2006-2007) of the Aichi Cancer Center Research Institute

Aichi Cancer Center Research Institute

Scientific Report 2006 – 2007

Chikusa-ku, Nagoya 464-8681 Japan

(The Cover) Looking at the North Building of Aichi Cancer Center Research Institute over the flower garden in bloom.

Published by 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. Tatsuya Tsurumi (Division of Virology) Dr. Keiichiro Sakuma (Division of Molecular Pathology) Dr. Hiroshi Kumimoto (Division of Central Laboratory & Radiation Biology) Dr. Malcolm A. Moore, English Editor Ms. Hiromi Tamaki (Director's Office)

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

Contents

Preface Kazuo Tajima 1

Organization of the Aichi Cancer Center Research Institute 2

SCIENTIFIC REPORTS Division of Epidemiology and Prevention General summary 5 1. Epidemiological studies using data from Aichi Cancer Registry Matsuo, K., Kawase, T., Ito, H., Tanaka, H. and Tajima, K. 6 2. The hospital-based epidemiologic research program at Aichi Cancer Center (HERPACC) Matsuo, K., Hiraki, A., Suzuki, T., Wakai, K., Hirose, K., Ito, H., Saito, T., Kuriki, K., Hasegawa, Y., Kanemitsu, Y., Hirai, T., Kato, T., Hamajima, N. and Tajima, K. 7 2.1. Gene-gene interactions between polymorphisms in alcohol-metabolizing enzyme genes regarding the risk of alcohol-related cancers 2.2. Impact of polymorphisms in alcohol-metabolizing enzyme genes on drinking behavior 2.3. Folate and folate-related enzyme polymorphisms and cancers of the head and neck, lung and breast 2.4. Family history and cancer risk 2.5. Breast and stomach cancer risk and n-3 highly unsaturated fatty acids in erythrocytes 3. Clinical applications of epidemiology 3.1. Identification of HLA allele mismatch combinations and amino acid substitution positions associated with GVL effects after unrelated HSCT: Analysis from the Japan Marrow Donor Program Kawase, T., Matsuo, K., Kashiwase, K., Inoko, H., Saji, H., Kato, S., Sasazuki, T., Kodera, Y. and Morishima, Y. 10 3.2. Exploration of risk factors of non-small-cell lung cancer with or without epidermal growth factor receptor (EGFR) mutations Matsuo, K., Ito, H., Suzuki, T., Hiraki, A., Yatabe, Y. and Mitsudomi, T. 11 4. Comparative epidemiological study of cancers on the increase, focusing on Korea, Japan and China (KOJACH study) Tajima, K., Matsuo, K., Hiraki, A., Kuriki, K., Wakai, K., Hirose, K., Gao C-M., Yoo K-Y., Ahn Y-O., Zhou Z-Y. 12 5. Ethnoepidemiologic studies on virus-related cancer Tajima, K., Sonoda, S., Chiba, H., Senoo, H. 13

Division of Oncological Pathology General summary 15 1. Loss of MUC2 expression correlates with progression along the adenoma- carcinoma sequence pathway as well as de novo carcinogenesis in the colon Mizoshita, T., Tsukamoto, T., Inada, K., Hirano, N., Tajika, M., Nakamura, T., Ban, H. and Tatematsu, M. 16

i 2. Inhibitory effects of nordihydroguaiaretic acid, a plant lignan, on Helicobacter pylori-associated gastric carcinogenesis in Mongolian gerbils Tatematsu, M., Toyoda, T., Tsukamoto, T., Mizoshita, T., Nishibe, S., Deyama, T., Takenaka, Y., Hirano, N., Tanaka, H., Takasu, S., Ban, H.,

Kumagai, T., Inada, K. and Utsunomiya, H. 16 3. 4-Vinyl-2,6-dimethoxyphenol (canolol) suppresses oxidative stress and gastric carcinogenesis in Helicobacter pylori-infected carcinogen-treated Mongolian gerbils Tsukamoto, T., Cao, X., Seki, T., Tanaka, H., Morimura, S., Cao, L., Mizoshita, T., Ban, H., Toyoda, T., Maeda, H. and Tatematsu, M. 17 4. Synergistic upregulation of inducible nitric oxide synthase and cyclooxygenase-2 in gastric mucosa of Mongolian gerbils by a high-salt diet and Helicobacter pylori infection Toyoda, T., Tsukamoto, T., Hirano, N., Mizoshita, T., Kato, S., Takasu, S., Ban, H. and Tatematsu, M. 17 5. Role of IKK and oscillatory NFkappaB kinetics in MMP-9 gene expression and chemoresistance to 5-fluorouracil in RKO colorectal cancer cells Fukuyama, R., Ng, K.P., Cicek, M., Kelleher, C., Niculaita, R., Casey, G., Sizemore, N. 18 6. A new diagnostic and therapeutic strategy targeting micrometastasis for prevention of peritoneal recurrence in gas-tric cancer patients Nakanishi, H., Ito, S., Kodera, Y., and Tatematsu, M. 18

Division of Molecular Oncology General summary 20 1. Genomic profiling of malignant pleural mesothelioma with array-based comparative genomic hybridization shows frequent non-random chromosomal alteration regions including JUN amplification on 1p32 Taniguchi, T., Karnan, S., Fukui, T., Yokoyama, T., Tagawa, H., Yokoi, K., Ueda, Y., Mitsudomi, T., Horio, Y., Hida, T., Yatabe, Y., Seto, M., Sekido, Y. 20 2. CLCP1 interacts with semaphorin 4B and regulates motility of lung cancer cells Osada, H., Nagai, H., Sugito, N., Matsubara, H., Tatematsu, Y., Hida, T., Sekido, Y., Nagino, M., Nimura, Y., Takahashi, Ta. 21 3. Variable DNA methylation patterns associated with progression of disease in hepatocellular carcinomas Gao, W., Kondo, Y., Shimizu, Y., Sano, T., Yamao, K., Goto, Y., Ito, M.,

Murakami, H., Osada, H., and Sekido, Y. 21

Division of Molecular Medicine General summary 24 1. Genetic abnormalities involved in t(12; 21) TEL-AML1 acute lymphoblastic leukemia: analysis by means of array-based comparative genomic hybridization Tsuzuki, S., Karnan, S. and Seto, M. 25 2. Isoform-specific potentiation of stem and progenitor cell engraftment by AML1/RUNX1 Tsuzuki, S., Matsuo, K. and Seto, M. 25 3. Chromosomal imbalances are associated with outcome of Helicobacter ii pylori eradication in t(11;18)(q21;q21) negative gastric MALT lymphomas Fukuhara, N., Nakamura, T., Nakagawa, M., Tagawa, H., Takeuchi, I., Yatabe, Y., Morishima, Y., Nakamura, S. and Seto, M. 25 4. Identification of the target gene at chromosome deletion 6q23.3-24.1 in ocular adnexal mucosa associated lymphoid tissue lymphomas Honma, K., Kim, W-S., Ko, Y-H. and Seto, M. 26 5. Array CGH analysis of PTCL-U for identification of a distinct subgroup Nakagawa, M., Nakagawa-Oshiro, A., Karnan, S., Nakamura, S., Takeuchi, I., Ohshima, K. and Seto, M. 26

