2004-2005) of the Aichi Cancer Center Research Institute

Aichi Cancer Center Research Institute

Scientific Report 2004 – 2005

Chikusa-ku, Nagoya 464-8681 Japan

(The Cover) Looking the front entrance and east face of the Main Building of Aichi Cancer Center Research Institute over the drooping cherries in bloom.

Published by Dr. Toshitada Takahashi Dr. Kazuo Tajima Director Aichi Cancer Center Research Institute 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan Telephone: 052-762-6111 Facsimile: 052-763-5233

Editorial Committee Dr. Reiji Kannagi, Chief (Division of Molecular Pathology) Dr. Kenji Wakai (Division of Epidemiology and Prevention) Dr. Hirotaka Osada (Division of Molecular Medicine) Dr. Hiroshi Kumimoto (Division of Central Laboratory & Radiation Biology) Dr. Malcolm A. Moore, English Editor

Printed by Nagoya University COOP 1 Furoucho, Chikusa-ku, Nagoya 464-0814, Japan

Contents

Preface Takahashi Toshitada 1

Organization of the Aichi Cancer Center Research Institute 2

SCIENTIFIC REPORTS Division of Epidemiology and Prevention General summary 5 1. Descriptive epidemiologic studies on cancer incidence and mortality Ito, H., Hirose, K., and Tajima, K. 5 2. The hospital-based epidemiologic research program at Aichi Cancer Center (HERPACC) study Hirose, K., Matsuo, K., Wakai, K., Ito, H., Saito, T.,Suzuki, T., Kuriki, K., Yang, C. X., Shinoda, M., Hatooka, S., Kanemitsu, Y., Hirai, T., Kato, T., Toyama, T., Iwata, H., Niwa, Y., Nakanishi, T., Morishima, Y., Nakamura, S., Yatabe, Y., Mitsudomi, T., Sugiura, T., and Tajima, K. 7 3. Nutritional factors and the risk of colorectal cancer: findings from the JACC Study Wakai, K. and the JACC Study Group 11 4. Comparative epidemiological study on increasing cancers focusing on Korea, Japan and China (KOJACH study) Matsuo, K., Wakai, K., Hirose, K., Kuriki, K., Huang, X-E., Yang, C. X., Takezaki, T., Hamajima, N., Gao, C-M., Mo, B-Q., Yoo, K-Y., Ahn, Y-O., Kim, J-S., Zhou, Z-Y., Cao, J., Li, C., Gao, F-C., Li, K., Tokudome, Y., and Tajima, K. 12 5. Ethnoepidemiologic study on virus-related cancer Tajima, K., Matsuo, K., Sonoda, S., Chiba, H., Senoh, H., Tretli, S., and Dobrodeeva, L.K. 12

Division of Oncological Pathology General summary 14 1. High salt diets dose-dependently promote gastric chemical carcinogenesis in Helicobacter pylori-infected Mongolian gerbils associated with a shift in mucin production from glandular to surface mucous cells Tatematsu, M., Tsukamoto, T., Mizoshita, T., Kato, S., Cao, X., Hirata, A., and Takasu, S. 15 2. HER2-driven constitutive activation of PI3K-AKT pathway is a new potential molecular target of gefitinib (Iressa) in human gastric cancer liver metastasis Nakanishi, H., Yokoyama, H., Ikehara, Y., Kodera,Y., Ikehara, S. and

Tatematsu, M. 15 3. Sox2 expression in human stomach adenocarcinomas with gastric and gastric-and-intestinal-mixed phenotype Tsukamoto, T., Mizoshita, T., Takenaka, Y., Ogasawara, N. and Tatematsu, M. 16

i 4. A carbohydrate recognition based drug delivery and controlled release system using intraperitoneal macrophages as a cellular vehicle Ikehara, Y., Nakanishi, H., Niwa, T., Biao, L., Ikehara, S., Ohashi, N., Kobayashi, T., Simizu, Y., Kojima, N. and Tatematsu, M. 16 5. Colonic and small-intestinal phenotypes in gastric cancers: relationships with clinicopathologic findings Mizoshita, T., Tsukamoto, T., Tanaka, H., Otsuka, T., Hirano, N., and Tatematsu, M. 17

Division of Molecular Oncology General summary 19 1. EGFR mutation is frequently detected in non-small cell lung cancer with occasional genetic events of second mutation or amplification Yokoyama, T., Kondo, M., Goto, Y., Fukui, T., Sato, N., Taniguchi, T., Kondo, Y., Osada, H., Yokoi, K., T. Shimokata, K., and Sekido, Y. 20 2. The ASH1 gene is a specific therapeutic target for lung cancers with neuroendocrine features Osada, H., Tatematsu, Y., Yatabe, Y., Horio, Y., and Takahashi, Ta. 20 3. A polycistronic miRNA cluster, miR-17-92, is overexpressed in human lung cancers and enhances cell proliferation Hayashita, Y., Osada, H., Tatematsu, Y., Yamada, H., Yanagisawa, K., Tomida, S., Yatabe, Y., Kawahara, K., Sekido, Y., and Takahashi, Ta. 21 4. Establishment and characterization of malignant pleural mesothelioma cell lines from Japanese patients Fukui, T., Taniguchi, T., Usami, N., Yokoyama, T., Hida, T., and Sekido, Y. 21

Division of Molecular Medicine General summary 24 1. Anti-apoptotic function of API2-MALT1 fusion protein involved in t(11;18)(q21;q21) MALT lymphoma Hosokawa, Y., Suzuki, H. and Seto, M. 24 2. Molecular pathways leading to t(12; 21) TEL-AML1 associated leukemias Tsuzuki, S. and Seto, M. 25 3. Cloning of a translocation partner of t(1;14)(p33;q32) in diffuse large B-cell lymphoma Suzuki, R., Nakamura, S. and Seto, M. 25 4. Comparison of genome profiles for identification of distinct subgroups of diffuse large B-cell lymphoma Tagawa, H., Suguro-Katayama, M., Tsuzuki, S., Morishima, Y. and Seto, M. 26 5. Genome-wide array-based comparative genomic hybridization of natural killer cell lymphoma/leukemia: different genomic alteration patterns of aggressive NK-cell leukemia and extranodal NK/T-cell lymphoma, nasal type Nakashima, Y., Tagawa, H., Suzuki, R., Karnan, S., Karube, K., Ohshima, K., Muta, K., Nawata, H., Morishima, Y., Nakamura, S. and Seto, M. 26

ii Division of Immunology General summary 29 1. Identification of an epitope from the epithelial cell adhesion molecule eliciting HLA-A*2402-restricted cytotoxic T lymphocyte responses Tajima, Ko., Demachi-Okamura, A., Ito, Y., Nishida, K., Akatsuka, Y., Tsujimura, K., Kuwano, H., Mitsudomi, T., Takahashi, To. and Kuzushima, K. 30 2. Three immunoproteasome-associated subunits cooperatively generate a CTL epitope of the EBV-LMP2A by overcoming specific structures resistant to epitope liberation Ito, Y., Kondo, E., Demachi-Okamura, A., Akatsuka, Y., Tsujimura, K., Tanimoto, M., Morishima, Y., Takahashi, To. and Kuzushima, K. 30 3. A novel HLA-A*3303-restricted minor histocompatibility antigen encoded by an unconventional open reading frame of the human TMSB4Y gene Torikai, H., Akatsuka, Y., Miyazaki, M., Warren, E.H., Oba, T., Tsujimura, K., Motoyoshi, K., Morishima, Y., Kodera, Y., Kuzushima, K. and Takahashi, To. 31 4. Combination of bortezomib and interferon-γ induces presentation of a newly identified HLA-A24-restricted human papillomavirus type 16 E6-specific cytotoxic T cell epitope Morishima, S., Akatsuka, Y., Nawa, A., Kondo, E., Kiyono, T., Torikai, H., Nakanishi, T., Ito, Y., Tsujimura, K., Iwata, K., Ito, K., Kodera, Y., Morishima, Y., Kuzushima, K. and Takahashi, To. 32 5. Immunity against the mouse thymus-leukemia antigen (TL) protects against development of lymphomas induced by a chemical carcinogenic agent, N-butyl-N-nitrosourea Tsujimura, K., Obata, Y., Matsudaira, Y., Taguchi, O., Nishida, K., Okanami, Y., Akatsuka, Y., Kuzushima, K. and Takahashi, To. 33

Division of Virology General summary 35 1. Epstein-Barr virus lytic replication elicits ATM checkpoint signal transduction while providing an S-phase-like cellular environment Kudoh, A. and Tsurumi, T. 35 2. Architecture of replication compartments formed during Epstein-Barr virus lytic replication Daikoku, T. and Tsurumi, T. 35 3. Activation of ATM DNA damage checkpoint signal transduction elicited by herpes simplex virus infection Shirata, N. and Tsurumi, T. 36 4. Purification of the product of the Epstein-Barr virus BZLF1 gene Nakasu, S. and Tsurumi, T. 37 5. Two Sp1/Sp3 binding sites in the major immediate-early proximal enhancer of human cytomegalovirus genes is necessary for transcriptional activation and viral replication Isomura, H. and Tsurumi, T. 37

iii Division of Molecular Pathology General summary 40 1. Mechanism of loss of disialyl Lewis A and induction of sialyl Lewis A expression in early stage human cancers Miyazaki, K. Ohmori, K., Izawa, M., Koike, T. and Kannagi, R. 41 2. Tumor hypoxia augments sialyl Lewis X and sialyl Lewis A expression in locally-advanced human cancers Koike, T., Kimura, N., Miyazaki, K., Chen, G.Y., Yin, J., Kojima, T., Takematsu, H., and Kannagi, R. 42 3. Study on L-selectin ligand mediated homing of naïve T-lymphocytes using gene-disrupted mice Izawa, M., Kimura, N. Uchimura, K., Ohmori, K, Muramatsu, T., Rosen, S.D. and Kannagi, R. 42 4. ATRA-induced apoptosis in the human neuroblastoma cell line, SH-SY5Y, is accompanied by alteration of ceramide species: Appearance of ceramide containing hydroxy fatty acids Hagiwara, K., Sobue, S., Kyogashima, M., Tamiya-Koizumi, K., Tadano-Aritomi, K., Hara, A., Aoyama, T., Murate, T. and Kannagi, R. 44 5. Rapid demonstration of diversity of sulfatide molecular species from biological materials by MALDI-TOF MS Kyogashima, M., Tamiya-Koizumi, K., Goto, Y., Hara, A., Aoyama, T. and Kannagi, R. 44 6. Expression cloning of a cDNA encoding sialic acid cyclase, which generates cyclic sialic acid containing glycoconjugates, and characterization of the produced enzyme Kanamori, A., Yamaguchi, M., Ishida, H., Kiso, M. and Kannagi, R. 44 + 7. Immune responses to retinal self-antigens in CD25 CD4+ regulatory T-cell- depleted mice Takeuchi, M., Keino, H., Kezuka, T., Usui, M. and Taguchi, O. 45

Division of Biochemistry General summary 47 1. Formation of Plk1 and INCENP complexes required for metaphase-anaphase transition Goto, H., Kiyono, T., Tomono, Y., Kawajiri, A., Urano, T., Furukawa, K., Nigg, E.A. and Inagaki, M. 47 2. Phosphorylation by Cdk1 induces Plk1-mediated vimentin phosphorylation during mitosis Yamaguchi, T., Goto, H., Yokoyama, T., Silljé, H., Hanisch, A., Uldschmid, A., Takai, Y., Oguri, T., Nigg, E.A. and Inagaki, M. 48 3. Mitotic Chk1 phosphorylation at novel sites regulated by cyclin-dependent kinase 1 (Cdk1) Shiromizu, T., Goto, H., Tomono, Y., Bartek, J., Totsukawa, G., Inoko, A., Nakanishi, M., Matsumura, F. and Inagaki, M. 48 4. Functional analysis of the cytoskeleton Izawa, I., Nishizawa, M. and Inagaki, M. 48 5. Characterization and functional analysis of novel keratin filament-binding proteins, trichoplein and Fbf-1 Inoko, A., Zou, P., Sugimoto, M., Hayashi, Y., Kiyono, T., Izawa, I. and iv Inagaki, M. 49 6. Vimentin-Ser82 as a memory phosphorylation site in astrocytes Oguri, T., Inoko, A., Shima, H., Izawa, I., Arimura, N., Yamaguchi, T., Inagaki, N., Kaibuchi, K., Kikuchi, K. and Inagaki, M. 49

Division of Central Laboratory & Radiation Biology General summary 51 1. A single nucleotide polymorphism of the MDM2 gene in Japanese esophageal and oral cancer patients Kumimoto, H., Sugimura, T., Furue, H., Shinoda, M., Hatooka, S. and Ishizaki, K. 51 2. DNA repair defects in AT cells and their hypersensitivity to low-dose-rate radiation

Nakamura, Hid., Yasui, Y., Saito, N. and Ishizaki, K. 52

Central Service Unit General summary Nakamura, Hir., Terashima, M., Tokumasu, S., Nishizawa, M., Yamamoto, M., Hagino, M., Mizuno, M. and Nishi, Y. 55 Librarians Yasuda, T., Teratani, M., Adachi, K. and Ieda, T. 56

Researches Supported by Special Project Programme 1. Defining second-hit genetic abnormalities involved in generation of t(12; 21) TEL-AML1 acute lymphoblastic leukemias by array-based comparative genomic hybridization Tsuzuki, S., Karnan, S., Horibe, K., Matsumoto, K., Kato, K., Inukai, T., Goi, K., Sugita, K., Nakazawa, S., Ueda, R., and Seto, M. 57

Publications 1 Journals 58 2 Reviews and books 80 3. Abstracts for international conferences 83

Records of seminars 88

Records of symposia 90

Author index for research reports and publications 99

From left to right The first row; Dr. M. Tatematsu (Associate Director, and Division chief of Oncological Pathology), Dr. To.Takahashi (Director, and President as of April 2005), and Dr. K. Tajima (Associate Director as of April 2005, and Division chief of Epidemiology and Prevention). The second row; Ms. E. Kaede, Ms. H. Tamaki, and Mrs. M. Hosokawa (Adachi).

Preface ______

It is my pleasure to share with you the 19th Scientific Report (2004-2005) of the Aichi Cancer Center Research Institute. Since the establishment of the Institute in 1965, Scientific Reports have been published biennially to document major research activities and highlight progress in and contributions to cancer research worldwide. As illustrated on the following page, the organization of the Research Institute was remodeled in 2000 to provide for 9 Divisions, consisting of three study groups: cancer prevention/ epidemiology; preclinical/ experimental therapy; and carcinogenesis/ molecular biology. A total of 60 full-time staff members, 44 researchers and 16 research assistants, as well as 12 research residents, are now conducting a wide range of studies, together with 6 graduate school students affiliated with Nagoya University School of Medicine, Nagoya, and approximately 30 visiting research fellows. The major areas being pursued are as follows: - descriptive and analytical epidemiology of cancers - primary and secondary prevention of cancer - molecular pathogenesis of gastrointestinal cancers - molecular oncology of lung cancer - molecular biology of translocation-junction genes of hemtopoietic tumors - basic studies for cancer immunotherapy - oncogenicity, molecular biology and immunology of DNA tumor viruses - glycobiology of cancer cells in relation to metastasis - molecular mechanisms of cell proliferation and movement - involvement of repair mechanisms in carcinogenesis More detailed descriptions of the research topics of each Division appear in the contents of the report. It is our sincere hope that the activities of the Institute will make a major contribution to elucidation of the mechanisms of carcinogenesis and to development of novel clinical applications in cancer diagnosis, treatment and prevention. Finally, I would like to express my deep appreciation to the Aichi Prefectural Government for the continuous support received, since this Institute was founded in 1964. Granting support from the Ministry of Education, Science, Sports, Culture and Technology, the Ministry of Health, Labor, and Welfare, the Ministry of Economy, Trade and Industry, Japan, and other related organizations is also gratefully acknowledged.

February, 2006

Toshitada Takahashi, M.D., D.Med.Sci. Acting Director of the Research Institute President of the Aichi Cancer Center

1 0)rganization ofthe Aichi Cancer Center Research institute

Cheif AdministRatOr M.Ban tun宙l March,2005) 工Aoki (asof April,2005)

President Director R.Ohno R.Ohno(un付 l DecembeL 2003) 工 Kato(aS Of January9 2004) (un宙lMaに h,2005) Head) To.Takahashi (Chief′ (as of Ap付:,2005) ―,Division of Epidemiology and Preventton(K.Taiima)

一 DivisionofOncological Patllology(M.Tatemaほ u)

一 DMdon J MdettlarOn∞ ,o9y将 M) 柔挺商謎混譜甘拭輪‖憂ぽ畢一 Director DivisionofMolecular Medicine(M.SetO) To.Takahashi 一 Divisionoflmmuno:ogy(K.Kuzushima) Associate Directors 一 M.Tatematsu DMtton ofVirologytt Tsuttmり K.千 ima 封一 Division of Molecular Pathology(R.Kannagi) (as ofAp甫 12005) ―B Division of Blochemisw (M・inagaki) 一 CentralLaboratory&Radiatton Biclogy(ェ ishiZaki)

_Central Service Un忙 (HぃNakamura) 一 Animal Fadnty(M.Tatematsu)

一 Laboratory of Transiational Research

SCIENTIFIC REPORTS

From left to right First row: Dr. H. Ito, Dr. K. Matsuo, Dr. K. Wakai, Dr. K. Tajima, Dr. K. Hirose, and Ms. T. Saito. Second row: Ms. C. Yoshida, Ms. S. Hiraiwa, Ms. T. Sato, Ms. M. Watanabe, Ms. Y, Yamauchi, and Ms. M. Nakano. Third row: Dr. T. Suzuki, Dr. K. Kuriki, Ms. M. Sato, Mr. N. Ito, Ms. K. Hasegawa, Ms. K. Mizutani, Ms. H. Achiwa, and Ms. S. Inui. Inset: Ms. H. Fujikura, Ms. K. Fukaya, Ms. Y. Kamori, Ms. K. Tomita, Ms. T. Nishiwaki, Ms. C. Kanto, and Ms. K. Suganuma.

4 Division of Epidemiology and Prevention ______

Kazuo Tajima, M.D., D.M.Sc., M.P.H. Chief Kenji Wakai, M.D., PhD. Section Head Kaoru Hirose, B.P., D.M.Sc. Senior Researcher Keitaro Matsuo, M.D., PhD., S.M. Researcher Hidemi Ito, M.D., PhD Researcher (As of April 2004) Takeshi Suzuki, M.D. Research Resident (As of April 2005) Toshiko Saito. Research Assistant Visiting Trainees Kiyonori Kuriki, B.P., D.M.Sc. Postdoctoral Fellow (Until March 2005) and Research Resident of Foundation for Promotion of Cancer Research (As of April 2005) Chuanxia Yang, M.D., West China University of Medical School, Chengdu, China (April 2004-March 2005) Takeshi Suzuki, M.D. Nagoya City University Medical School (December 2004- March 2005) Xinen Huang, M.D. Nagoya City University Medical School (Until March 2004) Kosuke Amano, M.Ed. Showa University School of Medicine Sanae Ikehara, Dept. of Epidemiology, Nagoya University Graduate School of Medicine

General Summary The current research activities of the Division of Epidemiology and Prevention cover the following four subjects: 1) descriptive epidemiology of cancer incidence and mortality, with special reference to improvement of the Aichi Prefectural Cancer Registry; 2) analytical epidemiology based on the hospital-based epidemiologic research program at Aichi Cancer Center (HERPACC) to determine risk and protective factors for main sites of cancer, with a particular focus on gene-environment interactions; 3) a multi-institutional collaborative cohort study on nutritional factors and cancer risk in Japan; 4) a Korea, Japan and China (KOJACH) collaborative study on increasing cancers for establishment of future prevention programs in each country; 5) ethnoepidemi- ology of tumor viruses among Mongoloids in the Asian-Pacific area. Descriptive epidemiologic studies are necessary for nation-wide statistics like “Vital Statis- tics Japan”. With the new model regional cancer registry in Aichi Prefecture established in 1999, a well organized data collection system and a risk evaluation trial of cancer in smokers and/or drinkers are now in operation. A large-scale HERPACC study was completed for more than 130,000 new outpatients and a third version of HERPACC incorporating the Japan Multi-institutional Collabora- tive Cohort (J-MICC) was started in 2005 to clarify gene-environment interactions for modification of carcinogenicity. In addition to local epidemiologic studies, international collaborative studies in Northeast Asian countries (KOJACH Study) and the North Pole region are ongoing to obtain ethnoepidemiologic evidence of cancer risk among selected Asian populaces. Furthermore, primary prevention trials are being conducted for control of obesity by improvement of dietary habits and promotion of daily exercise. Cancer prevention is the final goal of our epidemiological studies. Recently, we have been concentrating attention on molecular epidemiology to clarify interactions between host-specific characters and lifestyle exposure to risk factors, particularly with regard to actual functions of metabolic and detoxifying enzymes associated with genetic polymorphisms.

1. Descriptive epidemiological studies on Registry. The purpose of this study was to examine cancer incidence and mortality the risk impact of habitual smoking on cancer stage at diagnosis by site using data registered of the Ai- Ito, H., Hirose, K., and Tajima, K. chi Cancer Registry, Japan. The study subjects were Impact of habitual smoking on cancer stage registered cancer cases aged 20 or over and diag- at diagnosis based on Aichi cancer registry data: nosed between 1999 and 2003. The relationship Since 1999, information on smoking habits of can- between cancer stage at diagnosis (local, regional or cer patients has been collected by Aichi Cancer metastatic) and smoking history (never or ever) was

Figure 1. Smoking history and risk of metastatic and regional spread of cancer by site. ORs of smoker group relative to non-smoker group. assessed using a simple comparative approach. A evaluate relationships to cancer stage at diagnosis. total of 66,400 cancer patients with smoking history Ever smoking appears to be a risk factor for ad- and cancer stage at diagnosis were registered up un- vanced cancers in a wide range of body sites. til 2004. Fifty-six percent were male and 44% were Trends in cancer mortality in Japan: Cancer female. The mean age was 64 years. Ever smokers mortality statistics in Japan (1950-2003) were cal- had increased risks of regional (odds ratio (OR), culated from the ‘Vital Statistics, Japan’ series. 1.27; 95% confidence interval (CI), 1.21 to 1.32; The age-standardized death rate for all sites of can- p<0.001) and metastatic (OR, 1.30; 95% CI, 1.24 to cer has been stable in males in recent years, and 1.36; p<0.001) disease. The increase in regional gradually decreasing in females (Figure 2). The disease was most evident for oral & pharynx (OR, mortality rate from stomach cancer has been de- 1.38; 95%CI, 1.10-1.73), larynx (OR, 1.66; 95%CI, creasing in both sexes, while that from lung cancer 1.01-2.73), lung (OR, 1.74; 95%CI, 1.50-2.02), has been increasing, becoming stable more recently. breast (OR, 1.31; 95%CI, 1.12-1.53), and uterus In 2003 the numbers of all deaths from malignant (OR, 1.38; 95%CI, 1.09-1.75) cancers. Increase in neoplasms were 186,912 for men and 122,631 for metastatic disease was most evident for the lung women. Regarding cancer causing, lung cancer is (OR, 1.46; 95%CI, 1.26-1.69) and breast (OR, 1.80; the most common, accounting for 22.3% of all can- 95%CI, 1.35-2.40) cancer (Figure 1). The data for cer deaths for males, followed by stomach cancer smoking status of cancer patients collected by re- with 17.2% and hepatic cancer with 12.5%, while gional cancer registration can thus be utilized to colorectal cancer is the most common for females

Figure 2. Time trends in age-adjusted mortality rate for cancer in Ja- pan, 1960-2003 (Standard population: 1985 model population of Japan).

6 with 14.6%, followed by stomach cancer with cantly decreased esophageal cancer risk (odds ratio 14.2% and lung cancers with 12.3%. (OR)=0.27, 95% confidence interval (CI): 0.09-0.76). The OR for the gene-environment in- 2. The hospital-based epidemiologic re- teraction between heavy drinking and the 677TT search program at Aichi Cancer Center genotype with a case-only design was 0.31 (HERPACC) study (0.10-0.94), indicating risk with heavy drinking to be 69% decreased in individuals harboring the Hirose, K., Matsuo, K., Wakai, K., Ito. H, Saito, T., 677TT genotype. We failed to find any significant Suzuki, T., Kuriki, K., Yang, C. X., Shinoda, interaction between either of the polymorphisms M.*1,Hatooka, S.*2, Kanemitsu, Y.*3, Hirai, T.*3, Kato, and smoking. T.*3, Toyama, T.*4, Iwata, H.*4, Niwa, Y.*5, Nakanishi, 2) Colon cancer: One-carbon metabolism, in which T.*5, Morishima Y.*6, Nakamura, S.*7, Yatabe, Y.*8, folate plays an essential role, is involved in DNA Mitsudomi, T.*9, Sugiura, T.*10, and Tajima, K. methylation and synthesis and is suspected of im- Molecular epidemiology of folate and gastroin- pacting on colorectal carcinogenesis. Alcohol is testinal tract cancer risk: well recognized as a risk factor for colorectal cancer 1) Esophageal cancer: Folate takes part in two bio- (CRC) and interactions with one-carbon metabo- logical pathways involved in DNA methylation and lism have also been suggested. Therefore, func- synthesis and a potential protective influence tional polymorphisms in genes encoding members against carcinogenicity is now recognized in several of this pathway, MTHFR C677T and A1298C, MTR sites, including the esophagus. Therefore, functional A2756G and TS tandem repeats polymorphisms, polymorphisms in encoding genes in folate metabo- have attracted attention. We here conducted a lizing enzymes, MTHFR C677T and MTR A2756G, matched case-control study with 257 incident CRC might be suspected of impacting on esophageal cases and 771 non-cancer controls at Aichi Cancer cancer risk. To test this hypothesis we conducted a Center to clarify associations among folate intake matched case-control study with 165 esophageal and four polymorphisms with reference to CRC risk. cancer cases and 495 non-cancer controls to clarify Gene-environment interactions between polymor- associations among folate intake, MTHFR C677T phisms, drinking and folate consumption were also and MTR A2756G polymorphisms and cancer risk. evaluated. None of the polymorphisms was associ- Gene-environment interactions between the two ated with any significant impact on CRC risk by polymorphisms and drinking and smoking were genotype alone, but when combined with alcohol also evaluated. Folate consumption and MTHFR consumption, the MTHFR 677CC type showed a 677TT were associated with a non-significant ten- significantly reduced risk (OR=0.45, 95% confi- dency for decreased risk while MTR genotypes did dence interval (CI): 0.23-0.86) (p=0.01). MTR GG not show themselves demonstrate any significant increased risk only among drinkers (OR=3.35, influence; further, when analysis was limited to 1.40-8.05) (p=0.047). The TS polymorphism did heavy drinkers, the MTHFR TT genotype signifi- not have a significant impact by genotype alone, but

Figure 3. Dietary factors inversely associated with the risk of (a) colon and (b) rectal cancer (Q1-Q4: quartiles 1-4) in the HERPACC II Study.

7 interaction with drinking was evident (p=0.028), habits: Fish consumption rich in n-3 polyunsatu- even after stratification by daily folate consumption rated fatty acids (PUFAs), such as docosahexaenoic and drinking habit. acid (DHA), is suggested to reduce colorectal can- Dietary factors and colorectal cancer risk (Fig- cer risk through inhibiting the arachidonoic acid ure 3): In Japan, the incidence rate for colon cancer (AA) cascade related to tumorigenesis and cell pro- has more rapidly increased than that for rectal can- liferation. High intake of saturated fatty acids cer. To compare dietary risk factors between colon (SFAs), in contract, appears to increase the risk. To and rectal cancers, we undertook a case-control examine associations between colorectal cancer risk study using data from the Hospital-based Epidemi- and the fatty acid composition of erythrocyte mem- ologic Research Program at Aichi Cancer Center branes, reflecting dietary intake of fish, fat and fatty (HERPACC). Subjects included 507 men and acids, we conducted a case-control study with 74 women with newly diagnosed colon (n = 265) and incident cases and 221 non-cancer controls rectal (n = 242) cancers, and 2,535 age- and gen- (matched by age, sex and season of data collection). der-matched, cancer-free outpatients (controls). In- Erythrocyte fatty acids were measured by a takes of nutrients and food groups were assessed semi-automatic method using accelerated solvent with a food frequency questionnaire, and multivari- extraction and gas-liquid chromatography. Colo- ate-adjusted odds ratios (ORs) were estimated using rectal cancer incidence risk had no association with conditional logistic models. We found a decreasing dietary intake of meat, fish, fat and fatty acids. risk of colon cancer with increasing intakes of vi- However, the risk was inversely associated with tamin C and fruit; the ORs across quartiles of intake erythrocyte compositions of PUFAs, AA and DHA were 1.00, 0.90, 0.72, and 0.72 (95% CI: 0.47-1.11; [highest to lowest tertile, odds ratios (ORs) = 0.15, trend p = 0.089) for vitamin C, and 1.00, 0.85, 0.67, 0.42 and 0.36, 95% confidence intervals (CIs) = and 0.68 (95% CI: 0.45-1.03; trend p = 0.036) for 0.05 to 0.46, 0.18 to 0.95 and 0.14 to 0.93, Ptrend fruit. For rectal cancer, higher consumption of meat <0.05 to 0.005], and positively with those of SFAs, and green-yellow vegetables was associated with a palmitic acid and the ratio of SFAs/PUFAs (ORs = reduced risk; the ORs for the highest versus lowest 8.20, 6.46, and 9.45, 95% CIs = 2.86 to 23.52, 2.41 quartile were 0.65 (95% CI: 0.42-1.02) and 0.62 to 17.26, and 2.84 to 31.43, Ptrend <0.005 to 0.0001, (95% CI: 0.39-0.99), respectively. A decreased risk Figure 4). We thus demonstrated colorectal cancer associated with higher intakes of insoluble dietary risk to be clearly related to fatty acid composition in fiber was observed for colon cancer (OR for the erythrocyte membranes, but further studies are re- highest quartile: 0.57; 95% CI: 0.37-0.89) but only quired to clarify the apparent discrepancy that a suggested for rectal cancer. Carbohydrate intake high erythrocyte composition of AA may also re- was correlated particularly with the risk of female duce risk. rectal cancer. In conclusion, differences in secular Protective and risk factors for hormone related trends and in international distributions of incidence cancer in women: between the two colorectal sites may partly be at- 1) Soybean products and reduction of breast cancer tributable to variation in dietary risk factors. risk: Components of the Japanese diet which might Colorectal cancer risk and erythrocyte compo- contribute to the relatively low breast cancer inci- sition of fatty acids as biomarkers for dietary dence rates in Japan, have not been clarified in de-

Figure 4. Decreased (left) and increased (right) risks for colorectal cancer according to associations with fatty acid compositions in erythrocytes.

