AGE-RELATED HYPERMETHYLATION OF THE hMLH1 PROMOTER IN GASTRIC CANCERS Tomoko NAKAJIMA 1,2 , Yoshimitsu AKIYAMA 1 , Junichi SHIRAISHI 3 , Tomio ARAI 4 , Yuka YANAGISAWA 1 , Miyuki ARA 1 , Yoshiharu FUKUDA 5 , Motoji SAWABE 4 , Kiyoshi SAITOH 6 , Ryuichi KAMIYAMA 2 , Katsuiku HIROKAWA 3 and Yasuhito YUASA 1 * 1 Department of Molecular Oncology, Tokyo Medical and Dental University, Tokyo, Japan 2 School of Allied Health Sciences, Tokyo Medical and Dental University Faculty of Medicine, Tokyo, Japan 3 Department of Pathology and Immunology, Tokyo Medical and Dental University, Tokyo, Japan 4 Department of Pathology, Tokyo Metropolitan Geriatric Hospital, Tokyo, Japan 5 Department of Health Promotion, Tokyo Medical and Dental University, Tokyo, Japan 6 Department of Pathology, International Medical Center of Japan, Tokyo, Japan To determine whether methylation of the hMLH1 pro- moter is related to increasing age and gastric carcinogenesis, we examined hMLH1 methylation and expression in 100 gas- tric cancers. hMLH1 methylation and aberrant protein ex- pression were observed in 9 and 13 cancers, respectively. Normal and intestinal metaplastic tissues adjacent to cancers with hypermethylation did not exhibit any hMLH1 methyl- ation, indicating that it may be specific to gastric cancers. The frequency of hMLH1 methylation significantly increased with age. These results suggest that hMLH1 methylation plays an important role in gastric carcinogenesis in old peo- ple. © 2001 Wiley-Liss, Inc. Key words: aging; methylation, hMLH1; gastric cancer Gastric cancer is 1 of the most common malignancies world- wide and the leading cancer in several countries. Gastric cancer is generally classified into 2 histological types, differentiated (intes- tinal) and undifferentiated (diffuse). 1 The incidence of gastric cancers in old people has been increasing in some countries including Japan because of extension of the life span in the general population. 2 Many gastric cancers in old people are of the differ- entiated type. 3 The molecular mechanisms underlying gastric car- cinogenesis including those in old people have not been clarified yet. Aberrant CpG island methylation is a powerful mechanism for the inactivation of gene activity, and is observed in various can- cers. 4 A correlation between aberrant DNA methylation of the hMLH1 promoter and its reduced protein expression has been reported in colorectal and gastric cancers with microsatellite in- stability (MSI). 5–7 Several other gene promoters, such as estrogen receptor and MyoD, were hypermethylated in normal colonic mucosae, which has been linked to aging. 8, 9 To determine whether or not methylation of hMLH1 is related to increasing age and gastric carcinogenesis, we examined hMLH1 methylation and expression in gastric cancers. MATERIAL AND METHODS Tissue samples A total of 100 human primary gastric cancers was collected at Tokyo Metropolitan Geriatric Hospital and the International Med- ical Center of Japan. Patients were 63 male and 37 female with a mean age of 69.5  11.9 (mean  SD) years (range 43–99 years). Genomic DNAs of primary cancers were extracted from frozen or paraffin-embedded tissues as described previously. 10, 11 Immunohistochemistry Sections of formalin-fixed and paraffin-embedded primary can- cer tissues were de-paraffinized in xylene and then re-hydrated in graded ethanol solutions. Immunohistochemistry (IHC) was car- ried out with an ENVISION system (DAKO Japan Co., Ltd., Kyoto, Japan). 12 Endogenous peroxidase activity was blocked by incubation with 0.03% hydrogen peroxide in methanol for 20 min. Antigen retrieval was accomplished by microwave irradiation. Slides were placed in 10 mM citric acid buffer and then in a microwave oven for 10 min. To block non-specific protein bind- ing, sections were treated with 10% normal goat serum for 15 min. Monoclonal anti-hMLH1 antibody (clone G168-15; PharMingen, San Diego, CA) was diluted at 1: 75 and incubated for 12 hr at 4°C. Sections were treated with ENVISION labeled polymer re- agent (DAKO) for 1 hr, and then incubated with 0.02% hydrogen peroxide and 0.6 mM 3, 3'-diaminobenzidine in phosphate-buff- ered saline. Sections were counterstained with hematoxylin and dehydrated in graded ethanol solutions, then cleared in xylene. Adjacent normal and intestinal metaplastic tissues were used as internal controls. Combined bisulfite restriction analysis Bisulfite treatment was performed using a CpGenome DNA Modification Kit (Oncor, Gaithersburg, MD) according to the manufacturer’s instructions. The Combined bisulfite restriction analysis (COBRA) protocol was performed as described. 13 Bisul- fite-modified DNA was amplified by nested PCR with specific primers for the hMLH1 promoter. The primer sequences used for primary and secondary PCR amplification of hMLH1 were as follows: primary PCR forward, 5'-AGTCGTTTTAGGGAGG- GA(C/T)GAAG-3'; primary PCR reverse, 5'-CCGAATAAC- CCCTACCAC(A/G)AAC-3'; secondary PCR forward, 5'-TGT(C/ T)GTTGAAGGGTGGGGTTGG-3'; and secondary PCR reverse, 5'-ACCTTCAACCAATCACCTCAATAC-3'. 14 Primary PCR was performed in 25 l reaction mixtures, as follows: 94°C for 2 min, followed by 35 cycles at 94°C for 1 min, 55°C for 2 min and 72°C for 1 min, and then a final 10 min extension at 72°C. Secondary PCR was performed in 25 l reaction mixtures, as follows: 94°C for 2 min, followed by 35 cycles at 94°C for 1 min, 60°C for 2 min and 72°C for 1 min, and then a final 10 min extension at 72°C. The PCR products were then digested with a specific restriction enzyme, Rsa I, for at least 2 hr and then electrophoresed on 15% polyacrylamide gels. 15,16 Statistical analysis Comparisons among continuous and categorical variables in hMLH1 expression and methylation status were made using the Mann-Whitney test, and the chi-square or Fisher’s exact probabil- Grant sponsor: Ministry of Education, Science, Sports and Culture, Japan; Grant sponsor: Ministry of Health and Welfare of Japan; Grant sponsor: Foundation for Promotion of Cancer Research in Japan. *Correspondence to: Department of Molecular Oncology, Tokyo Med- ical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. Fax: +81-3-5803-0125. E-mail: yuasa.monc@tmd.ac.jp Received 18 December 2000; Revised 19 March 2001; Accepted 12 April 2001 Published online 3 August 2001; DOI 10.1002/ijc.1454 Int. J. Cancer: 94, 208 –211 (2001) © 2001 Wiley-Liss, Inc. Publication of the International Union Against Cancer