Division of Immunology General summary 28 1. The Human Cathepsin H Gene Encodes Two Novel Minor Histocompatibility Antigen Epitopes Restricted by HLA-A*3101 and -A*3303 Torikai, H., Akatsuka, Y., Miyazaki, M., Tsujimura, A., Yatabe, Y., Kawase, T., Nakao, Y., Tsujimura, K., Motoyoshi, K., Morishima Y., Kodera Y., Kuzushima, K., and Takahashi, To. 29 2. Identification of a novel minor histocompatibility antigen generated by alternative splicing due to an intronic SNP in HMSD Kawase, T., Akatsuka, Y., Torikai, H., Morishima, S., Oka, A., Tsujimura, A., Miyazaki, M., Tsujimura, K., Miyamura, K., Ogawa, S., Inoko, H., Morishima, Y., Kodera, Y., Kuzushima, K., Takahashi, To. 29 3. The HLA-A*0201-restricted minor histocompatibility antigen HA-1H peptide can also be presented by another HLA-A2 subtype, A*0206 Torikai, H., Akatsuka, Y., Miyauchi, H., Terakura, S., Onizuka, M., Tsujimura, K., Miyamura, K., Morishima, Y., Kodera Y., Kuzushima, K., and Takahashi, To. 30 4. Epstein-Barr virus (EBV) nuclear antigen 1-specific CD4+ T cells directly kill EBV-carrying NK and T cells Demachi-Okamura, A., Ito, Y., Akatsuka, Y., Tsujimura, K., Morishima Y., Takahashi, To., and Kuzushima, K. 31 5. Effective antigen presentation to EBNA1-specific CD4+ T lymphocytes Watanabe, Y., Demachi-Okamura, A., and Kuzushima, K. 31

Division of Virology General summary 33 1. Phosphorylation of MCM4 at sites inactivating DNA helicase activity of the MCM4-6-7 complex during Epstein-Barr virus productive replication Kudoh, A. and Tsurumi, T. 33 2. Expression of Epstein-Barr virus BZLF1 immediate-early protein induces p53 degradation independent of MDM2, leading to repression of p53- mediated transcription Sato, Y. and Tsurumi, T. 33 3. Enhanced phosphorylation of transcription factor Sp1 in response to herpes simplex virus type 1 infection is dependent on the ataxia telangiectasia- mutated protein Iwahori, S. and Tsurumi, T. 34 4. The purification of the product of Epstein-Barr virus BZLF1 gene Nakasu, S. and Tsurumi, T. 34

iii 5. A cis-element between the TATA box and the transcription start site of the major immediate-early (MIE) promoter of human cytomegalovirus determines efficiency of viral replication Isomura, H. and Tsurumi, T. 35

Division of Molecular Pathology General summary 37 1. Significance of interconversion between carbohydrate ligands for selectins and siglecs expressed on normal colonic epithelium and cancer cells Miyazaki, K. Izawa, M., Ohmori, K., Hashimoto, Y. and Kannagi, R. 38 2. Tumor hypoxia induces expression of gangliosides containing abnormal sialic acid on cancer cells Yin, J., Izawa, M., Miyazaki, K., Kitajima, K. and Kannagi, R. 38 3. Studies on cell adhesion activity of CD44 3.1. Roles of carbohydrate side chains of the CD44 molecule Lim, K.T., Miyazaki, K., Kimura, N. Murakami, Y. and Kannagi, R. 39 3.2. Potentiation of CD44-mediated leukocyte adhesion to the hyaluronan substratum by SHAP Zhuo, L.S., Kanamori, A., Kannagi, R. Itano, N., Wu, J.W., Hamaguchi, M., Ishiguro, N., and Kimata, K. 40 4. Diversity of sulfatide molecular species from biological materials by MALDI-TOF MS Kyogashima, M., Tadano-Aritomi, K., Murate, T., Tamiya-Koizumi, K., Hara, A., Aoyama, T. and Kannagi, R. 40 5. Molecular profiling of heparan sulfate glycosaminoglycans in human cancers using monoclonal antibodies Yusa, A., Fujii, M., Goto, Y., Suzuki, K., Ishimaru , T., Yamamoto, K., Kim, Y., Kimata, K., Iwata, H., Kyogashima, M., and Kannagi, R. 41 6. Studies on molecules involved in immune regulation 6.1. Interaction of GATA-3/T-bet transcription factors regulates expression of selectin ligands in human leukemic cells Chen, G.Y., Osada, H., Santamaria-Babi, L.F., and Kannagi, R. 41 6.2. Identification of cutaneous lymphocyte antigen as sialyl 6-sulfo-Lewis X, a selectin ligand expressed on a subset of skin-homing helper memory T cells Ohmori, K., Sakuma, K., Kiso, M., Imai, T., Yoshie, O., Hasegawa, H., Matsushima, K. and Kannagi, R. 42 6.3. Identification of α2-6 sialylated 6-sulfo- LacNAc as a preferred ligand for CD22/siglec-2 on human B-lymphocytes Kimura, N., Ohmori, K., Miyazaki, K., Izawa, M., Matsuzaki, Y., Yasuda, Y., Takematsu, H., Kozutsumi, Y., Moriyama, A., and Kannagi, R. 43 6.4. α-Internexin as a corneal autoantigen in a spontaneous auto-immune keratitis mouse model Hattori, T., Takeuchi, M., Usui, M. and Taguchi, O. 43

Division of Biochemistry General summary 46 1. Chk1 phosphorylation by cyclin-dependent kinase 1 controls mitotic entry through cytoplasmic sequestration of Chk1 Enomoto, M., Goto, H., Kasahara, K., Ikegami, Y., Yamaguchi, T., Shiromizu, T., Tomono, Y., Tsujimura, K., Kiyono, T. and Inagaki, M. 46 iv 2. Spatial regulation of Chk1 phosphorylation in checkpoint response Kasahara, K., Ikegami, Y., Goto, H., Enomoto, M., Tomono, Y., Tsujimura, K., Kiyono, T. and Inagaki, M. 47 3. Palmitoylation of ERBIN is required for its plasma membrane localization Izawa, I., Nishizawa, M., Hayashi, Y. and Inagaki, M. 47 4. Trichoplein and Albatross: unexpected passenger among keratin filaments, centriole and cell-cell contacts Inoko, A., Zou, P., Ohmuro-Matsuyama, Y., Shiromizu, T., Ibi, M., Hayashi, Y., Yonemura, S., Nakayama, M., Kaibuchi, K., Kiyono, T., Izawa, I. and Inagaki, M. 48