8 tail. Since soybean products are widely consumed leaner women (BMI=<22). In summary, the re- in Japan a case-control study taking account of the sults of the present study suggested that coffee menopausal status was conducted using data from consumption reduces the risk of endometrial cancer the hospital-based epidemiologic research program in Japanese, especially in relatively lean women. at Aichi Cancer Center (HERPACC). In total, 167 3) The CYP19 gene codon39 Trp/Arg polymor- breast cancer cases were included and 854 women phism increases breast cancer risk in subsets of confirmed as free of cancer were recruited as the premenopausal Japanese: The production of estro- control group. Odds ratios (OR) and 95% confi- gen from androgen via the estrogen biosynthesis dence intervals (95%CI) were determined by multi- pathway is catalyzed by aromatase P450 (CYP19). ple logistic regression analysis. Reduction in risk To assess the association between breast cancer risk of breast cancer was associated with high intake of and a polymorphism at codon 39 ( Trp to Arg ) of soybean products among premenopausal women, the encoding gene, a case-control study was con- the adjusted OR for the top tertile intake of tofu ducted at Aichi Cancer Center Hospital in Japan. (soybean curd) being 0.51 (95%CI, 0.26-0.98) Subjects were 248 histologically confirmed breast compared with women in the lowest tertile. A cancer patients and 603 hospital controls without significant decrease in premenopausal breast cancer cancer. Odds ratios (ORs) and 95% confidence risk was also observed for increasing consumption intervals (95%CI) were determined by logistic re- of isoflavones (OR=0.45, 95% CI:0.23-0.89 for gression analysis. The allele frequency among highest vs. lowest tertile, p for trend=0.02). The controls was 3.8% for the C allele and the OR of the present study found a statistically inverse associa- polymorphism relative to the TT-genotype was 1.21 tion between tofu or isoflavone intake and risk of (95% CI:0.69-2.14) for the TC/CC-genotypes com- breast cancer in Japanese premenopausal women bined. There was no association between the while no statistically significant association was CYP19 gene polymorphism and breast cancer risk evident with the risk among postmenopausal in the study group as a whole but homozygous and women. heterozygous carriers of the variant Arg allele 2) Coffee consumption and reduction of endo- showed a significantly increased risk of breast can- metrial cancer risk: Coffee has become a popular cer among premenopausal women with a late age at beverage worldwide and because it contains large first-full term pregnancy (OR=7.31, 95%CI: amounts of antioxidants, such as chlorogenic acids, 1.88-28.5) or a high BMI (OR=2.77, 95%CI: there has been increasing interest in possible bene- 1.12-6.87). Additional larger studies should be ficial health effects. Caffeine, a major ingredient performed to confirm that the rare CYP19 variant of coffee, has been proposed to modulate circulat- increases the risk of breast cancer among premeno- ing estrogen levels and therefore may also be of pausal Japanese women. importance for cancer development. To test this, Hematopoietic cancer: Recent increase in the in- we examined the relationship between intake of cidence of malignant lymphoma (ML) suggests coffee and hormone related cancer risk among possible involvement of environmental factors in its Japanese women using data from the hospital-based genesis. Medical conditions could be one potential epidemiologic research program at Aichi Cancer candidates and their identification may provide Center (HERPACC). In total, 2,122 breast, 539 clues for future prevention. We focused on peptic cervical, 229 endometrial and 166 ovarian cancer ulcer history on ML risk and conducted a cases were included, and 12,425 women, confirmed case-control study with 645 patients histologically as free of cancer, were recruited as the control diagnosed as having malignant lymphomas and group. A statistically significant inverse associa- 3225 non-cancer controls. Plasma H. pylori IgG tion between risk of endometrial cancer and coffee status was assessed for subgroups for which blood consumption was noted in our Japanese women, samples were available (116 cases and 114 con- with no clear associations evident for breast, cervi- trols). An association with a history of gastric, but cal or ovarian cancer risk. The ORs for daily not duodenal ulcers was found for gastric lympho- drinking of 1-2 cups and 3 or more cups per day for mas [odds ratio (OR) = 5.41, 95% confidence in- endometrial cancer were 0.64(95%CI:0.43-0.94) terval (CI): 3.12 - 9.39]. On examination according and 0.41 (95%CI:0.20-0.88), respectively, and the to histological subtype, the Ors were high for both linear trend was also statistically significant gastric mucous-associated lymphoid tissue (MALT) (p<0.01). The effect of coffee intake on risk of lymphoma (OR = 5.54, 95% CI: 2.56 - 12.01) and endometrial cancer was most prominent among diffuse large B-cell lymphoma (DLBCL) (OR =

9 7.23, 95% CI: 2.62 - 19.90). Further, on subgroup behavior in Japanese: Accumulating evidence in- analysis of subjects with H. pylori infection, gastric dicates that the genotype may impact on smoking ulcer history, but not duodenal ulcer history was behavior and a deeper understanding of the mo- associated with the risk of gastric lymphoma (OR = lecular basis could lead to more effective strategies 4.15, 95% CI: 1.02 - 16.89). This observation is for preventing initiation of the habit and for helping quite similar to the association between gastric smokers to quit. Since individual variation in air- cancer and peptic ulcer history, suggesting a similar way responsiveness to cigarette smoke might have mechanism underlying effects on gastric cancer and an important influence, we have focused on asso- gastric lymphoma development. ciation between smoking behavior and polymor- Molecular epidemiology of lung cancer: APE1 phisms affecting the inflammatory cytokine, IL-8. (apurinic/apyrimidinic endonuclease 1) and XRCC1 In the present study, 453 Japanese non-cancer out- (X-ray cross-complementing group 1) are DNA re- patients (191 males and 262 females) who visited pair proteins that play important roles in the base Aichi Cancer Center Hospital were genotyped for excision repair (BER) pathway. Polymorphisms in the IL8 -251T/A polymorphism and age- and sex- their encoding genes are associated with altered adjusted odds ratios (aORs) for smoking were esti- DNA repair capacity and thus may impact on can- mated by a logistic regression model. The aORs for cer risk. In a case-control study with 178 Japanese IL8 251-TA and AA combined, genotypes associated incident lung cancer cases and 449 age- and sex- with high production of IL-8, were 0.52 (95% CI matched controls, we therefore investigated 0.33-0.82, p=0.004) for being an ever smoker and gene-environment interactions among APE1 0.55 (0.55, 0.33-0.92, p=0.023) for being a current Asp148Glu, XRCC1 Arg399Gln, and smoking habit smoker. Our results suggest that the inflamma- in lung cancer risk. The results were analyzed using tory-prone genotype of IL8 may act to deter initia- conditional logistic regression models, adjusted for tion or characteristics of the smoking habit. age, sex and smoking status. The adjusted odds ra- 2) Smoking cessation inducement by providing in- tio for the current smokers with APE1 148Asp/Asp, formation on the L-myc genotype: To evaluate Asp/Glu and Glu/Glu genotypes as compared with whether feedback of genetic information regarding the never smokers with the Asp/Asp genotype were an L-myc polymorphism, identified as impacting on 3.01 (95% CI 1.39-6.51, p= 0.005), 2.73 (1.29-5.77, tobacco-related cancer risk, has an influence on p=0.008) and 7.33 (2.93-18.3, p<0.001), respec- smoking cessation, an intervention study was con- tively. The gene-environment interaction between ducted. We recruited smokers from first-visit outpa- current smoking and APE1 148Glu/Glu genotype tients at Aichi Cancer Center Hospital. Six hundred was statistically significant (OR 3.59, 1.28-10.1, and seventeen participated and were allocated into p=0.015). When APE1 Asp148Glu and XRCC1 two groups: the biomarker-feedback group (BF) and Arg399Gln polymorphisms were evaluated together, the follow-up smoking-status group (FS). The sub- the adjusted odds ratios for current smokers with jects were asked for their smoking status at enrol- 0-1, 2 and 3-4 of APE1 148Glu or XRCC1 399Gln ment and after 3- and 9-months follow-up. BF sub- alleles as compared with never smokers with the jects were notified about their L-myc genotype. The rare of these alleles were 2.96 (1.57-5.58, p=0.001), smoking cessation rate at 9-months follow-up was 3.86 (1.85-8.05, p<0.001) and 6.01 (2.25-16.1, essentially the same for both BF and FS cases, at p<0.001), respectively. The gene-environment in- 18.8% and 17.0%, respectively (p=0.798). However, teraction between current smoking and 3 or more a difference in the rate was evident with non-cancer APE1 148Glu or XRCC1 399Gln alleles was statis- subjects (12.7% and 8.4%, respectively, p=0.237), tically significant (OR 2.44, 1.00-9.22, p=0.049). especially in females (15.0% and 4.2%, respectively, The OR for the gene-environment interaction of the p=0.024). The non-cancer subjects informed of Glu/Glu genotype of the APE1 codon 148 with their genotype were more likely to quit smoking heavy smoking was 1.04 (0.38-2.90, p=0.936) and than the FS patients; particularly in those having a that with light smoking was 2.67 (1.00-7.68, risky genotype this was significant (odds ratio: 2.87, p=0.049). These results suggest that APE1 p=0.003). Again it was most prominent in females. Asp148Glu and XRCC1 Arg399Gln polymorphisms In conclusion, feedback regarding an L-myc poly- might modify the risk of lung cancer attributable to morphism did not impact on smoking cessation cigarette smoking exposure. overall, but appeared to benefit smokers without Smoking behavior modification: cancer. In addition, gender could affect the response 1) Interleukin 8 (IL8) polymorphisms and smoking to the feedback.

10 Introduction of the J-MICC Study: The Japan 3. Nutritional factors and the risk of colo- Multi-Institutional Collaborative Cohort (J-MICC) rectal cancer: findings from the JACC Study is a new multi-center approach to elucidate Study environmental and genetic risk factors for cancer Wakai, K. and the JACC Study Group and other lifestyle-related diseases. In this project, three types of studies are planned: 1) follow-up to The Japan Collaborative Cohort (JACC) Study clarify associations between development of disease is a large-scale prospective study conducted in 45 and lifestyle factors, genotypes, and other bio- areas throughout Japan. The cohort was established markers, or their combinations; 2) search for bio- from 1988 to 1990, when 110,792 inhabitants aged markers useful for early detection of disease par- 40 to 79 years completed a questionnaire on life- ticularly of cancer; and 3) cross-sectional studies style and medical history. About 35% of the par- relating to lifestyle, genotypes, and biomarkers. ticipants donated blood samples. To elucidate the Men and women aged 35 to 69 years are to be re- role of nutritional factors in the etiology of colorec- cruited for the study by about ten institutions tal cancer, we analyzed data from the study. Major throughout Japan from the general population, ex- findings of the analyses are as follows. (a) The as- aminees of health check-ups, or hospital patients, sociation of serum carotenoid levels with colorectal with a targeted number of 100,000. The subjects are cancer risk may be modified by sex. In a being requested to complete a questionnaire on life- nested-case control study, a higher level of serum style and medical factors and also to donate blood total carotenoids was associated with a decreased samples including buffy coat, plasma, and serum. risk in men (the odds ratio [OR] for the highest They will be followed up for death and cancer inci- versus the lowest tertile: 0.34; 95% CI: 0.11-1.00; dence by review of death certificates or medical re- trend P over tertiles: 0.040), whereas its higher cords, linkage with cancer registries, mail surveys, level was related to a somewhat increased risk in etc., until March 2025. After hot discussion on the women (the corresponding OR: 2.47 [95% CI: protocol, especially on its ethical issues, the study 0.73-8.34]; trend P: 0.064). (b) Male ex- or current was launched in October 2005. We, the Division of drinkers demonstrated a twofold risk for colon can- Epidmiology and Prevention, are taking part in the cer compared with nondrinkers: the incidence rate J-MICC Study as a study center and have been re- ratio (IRR) was 2.01 (95% CI: 1.09-3.68) for cruiting participants of the study together with those ex-drinkers and 1.97 (95% CI: 1.28-3.03) for cur- of the HERPACC at the Aichi Cancer Center Hos- rent drinkers. (c) An inverse correlation was ob- pital since November 2005. served between intake of dietary fiber and the risk of colon cancer; the IRRs across quartiles were 1.00, *1 Department of Rehabilitation, Aichi Cancer Center 1.18, 0.50, and 0.68 (95% CI: 0.38-1.21; trend P: Hospital 0.049) in men and 1.00, 0.69, 0.67, and 0.59 (95% *2 Department of Thoracic Surgery, Aichi Cancer Cen- CI: 0.34-1.01; trend P: 0.061) in women. (d) Intake ter Hospital of fat, particularly that of animal or saturated fat, *3 Department of Gastroenterological Surgery, Aichi was associated with an increased risk of colon can- Cancer Center Hospital cer. In men, the IRRs over quartiles were 1.00, 1.25, *4 Department of Breast Oncology, Aichi Cancer Center 1.86, and 1.81 (95% CI: 1.05-3.11) for total fat Hospital (trend P: 0.015), 1.00, 1.41, 1.75, and 2.06 (95% *5 Department of Gynecologic Oncology, Aichi Cancer CI: 1.24-3.41) for animal fat (trend P: 0.003), and Center Hospital 1.00, 1.98, 2.83, and 2.38 (95% CI: 1.35-4.20) for *6 Department of Hematology and Cell Therapy, Aichi saturated fat (trend P: 0.001). Although we found Cancer Center Hospital no significant dose-response relationship for total or *7 Department of Pathology, Graduate School of Medi- saturated fat in women, an increasing trend in colon cal Sciences, Nagoya City University cancer risk was detected with an increasing intake *8 Department of Pathology and Molecular Diagnostics, of animal fat. The IRRs across quartiles of intake Aichi Cancer Center Hospital were 1.00, 1.08, 1.33, and 1.65 (95% CI: 1.02-2.67; *9 Department of Thoracic Surgery, Aichi Cancer Cen- trend P: 0.035). ter Hospital *10 Department of Thoracic Oncology, Aichi Cancer Center Hospital

11 4. Comparative epidemiological study on 5. Ethnoepidemiologic study on virus-re- increasing colorectal and breast can- lated cancer in Mongoloids cers focusing on Korea, Japan and Tajima, K., Matsuo, K., Sonoda, S.*1, Chiba, H.*2, Senoh, China (KOJACH study) H.*3, Tretli, S.*4 , and Dobrodeeva, L.K. *5 Matsuo, K., Wakai, K., Hirose, K., Kuriki, K., Huang, Human T-cell leukemia virus type 1 (HTLV-1), X-E., Yang, C. X., Takezaki T., Hamajima N., Gao the main cause of adult T-cell leukemia/lymphoma, *1 *2 *2 *3 *4 C-M. , Yoo K-Y. , Ahn Y-O. , Cao J. , Pan I-M. is found throughout the world but with micro- *5 *6 Li K. , Tokudome Y. , and Tajima, K. geographical clusters of hyperendemicity. Epidemi- To establish a basis for cancer prevention in the ologic studies among Mongoloids showed that Asian Pacific region, we started a case-referent HTLV-1 is highly endemic in South Japan (one study on increasing cancers since 2000, based on a million carriers) and in the Andes district of South standardized epidemiological approach, in Korea America. In contrast, HTLV-II (also a risk factor (Seoul), Japan (Nagoya) and China (Nanjing, for adult T-cell leukemia/lymphoma) is broadly Chongqing, Benxi and Shantou), the so called distributed in all of South America, except the An- KOJACH Study. In Korea they developed their own des line. After sero-epidemiologic studies on SQFFQ and we established semi-quantitative food HTLV-I antibodies among Tibetan people in China frequency questionnaires (SQFFQs) in the four cit- and Sahme people in North Norway in 2001 and ies in China. The validity and reproducibility of all 2003 respectively, we conducted field work in the SQFFQs established in Korea and China were Nenets Autonomous District of the Arkhangelsk evaluated for further epidemiologic studies. We Region in northwestern Russia. In total, 105 blood have collected lifestyle data by standardized samples from Nenets people of Krasnoe were col- SQFFQs and blood samples for plasma and DNA lected in collaboration with Institute of Physiology after obtaining informed consent from more than of Adaptations to Environment, the Ural Branch of 1,600 colorectal cancer cases in each area in Korea, the Russian Academy of Sciences. No individuals Japan and China. The same number of referents positive for anti-HTLV-I were detected in the in- matched by age and sex were recruited from hospi- digenous Nenets people, in line with the HTLV-I/II tal patients in Korea and Japan and from the general clusters among Mongoloids in other areas of the population in China. A detailed analysis is now Asian Pacific. ongoing to clarify risk and protective factors for *1 colorectal cancer in each of the three countries and Department of Virology, Faculty of Medicine, Ka- areas of China using data for lifestyle, genetic goshima University, Kagoshima, Japan *2 polymorphisms and interactions. Furthermore, other Department of Laboratory Medicine, Hokkaido case-referent studies on breast cancer were also ini- University School of Medicine, Sapporo, Japan *3 tiated in June 2005. Department of Anatomy, Akita University School of Medicine, Akita, Japan, *4 *1 Division of Epidemiology, Cancer Institute of Ji- Institute of Population-based Cancer Research, Oslo, angsu Province, Nanjing, China Norway *5 *2 Department of Preventive Medicine, College of Department of Ecological Immunology, Institute of Medicine, Seoul National University, Seoul, Korea Physiology of Adaptations to Environment, the Ural *3 Laboratory of Molecular Toxicology, Third Military Branch of the Russian Academy of Sciences, Ark- Medical University, Chongqing, China hangelsk, Russia *4 Bengan General Hospital, Benxi, China *5 Department of Epidemiology, Shantou University, Shantou, Chian *6 Life Science, Nagoya Bunri College

From left to right First row: Dr. T. Mizoshita, Dr. T. Tsukamoto, Dr. M. Tatematsu, Dr. H. Nakanishi and Dr. Y. Ikehara Second row: Mrs. C. Ikedo, Dr. N. Hirano, Dr. Y. Takenaka, Mr. H. Tanaka, Mr. H. Asahara, Mrs. H. Ban

13 Division of Oncological Pathology ______

Tatematsu Masae, M.D. Chief Hayao Nakanishi, M.D. Section Head Tetsuya Tsukamoto, M.D. Section Head Yuzuru Ikehara, M.D. Senior Researcher (until Mar, 2006) Tsutomu Mizoshita, M.D. Researcher Harunari Tanaka, B.P., Research Assistant Hisayo Ban, Semi-regular Employee Mikako Asai Semi-regular Employee (until Nov, 2005) Visiting Scientists Malcolm A. Moore, Ph.D. Asian Pacific Organization for Cancer Prevention Kato Kazuo, M.D., Fujita Health University School of Medicine Visiting Trainees Xueyuan Cao, M.D., Dept. of Gastrointestinal Surgery, The University of Tokyo Yoshiharu Takenaka, M.D., Dept. of Gastrointestinal Surgery, The University of Tokyo Takasuke Yamachika, M.D., Kita Hospital Akihiro Hirata, D.V.M. Dept. of Veterinary Pathology, Gifu University Shinji Takasu, D.V.M. Dept. of Veterinary Pathology, Gifu University Yoshiyuki Yokoyama, M.D. Dept. of Surgery II, Nagoya University School of Medicine Norifumi Ohashi, M.D. Dept. of Surgery II, Nagoya University School of Medicine Kenji Tsuboi, M.D. Dept. of Surgery II, Nagoya University School of Medicine Yuichi Ito M.D. Dept. of Surgery II, Nagoya University School of Medicine Sanae Ikehara, Dept. ofEpidemiology, Nagoya University Graduate School of Medicine Toru Niwa, M.D., Dept. of Internal medicine II, Wakayama Medical College Takafumi Otsuka, M.D., Dept. of Internal medicine I, Toho University School of Medicine Naoki Hirano M.D., Dept. of Internal medicine I, Toho University School of Medicine Sousuke Katoh, M.D., Dept. of Internal medicine III, Hokkaido Univesity School of Medicine Naotaka Ogasawara, M.D., Dept. of Internal Medicine and Bioregulation, Nagoya City University of Medicine. Yoshikazu Hirata M.D., Dept. of Internal Medicine and Bioregulation, Nagoya City University of Medicine. Masayasu Hara, M.D., Dept. of Gastroenterological Surgery, Nagoya City University of Medicine. Mistuo Gotoh, D.D.S., School of Dentistry, Aichi-Gakuin University Yasushi Seki, D.D.S., School of Dentistry, Aichi-Gakuin University Fumi Ono, D.D.S., School of Dentistry, Aichi-Gakuin University Atsutaka Kinoshita, D.D.S., School of Dentistry, Aichi-Gakuin University Shinya Satoh, M.D., Aichi Cancer Center Hospital Chihiro Aoki, College of Bioscience and Biotechnology, Chubu University Hisayoshi Asahara, College of Bioscience and Biotechnology, Chubu University

General Summary The responsibility of the Division of Oncological Pathology includes autopsy and research activities. From the establishment of this laboratory in 1965 to the end of 2005, the number of autopsy cases amounted to 2568. Postmortem examinations are a source of valuable information on the behavior of neoplasms and their response to therapy. Autopsy findings also supply the basis for total evaluation of the course of disease, including the accuracy of clinical diagnosis, effectiveness or failure of drugs, irradiation and surgery, and the appearance of complications such as op- portunistic infection and hemorrhage during treatment. Main theme of this laboratory is the carcinogenesis and the progression of gastrointestinal malignancies. During 2004-2005, the research activities are divided into the following four main areas. The first deals with the molecular basis and detection of chemical carcinogenesis and initiation and promotion activities. In vivo five-week initiation assay model revealed heterocyclic amines, food-derived carcinogen, revealed interference each other. The second concerns gastric cancers and

14 their precancerous lesions along with the mechanisms regulating differentiation of stomach epithelium in humans as well as rodent models. High salt diet and younger acquisition of Helicobacter pylori (Hp) exacerbate inflammation and incidence of stomach adenocarcinomas. On the other hand, earlier eradication well suppressed stomach carcinogenesis. Molecular mechanism of intestinal metaplasia, major complication of Hp infection, includes alteration of gastric and intestinal transcription factors. The third research area involves basic research on tumor progression and metastasis, especially micrometastasis and its clinical application for prevention of recurrent disease after surgery. A new potential molecular therapy targeting to HER2 overexpression using gefitinib in gastric cancer liver metastasis is currently ongoing. In line with this therapeutic strategy, the fourth issue includes the development of a novel drug-delivery system using the carbohydrate-coated liposomes which are selectively accumulated at a milky spot, a preferential site for intraperitoneal metastasis of gastric cancer.

1. High salt diets dose-dependently pro- ronment. Reduction of salt intake could thus be mote gastric chemical carcinogenesis one of the most important chemopreventive meth- in Helicobacter pylori-infected Mongo- ods for human gastric carcinogenesis. lian gerbils associated with a shift in 1 mucin production from glandular to * Dept. of Gastroenterology, Hokkaido University surface mucous cells Graduate School of Medicine, Sapporo, Japan *2 Dept. of Veterinary Pathology, Gifu University, Gifu, Tatematsu, M., Tsukamoto, T., Mizoshita, T., Kato, S.*1, Japan Cao, X., Hirata, A.*2, and Takasu, S.*2

Intake of salt and salty food is known as a risk 2. HER2-driven constitutive activation of factor for gastric carcinogenesis. To examine the PI3K-AKT pathway is a new potential dose-dependence and the mechanisms underlying molecular target of gefitinib (Iressa) in enhancing effects, Mongolian gerbils were treated human gastric cancer liver metastasis with N-methyl-N-nitrosourea (MNU), Helicobacter Nakanishi, H., Yokoyama, H. *1, Ikehara, Y., Kodera, pylori (H. pylori), and food containing various Y.*1, Ikehara, S. and Tatematsu, M. concentrations of salt and sacrificed after 50 weeks. Among gerbils treated with MNU and H. pylori, the HER2 overexpressing gastric cancer is incidences of glandular stomach cancers were 15% known to lead to a poor patient outcome, but in the normal diet group and 33%, 36%, and 63% in there is virtually no efficient therapeutic mo- the 2.5%, 5%, and 10% NaCl diet groups, showing dality. The purpose of the present study is to dose-dependent increase (P<0.01). Intermittent investigate the possibility of molecular therapy intragastric injection of saturated NaCl solution, in targeting to HER2 overexpression in the gastric contrast, did not promote gastric carcinogenesis. cancer patients. We found that gastric cancers In gerbils infected with H. pylori, a high salt diet metastasized to the liver overexpressed HER2 was associated with elevation of anti-H. pylori an- at significantly higher incidence than primary tibody titers, serum gastrin levels, and inflamma- gastric cancers. We developed three new HER2 tory cell infiltration in a dose-dependent fashion. overexpressing gastric cancer cell lines Ten percent NaCl diet upregulated the amount of (GLM-1, GLM-2, GLM-4) without EGFR mu- surface mucous cell mucin (P<0.05), suitable for H. tations derived from such liver metastasis, two pylori colonization, despite no increment of of which had HER2 gene amplification. Inter- MUC5AC mRNA, while H. pylori infection itself estingly, all these GLM series were highly sen- had an opposing effect, stimulating transcription of sitive to gefitinib, a specific inhibitor of epi- MUC6 and increasing the amount of gland mucous dermal growth factor receptor (EGFR) tyrosine cell mucin. High salt diet, in turn, decreased the kinase (ZD1839, “Iressa”) with IC50 less than amount of gland mucous cell mucin, which acts 0.1 µM, but not Herceptin, whereas most of against H. pylori infection. In conclusion, the HER2-negative counterparts were not present study demonstrated dose-dependent en- (IC50>10 µM). Gefitinib induced strong apop- hancing effects of salt in gastric chemical carcino- tosis depending on caspase 3 and exhibited genesis in H. pylori-infected Mongolian gerbils as- anti-tumor activity against these sociated with alteration of the mucous microenvi- HER2-overexpressing cancer cell lines both in

15 vitro and in vivo. In GLM-1, GLM-2 and carcinoma cell line, KATOIII, demonstrated both GLM-4 cells, Akt, but not ERK1/2, was con- MUC5AC and Sox2, although MUC5AC mRNA stitutively phosphorylated without loss of was not detected in the Sox2-positive AGS cell line. PTEN expression, and gefitinib efficiently in- Conclusions: Sox2 may play an important role hibited this HER2-driven Akt phosphorylation. in maintaining a gastric phenotype in stomach can- However, gefitinib failed to inhibit constitutive cers as well as in normal tissue, in cooperation with phosphorylation of Akt in HER2-negative gas- other cofactor(s). tric cancer cell lines. On the other hand, gefitinib-resistant cells (GLM-1R), exhibited in- *1 Dept. of Gastrointestinal Surgery, The University of creased EGFR expression, followed by consti- Tokyo, Tokyo, Japan tutive activation of MAPK pathway. These re- *2 Dept. of Internal Medicine and Bioregulation, Na- sults suggest that the anti-tumor effect of gefit- goya City University of Medicine, Nagoya, Japan. inib is due to the effective inhibition of HER2-driven constitutive activation of 4. A carbohydrate recognition based drug PI3K/Akt pathway and that the acquired resis- delivery and controlled release system tance to gefitinib is due to the constitutive acti- using intraperitoneal macrophages as a vation of Ras/MAPK pathway in compensation cellular vehicle for PI3K/Akt pathway. Gastric cancer liver 1 2 metastasis with HER2-overexpression would Ikehara, Y., Nakanishi, H., Niwa, T.* , Biao, L.* , Ikehara, S., Ohashi, N.*3, Kobayashi, T. *1, Simizu, Y.*1, be a potential molecular target for gefitinib. Kojima, N. and Tatematsu, M.

*1 Department of Surgery II, Nagoya University School We developed a new technology using the car- of Medicine, Tsuruma, Shouwa-ku, Nagoya, Japan bohydrate recognition by macrophages that are applied to use as a cellular vehicle in a drug delivery system. The lymphoid tissue in the omentum, called 3. Sox2 expression in human stomach for milky spots, is known as an initial place for dis- adenocarcinomas with gastric and gas- seminated cancer cells to develop into solid tu- tric-and-intestinal-mixed phenotypes mours. Intraperitoneal macrophages significantly Tsukamoto. T., Mizoshita, T., Takenaka, Y.*1, took up Oligomannose-coated liposomes (OML) Ogasawara, N.*2 and Tatematsu, M. that were injected into peritoneal cavity, and then gradually accumulated in the omentum and the Aims: Other than ectopic expression of intesti- other lymphoid tissues within 24 h of intraperito- nal transcription factors, Cdx1 and Cdx2 , molecular neal injection of OMLs. When 5-fluorouracil mechanisms underlying gastric and intestinal phe- (5-FU) was encapsulated in the OMLs, more than notypes of human stomach adenocarcinomas have 60% of administered 5-FU accumulated in the yet to be clarified in detail. We have reported that omentum. Treatment of macrophages at 39 °C for Sox2, an HMG-box gastric transcription factor, is 30 min led to the release of 5-FU from the macro- expressed in normal gastric mucosa and phages, suggesting that controlled release from down-regulated in intestinal metaplasia. macrophages could be achieved by mild hyperther- Methods and Results: We analyzed mRNA lev- mia. We encased magnetic nanoparticles, which are els of Sox2 and other differentiation markers in fifty known to convert electromagnetic energy to heat, in surgically resected stomach adenocarcinomas, im- the OMLs to achieve in vivo hyperthermia at the munohistochemically classified into gastric (G), site. Using this system in a mouse intraperitoneal gastric-and-intestinal (GI)-mixed, solely intestinal metastasis model, we successfully controlled tu- (I), and null (N) types. Sox2 was found to be mour development by co-administration of transcribed in G and GI-mixed type adenocarcino- OML-encased 5-FU and OML-encased magnetic mas in accordance with MUC5AC and MUC6 ex- nanoparticles, followed by treatment with an alter- pression, while Cdx1 and Cdx2 were up-regulated nating magnetic field. No apparent reduction was in GI-mixed and I types along with the expression seen in tumour growth with the administration of of MUC2 and villin. In the N type, both gastric OML-encased magnetic nanoparticles or and intestinal transcription factors were suppressed. OML-encased 5-FU alone. Thus, we have estab- Immunohistochemistry confirmed expression of lished the use of intraperitoneal macrophages as a Sox2 in MUC5AC positive lesions and Cdx2 local- novel drug-delivery system for the control of cancer ization together with MUC2. A stomach adeno-

16 metastatic to milky spots. testine specific. Of 86 gastric cancers, sucrase and CA1 expression was observed in 12 (14.0%) and *1 Department of Applied Biochemistry and The Insti- only 2 (2.3%) cases, respectively, associated with tute of Glycotechnology, Tokai University, 1117 Ki- other intestinal markers such as villin and MUC2. takaname, Hiratsuka-shi, Kanagawa, 259-1292 Japan In the sucrase cases, expression appeared inde- *2 EP, pendent of the stage. However, CA1 expression *3 Department of Biological Chemistry, College of Bio- was observed only in two advanced cases. No as- science and Biotechnology, Chubu University sociation was observed between colonic and small-intestinal phenotypes and lymph node metas- 5. Colonic and small-intestinal pheno- tasis and postoperative survival in the advanced types in gastric cancers: relationships gastric cancer cases with intestinal phenotypic ex- with clinicopathologic findings pression. Cdx2 appeared linked to upregulation of both CA1 and sucrase. In conclusion, our data Mizoshita, T., Tsukamoto, T., Tanaka, H., Otsuka, T,*1. suggest that colonic phenotype occur rarely in gas- Hirano, N.*1, and Tatematsu, M. tric carcinogenesis. Colonic and small intestinal The clinicopathologic significance of colonic phenotypes appear with expression of several intes- and small-intestinal phenotypes has hitherto retinal phenotypic markers under the control of Cdx2 mained unclear in gastric cancers. In the present and presumably other related transcription factors. study, we therefore examined 86 gastric carcinomas histologically and phenotypically using several *1 Dept. of Internal Medicine I, Toho University School phenotypic markers, including colon specific car- of Medicine, Tokyo, Japan. bonic anhydrase 1 (CA1) and sucrase as small in-

From left to right Front row: Dr. Y. Kondo, Dr. Y. Sekido, Dr. H. Osada, and Dr. T. Fukui, Second row: Dr. T. Yokoyama, Dr. N. Sato, Dr. T Taniguchi, and Mr. Y. Tatematsu, Inset: Dr. Y. Goto.

