/SET (Suvar3-9, Enhancer of zeste, Tritho- rax) domain is involved in chromatin-mediated gene expres- sion 19,20 and plays an important role in human cancers as evi- denced by genetic mutations of several family members. 21 The RIZ gene produces 2 mRNA and protein products through alternative promoters. RIZ1 contains the PR domain, but RIZ2 lacks this domain. 22 The RIZ gene is located on human chromosome 1p36, a region frequently deleted in many human cancers, including gas- tric cancer. 23,24 Expression of RIZ1 but not RIZ2 is frequently silenced in many human cancers, including carcinomas of the breast, colon and liver. 25–27 The RIZ gene is also a target for frameshift mutations in microsatellite-unstable cancers of the co- lon, stomach, endometrium and pancreas. 27–29 Missense mutations of RIZ1 are common in human diffuse large B cell lymphoma but not in other tumors, including gastric carcinoma. 30 RIZ1 is con- sidered to be a tumor suppressor gene because RIZ1 can induce G 2 -M arrest and apoptosis in breast cancer, liver cancer and microsatellite-unstable colon cancers. 25–27,31 Moreover, a knock- out study showed that RIZ1 is a tumor susceptibility gene in mice. 30 RIZ1 and p53 deficiencies are likely to cooperate in tumor formation in mice and are expected to occur in human cancers as well. 30 Many sporadic human cancers carry both p53 mutation and silenced RIZ1 gene. 25,30 Recently, methylation of the RIZ1 pro- moter CpG island has been shown to be a common mechanism in inactivating the RIZ1 gene in human liver and breast cancers. 32 Although frameshift mutations of RIZ have been found in some microsatellite-unstable gastric cancers, 28,29 little is known about correlation between RIZ1 expression, and RIZ1 methylation status, clinicopathological features and p53 mutation status in gastric carcinoma. Gastric carcinomas frequently have CpG island methylator phe- notype. 33 Another common phenotype after the CpG island methy- lator phenotype (CIMP) is CIMP in bracelets. These gastric car- cinomas, designated for the CIMP-positive, show methylation at more than 3–5 loci (methylated in tumors [MINT]1, MINT2, MINT12, MINT25 and MINT31). CIMP-positive gastric carcino- mas are frequently associated with promoter methylation of p16 INK4a 33 and hMLH1, 34 suggesting that CIMP is an important pathway involved in stomach carcinogenesis. Association between promoter hypermethylation of RIZ1 and CIMP was found in colon carcinoma, 35 although it remains unclear in gastric carcinoma. We investigated promoter methylation status and expression levels of the RIZ1 gene in primary gastric carcinoma tissues as well as corresponding non-neoplastic mucosa and gastric carci- noma cell lines. We also examined promoter methylation status of the RIZ1 gene in normal gastric mucosa obtained endoscopically from young healthy individuals to investigate whether methylation of the RIZ1 gene is associated with aging. 1,2,36,37 To determine whether transcriptional silencing of the RIZ1 gene is caused by promoter hypermethylation, we compared the methylation status Grant sponsor: Ministry of Education, Culture, Science, Sports, and Technology of Japan; Grant sponsor: Ministry of Health, Labor, and Welfare of Japan. *Correspondence to: Department of Molecular Pathology, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3 Kasumi, Mi- nami-ku, Hiroshima, 734-8551, Japan. Fax: +81-82-257-5149. E-mail: wyasui@hiroshima-u.ac.jp Received 17 September 2003; Revised 5 November 2003; Accepted 24 November 2003 DOI 10.1002/ijc.20090 Published online 19 February 2004 in Wiley InterScience (www. interscience.wiley.com). Int. J. Cancer: 110, 212–218 (2004) © 2004 Wiley-Liss, Inc. Publication of the International Union Against Cancer