Division of Central Laboratory & Radiation Biology General summary 50 1. Mitochondria DNA instability due to ionizing radiation Kumimoto, H., Saito, N. and Ishizaki, K. 50 2. Role of ATM in repair of the double-strand break induced by low-dose-rate radiation

Nakamura, Hid., Yasui, Y., Kitajima, S., Saito, N. and Ishizaki, K. 51 3. Lethal effects of a heavy-ion beam on AT-heterozygote cells

Kitajima, S., Nakamura, Hid., Yasui, Y. and Ishizaki, K. 52

Central Service Unit General summary Nakamura, Hir., Nishizawa, M., Yamamoto, M. and Tanaka, Har. 54 Librarians Yasuda, T., Shibata, T., Adachi, K. and Horayama, K. 55

Researches Supported by Special Project Programme 1. Roles of mitotic kinases in carcinogenesis Goto, H., Enomoto, M., Kasahara, K., Ikegami, Y., Yamaguchi, T., Shiromizu, T., Tomono, Y., Tsujimura, K., Kiyono, T. Kawajiri, A., Hayashi, Y., Urano, T., Furukawa, K., Nigg, E.A., Hayashi, N. and Inagaki, M. 56 2. Array CGH analysis of malignant lym-phomas and its application for molecular diagnosis Tagawa, H. 57

Publications 1 Journals 58 2 Reviews and books 76 3. Abstracts for international conferences 78

Records of seminars 84

Records of symposia 87

Author index for research reports and publications 97

From left to right Ms. H. Tamaki, Dr, K. Tajima, and Mrs. J. Onishi.

Preface ______

First of all, let me introduce myself to you. I had been engaged in epidemiological studies at the Division of Epidemiology and Prevention during the last 30 years. I was promoted to Director of the Institute on April, 2006, as the former director, Dr. Toshitada Takahashi, M.D., D.M.Sci, became President of the Aichi Cancer Center. It is my pleasure to share with you the 20th Scientific Report (2006-2007) of the Aichi Cancer Center Research Institute. Since its establishment 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 pages, 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 54 full-time staff members, 42 researchers and 12 research assistants, as well as 16 research residents, are now conducting a wide range of studies, together with 7 graduate school students affiliated with Nagoya University School of Medicine, Nagoya, and approximately 40 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, 2008

Kazuo Tajima, M.D., M.P.H., D.M.Sci. Director

1 Organization ofthe Aichi Cancer Center Research institute

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SCIENTIFIC REPORTS

From left to right First row: Dr. T. Kawase, Dr. K. Matsuo, Dr. H. Tanaka, Dr. A. Hiraki, Dr. T. Suzuki Second row: Dr. J. Kanda, Dr. K. Kidokoro, Ms. S. Hiraiwa, Ms. M. Nakahama, Ms. Y. Yamauchi, Ms. T. Ito Third row: Ms. S. Inui, Dr. S. Hosono, Ms. K. Mizutani, Ms. C. Taniguchi, Dr. T. Okasaka Inset: Dr. K. Kuriki, Ms. M. Watanabe, Ms. S. Mano, Ms. K. Hasegawa, Ms. T. Nishiwaki, Ms.S. Irikura, Ms. K. Suganuma, Dr. M. Nishira, Dr. T. Paku, Ms. K. Fukaya, Ms. M. Ishida, Ms. C. Kanto, Ms. R. Saito

4 ______Division of Epidemiology and Prevention

Kazuo Tajima, M.D., D.M.Sc., M.P.H. Chief (until March 2006) Hideo Tanaka, M.D., PhD. Chief (as of October 2007) Kenji Wakai, M.D., PhD. Section Head (until September 2006) Akio Hiraki, M.D., PhD. Section Head (as of April 2006) Kaoru Hirose, B.P., D.M.Sc. Senior Researcher (until March 2006) Hidemi Ito, M.D., PhD. Senior Researcher (until July 2006) Keitaro Matsuo, M.D., PhD., S.M. Senior Researcher Takeshi Suzuki, M.D, PhD. Senior Researcher (as of April 2007) Takakazu Kawase, M.D., PhD. Senior Researcher (as of April 2007) Toshiko Saito, Research Assistant (until March 2006) Miki Watanabe, Research Assistant (as of April 2006)

Visiting Trainees Kiyonori Kuriki, B.P., D.M.Sc. Research Resident of Foundation for Promotion of Cancer Research Satoyo Hosono, M.D. Nagoya University Graduate School of Medicine (as of May 2007) Kumiko Kidokoro, M.D. Nagoya University Graduate School of Medicine (as of May 2007) Masao Nishira, M.D. Nagoya University Graduate School of Medicine (as of April 2007) Chong Park, M.D. Nagoya University Graduate School of Medicine (as of April 2007) Toshiki Okasaka, M.D. Nagoya University Graduate School of Medicine (as of April 2007) Junya Kanda, M.D. Kyoto University Graduate School of Medicine (as of April 2007)

General Summary The current research activities of the Division of Epidemiology and Prevention cover the following five subjects: 1) descriptive epidemiology of cancer incidence and mortality using data from 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 cancer, with a particular focus on gene-environmental interactions; 3) clinical epidemiology for elucidating better cancer treatment and prevention; 4) a Korea, Japan and China (KOJACH) collaborative study focusing on those cancers which are on the increase for establishment of future prevention programs in all three countries; and 5) ethnoepidemi- ology of tumor viruses among Mongoloids in the Asian-Pacific area. We are engaged in analyzing the data of the Aichi Cancer Registry to obtain estimated annual percent change of incidence rates in designated cancer sites in order to optimize cancer control programs in Aichi Prefecture. Several case-control studies using HERPACC data demonstrated that: 1) there are gene-gene interactions between polymorphisms in genes encoding alcohol-metabolizing enzymes and risk of head and neck, as well as colorectal cancers; 2) interactions exist between alcohol drinking habits and polymorphisms in the folate metabolism pathway regarding head and neck cancer; 3) there are significant associations between risk of prostate and thyroid cancer and a family history of neoplasia at the same sites; 4) intake of n-3 highly unsaturated fatty acids is associated with a lowered risk of breast cancer and gastric cancer. In collaborative studies with clinical departments, we have identified HLA allele mismatch combinations and amino acid substitution positions associated with graft-versus-leukemia (GVL) effects after unrelated hematopoietic stem cell transplantation, and different impacts of smoking on the risk of non-small cell lung cancer by types of epidermal growth factor receptor (EGFR). In addition, international collaborative case-referent studies in Northeast Asian Countries (KOJACH Study) in Seoul, Nagoya, Nanjing, Chongqing, Benxi and Shantou are still ongoing to establish a basis for cancer prevention in the Asian Pacific region. Sero-epidemiologic investigations on HTLV-1/-2 infection among Mongoloids are also continuing in the North Pole region i.e. Greenland.