18 Division of Molecular Oncology ______

Takashi Takahashi, M.D., Ph.D., Chief (until June 2004) Yoshitaka Sekido M.D., Ph.D., Chief (as of April 2005) Hirotaka Osada, M.D., Ph.D., Section Head Kiyoshi Yanagisawa, M.D., Ph.D., Senior Researcher (until June 2004) Yutaka Kondo, M.D., Ph.D., Senior Researcher (as of July 2005) Shuta Tomida, Ph.D., Researcher (until March 2005) Yoshio Tatematsu, B.S., Research Assistant Tomoko Harano, B.S., Research Assistant (until March 2004) Postdoctoral Fellows Toshiyuki Takeuchi, Ph.D. (until June 2004) Research Resident Yoji Hayashita, M.D., (until March 2005) Visiting Trainees Hidemasa Nagai, M.D., Nagoya University School of Medicine (until June 2004) Yoko Karube, M.D., Dokkyou University School of Medicine (until June 2004) Junichi Takamizawa, M.D., Nagoya University School of Medicine (until June 2004) Ken Maeno, M.D., Nagoya City University School of Medicine (until June 2004) Hideki Yamada, M.D., Nagoya University School of Medicine (until June 2004) Hisaaki Tanaka, M.D., Okayama University School of Medicine (until June 2004) Nobuyoshi Sugito, M.D., Nagoya City University School of Medicine (until Sep. 2004) Hiromichi Ebi, M.D., Nagoya City University School of Medicine (until March 2005) Takayuki Fukui, M.D., Nagoya University School of Medicine (as of April 2005) Toshihiko Yokoyama, M.D., Nagoya University School of Medicine (as of April 2005) Naohito Sato, M.D., Nagoya University School of Medicine (as of April 2005) Tetsuo Taniguchi, M.D., Nagoya University School of Medicine (as of May 2005) Yasuhiro Goto, M.D., Nagoya University School of Medicine (as of Aug. 2005)

General Summary Our goal is to determine the genetic lesions giving rise to human solid cancers and use this information for prevention, diagnosis, and treatment of these diseases. Currently, we are focusing on lung cancer, malignant mesothelioma, colon cancer, and hepatomas. Our studies also provide opportunities to dissect biochemical and pathological pathways of malignant phenotypes, related to dysregulated cell growth, differentiation, invasion, and metastasis. Human cancers arise because of genetic mutations in protooncogenes and tumor suppressor genes, and our approach is to focus on candidate genes, systematically analyses of molecular biochemical pathways, and apply microarray analysis of global gene expression and comparative genomic hybridization technique for identification of chromosomal abnormalities. Epigenetic changes due to DNA methylation and histone modification are also important mechanisms of inactivation of tumor suppressor genes. We also func- tionally analyze candidate genes by transfecting wild type copies into human cancer cells and testing for their ability to suppress malignancy in vitro and in vivo as well as characterizing their protein products biochemically. Alternatively, we inactivate their expression using RNA interference (RNAi) in either tumor or normal cells and then study their phenotype. Understanding the functions of genes which are mutated and the signaling pathways disrupted is necessary to provide a firm foundation for a translational research approach to human malignancies, from bench to bed- side.

19 1. EGFR mutation is frequently detected in there were no tumors that had two or more EGFR, non-small cell lung cancer with occa- HER2, and KRAS mutations. Our study further sional genetic events of second muta- demonstrated that a double genetic event of EGFR tion or amplification can occasionally occur in lung cancer, thus providing new clues to the understanding of the involve- Yokoyama, T., Kondo, M.*1, Goto, Y., Fukui, T., Sato, ment of EGFR signaling cascades in the pathogene- N., Taniguchi, T., Kondo, Y., Osada, H., Yokoi, K. *2, T. sis of NSCLCs. Shimokata, K.*1, and Sekido, Y.

Non-small cell lung cancer (NSCLC) is one of *1 Department of Respiratory Medicine, Nagoya Uni- the leading causes of death from neoplasia in Japan versity Graduate School of Medicine and western countries. As chemotherapy can only *2 Division of General Thoracic Surgery, Nagoya Uni- marginally prolong survival among patients with versity Graduate School of Medicine advanced disease, molecular target therapy appears the most promising clinical option. The epidermal 2. The ASH1 gene is a specific therapeutic growth factor receptor (EGFR), one of the ERBB target for lung cancers with neuroen- family of receptors, is a particularly promising tar- docrine features get, because of its frequent overexpression (range, Osada, H., Tatematsu, Y., Yatabe, Y.*1, Horio, Y.*2, and 40-80%) in NSCLCs and relation to a poor progno- Takahashi, Ta.*3 sis. Small molecule tyrosine kinase inhibitors (TKIs) of EGFR such as gefitinib and erlotinib have Lung cancers with neuroendocrine (NE) fea- been shown to have anti-tumor activity and several tures are usually aggressive, although the underly- clinical studies have revealed that Japanese, female, ing molecular mechanisms largely remain to be de- never-smoking patients with adenocarcinoma have termined. The basic-helix-loop-helix protein, a high response rate to gefitinib. We have analyzed achaete-scute complex-like 1 (ASCL1)/ mutation and/or amplification of EGFR, HER2, and achaete-scute homolog 1 (ASH1), is expressed in KRAS, which are involved in EGFR signaling cas- normal fetal pulmonary NE cells and lung cancers cades, among resected primary NSCLCs from with NE elements, and is suggested to be involved Japanese patients and determined whether there is a in lung carcinogenesis. We have shown inhibition correlation with clinicopathological factors. of ASH1 expression by plasmid-based RNAi to EGFR mutations were found in 102 (29%) of a total significantly suppress growth of lung cancer cells of 349 tumors, and 7 tumors had two missense mu- with ASH1 expression through G2/M-cell cycle ar- tations. Reverse transcriptase-polymerase chain rest and accumulation of sub-G1 populations, pos- reaction of EGFR and subsequent subcloning sibly linked to cleavage of caspase-9 and caspase-7. analyses idemonstrated the double mutations in the However, lung cancer cell lines without ASH1 ex- same allele, since we found both mutations in most pression and an immortalized normal BEAS-2B mutation-positive cDNA clones. Furthermore, in bronchial epithelial cells were not affected. The 202 NSCLCs analyzed by Southern blotting, 11 RNAi-resistant mutant ASH1 clearly induced res- (5.4%) exhibited amplification of EGFR, with 8 cue from G2/M-arrest, suggesting a target-specific tumors containing an EGFR mutation. Sequence effect of RNAi. An ASH1-RNAi adenovirus was analysis detected only weak or no signals of the also established, and shown to significantly inhibit wild-type allele in the 8 tumors, strongly suggesting not only in vitro cell proliferation but also in vivo the mutated allele to be selectively amplified. xenograft growth of ASH1-positive NCI-H460 cells. These findings indicate that a dual genetic change Elevated levels of apoptosis were also observed in of EGFR can occur in the same allele either with a NCI-H460 xenografts with the possible second-hit mutation or amplification, ASH1-RNAi-adenovirus. Our present studies which may imply a more selective growth advan- therefore suggest that ASH1 plays a crucial role in tage in cancer cells. At the same time, HER2 mu- lung cancer development and may be an effective tation and amplification were found in 6 (1.7%) of therapeutic target in lung cancers with NE features. 349 tumors and 3 (1.5%) of 202 tumors, respectively, and KRAS mutations in 21 (6%) of 349 tu- *1 Department of Pathology and Molecular Diagnostics, mors. Mutations of the EGFR and HER2 genes Aichi Cancer Center Hospital were more frequently found in female, never or *2 Department of Thoracic Oncology, Aichi Cancer light smoking patients with adenocarcinomas, and Center Hospital

20 *3 Division of Molecular Carcinogenesis, Center for sive form, SCLC, and that the C13orf25 gene may Neurological Diseases and Cancer, Nagoya Univer- well be serving as a vehicle in this regard. sity Graduate School of Medicine. *1 Department of Oncological Science (Surgery II), 3. A polycistronic miRNA cluster, miR-17- Oita University Faculty of Medicine *2 92, is overexpressed in human lung Division of Molecular Carcinogenesis, Center for cancers and enhances cell proliferation Neurological Diseases and Cancer, Nagoya Univer- sity Graduate School of Medicine, Hayashita, Y.*1, Osada, H., Tatematsu, Y., Yamada, H.*2, *3 Department of Pathology and Molecular Diagnostics, Yanagisawa, K.*2, Tomida, S.*2, Yatabe, Y.*3, Kawahara, Aichi Cancer Center Hospital. K.*1, Sekido, Y., and Takahashi Ta.*2.

MicroRNAs (miRNAs) are small, non-coding 4. Establishment and characterization of RNAs, thought to be involved in physiological and malignant pleural mesothelioma cell developmental processes by negatively regulating lines from Japanese patients expression of target genes. We have previously Fukui, T., Taniguchi, T., Usami, N.*1, Yokoyama, T., reported frequent down-regulation of the let-7 Hida, T.*2, and Sekido, Y. miRNA family in lung cancers and in the present study assessed alteration in a panel of 19 lung can- Malignant mesothelioma (MM) is an aggressive cer cell lines. Using Northern blot and quantita- neoplasm arising from mesothelial cells, most often tive RT-PCR analyses, we found for the first time occurring in the pleural cavity as malignant pleural that the miR-17-92 cluster, which comprises six mesothelioma (MPM). Owing to the long latency miRNAs (miR-17, miR-18a, miR-19a, miR-20a, period and the widespread use of asbestos fibers for miR-19b-1, miR-92-1) and resides in intron 3 of the many years, the incidence of MPM is projected to C13orf25 gene at 13q31.3, is markedly overex- rise sharply worldwide in the next two decades. In pressed in lung cancers, especially examples with Japan, 500 patients with MM died in 1995, and the small cell lung cancer (SCLC) histology. The number increased to approximately 900 patients in paralogous clusters, miR-106a-92 (Xq26.2) or 2003. MM has been demonstrated to be resistant miR-106b-25 (7q22), did not show significant alteration of their expression in lung cancers. Southern blot analysis revealed the presence of increased gene copy numbers of the miRNA cluster in a fraction of lung cancer cell lines with overexpression. In addition, we were able to show predominant localization of C13orf25 transcripts within the nuclei, introduction of the expression construct of the miR-17-92 cluster, but not the putative open reading frame of C13orf25, enhancing lung cancer cell growth. These findings clearly suggest that marked overexpression of the miR-17-92 cluster with occasional gene amplification may play a role in the development of lung cancers, especially in their most aggres-

21 to all of conventional therapy regimens including characteristics of MPM, showing epithelial and chemotherapy, radiotherapy and surgery, and the sarcomatous phenotypes in cell culture. Mutation prognosis of patients remains very poor. However, and expression analyses demonstrated that the tu- the discrepancy between the rising incidence of mor suppressor gene of NF2, which is known to be MM and the lack of success of new more effective one of the most frequently mutated in MPMs, is therapeutic strategies seems to be related at least in mutated in ACC-MESO-1. We detected homo- part to inadequate knowledge of the biological zygous deletion of p16 INK4A / p14 ARF in all four properties of this tumor. It is hoped that a better MPM cell lines. We also analyzed genetic altera- understanding of MM biology may provide the ra- tions of six other MPM cell lines and confirmed tionale for new therapeutic strategies. In this re- frequent mutations of NF2 and p16 INK4A / p14 ARF. gard, the development of tumor cell lines has been To characterize biological differences between an important tool in setting up suitable in vitro Y-MESO-8A and -8D, we performed cDNA mi- models for studying the biological properties of croarray analysis and detected genes that are dif- many tumors and to assess tumor sensitivity to ferentially expressed in these two cell lines. Thus, various drugs or biological response modifiers. our new MPM cell lines seem to be useful as mod- However, as opposed to lung cancer for example, els for studying various aspects of the biology of where several hundred cell lines have been estab- human MPM as well as materials for development lished, only a relatively small number of MPM cell of future therapy. lines are available. Furthermore, only a few cell lines have been established from Japanese patients *1 Division of General Thoracic Surgery, Nagoya Uni- with MPM. versity School of Medicine We therefore have established four MPM cell *2 Department of Thoracic Oncology, Aichi Cancer lines, ACC-MESO-1, ACC-MESO-4, Y-MESO-8A, Center Hospital and Y-MESO-8D from Japanese patients, with the latter two from the same patient with biphasic-like

First row (from left to right): Dr. Y. Hosokawa, Ms. Y. Kasugai, Dr. M. Sato, Ms. H. Suzuki, Dr. S. Tsuzuki. Second row (from left to right): Dr. Y. Kameoka, Dr. N. Hukuhara, Dr. Y. Nakashima, Dr. A. Oshiro. Third row (from left to right): Dr. H. Tagawa, Dr. R. Suzuki, Dr. M. Nakagawa, Ms. S. Sato, Mr. S. Karnan. Insets (from left to right): Dr. X. Zhang, Dr. K. Mayama, Dr. M. Suguro-Katayama.

23 Division of Molecular Medicine ______

Masao Seto, MD., PhD. Chief Yoshitaka Hosokawa, M.D., Ph.D. Section Chief (Until March, 2006) Shinobu Tsuzuki, M.D., Ph.D. Section Chief Ritsuro Suzuki, M.D., Ph.D. Senior Researcher (Until December, 2005) Hiroyuki Tagawa, M.D., Ph.D. Senior Researcher Hiroko Suzuki, B.P. Senior Research Assistant Yumiko Kasugai, B.S. Research Assistant Visiting Trainees Karnan Sivasundaram, Graduate School of Medical Sciences, Nagoya City University Miyuki Suguro-Katayama, MD. The Second Department of Internal Medicine, Mie University School of Medi- cine (Until August, 2004) Koh Mayama, MD. The Third Department of Internal Medicine, Hirosaki University School of Medicine (Until September, 2004) Yoshihiro Kameoka, MD. The Third Department of Internal Medicine, Akita University School of Medicine (Until August, 2005) Masao Nakagawa, MD. The Third Department of Internal Medicine, Hokkaido University School of Medicine (Until September, 2005) Xiaohua Zhang, M.D. Department of Pathology, Yan’an University School of Medicine (Until January, 2004) Yasuhiro Nakashima, MD. Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University Aya Oshiro, MD. Second Department of Internal Medicine, University Hospital, University of the Ryukyus Noriko Fukuhara, MD. Department of Rheumatology and Hematology, Tohoku University School of Medicine

General Summary Research in this laboratory is aimed at generating a better understanding of the genetic and molecular bases of human cancer, with eventual application of the acquired knowledge in the field of medical oncology. Our work has been mainly focused on hematologic malignancies, in cooperation with physicians of the Department of Hematology and Cell Therapy Aichi Cancer Center Hos- pital (Chief, Dr. Yasuo Morishima). Research on hematologic malignancies have several advantages for exploration of the molecular bases of neoplasia. Chromosomal abnormalities have been analyzed by a large number of researchers and the observed strong association between specific chromosome changes and specific hematopoietic tumors provides direct evidence that the resultant gene alterations play a pivotal role in the disease development. Over the last two years, we have concentrated attention on the following issues. The function of API2-MALT1 linked to mucosa-associated lymphoid tissue lymphomas is now being studied to identify target genes. An array-CGH (comparative genomic hybridization) that contains 2300 BAC clones on a slide glass that can scan the whole genome every 1.4 MB in average was applied to various kinds of hematopoietic malignancy. Differences in the genome profiles between NK cell leukemias and T/NK cell lymphomas were thereby clarified. Target genes in the 6p21 region amplicon were found to be either CCND3 or BYSL or both. TEL-AML1 leukemias were also analyzed by the array CGH approach and found to also have a characteristic genome profile. In vivo oncogenic function of the TEL-AML1 chimeric gene in combination with new oncogenes is being analyzed in a bone marrow transplantation system.

1. Anti-apoptotic action of the API2- mal translocation in the mucosa-associated lym- MALT1 fusion protein involved in phoid tissue (MALT) type lymphoma, which results t(11;18)(q21;q21) MALT lymphomas in fusion transcripts of APoptosis Inhibitor 2 (API2), also known as c-IAP2, and Mucosa-Associated Hosokawa, Y., Suzuki, H. and Seto, M. Lymphoid Tissue translocation gene 1 (MALT1). t(11;18)(q21;q21) is a characteristic chromoso- Although API2-MALT1 has been shown to enforce

24 activation of NF-κB signaling, the transcriptional TEL-AML1 (ETV6-RUNX1) fusion gene is the target genes of this fusion protein remain to be commonest structural chromosome change in identified. childhood cancer and is exclusively associated with Our analyses of API2-MALT transfectants have the common, B cell progenitor subset of acute suggested that one target gene may be the apoptotic lymphoblastic leukemias (ALLs). Evidence sug- inhibitor API2 gene. Luciferase reporter assays gests that the translocation usually occurs in utero with deletion and mutational constructs of the API2 during foetal haematopoiesis and most probably promoter and electrophoretic mobility shift assays constitutes an initiating or “first hit” mutation (EMSA) established that API2-MALT1 induces which is necessary but itself insufficient for the de- transcriptional activation of the API2 gene through velopment of overt, clinical leukemia. two NF-κB binding elements. Moreover, super- In our search for additional “second hit” muta- shift experiments indicated that these elements are tions that could be linked to leukemia development, recognized by the NF-κB p50/p65 heterodimer. we applied a genome-wide array-CGH technique to Taken together, our results strongly indicate that 24 TEL-AML1 leukemia samples and two cell lines API2-MALT1 possesses a novel mechanism of and found that at least three chromosomal imbal- self-activation by up-regulating its own expression ances were involved in all samples. The results in t(11;18)(q21;q21)-carrying MALT lymphomas, suggest that, in addition to TEL previously reported highlighting a positive feedback-loop pathway re- as lacking, genes involved in cell cycle regulation sulting in sustained NF-κB activation. (p16INK4a/ARF, BTG1), p53 pathways and apop- We also demonstrated that API2-MALT1 pos- tosis are also often deleted. sesses an anti-apoptotic effect, in part through direct To delineate the roles of deregulated cell cy- interactions with apoptotic regulators. These cle/p53/apoptosis pathways in leukemia develop- findings therefore have led us to hypothesize that ment, we have developed retroviral vectors to ex- the anti-apoptotic effect of API2-MALT1 may be press TEL-AML1 along with a second gene or mediated by its interaction with apoptotic regulators siRNAs. We are currently investigating the coop- on the one hand as well as by NF-κB-mediated erative roles of the “first” and “second” hits for upregulation of apoptotic inhibitor genes on the leukemia development in mice. other. We have also established that BCL10 and MALT1 shuttle between the nucleus and cytoplasm, and that MALT1 can regulate the subcellular loca- 3. Cloning of a translocation partner of tion of BCL10. t(1;14)(p33;q32) in diffuse large B-cell It is hoped that further studies will facilitate de- lymphoma velopment of therapeutic drugs that specifically in- Suzuki, R., Nakamura, S. *1 and Seto, M. hibit the antigen receptor signaling pathway. Several oncogenes, such as cyclin D1, BCL2, CARMA1 and MALT1 represent promising mo- BCL6, and c-Myc, are activated by the immu- lecular targets, because knock-out mice for these noglobulin heavy chain (IgH) gene in B-cell lym- molecules exhibit defects that are exclusively re- phomas. We have therefore focused on the translo- stricted to this pathway. Recent studies also indi- cation partner of the IgH gene in t(1;14)(p33;q32) cated that self-oligomerization of API2-MALT1 fu- identified as a novel translocation in diffuse large sion proteins results in deregulated ubiquitin ligase B-cell lymphoma. High molecular weight DNA was activity of MALT1, thereby leading to NF-κB acti- extracted from a frozen sample, and long distance vation. Thus, the development of specific inhibi- inverse PCR technique was employed for cloning. tors of API2-MALT1 ubiquitin ligase would be also The cloned genomic sequence matched the chro- beneficial for the treatment of MALT lymphoma. mosome 1p34 sequence on database search. No Such drugs would be expected to inhibit, with con- known gene existed around the breakpoint on 1p34, trollable side-effects, inappropriate growth and ex- but the expression of one EST was highly activated pansion of lymphoma clones. in the tumor sample. Performance of 5’- and

3’-RACE allowed a non-coding RNA spanning 2. Molecular pathways leading to t(12; 21) more than 20kb to be cloned as the partner. The TEL-AML1 associated leukemias presence of micro RNA was further investigated, Tsuzuki, S. and Seto, M. but we could not identify any known or novel micro RNA. Although the mechanism and the significance The t(12; 21) translocation which generates the of expression of non-coding RNA have yet to be

25 fully elucidated, lymphomagenesis by NK-cell leukemia and extranodal t(1;14)(p33;q32) apparently features a novel and NK/T-cell lymphoma, nasal type previously unrecognized mechanism, which war- Nakashima, Y., Tagawa, H., Suzuki, R., Karnan, S., rants further investigation. Karube, K.*1, Ohshima, K. *1, Muta, K. *2, Nawata, H. *2, Morishima, Y. *3, Nakamura, S. *4 and Seto, M. *1 Department of Pathology and Molecular Diagnostics, Aichi Cancer Center Hospital, Aichi, Japan Natural killer (NK) cell lymphomas/leukemias are highly aggressive lymphoid malignancies, but little is known about their genomic alterations, and 4. Comparison of genome profiles for thus there is an urgent need for identification and identification of distinct subgroups of analysis of NK cell lymphomas/leukemias. Re- diffuse large B-cell lymphoma cently, we developed our own array-based com- Tagawa, H., Suguro-Katayama, M., Tsuzuki, S., parative genomic hybridization (array CGH) with 1 Morishima, Y. * and Seto, M. an average resolution of 1.3 Mb. We performed an Diffuse large B-cell lymphoma (DLBCL) com- array CGH analysis for 27 NK-cell lym- prise distinct molecularly subgroups such as acti- phoma/leukemia cases that were classified into two vated B-cell-like (ABC) and germinal center disease groups based on the World Health Organi- B-cell-like (GCB) forms. We previously reported zation Classification (10 aggressive NK-cell leuke- that CD5+ and CD5-CD10+ DLBCL constitute mia cases and 17 extranodal NK/T-cell [NK/T] clinically relevant subgroups. To determine whether lymphomas, nasal type). We identified the differ- these two subgroups are related to ABC and GCB ences in the genomic alteration patterns of the two DLBCL, we analyzed the genomic imbalance of 99 groups. The recurrent regions characteristic of the cases (36 CD5+, 19 CD5-CD10+ and 44 aggressive NK-cell leukemia group compared with CD5-CD10-) using array-CGH. Some 46 of these those of the extranodal NK/T lymphoma, nasal type cases (22 CD5+, 7 CD5-CD10+ and 17 CD5-CD10-) group, were gain of 1q and loss of 7p15.1-p22.3 and were subsequently subjected to gene expression 17p13.1. In particular, gain of 1q23.1-q24.2 (P= profiling, resulting in their division into 28 ABC 0.041) and 1q31.3-q44 (P= 0.003-0.047), and loss (19 CD5+ and 9 CD5-CD10-) and 18 GCB (3 CD5+, of 7p15.1-p22.3 (P= 0.012 - 0.041) and 17p13.1 (P= 7 CD5-CD10+ and 8 CD5-CD10-) types. A com- 0.012) occurred significantly more frequently in the parison of genome profiles of distinct subgroups of former than in the latter group. Recurrent regions DLBCL demonstrated that: i) the ABC DLBCL is characteristic of the extranodal NK/T lymphoma, characterized by gain of 3q, 18q and 19q and loss of nasal-type group, compared with those of the other 6q and 9p21, and the GCB DLBCL by gain of 1q, group were gain of 2q, and loss of 6q16.1-q27, 2p, 7q and 12q; ii) the genomic imbalances charac- 11q22.3-q23.3, 5p14.1-p14.3, 5q34-q35.3, 1p36.23- teristic of the CD5+ and CD5-CD10+ groups are p36.33, 2p16.1-p16.3, 4q12, and 4q31.3-q32.1. Our similar to those of the ABC and GCB types, respec- results can be expected to provide further insights tively. These findings suggest that CD5+ and into the genetic basis of lymphomagenesis and the CD5-CD10+ subgroups are included, respectively, clinicopathologic features of NK-cell lympho- in the ABC and GCB types. Finally, on searching mas/leukemias. for genomic imbalances that affect patients’ prog- *1 nosis, we found that 9p21 loss (p16INK4a locus) Department of Pathology, School of Medicine, Fu- marks the most aggressive type of DLBCL. kuoka University, Fukuoka, Japan *2 Department of Medicine and Bioregulatory Science, *1 Department of Hematology and Cell Therapy, Aichi Graduate School of Medical Sciences, Kyushu Uni- Cancer Center Hospital, Nagoya, Aichi. versity, Fukuoka, Japan *3 Department of Hematology and Chemotherapy, Aichi Cancer Center Hospital, Aichi, Japan 5. Genome-wide array-based comparative *4 Department of Pathology and Molecular Diagnostics, genomic hybridization of natural killer Aichi Cancer Center Hospital, Aichi, Japan cell lymphoma/leukemia: different ge-

nomic alteration patterns of aggressive

The 11th Aichi Cancer Center International Symposium “Forefront of Cancer Prevention Strategy in Asia,” held on February 5, 2005.

From left to right First row: Dr. Y. Akatsuka, Dr. K. Kuzushima, Dr. K. Tsujimura, Ms. Y. Matsudaira Second row: Ms. T. Tsuboi, Dr. H. Torikai, Ms. F. Ando, Dr. Demachi-Okamura, Ms. K. Nishida, Dr. Y. Ito, Dr. T. Kawase, Ms. Y. Nakao, Dr. S. Morishima Insets: Dr. R. Ohta, Dr. M. Miyazaki, Dr. Y. Okanami, Dr. H. Miyauchi, Ms. K. Watanabe, Dr. S. Shi- mato, Ms. M. Nakayama

28 Division of Immunology ______

Kiyotaka Kuzushima, M.D. Chief Yoshiki Akatsuka, M.D. Section Head Kunio Tsujimura, M.D. Section Head Yoshinori Ito, M.D. Senior Researcher Ayako Demachi-Okamura, Ph.D. Research Resident Rieko Ohta, Ph.D. Research Resident (as of August 2004, until June 2005) Yasue Matsudaira, B.S. Senior Research Assistant Keiko Nishida, B.P. Senior Research Assistant Tomiko Tsuboi, Semi-regular Employee Yumi Nakao, Semi-regular Employee Fumiyo Ando, Semi-regular Employee (as of August 2004) Michiyo Nakayama, Semi-regular Employee (as of February 2004) Visiting Scientists Yuichi Obata, Ph.D. Department of Biological Systems, RIKEN BioResource Center Kazuhiro Yoshikawa, B.M.T., M.D. Second Department of Pathology, Aichi Medical University Visiting Trainees Mikinori Miyazaki, M.D. Department of Internal Medicine and Molecular Science, Nagoya City University Graduate School of Medical Sciences Hiroki Torikai, M.D. Third Department of Internal Medicine, National Defense Medical College Yuko Okanami, M.D. First Department of Surgery, Mie University School of Medicine (until March 2005) Satoko Morishima, M.D. Cancer Genetics, Nagoya University Graduate School of Medicine Takakazu Kawase, M.D. Cancer Genetics, Nagoya University Graduate School of Medicine Hidemasa Miyauchi, M.D. Blood Purification Center, JISHOKAI Health System, Inc. Kazue Watanabe, M.S. Medical & Biological Laboratories Co., Ltd. (as of June 2005) Shinji Shimato, M.D. Department of Neurosurgery, Nagoya University Graduate School of Medicine (as of September 2005)

General Summary The object of our research is to characterize and understand T lymphocyte responses to antigens expressed on cancer and virus-infected cells. The major projects undertaken over the past two years are summarized below. In the field of human immunology, four projects are continuing with the focus on epitope identification and fine characterization of processing of the epitopes recognized by cancer- and virus-targeting cytotoxic T lymphocyte (CTLs). Firstly, an HLA-A*2402-restricted CTL epitope was determined in the epithelial cell adhesion molecule, which is expressed in almost all carcinomas. Secondly, precise roles of interferon (IFN)-γ inducible immunoproteasome-associated molecules in generation of a CTL epitope derived from Epstein-Barr virus latent membrane protein 2A were elucidated. Unequivocal involvement of the immunoproteasome subunit low molecular weight protein 7 and 2 and the proteasome activator 28 α subunit was confirmed by means of RNA interference gene silencing. Thirdly, a novel HLA-A*3303-restricted male-specific minor histocompatibility antigen encoded by an unconventional open reading frame of human TMSB4Y gene was identified. Fourthly, an HLA-A*2402-restricted epitope recognized by CTL targeting the HPV-16 protein was characterized. Interestingly, combined treatment with proteasome inhibitors like bortezomib and IFN-γ dramatically augmented CTL-mediated lysis of SiHa cells carrying HPV. Thus, this may provide a novel approach for CTL-based immunotherapy in cervical cancer patients. To understand the roles of specific immunity to tumor progression in vivo, we have been conducting animal experiments employing the mouse thymus-leukemia antigen (TL) as a model. Both cellular and humoral immune responses to chemical carcinogen-induced lymphomas expressing the TL were extensively studied using T3b-TL transgenic and parental (B6 and C3H) strains.