5 1. Epidemiological studies using males and females. Stratified analysis also data from Aichi Cancer Registry showed significant increased risk of death regardless of the disease extent. The HRs for Matsuo, K., Kawase, T., Ito, H., Tanaka, H. and ever smokers compared with never smokers Tajima, K. were 1.22 (1.13-1.31), 1.29 (1.22-1.37), and Impact of habitual smoking on survival: 1.11 (1.05-1.17) for those whose extent of Since 1999, information on smoking habits disease were local, regional and metastatic, of cancer patients has been collected by Aichi respectively. In the cancer site specific Cancer Registry. The purpose was to exam- analysis, sites in which ever smokers showed ine the survival impact of habitual smoking HRs greater than unity were oral, pharyngeal, using data registered to the Aichi Cancer esophageal, colon, gallbladder, lung, skin, Registry, Japan. The study subjects were reg- uterus, breast and bladder. Lung, female istered cancer cases diagnosed between 1999 breast and uterine cancers showed statisti- and 2004. The relationship between survival cally significant increased risk of death. In and smoking history (never or ever) was as- contrast, only gastric cancer showed signifi- sessed using a Cox’s Proportional Hazard cantly lower risk of death among ever smok- model considering age, sex, extent of disease ers compared with never smokers. and site. We thus found a significantly increased A total of 72,789 cancer patients with risk of death among ever smokers compared smoking history and cancer stage at diagnosis with never smokers, providing evidence that were covered in the analysis. Overall, ever smoking continues to be harmful for people smokers were at higher risk of death com- even after having been diagnosed with can- pared with never smokers (Hazard ratio, HR: cer. 1.28; 95% confidence interval, CI: 1.23-1.32) and this was consistently observed in both

6 2. The Hospital-based epidemiologic demonstrated that this risk was associated research program at Aichi Cancer with significant gene-gene interactions be- Center (HERPACC) tween ADH2 and ALDH2 polymorphisms, as Matsuo, K., Hiraki, A., Suzuki, T., Wakai, K., well as gene-environment interactions be- Hirose, K., Ito, H., Saito, T., Kuriki, K., tween these polymorphisms and alcohol Hasegawa, Y.*1, Kanemitsu, Y.*2, Hirai, T.*2, drinking. Kato, T.*2, Hamajima, N.*3 and Tajima, K. 2) Colorectal cancer: Alcohol consumption 2.1. Gene-gene interactions between is recognized as a risk factor for colorectal polymorphisms in alco- cancer (CRC). Genetic polymorphisms, hol-metabolizing enzyme genes ALDH2 Glu487Lys and ADH2 His47Arg, regarding the risk of alco- which have a strong impact on alcohol me- hol-related cancers tabolism, are common in the Japanese popu- 1) Head and neck cancer: Alcohol con- lation but information on their significance sumption is a strong risk factor for squamous for CRC carcinogenesis has been limited. We cell carcinoma of the head and neck therefore conducted a matched case-control (SCCHN). The aldehyde dehydrogenase2 study with 257 incident CRC cases and 771 (ALDH2) Glu487Lys and alcohol dehydro- non-cancer controls at Aichi Cancer Center, genase 2 (ADH2) His47Arg genetic poly- including analysis of interactions among the morphisms, which have a strong impact on polymorphisms, alcohol exposure and folate alcohol metabolism, are common in the consumption. The ADH2 Arg allele was Japanese population. To clarify the signifi- found to be associated with increased risk, cance of these polymorphisms in SCCHN the odds ratios (ORs) being 1.35 (95% con- carcinogenesis, we conducted a matched fidence interval: 1.00-1.84) and 1.93 case-control study with 239 incident SCCHN (1.06-3.53) for the His/Arg and Arg/Arg subjects and 716 non-cancer controls. Both genotypes, respectively. In contrast, no ap- ADH2 Arg/Arg and ALDH2 Glu/Lys were parent links were observed with the ALDH2 found to be independently associated with genotypes. Individuals having ALDH2 increased risk, with odds ratios (OR) of 2.67 Glu/Glu with ADH2 Arg+, ALDH2 Lys+ (95% confidence interval [CI] 1.51-4.57) and with ADH2 His/His and ALDH2 Lys+ with 1.66 (1.20-2.31), respectively. Further, com- ADH2 Arg+ showed ORs of 0.10 (0.04-0.21), pared with subjects having both ADH2 0.10 (0.06-0.19) and 1.36 (0.94-1.97), re- His/His and ALDH2 Glu/Glu, the adjusted spectively, compared with ALDH2 Glu/Glu OR and its 95% CI for those with both with ADH2 His/His. A statistically signifi- ADH2 Arg/Arg and ALDH2 Glu/Lys was cant gene-gene interaction was found be- 5.00 (2.32-10.71) in all subjects. This com- tween the two polymorphisms for the risk of bination effect was evident in heavy drinkers CRC (p< 0.001). The impact of ALDH2 (11.3, 2.97-43.3) but not in moderate or Lys+ with ADH2 Arg+ was more evident in non-drinkers. Statistically significant low folate consumers (2.32, 1.19-4.55) than gene-environment interactions between the those with high consumption (1.38, two polymorphisms and the drinking level 0.80-2.38). In conclusion, while we failed to were seen (ADH2, p = 0.035, ALDH2, p = find any significant association with the 0.013). Furthermore, we also found a statis- ALDH2 polymorphism itself, significant in- tically significant gene-gene interaction be- teractions between ALDH2 and ADH2 tween the two polymorphisms (p = 0.042). In polymorphisms were observed. Replication conclusion, this case-control study showed a in future studies is warranted. significantly increased risk of SCCHN in subjects with the ADH2 Arg/Arg and 2.2. Impact of polymorphisms in alco- ALDH2 Glu/Lys polymorphisms in a Japa- hol-metabolizing enzyme genes nese population. In addition, our results on drinking behavior