29 1. Identification of an epitope from the processing and presentation of the peptide in a lung epithelial cell adhesion molecule elicit- cancer cell line could be confirmed by cold target ing HLA-A*2402-restricted cytotoxic T inhibition assays. The CTL clone was also lytic to lymphocyte responses normal bronchial epithelial cells but to a lesser extent at low effector : target ratios. All these data Tajima, Ko.*1, Demachi-Okamura, A., Ito, Y., Nishida, suggest that peptide-specific CTL responses may K., Akatsuka, Y., Tsujimura, K., Kuwano, H.*1, play roles in both anti-cancer and autoimmune reac- Mitsudomi, T.*2, Takahashi, To.*3 and Kuzushima, K. tions. Our peptide should prove useful to study Since the epithelial cell adhesion molecule anti-Ep-CAM CTL responses among populations (Ep-CAM) is expressed in almost all carcinomas possessing HLA-A*2402. and human leukocyte antigen (HLA)-A*2402 is the most common allele in many ethnic groups, includ- *1 Department of Surgery I, Gunma University Faculty ing Japanese, identification of peptide sequences of Medicine which elicit HLA-A*2402-restricted Ep-CAM- *2 Department of Pulmonary Medicine, Aichi Cancer specific CTL responses should facilitate specific Center Hospital immunotherapy for various histological types of *3 President, Aichi Cancer Center carcinomas. We have focused on identifying an epitope through the following steps: (a) com- 2. Three immunoproteasome-associated puter-based epitope prediction from the amino acid subunits cooperatively generate a CTL sequence of Ep-CAM; (b) MHC stabilization assays epitope of the EBV-LMP2A by over- to determine the affinity of predicted peptides with coming specific structures resistant to + HLA-A*2402 molecules; (c) stimulation of CD8 T epitope liberation cells with peptide-pulsed dendritic cells; (d) testing Ito, Y., Kondo, E.*1, Demachi-Okamura, A., Akatsuka, CTL specificity by enzyme-linked immunospot Y., Tsujimura, K., Tanimoto, M.*1, Morishima, Y.*2, (ELISPOT) assays, CTL assays and MHC/peptide- Takahashi, To.*3 and Kuzushima, K. tetramer staining (Fig. 1). Peripheral CD8+ T cells of 4 of 5 healthy donors after 3 rounds of stimula- Precise roles of interferon-γ inducible immuno- tion with the peptide Ep-CAM173-181 (RYQLDPKFI) proteasome-associated molecules in generation of were found to secrete IFN-γ in ELISPOT assays. A cytotoxic T lymphocyte (CTL) epitopes have yet to CTL clone specific for one peptide efficiently lysed be fully elucidated. We describe here a unique Ep-CAM-expressing cancer cell lines in an epitope derived from the Epstein-Barr virus (EBV) HLA-A*2402-restricted fashion and endogenous latent membrane protein 2A (LMP2A) presented by

Fig. 1. Tetramer staining of Ep-CAM peptide-specific poly- and monoclonal CTLs. A, Polyclonal CD8+ T cells after stimulation 4 times with an Ep-CAM derived peptide

RYQLDPKFI (Ep173), were stained with FITC-labeled anti-CD8 antibodies and PE-labeled HLA-A24-tetramers incorporating

Ep173 or a control peptide RYLRDQQLL

(ENV584), derived from HIV envelope protein. Percentages of tetramer-positive cells among total CD8+ T cells are shown at the upper right in each case. B, An

Ep173-specific CTL clone was stained as described above. The percentages of tetramer-positive cells among total CD8+ T cells are shown at the upper right in each case.

30 HLA-A*2402 molecules. Generation of the epitope, nor H (H-Y) antigens encoded by the Y chromo- designated LMP2A222-230, from the full length pro- some are elicited frequently. All previously identi- tein requires the immunoproteasome subunit low fied H-Y antigens have been found to be encoded molecular weight protein 7 (ip-LMP7) and the pro- by conventional open reading frames. teasome activator 28 α subunit, and is accelerated We here isolated an HLA-A*3303-restricted by ip-LMP2, as revealed by gene expression ex- CD8+ CTL clone termed 1B6 from a male patient periments using an LMP2A222-230-specific CTL after HSCT from his HLA-identical sister. By ex- clone as a responder in enzyme-linked immunospot amining the lytic patterns of EBV-transformed cell assays. Unequivocal involvement of all three com- lines carrying various Y chromosome terminal de- ponents was confirmed by means of RNA interfer- letions by clone 1B6, a narrow region containing 5 ence gene silencing. Interestingly, the candidate genes was localized. Further correlation LMP2A222-230 epitope could be efficiently generated studies between the lytic patterns of the cell lines by from incomplete EBV-LMP2A fragments produced 1B6 and mRNA expression levels from genes of the by puromycin treatment, or gene-engineered short- mapped region in these cell lines successfully iden- ened EBV-LMP2A lacking some of its hydrophobic tified a gene encoding the minor H antigen, domains. In addition, epitope generation was in- TMSB4Y. Minigene transfection and epitope recon- creased by a single amino acid substitution from stitution assays allowed determination of an 11-mer leucine to alanine immediately flanking the peptide, EVLLRPGLHFR, designated C-terminus, this being predicted by a TMSB4Y/A33. Surprisingly, its first amino acid web-accessible program to increase the cleavage was located 405 bp upstream of the TMSB4Y ini- strength. Taken together, the data indicate that gen- tiation codon (Fig. 2A). Analysis of the precursor eration of LMP2A222-230 is not only influenced by frequency of CTL specific for recipient minor H extrinsic influences such as immunoproteasomes, antigens in post-HSCT peripheral blood T cells us- but also by intrinsic factors such as the length of the ing limiting dilution methods revealed that a sig- EBV-LMP2A protein and proteasomal cleavage nificant fraction of the total donor CTL response in strength at specific positions in the source antigen. this patient was directed against the TMSB4Y epitope (30% and 10% at days 50 and 146 post-HCT, *1 Department of Internal Medicine II, Okayama Univer- respectively, Fig. 2B and C). Tetramer analysis sity Graduate School of Medicine and Dentistry continued to detect TMSB4Y/A33-specific CD8+ T *2 Department of Hematology and Cell Therapy, Aichi cells at least up to 700 days post-HSCT, indicating Cancer Center Hospital the TMSB4Y/A33 epitope to indeed be immuno- *3 President, Aichi Cancer Center genic. This finding underscores the in vivo immunological relevance of minor H antigen derived from unconventional open reading frame products. 3. A novel HLA-A*3303-restricted minor Because 1B6 preferentially lysed target cells of histocompatibility antigen encoded by hematopoiesis origin, despite relatively ubiquitous an unconventional open reading frame expression of TMSB4Y in various tissues, this mi- of the human TMSB4Y gene nor H antigen might be involved in GVL rather than Torikai, H.*1, Akatsuka, Y., Miyazaki, M.*2, Warren, GVHD. E.H.*3, Oba, T.*4, Tsujimura, K., Motoyoshi, K.*1, Morishima, Y.*5, Kodera, Y.*4, Kuzushima, K. and *1 Third Department of Internal Medicine, National De- Takahashi, To.*6 fense Medical College *2 Department of Internal Medicine and Molecular Sci- Minor histocompatibility (H) antigens are MHC ence, Nagoya City University Graduate School of (HLA in human)-associated peptides originating Medical Sciences mainly from polymorphisms in the genome that *3 Division of Immunology, Fred Hutchinson Cancer trigger T cell responses between MHC identical al- Research Center logeneic individuals. Graft-versus-host disease and *4 Department of Hematology, Japanese Red Cross Na- graft-versus-leukemia/lymphoma (GVL) effects in goya First Hospital hematopoietic stem cell transplant recipients are *5 Department of Hematology and Cell Therapy, Aichi initiated by donor T cell recognition of minor H an- Cancer Center Hospital tigens on recipient cells. In the setting of fe- *6 President, Aichi Cancer Center male-to-male hemopoietic stem cell transplantation

(HSCT), T cell responses against male-specific mi-

Fig. 2. A, Genomic organization of TMSB4Y and the relationship with its mRNA. E1 and E2 indicate exons 1 and 2, respectively. The conventional ORF is indicated below the mRNA as cORF and the location of identified epitope is shown below the 5'UTR of TMSB4Y cDNA. B and C, The proportions of CTL precursors specific for the identified TMSB4Y peptide among total CTLp against the recipient minor H antigens was quantitated using a standard limiting dilution assay. The CTLp frequencies against recipient PHA blasts (open circles) or donor PHA blasts pulsed with TMSB4Y peptide (closed triangles) or unpulsed (open triangles) were calculated with L-Calc software. 4. Combination of bortezomib and inter- HLA-A*2402 and HPV-16 E6, but could not rec- feron-γ induces presentation of a newly ognize the SiHa cervical cancer cell line positive for identified HLA-A24-restricted human HPV-16 and HLA-A*2402. However, SiHa cells papillomavirus type 16 E6-specific cy- transduced with the E6-E7 genes did become sus- totoxic T cell epitope ceptible to lysis, suggesting insufficient generation of antigenic peptides in the cell line. Interestingly, Morishima, S.*1, Akatsuka, Y., Nawa, A.*2, Kondo, E.*3, combined treatment with proteasome inhibitors, Kiyono, T.*4, Torikai, H.*5, Nakanishi, T.*2, Ito, Y., bortezomib or epoxomicin, and interferon (IFN)-γ, Tsujimura, K., Iwata, K.*6, Ito, K.*7, Kodera, Y.*8, fully restored CTL-mediated lysis of SiHa cells. Morishima, Y.*9, Kuzushima, K. and Takahashi, To.*10 Furthermore, pretreatment of three of four other Around 50% of cervical cancers are associated cervical cancer cell lines expressing HPV-16 E6 with human papillomavirus type 16 (HPV-16) and and HLA-A*2402 with bortezomib and IFN-γ in- since the HPV-16 E6 and E7 oncoproteins are con- duced cytokine production by specific CTLs. Un- stitutively expressed in tumor cells, they are attrac- expectedly, HPV-16 E6 levels were found to be de- tive targets for cytotoxic T lymphocyte creased with the combined treatment. These data (CTL)-mediated immunotherapy. Nevertheless, suggest that impaired presentation of the E649-57 only a limited number of HPV-16 E6 epitopes have epitope is most likely due to peptide destruction by so far been identified. Using reverse immunological proteasomes. Thus, combined treatment with a pro- methods, we have generated a CTL clone against teasome inhibitor and IFN-γ may further provide a the HPV-16 E649-57 epitope restricted by novel approach for CTL-based immunotherapy in HLA-A*2402, which is the most common allele in cervical cancer patients. Japan and worldwide. The CTL clone was found *1 Cancer Genetics, Nagoya University Graduate School able to lyse 293T cells transduced with of Medicine

32 *2 Department of Gynecology and Oncology, Aichi lymphocytes in these strains. When Cancer Center Hospital N-butyl-N-nitrosourea (NBU), a chemical carcino- *3 Department of Internal Medicine II, Okayama Univer- gen, was administered orally to B6 and C3H strains, sity Graduate School of Medicine and Dentistry lymphoma development was slower than in T3b-TL *4 Virology Division, National Cancer Center Research gene-transduced counterpart strains expressing TL Institute ubiquitously as self-antigens, suggesting that *5 Third Department of Internal Medicine, National De- anti-TL immunity may play a protective role. In fense Medical College addition, the development of lymphomas was *6 Iwata Hospital slightly slower in C3H than in B6, in accordance *7 Sakae Obstetrics and Gynecology Clinic with the results of skin graft experiments indicating *8 Department of Hematology, Japanese Red Cross Na- both cellular and humoral immunities against TL to goya First Hospital be stronger in the C3H strain. The interesting find- *9 Department of Hematology and Cell Therapy, Aichi ing that B lymphomas derived from a T3b-TL Cancer Center Hospital transgenic strain (C3H background) expressing a *10 President, Aichi Cancer Center very high level of TL were rejected in C3H, but not in H-2Kb transgenic mice (C3H background), raises 5. Immunity against the mouse thy- the possibility that TL-specific effector T cell mus-leukemia antigen (TL) protects populations are eliminated and/or anergized to a b against development of lymphomas in- certain extent by interacting with H-2K molecules. duced by a chemical carcinogenic 1 agent, N-butyl-N-nitrosourea * Department of Biological Systems, RIKEN BioRe- source Center Tsujimura, K., Obata, Y.*1, Matsudaira, Y., Taguchi, *2 Division of Molecular Pathology, Aichi Cancer Center O.*2, Nishida, K., Okanami, Y.*3, Akatsuka, Y., Research Institute Kuzushima, K. and Takahashi, To.*4 *3 First Department of Surgery, Mie University School Mouse thymus-leukemia antigens (TL) are of Medicine aberrantly expressed on T lymphomas in C57BL/6 *4 President, Aichi Cancer Center (B6) and C3H/He (C3H) mice, but not on normal T

From left to right First row: Dr. N. Shitara, Dr. S. Iwahori, Mr. K. Ohtake, and Dr. T. Tsurumi. Second row: Mr. J. Ohtsuka, Dr. A. Kudoh, Mr. L. Zhang, Dr. T. Daikoku, Dr. H. Isomura, Dr. S. Nakasu, and Mr. Y. Nishikawa.

34 Division of Virology ______

Tatsuya Tsurumi, M.D. Chief Shou Nakasu, PhD. Senior Researcher Hiroki Isomura, M.D. Senior Researcher Tohru Daikoku, Ph.D. Senior Researcher (until March 2006) Yutaka Sugaya, PhD. Research Resident (until March 2005) Noriko Shirata, PhD. Research Resident (until March 2006) Satoko Iwahori, PhD. Research Resident (as of April 2005) Yasuhiro Nishikawa. Research Assistant Ayumi Kudoh, Ph.D. Research Fellow of the Japanese Society for the Promotion of Science Visiting Trainees Zhan lumen. M.S. Department of Virology, Nagoya University Graduate School of Medicine

General Summary Approximately 15% of all human cancers have a viral etiology, but only six viruses have actually been implicated in their development. Among these the Ep- stein-Barr virus (EBV) is the object of our own studies. EBV is a ubiquitous gamma herpesvirus associated with several malignant diseases, including Burkitt’s lymphoma, nasopharyngeal lymphoma, a subset of Hodgkin’s lymphomas, some gastric cancers, and B cell lymphomas in immunosuppressed patients. Our research aims are to elucidate the molecular mechanisms of viral proliferation and oncogenesis of EBV as part of the world-wide effort to combat virus-infected cancers. During the period 2004-2005, our research interest was concentrated on the following issues: 1) Architecture of EBV replication compartments; 2) EBNA1 binding to oriP during EBV latent and lytic rep- lications; 3)Activation of DNA damage checkpoint signal transduction elicited by EBV genome replication; 4)Interaction between p53 and EBV-encoded immediate-early protein BZLF1; 5) Analysis of the human cytomegalovirus major immediate-early enhancer and promoter.

1. Epstein-Barr virus lytic replication elic- concentrated in replication compartments where it its ATM checkpoint signal transduction physically interacts with viral BZLF1 protein. De- while providing an S-phase-like cellular spite the activation of ATM checkpoint signaling, environment we found p53-downstream signaling to be blocked, with rather high S-phase CDK activity associated Kudoh, A. and Tsurumi, T. with progression of lytic infection. Therefore, al- When exposed to genotoxic stress, eukaryotic though host cells activate ATM checkpoint signal- cells demonstrate a DNA damage response with ing with response to the lytic viral DNA synthesis, delay or arrest of cell-cycle progression, providing we conclude that the virus can skillfully evade this time for DNA repair. We have established that in- host checkpoint security system and actively production of the Epstein-Barr virus (EBV) lytic promote an S-phase-like environment advantageous for gram elicits a cellular DNA damage response, with viral lytic replication. activation of the ataxia telangiectasia-mutated (ATM) signal transduction pathway. Activation of 2. Dynamics of Epstein-Barr virus EBNA1 the ATM-Rad3-related (ATR) replication check- protein binding to the viral genome and point pathway, in contrast, is minimal. The DNA the architecture of replication com- damage sensor Mre11-Rad50-Nbs1 (MRN) compartments formed during lytic replica- plex and phosphorylated ATM are recruited and re- tion tained in viral replication compartments, recogniz- Daikoku, T. and Tsurumi, T. ing newly synthesized viral DNAs as abnormal DNA structures. Phosphorylated p53 also becomes Epstein-Barr virus (EBV), a lymphotropic hu-

35 man herpesvirus, possesses two life styles; latent viral DNA. Inhibition of viral DNA replication with and lytic infections. During latent infection, EBV phosphonoacetic acid, a viral DNA Pol inhibitor, genomes are maintained as double-stranded DNA eliminated the DNA-bound form of the BMRF1 episomes, and replicated once per cell cycle during protein, although the protein was sufficiently ex- the S phase, following the rules of chromosome pressed in the cells. These observations together replication. The viral latent gene product, EBNA1, with the finding that almost all abundantly ex- binds directly to the latent replication origin, oriP, pressed BMRF1 proteins exist in the DNA-bound as a homodimer but lacks any activity predicted to form suggest that BMRF1 proteins not only act at be required for replication initiation. Lytic replica- viral replication forks as Pol processive factors but tion differs from the latent amplification state in also are widely distribute on newly replicated EBV that multiple rounds of replication are initiated genomic DNA. In contrast, the BALF5 Pol catalytic within the lytic replication origin, oriLyt, and the protein, the BALF2 single-stranded-DNA binding replication process has a greater dependence on protein, and the BBLF2/3 protein, a component of seven EBV-encoded lytic replication proteins. A the helicase-primase complex, were colocalized as low level of EBNA1 transcripts is still produced distinct dots distributed within replication com- during the viral productive cycle. Understanding partments, representing viral replication factories. protein-DNA interactions in vivo at origins of DNA Whereas cellular replication factories are con- replication throughout the cell cycle and lytic repli- structed based on nonchromatin nuclear structures cation may shed further insight on EBNA1 func- and nuclear matrix, Such viral replication factories tions on replication control. could be easily solubilized by DNase I treatment. Our focus has therefore been on EBNA1 bind- Thus, compared with cellular DNA replication, ing to the EBV genome-wide mapping through la- EBV lytic DNA replication factories appear simpler tent and lytic replication by ChIP assay. We thereby so that construction of the replication domain is fa- found that EBNA1 binds to the oriP region of the cilitated. EBV genome throughout the cell cycle. Even after induction of lytic replication EBNA1 still continues 3. Activation of ATM DNA damage check- to bind to oriP. From confocal microscopy analyses, point signal transduction elicited by lytic DNA replication occurs at discrete sites in nu- herpes simplex virus infection clei, called replication compartments, where viral Shirata,N. and Tsurumi, T. replication proteins are clustered. During lytic replication, no chromosomal DNA replication occurs. Eukaryotic cells are equipped with machinery to In latent infection EBNA1 could be shown to be monitor and repair damaged DNA. Herpes simplex distributed broadly in nuclei as fine punctate dots virus (HSV) DNA replication occurs at discrete with weak, diffuse stainig. After induction of lytic sites in nuclei, considering a replication compart- replication, EBNA1 was redistributed and clustered ment, where viral replication proteins cluster and to replication compartments with bright granular synthesize large amounts of viral DNA. In our re- spots. However, the spots of EBNA1 did not appear cent studies, HSV infection was found to elicit a to completely coincide with BrdU staining or viral cellular DNA damage response, with activation of replication proteins, but rather were located side by the ATM signal transduction pathway, as observed side with the viral replication proteins and viral by autophosphorylation of ATM and phosphoryla- progeny DNA. tion of multiple downstream targets including Nbs1, In our study we also performed comprehensive Chk2, and p53, while infection with a analyses of the architecture of the replication com- UV-inactivated virus or with a replication-defective partments were EBV productive DNA replication virus did not. Activated ATM and the DNA damage occurs. The BZLF1 oriLyt binding proteins showed sensor MRN complex composed of Mre11, Rad50, a fine, diffuse pattern of distribution throughout the and Nbs1 were recruited and retained at sites of vi- nuclei at immediate-early stages of induction and ral DNA replication, probably due to recognition of then became associated with the replicating EBV newly synthesized viral DNAs as abnormal DNA genome in the replication compartments during structures. These events were not observed in lytic infection. The BMRF1 polymerase (Pol) proc- ATM-deficient cells, indicating an essivity factor showed a homogenous, not dot-like, ATM-dependence for the underlying processes. In distribution in the replication compartments, which Nbs1-deficient cells, HSV infection induced an completely coincided with the newly synthesized ATM DNA damage response which was delayed,

36 suggesting a functional MRN complex requirement sedimented at a monomer position with sucrose for efficient ATM activation. However, ATM si- gradient centrifugation under low salt conditions lencing had no effect on viral replication in 293T (0.1 M NaCl), indicating existence as a free protein. cells. Our data open up the interesting question of Starting from 2 l culture, the protein composition of how the virus is able to complete its replication, the final fraction (0.2 ml) proved hardly visible by despite host cells activation of ATM checkpoint CBB staining of SDS page separation. Therefore a signaling in response to the HSV infection. larger scale purification procedure will be required for the further purification. 4. Purification of the product of the Ep- stein-Barr virus BZLF1 gene 5. Two Sp1/Sp3 binding sites in the major Nakasu, S. and Tsurumi, T. immediate-early proximal enhancer of human cytomegalovirus genes are The product of the BZLF1 gene (pBZLF1) of necessary for transcriptional activation the Epstein-Barr virus (EBV) is a nuclear protein and viral replication which is an activator of the lytic cycle in cells la- Isomura, H. and Tsurumi, T. tently infected with EBV. pBZLF1 has been suggested to play the role as a DNA binding protein HCMV is reactivated under immunosuppressive specific for the viral lytic origin of DNA replication conditions as with other herpesviruses and progress (ori lyt). of medical intensive care has made this virus an In order to understand the contribution of important pathogen that causes pneumonitis, hepa- pBZLF1 to induction of the lytic cycle, we focused titis, retinitis, and gastrointestinal diseases in im- on purification of the protein and characterization munocompromized individuals. The molecular of its biochemical features. We first tried to purify mechanisms of HCMV replication, especially in pBZLF1 from insect cells infected with baculovi- vivo, remain unclear. But it is clear that the major ruses which overproduced pBZLF1. But the par- immediate early (IE) genes, IE1 and IE2, are ex- tially purified protein tended to form aggregates and pressed without de novo protein synthesis like other was eluted with a wide range of salt concentrations auxiliary IE genes. Furthermore, IE2 is essential. from ion exchange chromatography columns. The Thus, the cis-elements of the MIE genes are thought results suggested an abnormal conformation of the to regulate the efficiency for viral replication. pBZLF1 produced in the insect cells so that we next We previously reported that replication of re- focused on pBZLF1 from B95-8 cells, a cell line combinant HCMV was less efficient when the en- latently infected with EBV in which the lytic cycle hancer was replaced by murine components, even can be infected. The pBZLF1 could be extracted though the human and murine enhancers are posi- from the induced cells with high salt buffer (0.6 to 1 tional homologues. Since both have similar ef- M NaCl) and purified more than 100 fold by hy- fects on the promoter in transient transfection as- drophobic, DEAE sephacel, phospho (P) cellulose says, the results of transfection experiments clearly and hydroxyapatite chromatographies. Most do not always coincide with expectation of the viral pBZLF1 appeared to be complexed with other pro- genome. teins in low salt buffer (< 0.12 M NaCl) and was In the present study our goal was to assess the eluted from columns with high salt buffer (1.5 M effect of the enhancer and the roles of its individual NaCl). The pBZLF1 was further purified with gel repeat elements on IE transcription and viral repli- filtration and sucrose gradient centrifugation under cation in the context of HCMV infection. high salt conditions (1.5 M NaCl) in which it be- HCMV has a genome over 200 kbp in length haved as a monomer. Though a pBZLF1 sample and therefore it is very difficult to make recombi- bound to P cellulose in 0.12 M NaCl buffer was ap- nant viruses on which specific target genes are mu- plied to gel filtration and sucrose gradient centrifu- tated. However, we recently have developed new gation, some proportion flowed through P cellulose genetic engineering in bacteria using homologous equilibrated with 0.12 M NaCl buffer after these recombination. The HCMV genome was cloned procedures (FT fraction). The pBZLF1 in the FT into the bacterial artificial chromosome (BAC) in fraction bound to DEAE sephacel stronger than be- 1999. This has allowed us to construct recombi- fore gel filtration and sucrose gradient centrifuga- nant infectious human CMV DNA containing BAC tion. In addition to these changes in ion exchange in vivo in E.coli., whereby mutagenesis can be ma- chromatographies, the pBZLF1 in the FT fraction nipulated. using double-stranded linear fragments

37 amplified by PCR. This is possible because the for viral replication, and a minimal requirement for bacteriophage-encoded recombination proteins exo, viral growth is the SpI binding site in the proximal beta, and gam efficiently recombine sequences with enhancer between –39 and –67 relative to the tran- homologies as short as 35 to 40 bases in the ab- scription start site of +1, newly discovered by sence of the E.coli RecA protein. We have con- EMSA and supershift assay with SpI antibodies in structed recombinant HCMVs featuring either en- the present study. Moreover, infection of NF-kB tire or partial deletion of the enhancer using this dominant negative cells with the recombinant vi- new rapid and reliable technology, and compared ruses revealed that two NF-kB and one CREB or the growth of recombinant and parental viruses. ATF binding sites between –-39 and –173 facilitate As a result, we found that different from the transcription from the MIE promoter. murine CMV case, the HCMV enhancer is essential

Figure Left panel; Accumulation of DNA damage checkpoint proteins such as ATM and MNR complex at sites of viral replication during EBV lytic infection. Right panel; Model for DNA damage signaling induced by EBV lytic replication.

From left to right First row: Dr. Keiko Tamiya-Koizumi, Dr. Osamu Taguchi, Dr. Reiji Kannagi, Dr. Akiko Kanamori and Ms. Yoshiko Goto. Second row: Ms. Naoko Kimura, Ms. Keiko Miyazaki, Dr. Lim Keh-Ti, Ms. Sasako Eguchi, Ms. Mineko Izawa, Ms. Akiko Nishioka, Mr. Hirokazu Yagi, Ms. Sachiko Kondo, Dr. Guo-Yun Chen, Mr. Jun Yin and Dr. Mamoru Kyogashima.

39 Division of Molecular Pathology ______

Reiji Kannagi, M.D., D.M.Sc., Chief Osamu Taguchi, D.M.Sc., Section Head Mamoru Kyogashima, M.D., D.M.Sc., Senior Researcher (as of April, 2004) Akiko Kanamori, Ph.D., Researcher (until March, 2005), Senior Researcher (as of April 2005) Takaaki Yaomura, M.D., Research Resident (until March, 2005) Masaru Ueda, M.D., Research Resident (until March, 2005) Mineko Izawa, B.A., Research Assistant Yoshiko Goto, D.V.M.S., Research Assistant Sasako Eguchi, Semi-regular Employee Akiko Nishioka, M.T., Semi-regular Employee Visiting Scientists Hiroshi Ikeda, M.D., D.M.Sc., Aichi Medical University Guo-Yun Chen, M.D., D.M.Sc., Japan Science and Technology Agency Keiko Miyazaki, M.T., Japan Science and Technology Agency Lim Keh-Ti, Ph.D., National Institute of Biomedical Innovation Ayako Hashimoto, B.A., Japan Science and Technology Agency (until October, 2005) Fathy Mohamed Mohamed El-Fasakhany, M.D., D.M.Sc., JSPS (until September, 2005) Takashi Murate, M.D., D.M.Sc., Nagoya University School of Health Sciences (as of April, 2004) Keiko Tamiya-Koizumi, Ph.D., Nagoya University School of Health Sciences (as of April, 2004) Visiting Trainees Naoko Kimura, B.P., Nagoya City University Jun Yin, M.T. Nagoya University School of Bioagricultural Sciences Tetsufumi Koike, M.D., Fukushima University School of Medicine Atsushi Akutagawa, M.D., Nagoya University School of Medicine (as of April 2004) Kazumi Hagiwara, M.T., Nagoya University School of Health Sciences (as of April 2004) Sayaka Sobue, M.T., Nagoya University School of Health Sciences (as of April 2005) Hirokazu Yagi, M.P. Nagoya City University (as of April 2005) Sachiko Kondo, M.E. Nagoya City University (as of April 2005) Masaru Ueda, M.D., Kyoto Prefectural University of Medicine (as of April 2005) Akinari Watanabe, M.D., Fukushima University School of Medicine (until March, 2005) Takaaki Hattori, M.D., Tokyo Medical University (until March, 2004)

General Summary Cell adhesion molecules called selectins and their specific carbohydrate ligands, namely sialyl Lewis X and sialyl Lewis A, are involved in hematogenous metastasis of cancers. Expression of sialyl Lewis X and sialyl Lewis A is markedly enhanced on cancer cells compared to normal epithelial cells. During the period 2004-2005, we have succeeded in elucidating the mechanism involved in the cancer-associated induction of sialyl Lewis X and sialyl Lewis A expression in human cancers. We found that normal epithelial cells express carbohydrate determinants that are more complex than the parent ligands. Good examples of such complex determinants are sialyl 6-sulfo Lewis X or disialyl Lewis A, which have additional modifications to sialyl Lewis X or sialyl Lewis A, respectively. Upon malignant transformation, intracellular synthesis of such complex carbohydrate determinants become partially impaired because of transcriptional suppression of some of the genes involved (we call this as "incomplete synthesis"), which results in accumulation of sialyl Lewis X and sialyl Lewis A with relatively simple structures in tumors at early stages. In tumor nests within locally advanced tumors, hypoxia-resistant cancer cells are clonally selected because of the hypoxic environment. Such cancer cells have a strong and sustained expression of a

40 transcription factor, hypoxia inducible factor-1α (HIF-1α), which induces expression of various genes which enable cancer cells to cope with or adapt to hypoxia. Included are several genes involved in sialyl Lewis X or sialyl Lewis A synthesis, and our studies have indicated that this further enhances expression of these carbohydrate determinants in hypoxia-resistant cancer cells, which selectively grow in advanced stages of cancers undergoing distant hematogenous metastasis. Selectin-mediated cell adhesion mediates homing of lymphocytes in healthy individuals, and during the period 2004-2005, we have clarified, using knock out mice, that the sialyl 6-sulfo Lewis X determinant contributes as a specific ligand for L-selectin in this normal homing. In these two years we have also concentrated attention on the role of regulatory T-cells in immune responses against self-antigens.

1. Mechanism of loss of disialyl Lewis A gesting that histone deacetylation and/or DNA me- and induction of sialyl Lewis A expres- thylation may be involved in the silencing of the sion in early stage human cancers. gene in cancers.