7 1) ADH2 polymorphisms: Although func- behavior was examined with regard to hap- tional effects of the ADH2 His(47) Arg lotypes of the ADH1B and ADH1C poly- polymorphism have been elucidated, the sig- morphisms. Strength of association was as- nificance for habitual drinking has been un- sessed by sex and ALDH2 adjusted ORs and clear. Here, we conducted a cross-sectional their 95% CIs for the haplotypes of the study of 2,299 nonalcoholic Japanese sub- ADH1B and ADH1C polymorphisms. jects (989 men and 1,310 women). Drinking The ORs for habitual drinking in males status, ethanol consumption, and physical re- were significant for ADH1B*2(rapid)- action to one glass of beer were examined ADH1C*2 (slow) (1.03; 95% CI: 1.01 -1.05), with regard to ADH2 and ALDH2 polymor- ADH1B*1 (slow) - ADH1C*1 (rapid) (1.15, phisms. Strength of associations was assessed 1.14-1.16), and ADH1B*1(slow)-ADH1C*2 with reference to age-, sex-, smoking status-, (slow) (1.31, 1.29-1.32) compared with and genotype-adjusted odds ratios and their ADH1B*2(rapid)-ADH1C*1(rapid). Simi- 95% confidence intervals. People with ADH2 larly, a significantly increased risk of heavy His/Arg and Arg/Arg genotypes showed drinking was observed for each haplotype higher risk of being habitual drinkers. Among compared with ADH1B*2(rapid) -ADH1C*1 men, ALDH2 genotype- and con- (rapid). In conclusion, this study showed a founder-adjusted ORs (95% CI) were 1.30 significant impact of the ADH1C polymor- (0.89-1.89) and 3.16 (1.03-9.70), and the phism on habitual drinking, regardless of the trend was significant (p = 0.024). A similar ADH1B/ALDH2 polymorphisms. trend was observed among women. The combination of the two polymorphisms 2.3. Folate and folate-related enzyme demonstrated a clear effect of the ADH2 Arg polymorphisms and cancers of allele among those with ALDH2 Glu/Lys in the head and neck, lung and both sexes (p(trend) = 0.007 for men and breast 0.024 for women). Physical reactions, such as A deficiency in folate is thought to in- flushing and palpitation, were significantly crease the risk of cancer by impairing DNA less common in those with Arg/Arg com- repair synthesis and disrupting DNA methy- pared with other ADH2 genotypes, and this lation, which may lead to protooncogene ac- was more marked when combined with tivation. Polymorphisms in critical enzymes ALDH2 Glu/Lys. Heavy drinker status was involved in the folate metabolism pathway, also strongly associated with ADH2 Arg al- methylenetetrahydrofolate reductase leles. In conclusion, this study showed strong (MTHFR C677T and A1298C), methionine effects of the ADH2 His(47)Arg polymor- synthase (MTR A2756G), methionine syn- phism on habitual drinking, regardless of the thase reductase (MTRR A66G) and thymidy- ALDH2 genotype. late synthase (TS) variable number of tandem

repeat (VNTR), play important and interre- 1) Haplotypes of the ADH1B and ADH1C lated roles in folate metabolism. We therefore polymorphisms: Linkage disequilibrium evaluated associations between dietary folate (LD) between the ADH1B and ADH1C intake and these polymorphisms as polymorphisms adds complexity to determi- gene-environment interactions impacting on nation of their significance for drinking be- the risk of head and neck, lung and breast havior and alcoholism. We have recently cancers. shown importance for the ADH1B polymorphism regarding habitual drinking in the 1) Head and neck cancer Japanese population; however, the issue of In total, 237 head and neck cancer cases LD between the ADH1B and ADH1C poly- and 711 age- and sex- matched non-cancer morphisms remained to be clarified. Here, we controls were included. Dietary folate intake conducted a cross-sectional study in 2,299 was found to be inversely associated with nonalcoholic Japanese individuals. Drinking head and neck cancer risk. The adjusted OR

8 for the top tertile intake of folate was 0.53 ORs and 95%CIs for family history of cancer (95%CI, 0.32-0.89) compared with individu- were determined by multiple logistic regres- als in the lowest tertile. Interactions between sion analysis, including stratification by fam- drinking habits and MTHFR C667T (p=0.04), ily history for 14 cancer sites. The associa- MTR A2756G (p=0.04) and MTRR A66G tions between family history and risk of can- (p=0.03) polymorphisms for head and neck cer were generally stronger at the same sites cancer risk were also observed. than across cancer sites. Risks to first-degree relatives at the same sites were found to be 2) Lung cancer significantly elevated with 8 of 14 cancer In total, 515 lung cancer cases and 1030 sites; especially high ORs being found for age- and sex- matched non-cancer controls prostate (OR=5.6; 95%CI, 1.5–20.5) and were included. Folate intake was not associ- thyroid cancers (13.3, 1.5–114.7). Some ated with lung cancer risk, but MTHFR 677T across-site associations were observed; and 1298C alleles was linked with reduced esophagus–liver, stomach–bladder, colorec- risk of squamous/small cell carcinoma tum–lymphoma, and pancreas–ovary. Fur- (p=0.029), especially among heavy smokers thermore, a significant reciprocal association (p=0.035). Furthermore, the MTHFR 677TT between breast and prostate cancer was genotype was linked to decreased risk of found. squamous/small cell carcinoma development among heavy drinkers (0.17, 0.03–0.98). 2.5. Breast and stomach cancer risk and n-3 highly unsaturated fatty 3) Breast cancer acids in erythrocytes In total, 456 breast cancer cases and 912 Breast cancer: Dietary intake of fish rich in age- and menopausal status- matched n-3 highly unsaturated fatty acids (HUFAs), non-cancer controls were included. Breast such as eicosapentaenoic acid (EPA) and cancer risk was inversely associated with docosahexaenoic acid (DHA), has been pro- consumption of dietary folate. The adjusted posed to decrease cancer risk. In contrast to OR was 0.65 (95% CI, 0.46–0.91) for the top results from laboratory studies, however, tertile of folate intake compared with the protective effects against breast cancer have lowest tertile of intake (trend P=0.010). An proved equivocal in epidemiological studies. increased risk of postmenopausal breast can- In the present case-control study, we exam- cer with the MTHFR 677TT genotype was ined associations between breast cancer risk also observed (1.83, 1.08–3.11). and fatty acid compositions of erythrocyte

membranes as biomarkers for intake. Dietary 2.4. Family history and cancer risk information and blood samples were col- A family history of cancers has consis- lected from 103 incident breast cancer cases tently been shown to increase the risk of neoplasia in several body sites. However, only a few studies of multiple cancer sites have been conducted. To assess familial cancer risk for common sites, we conducted a large-scale, case–control study. In total, 18,836 cancer cases (head and neck, esophagus, stomach, colorectum, liver, pancreas, lung, breast, uterus, ovary, prostate, bladder, thyroid, and lymphoma) and 28,125 age–matched and sex–matched controls, confirmed as being free of cancer, were recruited.

9 and 309 non-cancer controls (matched by age and season) and erythrocyte fatty acids were measured using accelerated solvent extraction and gas-liquid chromatography. Dietary intake of n-3 HU- FAs demonstrated a negative association with risk (the highest to the lowest tertile, odds ratio (OR)=0.51, 95% confidence interval (CI)=0.27–0.98; Ptrend < 0.05), but there was no association with those of saturated fatty acids (SFAs) and meat. Moreover, risk was inversely associated with erythrocyte compositions of EPA (0.27, 0.14–0.53; Ptrend < 0.0001), DHA gastric cancer, especially of well-differenti- (0.06, 0.02–0.16; Ptrend < 0.0001) and n-3 ated adenocarcinoma. Further studies are HUFAs (0.11, 0.05–0.24; Ptrend < 0.0001), needed to investigate mechanisms of action and positively with that of SFAs (12.29, of DHA relevant to antitumor effects in the 4.94–30.57; Ptrend < 0.0001) and the ratio of stomach. SFAs/n-3 HUFAs (14.65, 5.67–37.82; Ptrend < 0.0001). In conclusion, we could show that *1 Department of Head and Neck Surgery, Aichi erythrocyte compositions of specific fatty Cancer Center Hospital acids derived from fish intake, assayed as *2 Department of Gastroenterological Surgery, biomarkers, are associated with lower risk of Aichi Cancer Center Hospital breast cancer. Further studies are now needed *3 Department of Clinical Laboratory, Aichi to investigate mechanisms linked to the eti- Cancer Center Hospital ology.

Gastric cancer: Infection with Helicobacter 3. Clinical applications of epidemi- pylori is linked to inflammation and is the ology main cause of peptic ulcer, gastritis, and gas- 3.1. Identification of HLA allele mis- tric malignancies. DHA is related to match combinations and amino anti-inflammatory and apoptosis-inducing acid substitution positions asso- effects. To examine associations between ciated with GVL effects after un- gastric cancer risk and the erythrocyte com- related HSCT: Analysis from the position of DHA, we conducted a Japan Marrow Donor Program case-control study of 179 incident gastric Kawase, T., Matsuo, K., Kashiwase, K.*1, Inoko, cancer cases and 357 non-cancer controls. H.*2, Saji, H.*3, Kato, S.*4, Sasazuki, T.*5 , Kodera, Gastric cancer risk did not seem to be di- Y.*6 and Morishima, Y*7. rectly associated with dietary intake of fish and n-3 HUFAs, including DHA. However, Graft-versus-leukemia (GVL) effects are risk was inversely associated with erythro- considered to reduce relapse rates due to cyte compositions of n-3 HUFAs (0.39, eradication of residual leukemia cells after 0.23-0.68; Ptrend < 0.005) and DHA (0.47, allogeneic hematopoietic stem cell transplan- 0.28-0.79; Ptrend < 0.01). Particularly strong tation (HSCT). Segregation from graft- associations were noted for well-differenti- versus-host disease (GVHD) has been a ma- ated type lesions and n-3 HUFAs (0.10, jor issue clinically. We recently clarified 16 0.03-0.35; Ptrend = 0.0005) as well as DHA high-risk HLA mismatch combinations and (0.20, 0.07-0.58; Ptrend < 0.01) values. In con- eight high-risk specific amino acid substitu- clusion, the erythrocyte composition of DHA tion positions for severe acute GVHD in six was found to be negatively linked to risk of HLA loci. In the current study, we clarified

10 HLA allele mismatch combinations and responsible for acute GVHD. Responsible amino acid substitution positions associated amino acid substitutions on specific position with GVL effects. A total of 4,643 patients were also elucidated in HLA-C, but not in consecutive transplanted for hematological HLA-DPB1. These findings suggest that do- malignancy (ALL, AML, CML, MDS, MM nor selection according to these results could and ML) with T cell replete marrow from segregate GVL from acute GVHD. In addi- serologically HLA-A, -B and -DR anti- tion, we speculate that the molecular basis of gen-matched donors through the Japan Mar- GVL caused by HLA-DPB1 mismatch might row Donor Program were registered in this differ from that in HLA-C. cohort study. All HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 alleles were retrospec- *1Japanese Red Cross Tokyo Metropolitan Blood tively typed. The effect of HLA locus mis- Center match in allele level, the HLA allele mis- *2Division of Molecular Science, Tokai Univer- match combinations in HLA six loci and sity amino acid substitution positions on reduced *3HLA Laboratory relapse rates was analyzed using a multivari- *4 Department of Cell Transplantation & Regen- able competing risk regression model. As re- erative Medicine, Tokai University sults: (1) mismatches of HLA-C (p<0.0001) *5International Medical Center of Japan and HLA-DPB1 (p<0.0001) were strongly *6Department of Hematology, Japanese Red Cross reduced leukemia relapse, and HLA-A Nagoya First Hospital (p=0.9), HLA-B (p=0.91), HLA-DRB1 *7Department of Hematology and Cell therapy, (p=0.54) and HLA-DQB1 (p=0.54) were not; Aichi Cancer Center (2) a total of 10 HLA mismatch combinations were significantly associated with GVL ef- 3.2. Exploration of risk factors for fects, four in HLA-C alleles (donor non-small-cell lung cancer Cw*0303-patientCw*1502 (n=25), Cw* (NSCLC) with or without epidermal 0102-Cw*1402 (n=16), Cw*0801-Cw*0102 growth factor receptor (EGFR) (n=10) and Cw*1402-Cw*0304 (n=23)), and mutations six in HLA-DPB1 alleles (DP*0402- Matsuo, K., Ito, H., Suzuki, T., Hiraki, A., Yatabe, DP*0201 (n=66), DP*0501-DP*0201 Y. and Mitsudomi, T. (n=351), DP*0501-DP*0401 (n=53), DP*0501-DP*0402 (n=121), DP*0901- Recently, the epidermal growth factor re- DP*0201 (n=50) and DP*1301-DP*0201 ceptor (EGFR), which is a receptor tyrosine (n=21)), but none in HLA-A, -B, -DRB1 and kinase (TK) activating several signaling -DQB1 alleles (with the exception of two pathways resulting in cell proliferation, es- combinations in HLA-C, all were different cape from apoptosis, invasion or metastasis, from high-risk mismatches for severe acute all of which are associated with cancer phe- GVHD); (3) specific amino acid substitutions notypes, has been identified. Elevated levels at positions 9, 99, 156 in HLA-C molecule of EGFR are frequently seen in a variety of was elucidated to be significant factors re- epithelial tumours, including NSCLCs. Acti- sponsible for GVL effects and one of three vating mutations of EGFR have been re- was different from substitutions responsible ported in a subset of NSCLC usually highly for severe acute GVHD. As for HLA-DPB1, sensitive to kinase inhibitors. This implies no significant association between the posi- that NSCLCs can be classified into two dis- tions of specific amino acid substitutions and tinct groups, namely EGFR-mutated GVL were found. In conclusion, our large (EGFRmut) and EGFR-wild-type (EGFRwt) as scale comprehensive analysis made it possi- with the estrogen/progesterone receptor ble to identify 4 HLA-C and 6 HLA-DPB1 status in determining breast cancer treatment. mismatch combinations responsible for GVL Recent cross-sectional studies among effects, some of which differed from those NSCLC cases so far have revealed that the