Miyazaki, K. Ohmori, K.*1, Izawa, M., Koike, T. and *1 Department of Laboratory Medicine, Kyoto Univer- Kannagi, R. sity, School of Medicine. Expression of sialyl Lewis A, a ligand for E-selectin which mediates hematogenous metastasis, is known to be increased in cancers of digestive organs. In contrast, disialyl Lewis A, which has an extra sialic acid attached at the C6-position of the penultimate GlcNAc in sialyl Lewis A, is expressed preferentially on the surface of nonmalignant colonic epithelial cells, and is significantly down-regulated on malignant transformation. In- troduction of the gene for a 2-6 sialyltransferase responsible for disialyl Lewis A synthesis into colon cancer cells in our laboratory resulted in a marked increase in disialyl Lewis A expression and corresponding decrease in sialyl Lewis A expression. This was accompanied by complete loss of E-selectin binding of the cells. In contrast, transfected cells acquired significant binding activity to Siglec-7/p75/AIRM-1, an inhibitory receptor expressed on lymphoid cells (Fig. 1). These findings Fig. 1. Confocal microscopic analyses of epithelial indicate that the shift in carbohydrate determinants cells expressing disialyl Lewis A and leukocytes from a disialyl Lewis A-dominant status to a sialyl expressing Siglec-7 in human colonic mucosa. Lewis A-dominant status on malignant transforma- The distribution of Siglec-7, as detected by tion has a dual functional consequence: loss of polyclonal anti-Siglec-7 antibody, is shown in normal cell-cell recognition between mucosal green, and that of the disialyl Lewis A determi- epithelial cells and lymphoid cells on the one hand nant in red. Left panel, low magnification of and gain of E-selectin binding activity on the other. nonmalignant colonic mucosa, indicating adhesion of Siglec-7-expressing lymphoid cells to The transcription of a gene encoding the 2-6 sialyl- colonic epithelial cells (arrows). Right panels, transferase was found to be markedly higher magnification micrographs showing ad- down-regulated in cancer cells compared with hesion of Siglec-7-expressing lymphoid cells nonmalignant epithelial cells, in line with the de- (green) to nonmalignant colonic epithelial cells creased expression of disialyl Lewis A and in- expressing the disialyl Lewis A determinant creased expression of sialyl Lewis A. Treatment (red) through pseudopodia-like extensions (ar- of cancer cells with butyrate or 5-azacytidine rowheads). Bars, 10 µm. (Cancer Res., 64: strongly induced disialyl Lewis A expression, sug- 4498-4505, 2004)

2. Tumor hypoxia augments sialyl Lewis X The transcription of genes for fucosyltransferase and sialyl Lewis A expression in lo- VII (FUT7), sialyltransferase ST3Gal-I (ST3O), and cally-advanced human cancers. UDP-galactose transporter-1 (UGT1), which are all Koike, T., Kimura, N., Miyazaki, K., Chen, G. Y., Yin, J., known to be involved in the synthesis of the carbo- Kojima, T.*1, Takematsu, H.*2, and Kannagi, R. hydrate ligands for E-selectin, was significantly induced in cancer cells by hypoxic culture. In addi- Cancer cells undergo distinct metabolic changes tion, remarkable induction was detected for the to cope with their hypoxic environment, at least genes encoding syndecan-4 (SDC4) and α5-integrin partly through the action of transcriptional factors (ITGA5) (Fig. 2), cell-adhesion molecules involved called hypoxia-inducible factor-α and related in enhanced adhesion of cancer cells to fibronectin. molecules (HIFs). We have investigated gene ex- Transcriptional induction by hypoxia was repro- pression in human colon cancer cells cultured under duced in luciferase-reporter assays for these genes, hypoxic conditions with special reference to which were significantly suppressed by cell-adhesion molecules and carbohydrate determi- co-transfection of a dominant-negative form of HIF. nants having cell-adhesive activity using These results indicate that the metabolic shifts of DNA-microarray and RT-PCR techniques. Hy- cancer cells partly mediated by HIFs significantly poxic culture of colon cancer cells induced a enhance their adhesion to vascular endothelial cells, marked increase in expression of selectin ligands, through both selectin- and integrin-mediated path- the sialyl Lewis X and sialyl Lewis A determinants ways, and suggest that this enhancement further fa- at the cell surface, which led to a definite increase cilitates hematogenous metastasis and tumor an- in cancer cell adhesion to endothelial E-selectin. giogenesis.

*1 Department of Medical Technology, Nagoya Univer- sity School of Health Sciences. *2 Supra-Biomolecular System Research Group, RIKEN Frontier Research System.

3. Study on L-selectin ligand mediated homing of naïve T-lymphocytes using gene-disrupted mice. Izawa, M., Kimura, N. Uchimura, K.*1, Ohmori, K*2, Muramatsu, T.*3, Rosen, S.D.*1 and Kannagi, R. Selectin-mediated cell adhesion is involved in the routine homing of lymphocytes. Two kinds of carbohydrate ligands for selectin have so far been noted in humans, one is conventional sialyl Lewis X, and the other is sulfated sialyl Lewis X as repre- sented by sialyl 6-sulfo Lewis X. Conventional sialyl Lewis X is preferentially involved in the recruitment of leukocytes in inflammation, while sialyl 6-sulfo Lewis X primarily mediates routine homing of leukocytes. The sialyl 6-sulfo Lewis X determinant is expressed on the high endothelial Fig. 2. Hypoxia-induced syndecan-4 and α5- venules (HEVs) of peripheral lymph nodes and integrin expression. Signals including Peyer's patches, where it mediates syndecan-4 (left panel) and α5-integrin L-selectin-dependent homing of lymphocytes, and (right panel) in the DNA microarray are shown (white arrows). Cy3-dUTP is synthesized through the sequential action of (normoxia, red) or Cy5-dUTP (hypoxia, 6-O-sulfotransferase and fucosyltransferase. Two 1% O2 for 24 h: green) was incorporated 6-O-sulfotransferase isoenzymes are proposed to be into cDNA prepared from a human colon involved in the synthesis of sialyl 6-sulfo Lewis X cancer cell line, SW480. (Proc. Natl. in HEV, GlcNAc6ST-1 and -2. Mice with dis- Acad. Sci. U.S.A., 101: 8132-8137, 2004) rupted genes for GlcNAc6ST-1 or -2 are known to

have impaired lymphocyte homing, indicating both *1 Department of Anatomy, Program in Immunology, enzymes to be involved in the synthesis of sialyl Cardiovascular Research Institute, University of Cali- 6-sulfo Lewis X. Expression of sialyl 6-sulfo fornia. Lewis X in HEV of Peyer's patches and mesenteric *2 Department of Laboratory Medicine, Kyoto Univer- lymph nodes is decreased in GlcNAc6ST-1 KO sity, School of Medicine. mice, while its expression in peripheral lymph node *3 Department of Biochemistry, Nagoya University is decreased with GlcNAc6ST-2 KO. In line with Graduate School of Medicine. this, lymphocyte homing to Peyer's patches is partially impaired in the GlcNAc6ST-1 KO case, and 4. ATRA-induced apoptosis in the human that to peripheral lymph node is partially reduced in neuroblastoma cell line, SH-SY5Y, is GlcNAc6ST-2 KO mice. In double-KO mice for accompanied by alteration of ceramide both GlcNAc6ST-1 and -2, expression of sialyl species: Appearance of ceramide con- 6-sulfo Lewis X in HEV of all lymphoid tissues, taining hydroxy fatty acids. including Peyer's patches, peripheral and mesen- Hagiwara, K., Sobue, S., Kyogashima, M., teric lymph nodes, is almost completely abrogated Tamiya-Koizumi, K., Tadano-Aritomi, K.*1, Hara, A.*2, (Fig. 3), and marked reduction of lymphocyte Aoyama, T.*2, Murate, T. and Kannagi, R. homing is observed. These results confirmed our previous finding in humans that sialyl 6-sulfo Lewis All-trans retinoic acid (ATRA) and its related X is the major ligand for L-selectin in lymphocyte compounds are regarded as promising reagents for homing, and indicate that both GlcNAc6ST-1 and neuroblastoma treatment, but mechanisms of action -2 are involved in its synthesis. remain to be clarified. The purpose of our present study was to elucidate the molecular background to

Fig. 3. Sialyl 6-sulfo Lewis X expression in HEVs of wild-type and GlcNAc6ST-deficient lymphoid organs. Cry- ostat-cut sections of peripheral lymph nodes, mesenteric lymph nodes and Peyers' patches from wild-type and GlcNAc6ST-deficient mice were preincubated with sialidase. Sections were then stained with monoclonal antibody AG223, which recognizes 6-sulfo Lewis X. Arrows indicate HEVs lacking sialyl 6-sulfo Lewis X expression. Methyl green was used to counterstain the sections. Bars, 40µm. (Nat. Immunol., 6: 1105-1113, 2005)

ATRA-induced cell death in the human neuroblas- (d18:2) were readily detected. Finally, in addition toma cell line, SH-SY5Y. ATRA rapidly caused to SM4s, sulfatide SM3 (sulfated lactosylceramide) cell death in fetal calf serum-depleted culture, as and SM2 (sulfated gangliotriaosylceramide) were confirmed to be apoptosis by identification of DNA clearly present in renal tubule cells. The major ladder formation and inhibitory effects of a cas- SM4s was composed of ceramides possessing d18:1 pase-3 inhibitor. We focused our attention on with C22 hydroxy fatty acids (C22:0h), C23:0h, and sphingolipid metabolism as a signaling pathway of C24:0h, whereas the major SM3/SM2 forms were apoptosis. Metabolic labeling of sphingolipids composed of ceramides possessing t18:0 with C22 with [14C]-serine revealed that ATRA increased ra- normal fatty acids (C22:0), C23:0, C24:0. Namely, dioactivity in the ceramide fraction and decreased in these two series of sulfatides, either fatty acids or that of sphingomyelin. When we measured in vi- sphingoids were hydroxylated, and chain lengths of tro activities of sphingomyelinase, ceramidase, ser- these components were exactly the same, conse- ine palmitoyltransferase, and synthases of sphin- quently resulting in a similar polarity of ceramide gomyelin, ceramide and glycolipid, which may be moieties. These results demonstrate diversity of responsible for the increased of radioactive cera- sulfatide molecular species, not only with respect to mide, none of the enzymes examined showed dis- sugar- but also to ceramide moieties, which is tinct ATRA-dependent changes. However, analy- probably important for specific effective functions sis of the molecular species of radioactive ceramide in particular microenvironments, such as lipid by thin-layer chromatography detected ceramide membrane microdomains. containing hydroxy fatty acid in an ATRA-dependent manner. These results suggest *1 Department of Metabolic Regulation, Institute on that hydroxy fatty acid in ceramide may play a role Aging and Adaptation, Shinshu University Graduate in ATRA-induced apoptosis. Further detailed School of Medicine. analysis of ceramide species unique to ATRA-induced apoptosis is now in progress using 6. Expression cloning of a cDNA encoding mass spectrometry. sialic acid cyclase, which generates cyclic sialic acid containing glycoconju- *1 Department of Biochemistry, Teikyo University gates, and characterization of the pro- School of Medicine. duced enzyme. *2 Department of Metabolic Regulation, Institute on Kanamori, A., Yamaguchi, M.*1, Ishida, H.*1, Kiso, M.*1 Aging and Adaptation, Shinshu University Graduate and Kannagi, R. School of Medicine. Recently we have identified a metabolic path- 5. Rapid demonstration of diversity of way leading to modification of sialic acid so that a sulfatide molecular species from bio- novel ring form named "cyclic sialic acid", lacking logical materials by MALDI-TOF MS its carboxyl group, is generated. By the expression cloning method, we focused on isolating a Kyogashima, M., Tamiya-Koizumi, K., Goto, Y., Hara, cDNA encoding a "sialic acid cyclase", which could A.*1, Aoyama, T.*1 and Kannagi, R. catalyse this process. For this purpose, HEK-293T By combining the partition method for enrich- cells stably expressing sialyl 6-sulfo Lewis X were ment of sulfatides without any chromatographic used as the recipient cells. Using the G159 mono- procedures and a preparation method for lysosulfa- clonal antibody recognizing cyclic sialyl 6-sulfo tides, we have succeeded in analyzing sulfated gly- Lewis X antigen as a probe, a cDNA clone (indi- cosphingolipids from biological materials by cated as clone A) causing expression of a G159 an- MALDI-TOF MS within a single day. We found tigen was isolated. Interestingly, in order for the SM4s (galactosylsulfatide) to be composed of dif- G159 antigen to be produced in recipient cells, an- ferent species. While the exact composition de- other two cDNA clones needed to be co-transfected, pended on the source material, SM4s always con- whose encoded proteins appeared to contribute to tained hydroxy fatty acids to various degrees. In the intracellular localization of the clone A product. addition to the common sphingoid 4-sphingenine On the other hand, expression of clone A protein in (d18:1), uncommon/unusual sphingoids phyto- a HUT-102 derived mutant cell line lacking G159 sphingosine (t18:0), 4-eicosasphinganine (d20:0), antigen caused the expression of the latter. The 4-eicosasphingenine (d20:1), and sphingadienine expression levels of sialyl 6-sulfo Lewis X antigen

found to be in inverse proportion to those of cyclic autoantibodies to the retina were evaluated by indi- sialyl 6-sulfo Lewis X detected as the G159 antigen. rect immunofluorescence, and total IgG2a levels in Sialic acid cyclase activity of the recombinant clone sera were assessed by ELISA. The pathogenic abili- A protein could be assayed by an ELISA method ties of the splenic T cells were examined after and analysis of the effects of elements which influ- adoptive transfer to syngeneic nu/nu mice, and the ence its level is now in progress. Our conclusion proliferation responses and the secretion of granu- is that the enzyme protein encoded by clone A in- locyte-macrophage colony-stimulating factor deed possesses sialic acid cyclase activity. (GM-CSF), IFN-gamma, and IL-10 on stimulation by retinal self-antigens were also determined. *1 Department of Applied Bioorganic Chemistry, Gifu Autoantibodies to the retinal photoreceptor cell University, School of Agriculture. layer were detected in Tr-depleted mice, and the titers correlated well with the grades of inflammatory lesions. Splenic CD4(+) T cells of 7. Immune responses to retinal self-anti- Tr-depleted mice induced uveoretinitis in the re- + + gens in CD25 CD4 regulatory T-cell- cipients by adoptive transfer and exhibited prolif- depleted mice. erative responses and secretion of IFN-gamma, but Takeuchi, M.*1, Keino, H.*1, Kezuka, T.*1, Usui, M.*1 and not IL-10, by in vitro stimulation with S-Ag and in- Taguchi, O. terphotoreceptor retinoid-binding protein (IRBP). Moreover, the total IgG2a level in serum was Prior work has shown that autoimmune uveo- markedly and significantly augmented in retinitis develops spontaneously in CD25(+)CD4(+) Tr-depleted mice. The results suggest that in regulatory T-cell-depleted mice (Tr-depleted mice). Tr-depleted mice in which uveoretinitis develops, In our recent studies, the generation of autoanti- S-Ag and IRBP-specific T cells are spontaneously bodies and autoreactive T-cells specific to retinal sensitized and shifted to a Th1-phenotype. These antigens was examined in Tr-depleted mice with sensitized T cells may account for the development uveoretinitis, and the pathogenic and immunogenic of autoimmune uveoretinitis. abilities of the autoreactive T cells were evaluated.

Tr-depletion was achieved in (C57BL/6 x A/J) *1 Department of Ophthalmology, Tokyo Medical Uni- F1 (B6A) mice by thymectomy on day 3 of life fol- versity, Tokyo. lowed by intraperitoneal injection of an anti-CD25 monoclonal antibody. At 6 months of age,

From left to right First row: Ms. Y. Takada, Dr. A. Kawajiri, Ms. T. Yuhara, Ms. Y. Hayashi. Second row: Mr. T. Oguri, Dr. P. Zou, Mr. T. Siromizu, Ms. N. Saito, Ms. M. Nishizawa. Third row: Dr. M. Inagaki, Dr. A. Inoko, Dr. H. Goto, Mr. T. Yamaguchi, Dr. I. Izawa. Insets: Dr. K. Nagata, Dr. M. Sugimoto, Dr. T. Yokoyama, Dr. N. Hanai, Mr. M. Inoue.

46 Division of Biochemistry ______

Masaki Inagaki, M.D. Chief Koh-ichi Nagata, M.D. Section Head (until March, 2004) Ichiro Izawa, M.D. Senior Researcher Hidemasa Goto, M.D. Senior Researcher (as of April, 2004) Akihito Inoko, M.D. Researcher Miwako Nishizawa, B.P. Senior Research Assistant (until March, 2004) Yuko Hayashi, Ph.D. Research Assistant (as of April, 2004) Noriko Saito, B.M.T. Research Assistant (until March, 2004) Tomoya Yokoyama, M.D. Research Resident (until June, 2005) Zou Peng, M.D. Research Resident (as of April, 2005) Postdoctoral Fellows Nariko Arimura, Ph.D. (as of April, 2004, until August, 2005) Visiting Trainees Aie Kawajiri, Ph.D. Department of Pathology, Nagoya University School of Medicine (until April, 2004, as of September, 2005) Nobuhiro Hanai, M.D. Department of Otorhinolaryngology, Nagoya City University School of Medicine (until March, 2004) Masahiko Sugimoto, M.D. Department of Ophthalmology, Mie University School of Medicine (until June, 2005) Takashi Oguri, M.S. Department of Cancer Genetics, Nagoya University School of Medicine Takashi Siromizu, M.S. Department of Cancer Genetics, Nagoya University School of Medicine Tomoya Yamaguchi, M.S. Department of Cancer Genetics, Nagoya University School of Medicine (as of April, 2004) Masaki Inoue, Nagoya University School (as of April, 2003, until March, 2004)

General Summary Abnormalities in the cell cycle control and tissue architecture are considered to lead to uncontrolled proliferation, genetic instability, and cell invasion (metastasis), which are the characteristics of cancers. However, the precise processes of carcinogenesis remain largely unknown. Our research aim is to elucidate the mechanisms by which cell cycle (including cell cycle checkpoint) and tissue architecture (including intracellular cytoskeletal network) are controlled. Our attention is focused on 2 specific areas. (1) Identification and functional analysis of protein kinases involved in cell cycle control and checkpoint; (2) Regulation of the cytoskeletal protein (especially intermediate filaments) and its associated protein in the cell adhesion.

1. Complex Formation of Plk1 and INCENP known. Here we found that Cdk1 phosphorylates Required for Metaphase-Anaphase Thr59 and Thr388 on inner centromere protein Transition (INCENP), which regulates localization and kinase activity of Aurora-B, from prophase to metaphase. Goto, H., Kiyono, T.*1, Tomono, Y.*2, Kawajiri, A., INCENP depletion disrupts Plk1 localization spe- Urano, T.*3, Furukawa, K.*3, Nigg, E.A.*4 and Inagaki, cifically at the kinetochore. This phenotype is res- M. cued by the exogenous expression of INCENP wild Mitotic chromosomal dynamics is regulated by type (WT) and INCENP mutated at Thr59 to Ala the coordinated activities of many mitotic kinases, (T59A), but not at Thr388 to Ala (T388A). The re- such as cyclin-dependent kinase 1 (Cdk1), placement of endogenous INCENP with T388A re- Aurora-B or Polo-like kinase 1 (Plk1). The abnor- sulted in the delay of progression from metaphase malities in these protein kinases can lead to genetic to anaphase. These results suggested that INCENP instability, but its precise regulation remains un- phosphorylation by Cdk1 is necessary for the re-

47 cruitment of Plk1 to the kinetochore, and the complex formation of Plk1 and Aurora-B on INCENP 3. Mitotic Chk1 Phosphorylation at Novel may play critical roles in the regulation of chromo- Sites Regulated by Cyclin-dependent somal dynamics. kinase 1 (Cdk1)

Shiromizu, T., Goto, H., Tomono, Y.*1, Bartek, J.*2, *1 Virology Division, National Cancer Center Research Totsukawa, G.*3, Inoko, A., Nakanishi, M.*4, Matsumura, Institute F.*3 and Inagaki, M. *2 Division of Molecular and Cell Biology, Shigei Medical Research Institute Chk1 is phosphorylated at Ser317 and Ser345 by *3 Department of Biochemistry II, Nagoya University ATR in response to stalled replication and Graduate School of Medicine genotoxic stresses. This Chk1 activation is thought *4 Department of Cell Biology, Max-Planck Institute for to play critical roles in the prevention of premature Biochemistry, Germany. mitosis. However, the behavior of Chk1 in mitosis remains largely unknown. Here we reported that 2. Phosphorylation by Cdk1 induces Chk1 was phosphorylated in mitosis. The reduction Plk1-mediated vimentin phosphoryla- of this phosphorylation was observed at the meta- tion during mitosis phase-anaphase transition. Two-dimensional phos- phopeptide mapping revealed that Chk1 phos- Yamaguchi, T., Goto, H., Yokoyama, T., Silljé, H.*1, phorylation sites in vivo were completely over- Hanisch, A.*1, Uldschmid, A. *1, Takai, Y.*2, Oguri, T., lapped with the in vitro sites by cyclin-dependent Nigg, E.A.*1 and Inagaki, M. protein kinase (Cdk) 1 or by p38 MAP kinase. Several kinases phosphorylate vimentin, the most Ser286 and Ser301 were identified as novel phos- common intermediate filament protein, in mitosis. phorylation sites on Chk1. Treatment with Cdk in- Aurora-B and Rho-kinase regulate vimentin fila- hibitor butyrolactone I induced the reduction of ment separation through the cleavage fur- Chk1-S301 phosphorylation, although treatment row-specific vimentin phosphorylation. Cdk1 also with p38-specific inhibitor SB203580 or siRNA did phosphorylates vimentin from prometaphase to not. In addition, ionizing radiation (IR) or ultravio- metaphase, but its significance has remained un- let (UV) light did not induce Chk1 phosphorylation known. Here we demonstrated a direct interaction at Ser317 and Ser345 in nocodazole-arrested mi- between Plk1 and vimentin-Ser55 phosphorylated totic cells. These observations implied the regula- by Cdk1, an event that led to Plk1 activation and tion of mitotic Chk1 function through Chk1 phos- further vimentin phosphorylation. Plk1 phosphory- phorylation at novel sites by Cdk1. lated vimentin at ~ 1 mol phosphate/mol substrate, which partly inhibited its filament forming ability, *1 Division of Molecular and Cell Biology, Shigei in vitro. Plk1 induced the phosphorylation of Medical Research Institute. vimentin-Ser82, which was elevated from meta- *2 Danish Cancer Society, Institute of Cancer Biology, phase and maintained until the end of mitosis. This Department of Cell Cycle and Cancer, Denmark. elevation followed the Cdk1-induced *3 Department of Molecular Biology and Biochemistry, vimentin-Ser55 phosphorylation, and was impaired Rutgers University, USA. by Plk1 depletion. Mutational analyses revealed *4 Biochemistry, Nagoya City University Medical that Plk1-induced vimentin-Ser82 phosphorylation School. plays an important role in vimentin filaments seg- regation, coordinately with Rho-kinase and 4. Functional analysis of cytoskeleton Aurora-B. Taken together, these results indicated a Izawa, I., Nishizawa, M. and Inagaki, M. novel mechanism that Cdk1 regulated mitotic vimentin phosphorylation via not only a direct en- Intermediate filaments (IF) form the structural zyme reaction but also Plk1 recruitment to framework of cytoskeleton. Although the histopa- vimentin. thological detection of IF proteins are utilized for examining cancer specimens as reliable markers, *1 Department of Cell Biology, Max-Planck Institute for the molecular mechanisms how IFs are involved in Biochemistry, Germany. the biology of cancer cells are still unclear. We set *2 Department of Obstetrics and Gynecology, Saitama out to search for the binding partners with simple Medical Center. epithelial keratin 8/18, and have thus far identified

48 Mrj, TNF receptor type 1-associated death domain protein. But, we newly characterized this protein as protein (TRADD) and trichoplein. Mrj, a DnaJ/Heat a keratin filament-binding protein which mainly shock protein 40 (Hsp40) family protein, directly concentrated at cell-cell border, and we found that binds to keratin 18. Mrj may play an important role the amino acid sequence of Fbf-1 has similarity to in the regulation of the keratin 8/18 filament or- trichoplein because both have TPHD. Subsequently, ganization as a keratin 18-specific co-chaperone to we are going to clarify the function of Fbf-1 with work together with Hsp/c70. We found a direct as- RNA interfere experiments. sociation of keratin 18 with TRADD, an indispensable adaptor molecule for TNF receptor type 1 *1 Virology Division, National Cancer Center Research (TNFR1) signaling. Thus, keratin 18 may sequester Institute TRADD to attenuate the interaction of TRADD with activated TNFR1, leading to diminution of 6. Vimentin-Ser82 as a phosphorylation TNF-induced apoptosis. These results suggest that site that memorizes the activity of simple epithelial keratins may play a role in modu- CaMKII in astrocytes lating the response to some apoptotic signals. Oguri, T., Inoko, A., Shima, H.*1*2, Izawa, I., Arimura, Trichoplein has a domain that shows a low degree N., Yamaguchi, T., Inagaki, N.*3, Kaibuchi, K.*4, of sequence similarity between trichohyalin and Kikuchi, K.*1 and Inagaki, M. plectin, designated as trichohyalin/plectin homol- ogy domain (TPHD). Trichoplein may be involved In astrocytes, the PGF2α or ionomycin treat- in the organization of the keratin filament network, ment induces the phosphorylation at Ser38 and possibly affecting cell polarity in simple epithelial Ser82 of vimentin, a type III intermediate filament, cells. The identifications of IF-binding proteins and by Ca2+/calmodulin-dependent protein kinase II lessons from transgenic mouse models and human (CaMKII). We found here that vimentin phos- diseases have indicated that IF may affect cell pho-Ser82 was dephosphorylated much slower than growth, cell death and cell polarity through the in- phospho-Ser38. Vimentin phospho-Ser38 was teractions with a variety of non-structural proteins, dephosphorylated quickly by purified PP1 catalytic including kinases and adaptors for cell signaling. It subunit (PP1c) in vitro, whereas phospho-Ser82 is hence plausible that IF may play profound roles was insensitive to PP1c. Because PP1c directly in cancer development, invasion and metastasis. bound to vimentin through a VxF motif (Val83-Asp84-Phe85), the PP1c active site ap- 5. Characterization and functional analy- peared to be unable to approach phospho-Ser82, sis of novel keratin filament-binding leading to the prolongation of the phosphorylation proteins, trichoplein and Fbf-1 at Ser-82. In astrocytes, PP1cαwas in vivo associated with vimentin filaments. The repetitive treat- Inoko, A., Zou, P., Sugimoto, M., Hayashi, Y., Kiyono, ment by ionomycin at a short interval resulted in the T.*1, Izawa, I. and Inagaki, M. sustained elevation of Ser82 phosphorylation, lead- As described above, we searched for the binding ing to the marked disassembly of vimentin fila- partners with keratin 8/18 to reveal the function of ments. Taken together, these results suggest that cytoskeleton especially in the epithelial cells. And, phosphorylation state of vimentin is regulated by we identified a novel protein of trichoplein and PP1c in astrocytes, and vimentin-Ser82 may act as a Fbf-1 as keratin filament-binding proteins. phosphorylation site that memorizes the activity of Concerned about trichoplein, we newly found that CaMKII. trichoplein localized not only on keratin filament *1 but also at desmosome. This means that the Division of Biochemical Oncology and Immunology, trichoplein of keratin filament-binding protein has Institute for Genetic Medicine, Hokkaido University *2 Division of Cancer Chemotherapy, Miyagi Cancer potential to participate in the organization of Center Research Institute cell-cell contact. Next, to elucidate the function of *3 Division of Signal Transduction, Graduate School of trichoplein, we executed RNA interfere experiments. Biological Science, Nara Institute of Science and The results suggested that the decrease of Technology trichoplein causes morphological changes of cul- *4 Department of Cell Pharmacology, Graduate School tured cells. So, now we are investigating the mo- of Medicine, Nagoya University lecular mechanism of this alteration. Fbf-1 was previously reported as a Fas-binding

From left to right First row: Dr. H. Furue, Dr. Hid. Nakamura, Ms. N. Saito and Dr. K. Ijichi, Second row: Ms. S. Matsumoto, Dr. K. Ishizaki, Dr. Y. Yasui and Dr. H. Kumimoto

50 Division of Central Laboratory & Radiation Biology ______

Kanji Ishizaki, Ph.D. Chief Hiroshi Kumimoto, Ph.D. Researcher Yoshihiro Yamane, Ph.D. Research Resident (until March 2004) Hideaki Nakamura, Ph.D. Research Resident (as of April 2004) Hiroki Furue, D.D.S. Research Resident (as of April 2005) Yuko Hayashi, Ph.D. Research Assistant (until March 2004) Yoshihiro Yasui, Ph.D. Research Assistant Noriko Saito, Research Assistant (as of April 2004) Visiting Trainees Kei Ijichi, M.D. School of Medicine, Nagoya City University (as of April 2004) Tomotaka Sugimura, D.D.S. School of Medicine, Nagoya University (until March 2005) Makoto Adachi, D.D.S. Asahi University School of Dentistry

General Summary One of our main research projects is molecular genetic analysis of human esophageal and oral tumors. We are studying interactions between genetic polymorphisms and life-style factors in development of these tumors to find clues for effective prevention. So far, we have analyzed polymorphisms in the L-myc, CHK2, CYP1A1, CYP1B1, CYP2E1, XPA, XPC, XPD, XPF, OGG1, XRCC1, ERCC1, STK15, and MDM2 genes and found that specific alleles of the L-myc and STK15 genes are associated with induction of esophageal tumors by smoking and drinking. Poly- morphisms of the CYP2E1, XPA, and ERCCI genes were also shown to be related to oral cancers. Another research project is the study of genetic effects of low-dose-rate radiation on human cells. For this purpose we have established immortal cell lines derived from normal individuals and patients with ataxia telangiectasia (AT), a radiation sensitive genetic disease, by introducing the human telomerase gene. These cell lines are immortal but without any changes in the p53 and other genes that are involved in cellular signal transduction. Using these cell lines we have revealed that cytotoxic effects and mutation induction by low-dose rate radiation in normal cells is much lower than those by high-dose-rate radiation but that AT cell lines exhibit similar radiation sensitivity independent of the dose-rate. Using phosphorylated H2AX foci as indicators of DNA double strand breaks (DSBs), we have demonstrated that AT cells exhibit a partial defect in the repair of DSBs.