11 frequency in non-smokers is significantly trends and this result may partly explain the different between EGFRmut and EGFRwt sex difference in the acquisition of EGFR NSCLCs. mutations. In conclusion, our case-control The present study aimed to assess the im- study clearly demonstrated that impacts of pact of smoking and sex on the risk of smoking and sex on the risk of EGFRmut non-small-cell lung cancer (NSCLC) with or NSCLC are different from those for EGFRwt without EGFR mutations. We conducted a NSCLCs. Our findings provide clear evi- case-control study using 152 patients with dence that NSCLCs can be divided on the EGFR-mutated (EGFRmut) NSCLCs, 283 basis of their EGFR mutation status. with EGFR-wild-type (EGFRwt) NSCLCs and 2,175 age- and sex-frequency-matched 4. Comparative epidemiological controls. Smoking was a significant risk fac- wt study of cancers on the increase, tor for EGFR NSCLCs [odds ratio (OR) for focusing on Korea, Japan and ever-smokers: 4.05; 95% confidence interval: China (KOJACH study) 2.79-5.88] but not for EGFRmut NSCLCs Tajima, K., Matsuo, K., Hiraki, A., Kuriki, K., (OR=0.73, 0.46-1.14). Sex did not affect this Wakai, K.*1, Hirose, K.*2, Gao C-M.*3, Yoo association, which was consistently observed K-Y.*4, Ahn Y-O.*5, Zhou Z-Y.*6 across other smoking-related parameters including pack-years. Sex was the sole risk To establish a basis for cancer prevention factor for EGFRmut NSCLCs (OR for women in the Asian Pacific region, we started a relative to men: 2.19, 1.41-3.39) and there case-referent study on colorectal cancers in was no significant interaction between being 2000, based on a standardized epidemiologi- female and smoking. In contrast, sex, smok- cal approach, in Korea (Seoul), Japan (Na- ing and their interaction were significant in goya) and China (Nanjing, Chongqing, Benxi EGFRwt NSCLCs. The impact of sex on and Shantou), the so called KOJACH Study EGFR mutation status was assessed with ref- (Fig.4). In Korea they developed their own erence to several indicators of reproductive SQFFQ and we established semi-quantitative history among women. Total fertile years food frequency questionnaires (SQFFQs) in showed a significant positive association the four cities in China. The validity and re- with EGFRmut NSCLCs but not with EGFRwt producibility of all SQFFQs established in NSCLCs. Other indicators showed similar the four cities in China were evaluated for

12 further epidemiologic studies. We have col- 5. Ethnoepidemiologic studies on lected lifestyle data by standardized SQFFQs virus-related cancer and blood samples for plasma and DNA after Tajima, K., Sonoda, S.*1, Chiba, H.*2, Senoo, H.*3 obtaining informed consent from 400 colorectal cancer cases in Seoul, Nagoya, Nanjing Human T-cell leukemia virus type 1 and Chongqing, and in total more than 1,600 (HTLV-1), the main cause of adult T-cell cases in the three countries by 2005. The leukemia/lymphoma, is found throughout the same number of referents matched by age world but with microgeographical clusters of and sex were recruited from hospital patients hyperendemicity. Epidemiologic studies in Korea and Japan and from the general among Mongoloids showed that HTLV-1 is population in China. A detailed analysis on highly endemic in South Japan (one million risk and protective factors for colorectal can- carriers) and in the Andes district of South cer in each country was conducted and main America. In contrast, HTLV-II (also a risk results published in the several international factor for adult T-cell leukemia/lymphoma) is journals. Furthermore, detailed analysis of broadly distributed in all of South America, combined risk impact of lifestyles and ge- except the Andes line. After netic polymorphisms are ongoing in the three sero-epidemiologic studies on HTLV-I anti- countries. Furthermore, since 2005 we have bodies among Tibetan people in China, focused on breast cancer which is also rap- Sahme people in North Norway and Nenets idly increasing in all three countries. We are people in Northwest Russia in 2001, 2003 collecting a total of 1,800 breast cancer cases and 2005, respectively, we confirmed the with the same number of controls in Seoul, lack of HTLV-I/II clusters among Mongol- Nagoya and Nanjing by the same methods as oids in the Central Asia and North Scandina- conducted for colorectal cancer. We had col- vian and Russian polar areas. In 2006 we lected more than 70% of cases and controls tried to conduct field work in Northern Can- by the end of 2007 and should finish com- ada to study HTLV-I/II epidemic patterns pletely by the end of 2008 to allow analysis among Inuit people, but finally our study ofrisk impact for breast cancer in the three plan was not accepted by the Canadian gov- countries according to the same methods ernment. Now we are planning a new chal- conducted for colorectal cancer. lenge to clarify the HTLV-I/II distribution among Inuit people in Greenland who have *1 Department of Preventive Medicine, Nagoya migrated to the most Northeastern polar areas University of Medical School, Nagoya, Japan in the world and finally we hope to complete *2 Department of Planning and Information, Ai- our worldwide sero-epidemiologial survey of chi Prefectural Institute of Public Health, Na- HTLV-I/II among Mongoloid people in the goya, Japan world. *3 Division of Epidemiology, Cancer Institute of *1 Jiangsu Province, Nanjing, China Southern Region Hospital, Kagoshima, Japan *2 *4 National Cancer Center, Korea Department of Laboratory Medicine, Hok- *5 Department of Preventive Medicine, College kaido University School of Medicine, Sapporo, of Medicine, Seoul National University, Seoul, Japan *3 Korea Department of Anatomy, Akita University *6 Laboratory of Molecular Toxicology, Third School of Medicine, Akita, Japan, Military Medical University, Chongqing, China

From left to right First row: Dr. T. Toyoda, Dr. T. Tsukamoto, Dr. M. Tatematsu, Dr. H. Nakanishi, and Dr. R. Fukuyama Second row: Mr. S. Takasu, Ms. K. Inoue, Dr. M. Yamamoto, Ms. H. Miyamoto, Ms. I. Hanaoka, and Dr. X. Zhang Third row: Dr. H. Suzuki, Dr. S. Kondo, Dr. M. Matsui, and Dr. D. Takagi Inset: Dr. M. Ota, Ms. T. Hagiwara, Dr. Y. Ito, Mr. H. Tanaka, Ms. M. Yoshimura, and Dr. A. Hirata

14 ______Division of Oncological Pathology

Masae Tatematsu, 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 (until June, 2006) Ryuichi Fukuyama, M.D., Senior Researcher Masami Yamamoto, D.V.M., Ph.D. Research Assistant Harunari Tanaka, B.P., Research Assistant (until Mar, 2007) Takeshi Toyoda, D.V.M., Ph.D., Research Resident Hidenori Suzuki, M.D., Research Resident Hisayo Ban, Semi-regular Employee Kuniko Mizutani, Semi-regular Employee (until Jul, 2007)

Visiting Scientists Malcolm A. Moore, Ph.D., Asian Pacific Organization for Cancer Prevention

Visiting Trainees Shinji Takasu, D.V.M., Dept. of Veterinary Pathology, Gifu University Norifumi Ohashi, 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. of Epidemiology, Nagoya University Graduate School of Medicine Naoki Hirano, M.D., Dept. of Internal medicine I, Toho University School 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. 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 Kondo, M.D., Dept. of Internal Medicine, Nagoya University Xia Zhang, M.D., Tumor Immunology and Gene Therapy Center, Second Military Medical University, China Liyu Cao, M.D., Dept. of Pathology, Anhui Medical University, China Daishi Takagi, M.D., Dept. of Surgery II, Nagoya University School of Medicine Mitsuhiko Ota, D.D.S., School of Dentistry, Aichi-Gakuin University Makoto Matsui, Ph.D., Bio Med Core Inc. Suguru Yamada, M.D., Dept. of Surgery II, Nagoya University School of Medicine