1. A single nucleotide polymorphism of 591-602). the MDM2 gene in Japanese esophageal Since mutations of the p53 gene which is a and oral cancer patients target of the MDM2 protein, and amplification of the MDM2 gene are frequently observed in eso- Kumimoto, H., Sugimura, T.*1, Furue, H.*1, Shinoda, phageal cancer, and since oral cancer is developed M.*2, Hatooka, S.*2, and Ishizaki, K. in the same upper digestive tract area, risks with The human homologue of mouse double min- SNP309 for these cancers were analyzed by the po- ute 2 (MDM2) is a key negative regulator of p53. lymerase chain reaction-based restriction fragment A single nucleotide polymorphism (SNP309) has length polymorphism (PCR-RFLP) approach. been identified in the first intron of the MDM2 gene Total of 330 Japanese non-cancer outpatients and with functional consequences, since the G allele 165 patients with esophageal cancer at Aichi Can- shows higher promoter activity than the T allele. cer Center Hospital were the subjects. And also Thus the SNP309 can influence p53 tumor sup- PCR-RFLP analysis was performed 122 patients pressorion through its expression level. The G al- with oral cancer. The genotype distribution fitted lele of the SNP309 showed an increased expression with the Hardy-Weinberg equilibrium among level of the MDM2. And the GG genotype was non-cancer subjects; 106 with the GG genotype, reported to be associated with high cancer suscepti- 165 with the GT genotype and 61 with the TT bility and an early onset of soft tissue sarcoma de- genotype. While the T allele is dominant in the velopment (G.L. Bond et al., Cell 119 (2004) United States, the G allele was found to be most

51 OR to the result in the United States, while the risk of oral cancer did not significantly differ with the 1.4 genotype (Fig. 1). Interactions with smoking were also analyzed. Smoking and drinking rate did im- 1.2 pact on esophageal cancer but no genotype differ- 1 ences were significant. Risks of oral cancer, were also significantly influenced by smoking or heavy 0.8 drinking but again the genotype did not appear to 0.6 play any role. The average ages of onset of esophageal cancer were similar with each genotype and 0.4

standard standard this was also the case for oral cancer (Fig. 2). In 0.2 conclusion, the genotype distribution of the MDM2 gene among Japanese differ from that among 0 Americans and the risks of upper alimentary tract GG GT TT GG GT TT cancers with the GG genotype are not high compared with the TT genotype, suggesting that the esophageal cancer oral cancer contribution of the GG genotype of the MDM2 gene in cancer development might be, if anything, small Fig. 1. Age-sex-adjusted ORs for esophageal or in Japanese. oral cancer according to the genotype of the MDM2 gene. ORs for the GG and GT genotypes in esophageal cancer were significantly low com- *1 Nagoya University Graduate School of Medicine, 65 pared with the TT genotype. However, no sig- Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8550 nificant differences risks were found for oral can- *2 Department of Thoracic Surgery, Aichi Cancer Cen- cers. ter Hospital prevalent, with significance (p<0.001), suggesting that the genotype distribution of the SNP309 may 2. DNA repair defect in AT cells and their be different among each ethnic group. The risks hypersensitivity to low-dose-rate radia- of esophageal cancer in the GG and TG genotypes tion compared with the TT genotype were significantly Nakamura, Hid., Yasui, Y., Saito, N. and Ishizaki, K. low (OR=0.53 95%CI 0.32-0.88, and OR=0.48 95%CI 0.30-0.77, respectively), which is opposite To investigate the effects of low-dose-rate radiation on ataxia telangiectasia (AT) cells arrested age in the G0/G1 phase, AT and normal cells immortalized with the human telomerase gene were irradiated in non-proliferative condition at high- (2 60 Gy/min) or low-dose-rate (0.3 mGy/min) radiation. Figure 1 shows the survival curves obtained by colony formation assay with AT (AT1OS/T-n, 40 AT2KY/T-n and AT5KY/T-n) and normal (SuSa/T-n) cells exposed to high- or low-dose-rate radiation. While normal cells showed a higher re- 20 sistance after irradiation at a low-dose rate than a high-dose rate, AT cells showed virtually the same survival after low- and high-dose-rate irradiation. 0 Then, we used the micronucleus assay as a measure GG GT TT GG GT TT of induction of chromosomal aberrations. Although esophageal cancer oral cancer the frequency of micronuclei by low-dose-rate radiation showed a large reduction in normal cells, in AT cells it did not exhibit a significant reduction Fig. 2. The average ages of onset of esophageal and oral cancers with each genotype of the MDM2 (data not shown). Since recent studies showed a gene. The average ages of onset of esophageal close correlation between the number of γH2AX cancer in each genotype were similar each other. foci in a nucleus and the number of expected DSBs And those of oral cancer were also similar. after irradiation, we determined the number of

52 AT1OS/T-n HDR AT5KY/T-n HDR AT1OS/T-n HDR AT5KY/T-n HDR AT1OS/T-n LDR AT5KY/T-n LDR AT1OS/T-n LDR AT5KY/T-n LDR AT2KY/T-n HDR SuSa/T-n HDR AT2KY/T-n HDR SuSa/T-n HDR AT2KY/T-n LDR SuSa/T-n LDR AT2KY/T-n LDR SuSa/T-n LDR 40 1 35

0.1 20

Survival 15 H2AX foci/cells γ 10

0.01 0 0123456 01234567 Dose (Gy) Dose (Gy) FIG. 1. Dose-dependent survival curves of AT and FIG. 3. Dose-response curves for γH2AX focus in- normal cells exposed to high- (HDR: open symbol, 2 duction in AT and normal cells after high- (HDR: Gy/min) or low-dose-rate (LDR: closed symbol, 0.3 open symbol, 2 Gy/min) or low-dose-rate (LDR: mGy/min) radiation. Bars indicate standard deviations closed symbol, 0.3 mGy/min) radiation. For each (n = 3). dose, more than 100 nuclei are scored, and mean values with standard errors were shown.

DSBs and DSB repair activity by analysis of the focus formation of the γH2AX. Figure 2 shows representative focus formations in SuSa/T-n and AT2KY/T-n cells after high- or low-dose-rate irradiation. The numbers of foci on each nucleus were counted, and the mean numbers of foci induced by each dose of radiation are shown in Fig. 3. The number of γH2AX foci increased in proportion to the dose in both AT and normal cells after high-dose-rate irradiation. Although few γH2AX focus formations were observed by low-dose-rate radiation in normal cells, significant and dose-dependent γH2AX foci were observed in AT cells even after low-dose-rate irradiation, indicating that DNA damage was not completely repaired during low-dose-rate irradiation. These results suggest that AT cells might not be able to repair some FIG. 2. γH2AX foci in SuSa/T-n and AT2KY/T-n cells observed after irradiation at 5 Gy of high- fraction of DNA damage and are severely affected (HDR) or low-dose rate (LDR). Quiescent cells in the by low-dose-rate radiation. SlideFlask were irradiated at high- or low-dose rate and stained with a specific antibody to γH2AX. Nu- clei were also stained with DAPI (blue).

From left to right, First row: Dr, Hir. Nakamura and Mr. Y. Minoura Second row: Ms. M. Mizuno, Ms. M. Nishizawa and Ms. M. Yamamoto

54 Central Service Unit ______

Hiromu Nakamura, D.M.Sc. Section Head Morio Terashima, B.A. Senior Research Assistant (until March 2004) Sachiko Tokumasu, B.D. Research Assistant (until March 2004) Miwako Nishizawa, B.P. Senior Research Assistant (as of April 2004) Masami Yamamoto, D.V.M. Research Assistant Mikio Hagino Research Assistant (Animal care specialist)

Visiting Trainees Yoshimi Nishi, PhD. Nagoya University School of Medicine (until March 2004)

General Summary

The Central Service Unit fulfills many functions in assisting the investigations performed by the Institute and has responsibilities for the maintenance and operation of various instruments for biotechnology research. These are the DNA sequencer (ABI 3100), flow-cytometer (Becton- Dickinson FACS Calibur), imaging analyzers (Fujix BAS-2500Mac, Amersham-Pharmacia ImageMaster-CL and FluorImager-595), X-ray machine (Hitachi MBR-1520R3), electron microscopes (JEOL TEM and Hitachi SEM), confocal laser microscope (Bio-Rad Radiance), real-time PCR equipment (Roche Light Cycler), ultracentrifuges (Beckman and Hitachi), and computer systems for image treatment (Windows and Macintosh systems). Furthermore, we maintain and manage the radioisotope experimental facilities, SPF and conventional animal rooms, laboratories for translational research and technical photography and hazardous chemical storage, ultra-low temperature freezers, cold rooms, the liquid nitrogen storage room, security systems, air-conditioning, water purifying and waste water treatment systems, as well as the carbon dioxide gas supply, thereby contributing to many other of the Institute’s functions. During the last two years we replaced several instruments such as an ultracentrifuge (Beckman-Coulter Optima LE-80K), a molecular interaction analyzer (Biacore X-system), a flow-cytometer (Becton-Dickinson FACS Calibur) and a real-time PCR (ABI 7500 Fast Real-Time PCR System). Our activities thus provide essential background support for all the research carried out by the Research Institute.

55 Librarians ______

From Left to right Librarians, Ms. K. Adachi, Ms. T. Ieda, Ms. M. Teratani, Ms. T. Yasuda, supporting scientific and medical informations.

56 Researches Supported by Special Project Programme ______

1. Defining Second-Hit Genetic Abnormali- 11q22-q23 (13 %) and 19q13.11-q13.12 (13 %). ties Involved in Generation of t(12; 21) The two cell lines showed gain of 21q22.12-qter TEL-AML1 Acute Lymphoblastic Leuke- and loss of 2p11.2, 9p21.3, 12p13.2, and 12q21.3. mias by Array-based Comparative Ge- Among these, six regions of loss (2p11, 3p21, nomic Hybridization 4q31.23, 9p13.2, 12q21.3 and 19q13.12) have not been identified previously by conventional CGH in Tsuzuki, S., Karnan, S., Horibe, K.*1, Matsumoto, K.*2, TEL-AML1 leukemias. Representative genes Kato, K.*2, Inukai, T.*3, Goi, K.*3, Sugita, K.*3, involved in the regions of loss were Igkappa (2p11), Nakazawa, S.*3, Ueda, R.*4, and Seto, M. TEL (12p13.2), p16INK4a/ARF (9p21.3), Pax5 (9p13.2), BTG1 (12q21.3), LIMD1 (3p21), AIM1 The TEL (ETV6)-AML1 (RUNX1) chimeric gene and BLIMP1 (6q21), NR3C2 (4q31.23), ATM fusion is the most common genetic abnormality in (11q22-q23), and PDCD5 (19q13.11-q13.12), while childhood acute lymphoblastic leukemias (ALL). the region of gain at 21q contained RUNX1. These Evidence suggests that chimeric gene fusion usually findings suggest that, in addition to TEL previously occurs in utero during fetal hematopoiesis and most reported to be lacking, genes involved in cell cycle probably constitutes an initiating or first-hit regulation, p53 pathways and apoptosis are also mutation that is necessary but insufficient for the often deleted. Our array –CGH obtained data should development of overt, clinical leukemia. In our provide further insights into the molecular basis of search for additional secondary and postnatal TEL-AML1 leukemia development. genetic events that could be linked to leukemia development, we applied a genome- wide array- *1 Clinical Research Center, National Hospital CGH technique to 24 TEL-AML1 leukemia Organization Nagoya Medical Center samples and two cell lines (REH, KOPN41) and *2 Division of Hematology and Oncology, Children's found that at least three chromosomal imbalances Medical Center, Japanese Red Cross Nagoya First were involved in all patient samples and cell lines. Hospital Recurrent regions of chromosomal imbalance *3 Department of Pediatrics, Faculty of Medicine, (found in > 10% of clinical samples) were gain of University of Yamanashi chromosomes 10 (17 %) and 21q (25 %) and loss of *4 Department of Internal Medicine and Molecular chromosomes 2p11 (100 %), 12p13.2 (87 %), Science, Nagoya City University, Graduate School of 9p21.3 (29 %), 9p13.2 (25 %), 12q21.3, (25 %), Medical Sciences 3p21 (21 %), 6q21 (17 %), 4q31.23 (17 %),

57 Publications ______activities of chemicals in rat liver. J Appl Toxicol, Journals 25: 554-561, 2005. (PMID: 16208626)

J008. Azuma, T., Otsuki, T., Kuzushima, K., J001. Abe, T., Takano, K., Suzuki, A., Shimada, Froelich, C.J., Fujita, S. and Yasukawa, M.: Y., Inagaki, M., Sato, N., Obinata, T. and Endo, Myeloma cells are highly sensitive to the granule T.: Myocyte differentiation generates nuclear exocytosis pathway mediated by WT1-specific invaginations traversed by myofibrils associating cytotoxic T lymphocytes. Clin. Cancer Res. 10: with sarcomeric protein mRNAs. J.Cell Sci. 117: 7402-7412, 2004. (PMID: 15534117) 6523-6534, 2004. (PMID: 15572409) J009. Bork, S., Yokoyama, N., Ikehara, Y., J002. Akazawa, T., Masuda, H., Saeki, Y., Kumar, S., Sugimoto, C. and Igarashi, I.: Matsumoto, M., Takeda, K., Tsujimura, K., Growth-inhibitory effect of heparin on Babesia Kuzushima, K., Takahashi, To., Azuma, I., Akira, parasites. Antimicrob. Agents Chemother. 48: S., Toyoshima, K. and Seya, T.: Adjuvant-mediated 236-241, 2004. (PMID: 14693545) tumor regression and tumor-specific cytotoxic J010. Cao, X., Tsukamoto, T., Nozaki, K., response induction are impaired in Mizoshita, T., Ogasawara, N., Tanaka, H., MyD88-deficient mice. Cancer Res. 64: 757-764, Takenaka, Y., Kaminishi, M., and Tatematsu, M.: 2004. (PMID: 14744795) β-Catenin gene alteration in glandular stomach J003. Akiyama, Y., Kuzushima, K., Tsurumi, T. adenocarcinomas in N-methyl-N-nitrosourea- and Yamaguchi, K.: Analysis of HLA-A24- treated and Helicobacter pylori-infected Mongolian restricted CMVpp65 peptide-specific CTL with gerbils. Cancer Sci., 95: 487-490, 2004. (PMID: HLA-A*2402-CMVpp65 tetramer. Immunol. Lett. 15182428) 95: 199-205, 2004. (PMID: 15388261) J011. Cao, X., Tsukamoto, T., Nozaki, K., J004. Anumanthan, G., Halder, SK., Osada, H., Shimizu, N., Mizoshita, T., Kumagai, T., Takahashi, Ta., Massion, PP., Carbone, DP., and Kaminishi, M., and Tatematsu, M.: Eradication of Datta, PK.: Restoration of TGF-β signalling Helicobacter pylori induces apoptosis and inhibits reduces tumorigenicity in human lung cancer cells. proliferation of heterotopic proliferative glands in Br. J. Cancer. 93: 1157-1167, 2005. (PMID: infected Mongolian gerbils. Cancer Sci, 95: 16251876) 872-877, 2004. (PMID: 15546504) J005. Arimura, N., Ménager, C., Kawano, Y., J012. Chiba, H., Takezaki, T., Neupani, D., Kim, Yoshimura, T., Kawabata, S., Hattori, A., Fukata, J., Yoshida, S., Mizoguchi, E., Takeuchi, J., Y., Amano, M., Goshima, Y., Inagaki, M., Morone, Suzuki, J., Tanaka, Y., Ito, K., Kitamura, T., N., Usukura, J. and Kaibuchi, K.: Phosphorylation Kuriki, K., Wakai, K., Samejima, K., Sonoda, S. by Rho-kinase regulates CRMP-2 activity in growth and Tajima, K.: An epidemiological study of HBV, cones. Mol. Cell. Biol. 25: 9973-9984, 2005. HCV and HTLV-I in Sherpas of Nepal. Asian Pac J (PMID: 16260611) Cancer Prev, 5: 370-373, 2004. (PMID: 15546239) J006. Asano, N., Suzuki, R., Kagami, Y., Ishida, J013. Chiba, H., Tretli, S., Lund, E., Wakai, K., F., Kitamura, K., Fukutani, H., Morishima, Y., Takezaki, T., Senoo, H., Sonoda, S. and Tajima, Takeuchi, K. and Nakamura, S.: Clinicopathologic K.: Lack of HTLV-I carriers in the Sami, an ethnic and prognostic significance of cytotoxic molecule group living in the Arctic area in Norway. Asian expression in nodal peripheral T-cell lymphoma, Pac J Cancer Prev, 5: 50-53, 2004. (PMID: unspecified. Am. J. Surg. Pathol., 29:1284-1293, 15075005) 2005. (PMID: 16160469) J014. Daikoku, T., Kudoh, A., Fujita, M., J007. Asaoka, Y., Sakai, H., Takahashi, N., Sugaya, Y., Isomura, H. and Tsurumi, T.: In vivo Hirata, A., Tsukamoto, T., Yamamoto, M., Yanai, dynamics of EBNA1-oriP interaction during latent T., Masegi, T., and Tatematsu, M.: Intraperitoneal and lytic replication of Epstein-Barr virus. J. Biol. injection of D-galactosamine provides a potent cell Chem. 279: 54817-54825, 2004. (PMID: proliferation stimulus for the detection of initiation 15498777)

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"Bainiku-ekisu", Japanese apricot extracts, on motility and growth of Helicobacter pylori. In: A001. Akatsuka, Y., Kuzushima, K. and Tanaka, T., and Tsuda, H. (eds). Carcinogenesis Takahashi, To.: Identification of minor and Modification of Carcinogenesis, Research histocompatibility antigens involved in Signpost, Kerala, India,2005. graft-versus-leukemia effect and GVHD following allogeneic hematopoietic cell transplantation. R065. Wakai, K., Ando, M., Ozasa, K., Ito, Y., Abstract for US-Japan Cooperative Cancer Suzuki, K., Nishino, Y., Kuriyama, S., Seki, N., Research Program, 2005. Kondo, T., Watanabe, Y., Ohno, Y. and Tamakoshi, A.: Updated information on risk A002. Akatsuka, Y.: Identification of two novel factors for lung cancer: findings from the JACC minor histocompatibility antigens by linkage Study. J Epidemiol, 15 Suppl 2: S134-139, 2005. analysis and their clinical relevance, Abstract for (PMID: 16127225) the 2004 Tandem BMT Meeting (IBMTR/ABMTR and ASBMT), 2004. R066. Wakai, K.: The JICA training course, community-based cancer prevention for Asian A003. Daikoku, T., Kudoh, A. and Tsurumi, T.: Pacific countries, 2004 (Epidemiological approach). Dynamics of Epstein-Barr virus EBNA1 protein Asian Pac J Cancer Prev, 5: 231-236, 2004. (PMID: binding to viral genome and subcellular 15460556) redistribution from latent to lytic infection. EBV symposium 2004 The 11th Symposium of the R067. Yokoyama, T.: Methods for the preparation International Association for Research on of activated kinase in the insect expression system. Epstein-Barr Virus and Associated Diseases. 04.12 In: Protocols for post-translational protein Regensburg, Germany. 2004. modification (Inagaki, M. ed.), pp. 266-275, Tokyo: Yodosha, 2005 [in Japanese]. A004. Daikoku, T., Kudoh, A. and Tsurumi, T.: Postreplicative mismatch repair factors are recruited

to Epstein-Barr virus replication compartments. 5th 3R symposium P47, Awaji Yumebutai, Hyogo. 2005

A005. Fujiwara, K., Fujimoto, N., Tabata, K.,

Matsuo, K., Kozuki, A., Tokuda, Y., Kiura, K., Nishii, K., Ueoka, H. and Tanimoto, M.: Identification of aberrant promoter methylation in

serum DNA for early detection of lung cancer. Proceeding of Annual Meeting 2004, American Association of Cancer Research, 3959, 2004.

A006. Hirose, K., Tajima, K. and Tokudome, S.:

Soybean products and reduction of breast cancer risk ： A case-control study in Japan. The 3rd Regional Conference of Asian Pacific Organization

for Cancer Control, 26, 2005. A007. Hotta, K., Matsuo, K., Ueoka, H., Kiura, K., Tabata, M., Harita, S., Gemba, K., Yonei, T., Bessho, A. and Tanimoto, M.: Continued gefitinib treatment after disease stabilization prolongs survival of patients with advanced non-small-cell lung cancer. J Clin Oncol. 23: 642S, 2005.

A008. Ikehara, Y., Kojima, N., Nakanishi, H., Yoshii, T., Biao, L., Niwa, T., and Tatematsu, M.: Intra peritoneal macrophage is activated by uptake

83 of Mannose conjugated Liposome. American Mitsudomi, T., Sugiura, T., Sato, S., Ueda, R. and Society of Glycobiology, Poster, Hawaii, USA, Tajima, K.: Gene-environment interaction between 2004. smoking habit and DNA repair genes, APE1 Asp148Glu and XRCC1 Arg399Gln, in lung cancer A009. Ikehara, Y.,Sato, T., Niwa, T., Nakamura, risk among Japanese. The 6th joint conference of S., Goto,M. ,Ikehara, K., S., Kiyohara, K., Iwai, T., the American Association for Cancer Research and Hirabayashi, J., Nakanishi, H., Tatematsu, M. the Japanese Cancer Association, A59, 2004. and Narimatsu, H.: Apical Golgi localization of N,N'-diacetyllactosediamine synthase β A018. Ito, H., Masui, T. and Tajima, K.: Impact 4GalNAc-T3, is responsible for LacdiNAc of habitual smoking on cancer stage at diagnosis expression on gastric mucosa. American Society of based on regional cancer registry data for Aichi Glycobiology, Poster, Boston, USA,2005. prefecture, Japan. Final program and abstract book of 27th annual meeting of the International A010. Ikehara, Y.: Polymorphisms of two Association of Cancer Regisries, 60, 2005 fucosyltransferase genes (Lewis and Secretor genes) involving type I Lewis antigens are A019. Ito, H., Matsuo, K., Shinoda, M., Hatooka, associated with the presence of anti-Helicobacter S., Hirose, K., Saito, T., Wakai, K. and Tajima, K.: pylori IgG antibody. The 11th Aichi Cancer Center Esophageal cancer and polymorphisms of APE1 International Symposium, Nagoya, Japan,2005. Asp148Glu and XRCC1 Arg399Gln. The 2nd APOCP General Assembly Conference, 100, 2004. A011. Inagaki, M.: Identification and characterization of cleavage fullow kinases. Gordon A020. Iwata, S., Sato, C., Ando, H., Kiso, M., Research Conference on “Intermediate Filaments”. Kannagi, R., and Kitajima, K.: Studies on chemical Oxford, 2004. properties of cyclic sialic acid using their synthetic S- and O-glycosides as model compounds. A012. Inoko, A., Nishizawa, M., Izawa, I., US/Japan Glyco 2004 (Joint meeting of the Society Nagata, K. and Inagaki, M.: Identification and for Glycobiology and the Japanese Society of characterization of a novel keratin binding protein, Carbohydarate Research, (President, M.E. Etzler) trichoplein(trichohyalin and plectin like protein), as Hawaii, November 17-20, 2004. a desmosomal protein. Gordon Research Conference on “Intermediate Filaments”. Oxford, A021. Kakizaki, I., Kojima, K., Takagaki, K., 2004. Endo, M., Kannagi, R., Yasuda, T., Mita, S., Kimata, K., and Itano, N.: A novel inhibition A013. Ishizaki, K.: Effects of low-dose-rate mechanism of hyaluronan synthesis by radiation on HTERT-immortalized human cells. 3rd 4-methylumbelliferone. US/Japan Glyco 2004 International Workshop on Space Radioration (Joint meeting of the Society for Glycobiology and Reserch. NewYork, USA, May 2004. the Japanese Society of Carbohydrate Research, A014. Isomura , H., Stinski, M.F., Kudoh, A., (President, M.E. Etzler) Hawaii, November 17-20, Daikoku, T., Shirata, N. and Tsurumi, T.: Two 2004. SP1/SP3 binding sites in the major immediate-early A022. Kannagi, R.: Carbohydrate determinants proximal enhancer of human cytomegalovirus have involved in cell-cell interactions: Transcriptional a significant role in viral replication. 30th regulation of their expression. Human Disease Herpesvirus Workshop. 1.42, Turku, Finland. 2005. Glycomics/Proteome Initiative (HGPI) Workshop A015. Isomura, H., Tsurumi, T. and Stinski, M.F.: "Functional Glycomics in Disease", Chaired by N. The Role of the Proximal Enhancer in human Taniguchi, Osaka, Japan, August 23-24, 2004. Cytomegalovirus Replication. 29th Herpesvirus A023. Kannagi, R.: Regulation of gene Workshop, 1.16, Nevada, USA. 2004. expression in carbohydrate-mediated cell-cell A016. Ito, H. and Tajima, K.: Comparison of interactions. 2004 Glycolipid and Sphingolipid relative risk impact of habitual smoking and Biology Gordon Conference, Chaired by Suzuki, A. drinking for cancer by site based on regional cancer and Futerman, T., SPring-8, Hyogo, Japan, July registry data for Aichi prefecture, Japan. The 3rd 25-30, 2004. Regional Conference of Asian Pacific Organization A024. Kannagi, R.: Relationship between for Cancer Control, 14, 2005. enhanced selectin-mediated cell adhesion and A017. Ito, H., Hamajima, N., Matsuo, K., hypoxia-induced metabolic shift in human cancers.

Joint meeting of the Japanese and American A032. Kyogashima, M., Hara, A., Aoyama, T., Consortia for Glycomics (Chaired by Paulson JC Kannagi, R.: Determination of sulfatides and and Taniguchi N), Hawaii, November 21, 2004. complicated sulfated glycosphingolipids by MALDI-TOF MS. US/Japan Glyco 2004, Joint A025. Kannagi, R.: Sialoconjugates involved in meeting of the Society for Glycobiology and the cell-cell interactions. Sapporo Sphingolipid Japanese Society of Carbohydrate Research, Symposium, Chaired by Igarashi, Y., Sapporo, (President, M.E. Etzler) Hawaii, November 17-20, Japan, July 21 - 23, 2004. 2004. A026. Kim, D. H., Ahn, Y. O., Lee, B. H., Whang, A033. Matsuo, K. Folate metabolizing gene D. Y., Kono, S., Wakai, K., Matsuo, K., Hamajima, polymorphisms and risk of malignant lymphoma in N. and Tajima, K.: The effect of alcohol and Japan. Proceedings U.S.-Japan Meeting on Large aldehyde dehydrogenase polymophism on the risk Cohort Studies for Molecular Epidemiology, 19-20, of colorectal cancer. Proceedings of 18th Asia 2004. Pacific Cancer Conference-Cancer Research and Treatment, 161, 2005. A034. Matsuo, K. and Tajima, K.: Hepatitis C virus infection and risk of non-Hodgkin's A027. Kudoh, A. and Tsurumi, T.: Epstein-Barr lymphoma: Meta-analysis. The 2nd APOCP virus lytic replication evokes ATM checkpoint General Assembly Conference, 87, 2004. signal transduction while preventing p53- dounstream signaling. EBV symposium 2004 The A035. Matsuo, K., Hamajima, N. and Tajima, K.: 11th Symposium of the International Association Risk of esophageal cancer by alcohol drinking is for Research on Epstein-Barr Virus and Associated modified by genetic polymorphisms. Proceeding of Diseases. 04.18. Regensburg, Germany. 2004. The fourth Japan-China Joint Conference for Cancer Research, 8-9, 2005. A028. Kudoh, A., Daikoku, T., Ishimi, Y., Shirata, N., Iwahori, S. and Tsurumi, T.: A036. Matsuo, K., Hamajima, N., Mueller, N. E., Phosphorylation of MCM4 at sites inactivating Nakamura, S., Seto, M., Morishima, Y. and DNA hericase activity of the MCM4-6-7 complex Tajima, K.: Methylenetetrahydrofolate reductase during Epstein-Barr virus productive replication. 5th gene (MTHFR) polymorphisms and reduced risk of 3R symposium. P10, Awaji Yumebutai, Hyogo. malignant lymphoma. The 6th joint conference of 2005 the American Association for Cancer Research and the Japanese Cancer Association, A56,2004. A029. Kumimoto, H., and Ishizaki, K.: Frequent somatic mutations in a D-loop region of the A037. Matsuo, K., Tagawa, H., Tsuzuki, S., mitochondria! DNA in esophageal squamous cell Suzuki, R., Morishima, Y., Nakamura, S., Tajima, carcinoma. 6th Joint Conference of the American K. and Seto, M.: Different genomic gain pattern by Association for Cancer Research and the Japanese MTHFR C677T genotypes in diffuse large B-cell Cancer Association. Advances in Cancer Research. lymphoma. Proceeding of Annual Meeting 2005, Hawaii, USA, Jan. 2004. American Association of Cancer Research, 5793, 2005. A030. Kumimoto, H. and Ishizaki, K.: Novel polymorphisms in the L-myc 5' UTR showed A038. Matsuo, K., Yang, C. X., Ito, H., Hirose, different risks of smoking of drinking for K., Wakai, K., Kuriki, K. and Tajima, K.: esophageal cancer. The 9th Japan - Korea Cancer Gene-environment interaction between alcohol Research Wrokshop -Molecular signature of cancer drinking and MTHFR C677T polymorphism for cells and its application to diagnosis and treatment, esophageal cancer risk. The 3rd Regional pp 82-83, Gyeongju, Korea, Dec 2004. Conference of Asian Pacific Organization for Cancer Control, 15, 2005. A031. Kuriki, K., Matsuo, K., Ito, H., Hirose, K., Wakai, K., Saito, T. and Tajima, K.: Colorectal A039. Matsuo, K.: Challenging strategy of cancer cancer risk according to interactions between meat epidemiology in hospital. Aichi Cancer Center consumption and genetic polymorphisms of fat International Symposium XI, 6-7, 2005. metabolism related PPARgamma and CD36 among A040. Miyazaki, K., Ohmori, K., Izawa, M., Japanese. The 3rd Regional Conference of Asian Koike, T., Yamaji, T., Hashimoto, Y., Suzuki, A. Pacific Organization for Cancer Prevention and Kannagi, R.: Interconversion of carbohydrate (APOCP) GI Cancer Control, 17, 2005. ligands for siglecs and selectins on colonic

85 epithelial cells upon malignant transformation. APOCP Meeting, 3, 2004. Interlec 21, the 21st International Lectin Meeting, A050. Tajima, K.: Epidemic patterns and Chaired by K. Kasai, Shonan, Kanagawa,Japan, prevention strategies for GI tract cancers in the May 23-28, 2004. Asian Pacific. The 3rd Regional Conference of A041. Mizoshita, T., Tsukamoto, T., Takenaka, Asian Pacific Organization for Cancer Control, 2, Y., Ogasawara, N., Joh, T., Itoh, M., Ito, S., 2005. Nakamura, T., Yamamura, Y., and Tatematsu, M.: A051. Tajima, K.: National cancer control Cdx2 Correlates with Intestinal Phenotypic program. Proceedings of 18th Asia Pacific Cancer Expression and Prognosis in Advanced Gastric Conference-Cancer Research and Treatment, 40-41, Cancers. 6th International Gastric Cancer Congress, 2005. Oral 9, Yokohama, Japan,2005. A052. Tajima, K.: Virus related cancers in the A042. Nakanishi, H., Kodera, Y., Ito, S., Asian Pacific with special reference to ATL. Yamamura, Y., Jun, Q., Hara, T.,Hirai, T., Kato, Proceeding of the 36th international symposium of T., Tatematsu, M.: Comparison of peritoneal the Princess Takamatsu Cancer Research metastasis between gastric cancer and colorectal Foundation Developments in Cancer cancer:Clinical and experimental studies. The 3rd Epidemiology-Prospects for Cancer Control in the International Conference on Gastroenterological Asian Pacific-Region. 42-43, 2005. Carcinogenesis, Oral, Sapporo, Japan,2004. A053. Takematsu, H., Yamamoto, H., Okuno, Y., A043. Nakanishi, H., Yokoyama, Y., Ikehara, Y., Kannagi, R., Suzuki, A., Kozutsumi, Y.: DNA Kodera, M., tatematsu, M.: Anti-tumor effects of Microarray analysis of genes responsible for the EGFR tyrosine kinase inhibitor, gefitinib, on the expression of carbohydrate epitopes. US/Japan HER2-overexpressing human gastric cancer cell Glyco 2004, Joint meeting of the Society for lines derived from liver metastasis. International Glycobiology and the Japanese Society of Conference on Tumor Progression and Therapeutic Carbohydarate Research, (President, M.E. Etzler) Resistance. Poster, Philadelphia, USA,2004. Hawaii, November 17-20, 2004. A044. Shirata, N. and Tsurumi, T.: Activation of A054. Tatematsu, M.: Helicobacter pylori and ATM DNA damage checkpoint signal transduction gastric carcinogenesis in Mongolian gerbils. 6th elicited by herpes simplex virus infection. 5th 3R International Gastric Cancer Congress, Symposium symposium. P103, Awaji Yumebutai, Hyogo. 2005 5, Yokohama, Japan,2005. A045. Suzuki, T., Matsuo, K., Wakai, K., Ito, H., A055. Terakura, S., Murata, M., Nishida, T., Hirose, K., Sato, S., Nakamura, S., Ueda, R. and Emi, N., Akatsuka, Y., Riddell, S.R., Morishima, Tajima, K.: Past history of gastric ulcer and risk of Y., Kodera, Y. and Naoe, T.: Impact of malignant lymphoma. Proceedings of 18th Asia homozygous deletion of UGT2B17 on outcome of Pacific Cancer Conference-Cancer Research and allogeneic BMT. Abstract for the 46th Annual Treatment, 1827, 2005. Meeting of the American Society of Hematology, A046. Tajima, K. Overview of cancer statistics in 2004. (Abstract#1837) Asia. Proceeding of The 3rd Asia High-Technology A056. Terakura, S., Murata, M., Nishida, T., Network, 44, 2005 Emi, N., Akatsuka, Y., Riddell, S.R., Morishima, A047. Tajima, K. UICC programs for cancer Y., Kodera, Y. and Naoe, T.: Increased risk for prevention in Asia. The 2nd APOCP General treatment-related mortality of bone marrow Assembly Conference, 24, 2004. transplantation in GSTM1-positive recipients. Abstract for the 47th Annual Meeting of the A048. Tajima, K.: A model of hospital-based American Society of Hematology, 2005. (Abstract# epidemiologic research program for cancer control 1756) in Japan: case-referent studies, cohort study and prevention trial. Proceedings U.S.-Japan Meeting A057. Teshima, T., Matsuo, K., Matsue, K., on Large Cohort Studies for Molecular Kawano, F., Taniguchi, S., Hatanaka, K., Nakao, Epidemiology, 9-12, 2004. S., Tanimoto, M., Hara, M., Eto, T., Wake, A., Abe, Y., Ohno, Y., Takemoto, Y., Harada, M., A049. Tajima, K.: Cultural determinants for Takahashi, S., Ishida, Y., Kanda, Y., Imamura, M., cancer control. Proceeding of the 2nd Regional Kasai, M. and Takaue Y.: Impact of HLA

Mismatch on the Incidence of Acute GVHD and Serum carotenoids, retinol, and tocopherols and Rejection after Reduced-Intensity Conditioning colorectal cancer risk: a case-control study nested in Hematopoietic Stem Cell Transplantation (RICT). the Japan Collaborative Cohort (JACC) Study. The Blood. 104: 2758, 2004. 6th joint conference of the American Association for Cancer Research and the Japanese Cancer A058. Tsukamoto, T., Mizoshita, T., Ito, S., Association, C1, 2004. Yamamura, Y., Nakamura, T., Ushijima, T., and Tatematsu, M.: Alteration of gastric and intestinal A066. Wu, P-X., Kimura, N., Kannagi, R., and transcription factors in intestinal metaplasia and Sato, T.: Synthesis of sulfated oligosaccharides by adenocarcinomas of the human stomach. 6th sulfotransferase-transfected ECV304 cells using International Gastric Cancer Congress, Workshop 2, saccharide primer and structure analysis by Yokohama, Japan,2005. MALDI-TOF mass spectrometry. US/Japan Glyco 2004, Joint meeting of the Society for Glycobiology A059. Tsurumi, T. and Kudoh, A.: Epstein-Barr and the Japanese Society of Carbohydrate Research, virus lytic replication elicits ATM checkpoint (President, M.E. Etzler) Hawaii, November 17-20, signal transduction while providing an S-phase-like 2004. cellular environment. The Awaji International Forum on Infection and Immunity, Awaji A067. Yagi, H., Takahashi, N., Yamaguchi, Y., Yumebutai, Hyogo. 2004. Kimura, N., Kannagi, R., and Kato, K.: Development and application of multi-dimensional A060. Tsurumi, T. and Kudoh, A.: Epstein-Barr HPLC mapping of N-linked glycans. 2nd virus lytic replication elicits ATM checkpoint Pharmaceutical Sciences World Congress signal transduction while providing an S-phase-like (PSWC2004) Chaired by Sugiyama Y, Kyoto, cellular environment. The XIII International Japan, May 29-June 2, 2004. congress of Virology, P116, V-22, San Francisco, USA. 2005. A068. Yagi, H., Takahashi, N., Yamaguchi, Y., Kimura, N., Kannagi, R., and Kato, K.: A061. Uchida, A., Tabata, M., Matsuo, K., Ogino, Development of structural analyses of sulfated A., Fujiwara, K., Hotta, K., Shinagawa, K., Kiura, N-glycans by mass spectrometry and HPLC K., Ueoka, H. and Tanimoto M.: An increase in mapping. US/Japan Glyco 2004, Joint meeting of incidence of acute promyelocytic leukemia during the Society for Glycobiology and the Japanese gefitinib treatment for advanced non-small cell lung Society of Carbohydrate Research, (President, M.E. cancer. J Clin Oncol. 23: 667S, 2005. Etzler) Hawaii, November 17-20, 2004. A062. Uchimura, K., Singer, M.S, Tsay, D., A069. Yanada, M., Emi, N., Usui, N., Takeuchi, Kadomatsu, K., Kannagi, R., Muramatsu, T., and J., Sugiura, I., Takeuchi, M., Kobayashi, T., Rosen, S.D.: GlcNAc 6-O-sulfotransferase Yagasaki, F., Ohtake, S., Matsuo, K., Naoe, T. and (GlcNAc6ST)-1 and GlcNAc6ST-2 regulate Ohno, R.: Combination of intensive chemotherapy lymphocyte homing to lymph nodes. US/Japan and imatinib (IDEAMOP regimen) for the Glyco 2004, Joint meeting of the Society for treatment of newly diagnosed BCR-ABL positive Glycobiology and the Japanese Society of acute lymphoblastic leukemia; excellent efficacy Carbohydrate Research, (President, M.E. Etzler) without increasing toxicity. Blood, 2736, 2004. Hawaii, November 17-20, 2004. A070. Yanada, M., Takeuchi, J., Akiyama, H., A063. Wakai, K. Japan Collaborative Cohort Usui, N., Yagasaki, F., Emi, N., Miyazaki, Y., Study. Aichi Cancer Center International Ohtake, S., Jinnai, I., Matsuo, K., Naoe, T. and Symposium XI, 10-11, 2005. Ohno R.: High complete remission rate and A064. Wakai, K., Hirose, K., Matsuo, K., Ito, H., promising outcome by combination of imatinib and Kuriki, K. and Tajima, K.: Dietary factors and chemotherapy for newly diagnosed BCR-ABL- colorectal cancer risk in Japan: comparison between positive acute lymphoblastic leukemia. Blood. 1827, colon and rectal cancers. The 2nd APOCP 2005. General Assembly Conference, 88, 2004. A071. Yang, C. X., Matsuo, K., Wang, Z. M. and A065. Wakai, K., Suzuki, K., Kojima, M., Tajima, K.: Phase I/II enzyme gene polymorphisms Tamakoshi, K., Toyoshima, H., Watanabe, Y., and esophageal cancer risk: A Meta-analysis. The Hayakawa, N., Hashimoto, S., Tokudome, S., Ito, 2nd APOCP General Assembly Conference, 98, Y. and Tamakoshi, A. for the JACC Study Group.: 2004.

87 Record of Seminars ______

Invited Speakers

2004 Mar. 09 Kaneko, R., Kawaguchi, K. and Yashiro K. (Life Science Division, Merk Ltd. Japan) Expression and solubilization of proteins.

April 02 Moore, M. (APPOCP Coordination Director) A practical guide to the use of scientific English.

Jun. 22 Henderson, B. (University of Southern California) Inter-continental comparative study on breast cancer risk among Japanese with special reference to application of a genome wide scan for molecular epidemiology.

Jun. 30 Hanaoka, F. (Institute for Molecular and Cellular Biology, Osaka University) Function of DNA polymerase eta as a Xeroderma pigmentosum variant responsible gene.

July 16 Kanoh H (Department of Science, Tokyo University) Application of Raman spectroscopy for cell biology and medical science.

Oct. 07 Warren, E.H. (University of Washington, Fred Hutchinson Cancer Research Center) Rear- rangement and proteasome-mediated splicing of non-contiguous peptides encoded by the SP110 gene create a human minor histocompatibility antigen.

2005 Jan. 14 Tsubata, T. (Laboratory of Immunology, School of Biomedical Science, Tokyo Medical and Dental University) Regulation of the humoral immune response by membrane lectin molecules.

Mar. 29 Nomura, T. (Department of Gastrointestinal Surgery, Faculty of Medicine, University of Tokyo) Polypeptide (TFF2) expressing metaplasia (SPEM): From clonality to bone marrow homing.

Jun. 13 Uchimura, K. (Department of Anatomy, University of California, San Francisco, CA, USA) Regulation of lymphocyte homing to lymph nodes by cell surface sulfated glycoconjugates.

Dec. 14 Tauchi, H. (Faculty Science, Ibaragi University) NBS1 controls radiation induced DNA-damage response and its reapir.

Dec. 19 Cheng, A. (Human Cancer Genetics Program, Comprehensive Cancer Centre, Ohio State University, Columbus, OH, USA) Combinatorial regulation of estrogen signaling.

Institute Speakers

2004 Jan. 07 Ikehara, Y. (Oncological Pathology) Siglec-7 and Siglec-9 negatively regulate T cell receptor activation.

Mar. 04 Nakamura, Hid. (Central Laboratory & Radiation Biology) Effects of low-dose- rate radiation on human cells.

April 04 Hirose, K. (Epidemiology and Prevention) Risk and protective factors for breast cancer confirmed by HERPACC study - Obesity control against breast cancer risk among Japanese postmenopausal women.

Jun. 17 Osada, H. (Molecular Oncology) Novel therapeutic approaches with RNAi targeting ASH1 based on molecular characteristics of lung cancer cells.

Sep. 09 Ohta, R. (Immunology) Therapeutic inhibition of a complement regulator enhances antibody therapy in a model of mammary adenocarcinoma.

Nov. 30 Daikoku, T. (Virology) Molecular basis for Epstein-Barr virus genome replication.

2005 Jan. 24 Goto, H. (Biochemistry) Regulation of mitosis through the crosstalk among mitotic protein kinases.

Feb. 10 Adachi, M. (Central Laboratory & Radiation Biology) Molecular mechanisms of cisplatin resistance in human head and neck squamous cell carcinoma cell lines.

Mar. 03 Taguchi, O. (Molecular Pathology) Roles of the thymus in recognition of auto-antigens.

Mar. 23 Tsukamoto, T. (Oncological Pathology) Stomach cancer and phenotypic differentiation.

May 12 Matsuo, K. (Epidemiology and Prevention) Gene-environment interactions of MTHFR gene polymorphism with habitual drinking for gastrointestinal tract cancer risk.

Jun. 09 Ikehara, Y. (Oncological Pathology) A carbohydrate recognition based drug delivery system.

Sep. 08 Kondo, Y. (Molecular Oncology) Alterations of DNA methylation and histone modification in human cancer.

Dec. 21 Suzuki, R. (Molecular Medicine) Lymphomagenesis : Translocation, oncogene, microRNA, and beyond•••.

Dec. 27 Shirata, N. (Virology) 1. Interaction between Epstein-Barr virus immediate-early protein, BZLF1, and p53. 2. Activation of ATM DNA damage checkpoint signal transduction elicited by Herpes simplex virus infection.

89 ______Record of Symposia The 11th Aichi Cancer Center International Symposium “Forefront of Cancer Prevention Strategy in Asia”

Organizing Committee: Kazuo Tajima (Chairperson), Masae Tatematsu, Shigeo Nakamura, Kenji Wakai, Hayao Nakanishi, Hirotaka Osada, Kaoru Hirose, Keitaro Matsuo, Hidemi Ito, Hiro- shi Yamaguchi, Takanori Umeda

February 5, 2005, International Conference Hall, Aichi Cancer Center.

Program of symposium

Opening Remarks: Ryuzo Ohno (Aichi Cancer Center)

Comprehensive epidemiologic studies in hospital: A Challenging strategy of cancer epidemiology in hospital. Keitaro Matsuo (Aichi Cancer Center) Diet and risk in Mediterranean countries Carlo La Vecchia (Istituto di Ricerche Farmacologiche, Milan, Italy)

Cohort study and cancer risk assessment in Asia: The Japan Collaborative Cohort (JACC) Study. Kenji Wakai (Aichi Cancer Center) Korean Multi-center Cancer Cohort Studies for Genomic Epidemiology: Current Status and Perspectives Keun-Young Yoo（Seoul National University） The Self Defense Forces Cohort Study Suminori Kono (Kyushu University) A population-based prospective study on cancer and major chronic diseases: the JPHC Study Shoichiro Tsugane (National Cancer Center)

Infection and cancer prevention: Polymorphisms of two fucosyltransferase genes (Lewis and Secretor genes) involving type I Lewis antigens are associated with the presence of anti-Helicobacter pylori IgG antibodies. Yuzuru Ikehara (Aichi Cancer Center) Study of the Association of Human Papillomavirus Infection and Cervical Cancer in China Wang Yixun (Liaoning province Tumor Hospital and Institute, Liaoning, P. R China) Hepatitis virus and heapatocellular carcinoma Hideo Tanaka (Osaka Medical Center for Cancer and Cardiovascular Diseases) Liver flukes and Cholangiocarcinoma Petcharin Srivatanakul (National Cancer Institute, Bangkok, Thailand)

Forefront strategy of cancer prevetion: New strategies of individualized cancer prevention Nobuyuki Hamajima (Nagoya University) Breast cancer susceptibility and chemoprevention T.Rajkumar (Cancer institute, Chennai, India) Natural immunological host defense and cancer prevention Kei Nakachi (Radiation Effects Research Foundation) Forefront of cancer prevention in Asia Robert Burton (Strategic Leader, UICC, Melbourne, Australia)

Concluding Remarks: Toshitada Takahashi (Aichi Cancer Center)

90 Abstracts

A challenging strategy for cancer epidemi- ern Italy on over 12,000 cases of 20 cancer sites and ology in hospital. 10,000 controls. For most epithelial cancers, the risk decreased with increasing vegetable and fruit Keitaro Matsuo consumption, with relative risks (RR) between 0.3 Division of Epidemiology and Prevention, Aichi Cancer and 0.7 for the highest versus the lowest tertile. For Center Research Institute, Nagoya, Japan digestive tract cancers, the population attributable risks for low intake of vegetables and fruit ranged Population-based approaches have been the between 15 and 40%. A number of antioxidants gold standard of analytical epidemiologic study in (including carotenoids, lycopene and flavonoids) the field of cancer, however, but are not practical in and other micronutrients showed an inverse relation certain conditions; e.g., where there is a low inci- with cancer risk, but the main components respon- dence or a requirement for biomarkers. In such sible for the favourable effect of a diet rich in vege- conditions, hospital-based efforts are advantageous. tables and fruit remain undefined. Fish, and conse- We have developed a comprehensive cancer quently a diet rich in n-3 fatty acids, tended to be epidemiology study system called HERPACC another favourable diet indicator. In contrast, sub- (Hospital-based Epidemiologic Research Program jects reporting frequent red meat intake showed at Aichi Cancer Center), the first version (HER- RRs above unity for several common neoplasms. PACC-I) which was started in 1988. Every first Whole grain food (and hence possibly fiber) intake visit outpatient was systematically requested to en- was related to reduced risk of several cancers, par- roll in the program and fill out a common ques- ticularly of the upper digestive tract. In contrast, re- tionnaire covering lifestyle factors. Case status was fined grain intake and, consequently, glycemic load identified via hospital-based cancer registration. and glycemic index were associated to increased Until 2000, data for 12,500 cancer patients and risk of different types of cancers. 83,000 non-cancer outpatients were pooled in In conclusion, a low risk diet for cancer in the HERPACC-I. In a second version of HERPACC, Mediterranean would imply increasing fruit and started in 2001, we added a protocol for obtaining vegetables as well as avoiding increasing meat and blood samples and semi-quantitative food fre- refined carbohydrate consumption. Olive oil and quency questionnaire, and this is ongoing. other unsaturated fats, which are also typical as- We are now planning to conduct a cohort study pects of the Mediterranean diet, should also be pre- using non-cancer subject enrolled in HERPACC. ferred to saturated ones. Direct comparison of results of case-control studies and cohort study using same HERPACC population is a unique and challenging approach for cancer The Japan Collaborative Cohort (JACC) epidemiology. Study In this presentation, several results of HER- Kenji Wakai PACC-based epidemiological studies will be introduced. Division of Epidemiology and Prevention, Aichi Cancer Center Research Institute, Nagoya, Japan Diet and risk in Mediterranean countries The Japan Collaborative Cohort Study (JACC Study; Chairman of the study group: Akiko Tama- Carlo La Vecchia koshi at Nagoya University Graduate School of Istituto di Ricerche Farmacologiche“Mario Negri”- Mi- Medicine) is a nationwide, multicenter cohort study, lano and Istituto di Statistica Medica e Biometria, Uni- with 24 participating research institutions. The versita di Milano - Milano, Italy study started in 1988 to 1990, when 110,792 inhabitants aged 40 to 79 years completed a baseline Various aspects of the Mediterranean diet are questionnaire. They were enrolled from 45 study considered favourable not only cardiovascular dis- areas throughout Japan. ease, but also on several common epithelial cancers. The baseline questionnaire covered lifestyle These include frequent consumption of vegeta- factors including smoking and drinking habits, bles and fruit, which was analyzed using data from physical activity, and dietary habits, as well as a series of case-control studies conducted in North-

91 medical history, education, family history of cancer, diet, reproductive factors, and agricultural expo- height and weight, and occupation. In addition to sures were obtained through direct interview using completing the questionnaire survey, 39,242 par- a structured questionnaire. Anthropometric meas- ticipants donated peripheral blood samples at health urements and some clinical laboratory findings have screening check-ups. The serum samples were also been collected and stored in the web-based da- stored at -80. until analyzed for nested case-control tabase system. A biological materials bank with studies. For 61,557 subjects (55.6% of the total) in blood (serum, plasma, buffy coat, packed erythro- selected areas, we ascertained the incidence of can- cytes) stored at -70. and urine at -20. has been es- cer by means of linkage with the records of popula- tablished for the genome epidemiological studies on tion-based cancer registries, supplemented by a re- the cancer etiology. DNA yield study revealed the view of medical records. The vital and residential PCR products for β-globin from nearly all of the status of subjects was determined using resident samples (98%) from the long term-stored buffy coat registration records, and causes of death were iden- specimen in the KMCC. Follow-up for the cancer tified from death certificates. occurrence has been commencing based on an ac- During the follow-up through 1999 for death and tive surveillance system by health personnel in each through 1997 for cancer incidence, 4,528 cancer district, and a passive surveillance system through deaths (in all study areas) and 3,437 incident cases record linkages between the central cancer registry, of cancer (in the selected areas) were documented. the national death certificate, and the national health Members of the JACC Study Group have exten- insurance claim databases in Korea. As of August sively examined associations of lifestyle or other 2004, total number of observation for the cohort factors and serum components with the risk of can- with biologic specimen was 20,342. Until Decem- cer incidence and death. More than forty original ber 2002, total 382 incident cancer cases have been articles have been published from the study includ- identified by the passive surveillance and total ing papers from nested case-control studies utilizing number of follow-up was 64,999 person-years. Five the stored sera. The follow-up of subjects and the leading sites of cancer incidence were stomach, analysis of data and serum samples are still on go- lung, liver, colorectum, esophagus in men, and ing. uterine cervix and breast in women. A fundamental The JACC Study has generated a significant question about genomic cohort is how large should body of information for primary cancer prevention it be in order to estimate the main effect of a SNP or in Asia. It also may provide a good model for na- haplotype and to detect gene-environmental and tionwide or international multicenter cohort studies, gene-gene interactions. On this purpose, the Korean in which individual participating institutions are Genomic Epidemiology Society has recently been independent and maintain their originality in re- founded, and a few of new genomic cancer cohorts, search but all contribute to one large cohort. i.e. KOEX, KCDC, KNCC, etc., have been launched under the supervision of the Society. The recruitment goal and the design of each cohort will Korean Multi-center Cancer Cohort Studies briefly be introduced. Along with other cancer co- for Genomic Epidemiology: Current horts in Japan, the Korean Genomic Cancer Cohort Status and Perspectives could provide more convincing evidence on new Keun-Young Yoo etiologies of cancer and on the cancer prevention strategy in the Asian-Pacific region. Department of Preventive Medicine, Seoul National University College of Medicine, Seoul, Korea. Human genome epidemiology is the systematic The Self Defense Forces Cohort Study application of the epidemiologic method to the ge- Suminori Kono nome to assess the impact of genetic variation on Department of Preventive Medicine, Kyushu University health and disease. Cohort studies are the ultimate Faculty of Medical Sciences, Fukuoka, Japan application of human genome epidemiology. The Korean Multi-center Cancer Cohort (KMCC) is a Taking into account the advantage of the com- multi-center prospective cohort to meet the re- prehensive medical examination for retiring quirement of genome epidemiological studies on self-defense officials at the Self Defense Forces cancer etiology, which had been conducted since (SDF) hospitals, the author initiated the SDF Health 1993. Data on general lifestyle, physical activity, Study at the SDF Fukuoka Hospital in October of

92 the year 1986, when he was a part-time physician Lifestyle is closely related to occurrence of there. Sigmoidoscopy, abdominal ultrasonography, cancer and other chronic diseases. Biological and a 75-g oral glucose tolerance test were included specimens such as plasma and white blood cells are as a procedures in the preretirement health exami- expected to provide useful information on expo- nation during a 5-day admission. This health ex- sure-disease relations considering genetic suscepti- amination was not mandatory, but was received by bility using recent biochemical and molecular tech- almost all retiring officials. A lifestyle question- niques. To investigate factors associated with can- naire was introduced to inquire about smoking, al- cer and other chronic diseases in Japan where the cohol use, physical activity, and habitual consump- disease profile (coronary heart disease is a minor tion of limited items of foods and beverages. At the cause of death and the stomach continues to be the time of the inception of the SDF Health Study, most frequent cancer site) and diet are substantially much interest had been focused on increased risk of different from Western countries, we launched a colon or colorectal cancer associated with low population-based prospective study in 1990. Ap- blood cholesterol levels observed in prospective proximately 140,000 men and women aged 40-69 studies. Thus the first cancer-related study was to years were selected based on resident registration in examine the relation between serum lipids and co- 29 communities covered by 11 public health center lorectal adenomas, and the outcome was no material areas nationwide. At the baseline survey, a relation with serum total cholesterol. The SDF self-administered questionnaire including items on Health Study was once deployed at four SDF hos- simple food frequency, blood (3 aliquots of plasma pitals across the nation. and 1 buffy coat) and health check-up data (an- A series of studies have revealed increased risk thropometric measures, blood pressure, biochemical of colorectal adenomas associated with cigarette measures such as lipids and liver function test) were smoking, alcohol use, physical inactivity, abdomi- collected from 110,000 (80%), 49,000 (35%) and nal obesity, and non-insulin dependent diabetes 48,000 (34%) persons, respectively. At year six, a mellitus. Total colonoscopy was introduced as a follow-up survey (same survey items as those at routine procedure in the year 1995; at that time the baseline plus a semiquantitative food frequency study had retreated to Kyushu. This approach estab- questionnaire with validity information compared lished that cigarette smoking and alcohol use were with 4 season 7 day diet records) was conducted, associated with a greater risk of adenomas in the and 100,000 questionnaires, 35,000 blood samples distal segment of the colorectum while diabetes and 33,000 health check-up reports were collected. mellitus was more markedly related to an increased Further at year eleven, the self-administered ques- risk of proximal colon adenomas. It was also found tionnaire used at year six was repeated and col- that the association between cigarette smoking and lected from 97,000 persons. Mortality and immigra- colorectal adenomas did not vary with genetic tion, as well as disease incidence (cancer, cere- polymorphisms of CYP1A1, GSTM1, and GSTT1, brovascular disease, ischemic heart disease, diabe- which are key enzymes in the metabolism of to- tes, etc.), were treated as endpoints. Among all co- bacco-related carcinogens. The SDF Health Study hort subjects, 10,500 deaths, 8,900 cancers, 2,900 evolved to a prospective cohort study in April of the strokes and 600 myocardial infarctions were docu- year 2004. Informed consent was obtained as re- mented as of October 2004. Several findings from gards the follow-up health survey as well as for do- the JPHC study will be presented. nation of venous blood for genetic analysis. Details of the design of the cohort study and conduct of the baseline survey will be discussed. Polymorphisms of two fucosyltransferase genes (Lewis and Secretor genes) in-

volving type I Lewis antigens are asso- A population-based prospective study on ciated with the presence of anti- cancer and major chronic diseases: the Helicobacter pylori IgG antibodies JPHC Study Yuzuru Ikehara Shoichiro Tsugane Division of Oncological Pathology, Aichi Cancer Center Epidemiology and Prevention Division, Research Center Research Institute Nagoya, Japan for Cancer Prevention and Screening, National Cancer Recent progress in the molecular analysis of H. Center, Tokyo, Japan pylori infection has revealed that the bacteria attach

93 to the gastric mucosa through the blood group anti- types;moderate risk, other than low- or highrisk gen-binding adhesin, BabA and a clinical relevance group], the odds ratio relative to the low-risk group has been shown for the babA2 gene encoding BabA was 3.30 (95% confidence interval, 1.40-7.78) for adhesin with regard to H. pylori-related diseases. the moderate-risk group and 10.33 (95% confidence BabA binds to both Leb [Gal (α 1.2Fuc)β1.3 interval, 3.16-33.8) for the high-risk group (Figure GlcNAc(α1.4Fuc)-R] and H type I blood group 2). Immunohistochemical analysis supported the carbohydrate structures [H type I structures; Gal finding that Se and Le genotypes affected the ex- (α1.2Fuc)β1.3GlcNAc-R] expressed on the foveo- pression of H. pylori adhesin ligands. lar epithelium of the gastric mucosa. We conclude that Se and Le genotypes impact on We have been studying Lewis blood type anti- susceptibility to H. pylori infection. gens using biochemical and molecular biological methods and have obtained the following results concerning type I Le antigen synthesis. Individuals Study of the Association of Human Papil- homozygous for nonfunctional alleles of Le gene lomavirus Infection and Cervical Cancer (le/le) fail to express type I Le antigen (so called Le in China negative). In the human fucosyltransferase family, Wang Yixun only the Le enzyme (FUT3, Fuc-T III) exhibits GYN Oncology Department, Liaoning province Tumor fucose transfer activity toward a type I precursor Hospital and Institute, Liaoning, P. R China (Galβ1.3GlcNAc-R) or H type I structure with β1.4 linkage. The Se enzyme (FUT2, Fuc-T II) exhibits It was in 1980s, the relationship of cervical fucose transfer activity toward the type I precursor cancer to HPV started to be elucidated. HPV type with α1.2 linkage and is responsible for Leb expres- 16 and 18 were first isolated directly from cervical sion on erythrocytes, solely determining the secre- cancer in 1983. From that time the strong associa- tor status, and making a marked contribution to Leb tion between HPV and cervical neoplasms has been expression in colorectal tissues. reported worldwide. HPV DNA can be detected Individuals homozygous for nonfunctional al- from 99.7% of cervical cancer specimens. Ap- leles of the Se gene fail to express ABH blood an- proximately 90 types of HPV have been identified, tigens in secreted fluids (so called for some of which are oncogenic or high risk types. non-secretors), whereas those very rare individuals In order to investigate Human Papilloma virus homozygous for nonfunctional alleles of the H gene infection prevalence in China, The study of associa- (h/h) fail to express ABH blood antigens on eryth- tion between HPV infection and cervical cancer rocytes. Considering the type I Le antigen synthetic was conducted in a high incidence area of cervical pathway, it is possible that the type I precursor cancer -Shanxi province. It was found that for structure for acceptor substrate is used by Se and Le women from 35- 50, the high risk HPV infection enzymes, with some competition between the two. rate was 24%, which is much higher than 5%-10% The present study was performed to investigate reported from the world. For a group of 1997 mar- the possibility that Se and Le gene polymorphisms ried women aged 35-45 : exfoliated cells were col- alter the risk of H. pylori infection. Two hundred lected from cervix (by clinician) and from vagina and thirty-nine participants were genotyped for Se (by subject herself), Hybrid capture 2 assay, which and Le and tested for the presence of anti-H. pylori could detect 13 types HPV DNA of high risk, was IgG antibodies. Using the normal gastric mucosa carried out. HPV DNA detection rate was 20.8%. from 60 gastric cancer patients, we further assessed The infection rate increased with progression of immunohistochemically whether type I Le antigen cervical lesions. (X2=444.04,P=0.000). Comparison expression depended on the Se and Le genotypes. of 2 groups of aged 35-39 and 40- 45, there was no The H. pylori infection rate was positively as- significant difference of infection rate (20.9%; sociated with the number of Se alleles (se/se group, 20.6%, X2=0.03, P=0.86). While compared with 45.1%; Se/se group, 64.6%; and Se/Se group, normal subjects: the risk odds ratio HPV infection 73.3%) and negatively associated with the number with cervical cancer/high grade CIN and low grade of Le alleles (le/le group, 76.4%; Le/le group, CIN were 254.2 and 26.4 respectively, with attribu- 68.3%; and Le/Le group, 55.6%). tive risk percentage (ARP) of 98.1% and 83.6%. When the subjects were classified into three groups The sensitivity of the assay for high risk HPV DNA [low risk, (se/se, Le/Le) genotype; high risk, (Se/Se, from clinician collected sample was 98%, which le/le), (Se/Se, Le/le), and (Se/se, le/le) geno- was higher than self collected samples of

94 84%(X2=5.92 P=0.015. No significant difference the age group of 40-70 years has been under the can be seen in specificity. (86%:85%, X2=0.00, process of development by local municipal gov- P=0.997). We conclude that high risk HPV infec- ernments since 2002. The effectiveness of this tion in female genital tract was the major risk factor screening system for preventing HCC will be of cervical cancer and CINs in this area of China. evaluated in the future.

Hepatitis virus and hepatocellular carci- Liver flukes and Cholangiocarcinoma noma Petcharin Srivatanakul Hideo Tanaka Cancer Control Unit, National Cancer Institute, Bang- Department of Cancer Control and Statistics, Osaka kok,Thailand Medical Center for Cancer and Cardiovascular Diseases, The liver flukes, Opisthorchis viverrini, Opist- Osaka, Japan horchis felineus and Clonorchis sinensis, are bio- Chronic hepatitis B virus (HBV) infection is a logically similar, food-borne trematodes which major risk factor of hepatocellular carcinoma chronically infect the bile ducts and, more rarely, (HCC) in Asia. Immunization with HBV vaccine is the pancreatic duct and gall-bladder of human be- a most effective weapon against HBV infection and ings and other mammals. Infection is acquired by its consequences, although the modality differs eating raw or undercooked freshwater fish which among Asian countries. A recent cohort study in contain the infective stage (metacercaria) of flukes. Japanese blood donors showed coinfection with Immature flukes migrate up through the ampulla of HBV and hepatitis C virus (HCV) carried a super- Vater to the biliary tree, mature in the small intra- additive risk for HCC. hepatic ducts and produce eggs, which are passed in HCV is a blood-borne virus which causes a the faeces. If the eggs reach a water body and are wide spectrum of liver diseases, ranging from acute consumed by an appropriate species of snail, they hepatitis to HCC. Parenteral infection through hatch and undergo asexual multiplication to pro- blood transfusion, intravenous drug abuse (IVDU) duce free-swimming larvae, which can penetrate and tattooing, as well as occupational exposure to freshwater fish and become encysted metacercariae. blood, have been well defined as determinants of Infection with Opisthorchis viverrini is car- HCV transmission. Recently, the incidence of HCV cinogenic to humans (Group 1). Infection with O. infection among Japanese blood donors was esti- felineus is not classifiable as to its carcinogenicity mated as 2-5 per 105 person-years. to humans (Group 3). Infection with C. sinensis is Approximately 60% of persons infected with probably carcinogenic to humans (Group 2A). HCV become HCV carriers and about 75% of Primary cancers of the liver in adults are of two Japanese with HCC cases are associated with main histological types: hepatocellular carcinoma chronic HCV infection. Life time risk of developing which is derived from hepatocytes, and cholangio- HCC among HCV carriers has been estimated as carcinoma, (CCA) which is derived from the 30% for males and 6% for females, based on data epithelial lining of the intrahepatic bile ducts. for the age and sex specific incidence rates of HCC About 560,000 new cases of liver cancer, usually among HCV carriers in Osaka. An older age, being hepatocellular carcinoma, occur annually, and con- male, duration of HCV infection, type Ib infection, tribute significantly to cancer mortality worldwide. co-infection with HBV, having a high serum CCA is a relatively rare tumour in most popula- transaminase level, having a low platelet count, and tions but second among primary malignant liver heavy drinking and smoking are independent fac- tumours; about 15% of liver cancers are estimated tors associated with the development of HCC to be CCA. The geographic distribution worldwide among HCV carriers. Cohort studies have demon- coincides with endemic areas of the liver flukes O. strated that interferon therapy can significantly viverrini and C. sinensis. The interaction between lower the incidence of HCC among patients with genes and the environment and the interplay of en- chronic hepatitis C who showed normalization of vironmental factors, which include diet and lifestyle, the serum transaminase level after completion of illustrate the complexity in understanding the sus- the therapy. ceptibility to environmental exposures. In Japan, a nationwide community-based The highest incidence of CCA is found in areas anti-HCV and HBsAg screening system targeting of Laos and North and Northeast Thailand suffering

95 from endemic infection with the liver fluke, O. New strategies of individualized cancer pre- viverrini. In Khon Kaen (the Northeast Thailand) , vention 86.5% of liver cancer cases are CCA. In both en- Nobuyuki Hamajima demic and non-endemic areas, there have been no significant changes in the incidence of CCA in re- Department of Preventive Medicine / Biostatistics and cent years. It is less than 10 years since O. viverrini Medical Decision Making, Nagoya University Graduate drug therapy was initiated;since it probably takes 30 School of Medicine, Nagoya, Japan years for CCA development after the infection, the Generally, health information based on the in- trends of CCA are probably not likely to change in dividuals' background allows a stronger message to the next decade. Patients with CCA are elderly, induce behavior changes in lifestyle, supplement with no clear sex differences. CCA occurs at rather intake, health checkup motivation, and medical fa- older ages than hepatocellular carcinoma in most cility visits. The background for each individual is clinical series. made up of past exposure experiences, disease his- C. sinensis parasitizes the bile ducts of millions tory, current biomarker conditions, and genetic of individuals in the Far East, particularly China traits, as evidenced by a family history. In order to and Korea. In the C. sinensis endemic area in Ko- provide attractive cancer prevention methods for rea , there is also a high incidence of liver cancer. individuals, links to such individual background About 20% of liver cancers in Pusan , Korea are information are becoming important. CCA. To date, several individualized cancer preven- Chronic infection with the liver fluke, O. viver- tion models have been introduced in practice. Pre- rini is the major risk factor for the development of ventive measures to block mother-to-child trans- CCA. Carcinogenesis of CCA is probably related to mission of hepatitis B virus and thus liver cancer is the length and severity of infection, the host's im- provided for carrier mothers. Preventive mastec- mune response, and other variables such as inges- tomy against breast cancer is conducted for BRCA1 tion of dietary carcinogens, for example nitrosa- abnormal gene carriers. Intensive checkup and mines. In northeast Thailand, several carcinogenic counseling against colorectal cancer are provided N-nitroso compounds and their precursors exist at for those with FAP and HNPCC related genes. low levels in the daily diet. In addition, endogenous These specific cancer prevention models for carriers nitrosamine formation by liver fluke infection has are accepted in several societies. been reported. Increased levels of urinary nitrates Recent biomarker studies on the most common and salivary nitrites are found in O. viverrini in- cancers indicate possible new strategies. Eradica- fected individuals. The subjects living in high-risk tion of Helicobacter pylori seems effective to pre- areas for fluke infection who had antibodies to O. vent gastric cancer for individual with heavy infec- viverrini had a 10-fold greater potential for en- tion. Health services not covered by health insur- dogenous nitrosation, measured on the basis of uri- ance started in Japan for those who seek the test and nary levels of N-nitrosoproline after praline inges- medication for the eradication. Reported tion, than individuals who did not have antibodies. gene-environment interactions between genotypes Vitamin C is found to be effective inhibitor for and interventions also provide new models for indi- prevention of the formation of endogenous nitrosa- vidualized cancer prevention. For example, the in- tion. In several studies in hamsters infected with O. teraction between aspirin use and an A316G poly- viverrini and treated with various carcinogenic Nni- morphism of the ornithine decarboxylase gene sug- trosamines, induction of cholangiocarcinomas and gests a possibility to recommend aspirin intake of hepatocellular nodules was enhanced. These re- more strongly for A-allele-possessing patients at sults suggest that the interaction between chemical risk of colorectal adenoma/carcinoma. carcinogens, especially nitrosamines, and OV in- Genotype announcement may be a new strategy festation may play role in the development of CCA to induce behavior changes to a less risky lifestyle. in Thailand. Both exogeneous and in situ nitrosa- Awareness of susceptible genotypes or enhanced mine formation may lead to DNA alkylation and health consciousness through genotyping could deamination. It seems that the presence of parasites provide opportunities to correct high risk behavior, induces DNA damage and mutations as a conse- e.g., smoking and drinking. Concerning smoking, quence of the formation of carcinogens/free radicals the cessation rate was found to be higher for the and of cellular proliferation of the intrahepatic bile announced group in some studies, although not in duct epithelium. all cases.

96 Undoubtedly, the increasing number of avail- Natural immunological host defense and able biomarkers will contribute to the establishment cancer prevention of new modes of individualized cancer prevention. Kei Nakachi

Department of Radiobiology/Molecular Epidemiology, Breast cancer susceptibility and chemo- Radiation Effects Research Foundation, Hiroshima, Ja- prevention pan T. Rajkumar The concept of multi-stage carcinogenesis im- Department of Molecular Oncology, Cancer institute plies that cancer prevention with different strategies (WIA), Adyar, Chennai, India is feasible for each stage. Recently emphasis has Breast cancer is the second most common can- been placed on defense mechanisms existing in dif- cer among Indian women. The incidence of breast ferent stages of carcinogenesis, with the immune cancer has shown a trend towards gradual increase system as the body's last line of defense against in the Chennai Metropolitan area over the past two cancer development. Cancer immunosurveil- decades, with the current CIR of 19.9 /100,000. lance-routinely eliminating nascent transformed While hereditary breast cancers account for 5-10%, cells in the body-needs to be proven through inves- the vast majority is sporadic. tigations of general populations. BRCA1 and BRCA2 are the major breast cancer We previously reported an elevated incidence of susceptibility genes identified to date. However, cancer among individuals showing low levels of contrary to the initial predictions, they account for NK activity in peripheral blood lymphocytes, as only 15-20% of hereditary cancers. In our series of compared to medium or high levels, based on a 61 patients of Hereditary breast &/or ovarian can- prospective cohort study of a Japanese general cers, 8 deleterious mutations were detected. Of the population. Large differences were found among 24 Hereditary breast cancer families tested, four individuals in NK activity, and lifestyle factors were found to have a deleterious mutation (16%). seemed to explain only a part of these, so we inves- Only one of the thirteen families with Hereditary tigated the genetic factors underlying individually breast and ovarian families were detected to have a variation in NK activity. From the cohort members, deleterious mutation in our series. Two out of 20, of we selected two groups with low and high NK ac- the early onset breast cancers (<35 years of age) tivity, each of which consisted of gender-and tested were found to have a disease causing muta- age-matched individuals who had not experienced tion. any cancer. Next, a phenotype-genotype association Apart from the high-risk genes several low risk analysis was carried out, comparing these two genes could also contribute to the development of groups in terms of HLA class I genotypes and SNPs breast cancer. These low susceptibility genes in- in the NKG2D gene. clude those that are involved in carcinogen activa- We found that specific HLA-B and C genotypes tion and inactivation, estrogen metabolism, growth were associated with NK activity, implying a role factors and their receptors etc. Single nucleotide of HLA class I molecules in maintaining a stable polymorphisms in these genes could contribute to repertoire of NK cells; furthermore we succeeded in changes in their functional efficiency. Examples of identifying two haplotype blocks in the NKG2D these include Cyp19 (Trp39Arg), Cyp17 (T-34C) gene region, each of which generated two major GSTP1 (Ile462Val), GSTM1 (Present/Null), TGF haplotype alleles closely related to low and high β (Leu10Pro) and c-erbB2 (Ile655Val). NK activity (P<0.0001). In addition, these haplo- Chemo-prevention is likely to contribute sig- types were found to be significantly associated with nificantly in the prevention of several cancers in- cancer risk, in terms of a case-control study within cluding that of breast cancer. Tamoxifen for exam- this cohort. We previously found that selected life- ple has been shown to reduce the risk of contralat- style factors known as good health practices were eral breast cancers. The trials evaluating the role of associated with high NK activity. In our study, we steroidal and non-steroidal aromatase inhibitors are are looking for further evidence of “individualized already underway and should provide the critical immuno-prevention of cancer”: NK activ- answers to the efficacy of this approach in the ity-enhancing effects of lifestyle may differ among high-risk group. individuals with different NKG2D haplotypes.

97 Forefront of cancer prevention in Asia death and disability, and cancer is either the second Robert Burton or third commonest cause of death in most Asian countries. The major risk factors for NCD in Asia International Union Against Cancer, Geneva, Switzer- are tobacco use, unhealthy nutrition, physical inac- land, National Cancer Control Initiative, Melbourne, tivity and alcohol abuse. In some Asian countries Australia more than half of all adult males use tobacco, and Only primary prevention and early detection overweight/obesity is a rapidly developing problem and removal of pre-malignant lesions can reduce in many countries. Therefore integrated NCD pre- cancer incidence, although early detection of many vention programs are being developed and imple- cancers and effective treatment can cure the major- mented in a number of Asian countries, of which ity of cancer patients. The World Health Organiza- the Philippines is the leading example. tion has estimated that failure to implement effec- Vaccination (immunisation) is the great hope tive cancer prevention programs will result in the for prevention of the infectious cancers, and hepati- global burden of cancer increasing from 10 million tis B vaccination of newborn children has been new cases in the year 2000, to 15 million in 2020 progressively introduced in Asia since the early with about 10 million cancer deaths in that year. 1980's. Finally, cervical screening and removal of About 85 percent of cancer is caused by environ- premalignant lesions, using low technology screen- mental exposure, most of which are due to non- ing tests and simple surgical procedures preformed communicable diseases (NCD), and about 20 per- by trained health workers, has great potential to cent to communicable diseases. About half of the dramatically decrease cervical cancer mortality, 10 million new cases of cancer in the year 2000 which is the second commonest cancer of women in were preventable, using our current knowledge. Asia. In Asia, NCD are now the commonest causes of

98 Author index for research reports and publications ______

Adachi, K. 56 Hara, M. J256, A057 Akatsuka, Y. 30, 31, 32, 33, J066, J123, J124, Harada, H. J201 J179, J227, J243, J244, J255, J263, Harano, T. J247, J262 J269, R001, R002, R003, R004, R005, R006, R043, R044, A001, Hasegawa, Y. J034, J038, J073, J112, J126, J201 A002, A055, A056 Hashimoto, A. J306 Akazawa, T. J002 Hatooka, S. 7, 51, J133, J154, J298, J299, Aoki, K. R036 A019 Arimura, N. 49, J005 Hayashi, N. J279 Cao, X. 15, J010, J011, J157, J159, J160, Hayashi, Y. 49, J067, J175, J184, J203 J194, J254 Hayashita, Y. 21, J041, J174 Chen, G.Y. 42, J306 Hida, T. 21, J154, J244 Daikoku, T. 35, J014, J015, J068, J130, J131, Hirai, T. 7, J134, J135, J151, J153, A042 J224, R007, A003, A004, A014, A028 Hirata, A. 15, J007, J043, J044, J060, J148, J158, J272, J273, R059 Demachi-Okamura, A. 30, J243, J244 Hirose, K. 5, 7, 12, J037, J046, J047, J057, Fujii, K. J023 J151, J153, J161, J187, J188, J189, Fujita, M. J014, J015, J024, J130, J131, J224, J190, J191, J237, J261, J279, J298, J229, R062 J299, R014, R035, R036, A006, A019, A031, A038, A045, A064 Fukami, H. J133, J175, J272, J273 Hishida, A. J048, J049, J223 Fukui, F. J197, R027 Horio, Y. 20, J116, J154, J205, J206 Fukui, T. 20, 21, J026, J079, J126, J230 Hoshino, Y. J106, J109, J228 Gao, C. J030, J031 Hosokawa, Y. 24, J050, J051, J172, J239, J312, Goto, H. 47, 48, J020, J033, J035, J043, R015, R016 J064, J144, J198, J259, J293, J308, R008, R009, R010, R011, R012, Huang, X-E. 12, J057, J225 R022 Ichinose, M. J146, J185 Goto, M. J034 Ieda, T. 56 Goto, Y. 20, 44, J035, J036, R027 Iidaka, T. J059, J060 Gotoh, M. J062 Ikehara, S. 15, 16, J061, J062 Hagino, M. 55 Ikehara, Y. 15, 16, J009, J061, J062, J176, Hagiwara, K. 44 J185, J194, J199, J290, J307, R040, A008, A009, A010, A043 Hamajima, N. 12, J030, J031, J035, J036, J037, J047, J048, J049, J075, J076, J077, Imai, T. J129 J103, J108, J134, J135, J139, J149, Inada, K. J034, J043, J063, J146, J156, J158, J150, J161, J169, J179, J187, J188, J185, J195, J211, J212, J254, J270, J189, J190, J191, J192, J193, J198, R051, R064 J223, J258, J279, R040, A017, A026, A035, A036 Inagaki, M. 47, 48, 49, J001, J005, J020, J024, J033, J038, J043, J083, J087, J170,

99 J171, J184, J203, J226, J245, J267, Karnan, S. 26, 57, J092, J099, J177, J209, J293, J302, J308, J309, R008, J239, J240, J241, J242, J312 R009, R019, R020, R021, R025, Kasugai, Y. J101, J239 R026, R032, A011, A012 Kato, K. 57, J052, J081, J181, J182, J183 Inagaki, N. 49, J309 Kato, S. 15, J104, J158, J159, J160, J211, Inoko, A. 48, 49, J184, R022, A012 J254 Inoue, M. J065, J074, J108 Kato, T. 7, J134, J135, J151, J153, J162, Ishida, H. 44, J291, J292 A042 Ishida, T. J066 Kawajiri, A. 47, J024, J033, J038, J302, R032 Ishizaki, K. 51, 52, J067, J132, J133, J175, Kimura, N. 42, J118, J289, R029, A066, A067, J201, J224, J230, A013, A029, A068 A030 Kitamura, T. J012, J203 Isomura, H. 37, J014, J015, J016, J068, J069, Kiyono, T. 32, 47, 49, J033, J130, J224, J229 J130, J131, J224, R023, R024, A015 Kobayashi, T. 16, J169, J297, A069 Ito, H. 5, 7, J036, J040, J046, J057, J074, Kodera, Y. 31, 32, J058, J072, J103, J113, J075, J076, J077, J078, J151, J153, J114, J115, J123, J124, J162, J176, J161, J178, J191, J237, J258, J298, J179, J180, J196, J202, J236, J255, J299, A016, A017, A018, A019, J263, J307, R046, A042, A055, A031, A038, A045, A064 A056 Ito, S. J072, J113, J114, J115, J157, J162, Koike, K. J134, J135 R045, A041, A042, A058 Koike, T. 41, 42, J118, J155, R028, R029, Ito, Y. 30, 32, J019, J080, J105, J106, A040 J110, J111, J243, J244, Kojima, N. 16, A008 Iwata, H. 7, J046, J047, J085, J086, J164 Kondo, E. 30, 32, J123, J124, J125 Izawa, I. 48, 49, J083, J087, J117, J184, Kondo, M. 20, J026, J073, J079, J126, J144 J302, J308, J309, R025, R026, A012 Kondo, Y. 20 Izawa, M. 41, 42, J155, J278, J306, R028, Konishi, H. J021, J145, J247 R029, A040 Kontani, K. J127 Joh, T. J156, J158, J160, J195, J254, J258, Koshikawa, K. J039, J262 A041 Kozaki, K. J276 Kagami, Y. J006, J022, J048, J123, J150 Kudoh, A. 35, J014, J015, J016, J068, J130, Kameoka, Y. J092, J101, J173, J242 J131, J224, R024, R033, R061, Kanamori, A. 44, J291, J292, R013 R062, A003, A004, A014, A027, A028, A059, A060 Kanemitsu, Y. 7, J134, J135, J151, J153 Kumamoto, K. J118, J155, R034 Kannagi, R. 41, 42, 44, J045, J071, J091, J093, J094, J118, J138, J155, J166, J197, Kumimoto, H. 51, J077, J132, J133, J230, A029, J210, J222, J232, J257, J277, J278, A030 J289, J291, J292, J306, R027, Kuriki, K. 7, 12, J012, J088, J089, J090, J134, R028, R029, R030, R031, R034, J135, J136, J259, J260, R042, R041, A020, A021, A022, A023, R055, R056, A031, A038, A064 A024, A025, A032, A040, A053, A062, A066, A067, A068 Kuroishi, T. R035, R036

100 Kuzushima, K. 30, 31, 32, 33, J002, J003, J008, Morishima, Y. 7, 26, 30, 31, 32, J006, J048, J092, J032, J109, J123, J124, J179, J228, J099, J101, J123, J124, J149, J150, J243, J244, J246, J263, J268, J269, J177, J179, J208, J237, J240, J241, J287, R037, A001 J242, J244, J255, J263, J312, R002, A036, A037, A055, A056 Kuzuya, K. J124, J187, J188, J189, J190, J191 Murate, T. 44 Kyogashima, M. 44, J098, J138, J143, R038, A032 Nagata, K. J038, J170, J171, J184, J203, J226, J302, A012 Maeda, Y. J129 Nakagawa, M. J050, J172, J173 Maeno, K. J145, J174 Nakagawa, T. J174 Masuda, A. J145, J174, J200 Nakamura, Hir. 55 Masui, T. A018 Nakamura, Hid. 52, J067, J175 Matsudaira, Y. 33, J269 Nakamura, S. 7, 25, 26, J006, J029, J062, J064, Matsukage, A. J229 J088, J090, J092, J095, J099, J101, Matsuo, K. 7, 12, J023, J025, J031, J035, J046, J149, J150, J152, J156, J157, J177, J047, J048, J052, J053, J054, J055, J207, J208, J234, J235, J237, J240, J056, J057, J058, J075, J076, J077, J241, J242, J270, J271, J279, J312, J085, J086, J103, J129, J134, J135, A009, A036, A037, A045 J149, J150, J151, J152, J153, J164, Nakamura, T. 25, J064, J157, A041, A058 J188, J189, J191, J230, J231, J237, J240, J241, J242, J256, J258, J265, Nakanishi, H. 15, 16, J029, J062, J072, J113, J266, J276, J285, J295, J296, J297, J114, J115, J162, J176, J185, J194, J298, J299, J300, J301, J310, J196, J290, J307, J311, R045, R017, R018, R039, R040, R063, R046, A008, A009, A042, A043 A005, A007, A017, A019, A026, Nakanishi, T. 7, 32, J187, J188, J189, J190, J191 A031, A033, A034, A035, A036, A037, A038, A039, A045, A057, Nakashima, Y. 26, J177 A061, A064, A069, A070, A071 Nakasu, S. 37 Matsuzawa, K. J167 Nawa, A. 32, J124, J187, J188, J189, J190, Mitsudomi, T. 7, 30, J021, J075, J100, J116, J128, J191 J154, J204, J243, J244, J247, J262, Nishi, Y. 55 J303, J304, J305, R057, A017 Nishida, K. 30, 33, J095, J243, J244, J269 Miyazaki, K. 41, 42, J118, J155, J306, R029, R041, A040 Nishida, T. J179, J180, J255, A055, A056 Miyazaki, M. 31, J066, J263 Nishimoto, Y. J133 Mizoshita, T. 15, 16, 17, J010, J011, J034, J043, Nishio, M. J234 J063, J104, J148, J156, J157, J158, Nishizawa, M. 48, 55, J184, A012 J159, J160, J195, J211, J212, J254, J270, J271, R052, R053, R054, Niwa, T. 16, J049, J062, J185, J186, J290, A041, A058 A008, A009 Mizuno, M. 55 Nozaki, K. J010, J011, J156, J194 Mizutani, K. J298 Obata, Y. 33, J141, J269, R058 Mochizuki, Y. J072, J115, J162, R045 Ogasawara, N. 16, J010, J148, J158, J195, J254, A041 Moore, M. J260, R042, R056 Ogura, M. J048, J150 Morishima, S. 32, R043

101 Oguri, T. 48, 49, J040, J293 Sugimoto, M. 49 Ohashi, N. 16, J196, J307 Sugiura, T. 7, J075, A017 Okada, Y. J245 Suguro-Katayama, M. 26, J092, J099, J241, J242 Okanami, Y. 33, J269 Suzuki, H. 24, J050, J051 Okuma, K. J076, J077 Suzuki, R. 25, 26, J006, J085, J092, J099, Osada, H. 20, 21, J004, J021, J041, J100, J137, J149, J150, J165, J172, J173, J145, J174, J204, J205, J206, J247, J177, J180, J202, J207, J234, J235, J262 J236, J239, J240, J242, J312, Oshiro, A. J208 R050, A037 Ota, A. J092, J209, J240, J242 Suzuki, S. J081, J088, J089, J090, J120, J122, Otsuka, T. 17, J157, J211, J212 J142, J192, J193, J214, J233, J249, J252, J253, J259, J260, J281, J283, Ozeki, S. J269 J284, J288, R055, R056 Saito, H. J204 Suzuki, T. 7, J147, J151, J153, J200, J236, Saito, N. 52 J237, J296, J299, A045 Saito, T. 7, J075, J076, J077, J134, J135, Tagawa, H. 26, J092, J099, J101, J177, J208, J145, J151, J153, J187, J237, J298, J209, J238, J239, J240, J241, J242, J299, A019, A031 J312, A037 Sakai, H. J007, J043, J044, J059, J060, J218, Taguchi, O. 33, 45, J127, J248, J269 J272, J273 Taji, H. J048, J150, J180 Sato, N. 20, J001, J026 Tajima, Ka. 5, 7, 12, J012, J013, J018, J030, Sekido, Y. 20, 21, J026, J041, J049, J073, J031, J037, J046, J047, J048, J057, J079, J112, J126, J144, J163, J074, J075, J076, J077, J108, J134, R047 J135, J136, J139, J140, J147, J149, J150, J151, J152, J153, J161, J168, Seto, M. 24, 25, 26, 57, J022, J050, J051, J169, J187, J188, J189, J190, J191, J064, J092, J095, J099, J101, J125, J225, J230, J237, J258, J261, J279, J208, J238, J239, J149, J150, J172, J298, J299, J300, J301, J313, , J177, J209, J223, J240, J241, J242, R014, R035, R040, R042, R036, J274, J312, R015, R048, A036, A006, A016, A017, A018, A019, A037 A026, A031, A034, A035, A036, Shima, H. 49 A037, A038, A045, A046, A047, A048, A049, A050, A051, A052, Shimizu, N. J011, J194, J266 A064, A071 Shimizu, S. J040, J178 Tajima, Ko. 30, J021, J123, J243, J244 Shinoda, M. 7, 51, J133, J154, J298, J299, Takahashi, M. J039, J102, J144, J163 A019 Takahashi, Ta. 20, 21, J004, J021, J041, J042, Shirai, N. J059, J060 J084, J100, J116, J128, J145, J154, Shirata, N. 36, J015, J016, J068, J131, J224, J174, J200, J204, J205, J206, J247, A014, A028, A044 J262, J303, J304, J305, R057 Shiromizu, T. 48, J038, R049 Takahashi, To. 30, 31, 32, 33, J002, J066, J070, J123, J124, J179, J243, J244, J262, Sobue, S. 44 J263, J269, R058, A001 Sugaya, N. J109, J228 Takenaka, Y. 16, J010, J159, J160, J211, J271, Sugaya, Y. J014, J015, J016, J130, J131, J224 A041

102 Takezaki, T. 12, J012, J013, J030, J031, J057, A037 J140, J147, J168, J225, J279, Uchida, K. J276 J313, , R035, R036 Ueda, R. 57, J040, J064, J066, J075, J078, Tamiya-Koizumi, K. 44, J138 J095, J145, J178, J236, J237, Tanaka, H. 17, J010, J100, J104, J160, J185, A017, A045 J194, J212, J219, J254, J270, J271 Wakai, K. 7, 11, 12, J012, J013, J017, J046, Taniguchi, T. 20, 21 J057, J065, J077, J081, J082, J102, J107, J112, J119, J120, J121, J122, Tatematsu, M. 15, 16, 17, J007, J010, J011, J027, J141, J142, J151, J153, J167, J192, J028, J029, J034, J043, J044, J059, J193, J198, J213, J214, J215, J216, J060, J062, J063, J072, J096, J097, J217, J219, J220, J221, J233, J237, J104, J113, J114, J115, J146, J148, J249, J250, J251, J252, J253, J279, J156, J157, J158, J159, J160, J162, J280, J281, J282, J283, J284, J286, J176, J185, J186, J194, J195, J196, J288, J298, J299, R065, R066, J211, J212, J218, J254, J270, J271, A019, A026, A031, A038, A045, J272, J273, J290, J294, J307, J311, A063, A064, A065 R045, R046, R051, R052, R053, R054, R059, R060, R064, A008, Watanabe, A. J058 A009, A041, A042, A054, A058 Yabuta, T. J118 Tatematsu, Y. 20, 21, J041, J100, J204, J205, Yamachika, T. J290 J206 Yamada, N. J259 Terashima, M. 55 Yamaguchi, M. 44, J092, J099, J207, J242, J291, Teratani, M. 56 J292 Tokumasu, S. 55 Yamaguchi, T. 48, 49, J181, J182, J183, J293 Tomida, S. 21, J021, J041, J100, J247, J262, Yamamoto, M. 55, J007, J044, J059, J060, J273 R057 Yamamura, Y. J072, J113, J115, J156, J157, J158, Tominaga, S. J147, R035, R036 J162, J211, J270, J271, A041, Torikai, H. 31, 32, J263 A042, A058 Tsujimura, K. 30, 31, 32, 33, J002, J070, J123, Yamane, Y. J132, J133 J124, J179, J243, J244, J263, J269, Yanagisawa, K. 21, J041, J100, J145, J174, J247, R058 J262, R057 Tsukamoto, T. 15, 16, 17, J007, J010, J011, J027, Yang, C. X. 7, 12, J298, J299, J300, J301, J028, J034, J043, J044, J059, J060, A038, A071 J063, J096, J097, J104, J146, J148, J156, J157, J158, J159, J160, J185, Yasuda, T. 56 J186, J195, J211, J212, J254, J270, Yasui, K. J176, J290 J271, J272, J273, R051, R052, R053, R054, R059, R060, R064, Yasui, Y. 52, J067, J302 A041, A058 Yatabe, Y. 7, 20, 21, J021, J039, J041, J084, Tsurumi, T. 35, 36, 37, J003, J014, J015, J016, J100, J116, J128, J154, J204, J206, J032, J068, J069, J130, J131, J224, J247, J262, J303, J304, J305, J228, J229, R023, R024, R033, R057 R061, R062, A003, A004, A014, Yin, J. 42, J306 A015, A027, A028, A044, A059, A060 Yokoyama, N. J009, J199 Tsuzuki, S. 25, 26, 57, J092, J172, J209, J239, Yokoyama, T. 20, 21, 48, J126, J184, J219, J293, J241, J242, J264, J274, J275, J312, J308, R019, R067

103 Yokoyama, Y. A043 Yoshikawa, K. J125 Yonezumi, M. J172 Zhang, X. J312 Yoshida, T. J103 Zou, P. 49 Yoshikawa, A. J273

104