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 2007, the number of autopsy cases amounted to 2605. 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 opportunistic infection and hemorrhage during treatment. The main research theme of the laboratory is carcinogenesis and progression of gastrointestinal malignancies. During 2006-2007, the research activities were divided into the following three main areas. The first deals with links between clinicopathological findings and phenotypes using several gastric and intestinal phenotypic markers. Reduction of MUC2 expression may be associated with occurrence and progression of colorectal carcinomas arising though both the adenoma-carcinoma sequence pathway and de novo carcinogenesis. The second area concerns gastric cancers and their prevention. Nordihydroguaiaretic acid (NDGA) and 3. 4-vinyl-2,6-dimethoxyphenol (canolol) suppress gastric carcinogenesis in Helicobacter pylori (H. pylori) in-

15 fected Mongolian gerbils (Mgs) while a high-salt diet dose-dependently enhance H. pylori-associated gastritis and stomach carcinogenesis. A high-salt diet works synergistically with H. pylori infection to enhance iNOS and COX-2 expression in the gastric mucosa of Mgs, and our results support the hypothesis that excessive salt intake may be associated with progression of H. pylori-induced gastritis. The third research area involves basic research on tumor progression and metastasis, especially micrometastasis and related clinical applications. Selection of the patients with high-risk for peritoneal relapse detected by genetic diagnosis and subsequent intraperitoneal chemotherapy targeting peritoneal micrometastasis is now under clinical trial as a potential new therapeutic strategy for gastric cancer patients.

1. Loss of MUC2 expression correlates 2. Inhibitory effects of nordihydro- with progression along the adenoma- guaiaretic acid, a plant lignan, on carcinoma sequence pathway as well as Helicobacter pylori-associated gastric de novo carcinogenesis in the colon carcinogenesis in Mongolian gerbils Mizoshita, T., Tsukamoto, T., Inada, K., Hirano, N., Tatematsu, M., Toyoda, T., Tsukamoto, T., Mizoshita, T., Tajika, M.*1, Nakamura, T.*1, Ban, H. and Tatematsu, M. Nishibe, S.*1, Deyama, T.*2, Takenaka, Y., Hirano, N., Tanaka, H., Takasu, S., Ban, H., Kumagai, T.*3, Inada, We have previously demonstrated links between K.*4 and Utsunomiya, H.*5 clinicopathological findings and phenotypes using several gastric and intestinal phenotypic markers in Recent epidemiological studies have demon- stomach and pancreatic cancers. However, the strated that consumption of certain natural products clinicopathological significance of the phenotype can lower cancer risk in humans. For example, and Cdx2 expression has hitherto remained unclear plant-derived lignans have been shown to exert in colorectal carcinogenesis. We examined the cor- chemopreventive effects against cancer in vitro and relation between gastric and intestinal phenotypic in vivo. In the present study, the effects of three expression in 91 primary early carcinomas of the such lignans, termed arctiin, arctigenin, and nordi- colon. MUC2 expression demonstrated a significant hydroguaiaretic acid (NDGA), on the proliferation decrease from tubular/tubulovillous adenomas with of Helicobacter pylori and H. pylori-associated moderate atypia, through intramucosal carcinomas, gastric cancer development were investigated in to cancers with submucosal invasion (P < 0.0001). Mongolian gerbils. To examine the effects of arcti- Intramucosal de novo carcinomas (flat type carci- genin and NDGA on stomach carcinogenesis, spe- nomas without adenomatous components) exhibited cific pathogen-free male, 5-week-old gerbils were a greater decrease of MUC2 than intramucosal le- infected with H. pylori, administered 10 p.p.m. sions with adenomatous components. Expression of N-methyl-N-nitrosourea in their drinking water and MUC5AC also decreased significantly with pro- fed diets containing various concentrations of lig- gression according to the tubular/tubulovillous nans until they were killed after 52 weeks. At a die- adenoma-carcinoma sequence, carcinomas with tary level of 0.25%, NDGA significantly decreased villous adenomatous components having a higher the incidence of gastric adenocarcinomas. Arcti- level compared with their tubular adenomatous genin, in contrast, failed to attenuate neoplasia at a counterparts, suggesting differences in the pathway level of 0.1%. Both NDGA and arctigenin signifi- of malignant transformation. Cdx2 nuclear expres- cantly reduced serum 8-hydroxy-2'-deoxyguanosine sion was maintained in all of the adenomas and levels at doses of 0.25 and 0.05% (NDGA), and early carcinomas examined. Our data suggest that 0.1% (arctigenin). Administration of 0.25% NDGA reduction of MUC2 expression may be associated significantly suppressed the formation of intestinal with the occurrence and progression of colorectal metaplasia both in the antrum and the corpus. Al- carcinomas in both the adenoma-carcinoma se- though all three lignans dose-dependently inhibited quence pathway and de novo carcinogenesis. Tu- the in vitro proliferation of H. pylori, there were no mor-suppressive effects of Cdx2 may be preserved differences in the titers of anti-H. pylori antibodies during early stages of colorectal carcinogenesis. or the amount of the H. pylori-specific urease A gene among all H. pylori-infected groups. These *1 Department of Gastroenterology, Aichi Cancer Cen- results suggest that NDGA might be effective for ter Hospital, Chikusa-ku, Nagoya 464-8681, Japan prevention of gastric carcinogenesis. The possible mechanisms appear to be related to inhibitory ef-

16 fects on progression of gastritis and antioxidative gastric epithelial cell proliferation and gastric car- activity rather than direct antimicrobial influence. cinogenesis in H. pylori-infected Mongolian gerbils. Interestingly, the viable H. pylori count was not *1 Faculty of Pharmaceutical Sciences, Health Sciences changed by the canolol containing diet. Thus, the University of Hokkaido, Ishikari-Tobetsu, Hokkaido level of inflammation because of H. pylori rather 061-0212, Japan than the existence of the bacteria might be the de- *2 Central Research Laboratories, Yomeishu Seizo Co., termining factor. Importantly, canolol appears to Ltd, Kamiina-Minowa, Nagano 399-4601, Japan suppress induction of mRNAs for inflammatory *3 Central Clinical Laboratories, Shinshu University cytokines. Hospital, Matsumoto, Nagano 390-8621, Japan *4 Department of Pathology, Fujita Health University *1 Graduate School of Science and Technology, Ku- School of Medicine, Toyoake, Aichi 470-1192, Japan mamoto University, Kumamoto 860-8555, Japan *5 Department of Pathology, Wakayama Medical Uni- *2 Faculty of Pharmaceutical Science, Sojo University, versity, Kimiidera, Wakayama 641-0012, Japan Kumamoto 860-0082, Japan

3. 4-Vinyl-2,6-dimethoxyphenol (canolol) 4. Synergistic upregulation of inducible suppresses oxidative stress and gastric nitric oxide synthase and cyclooxy- carcinogenesis in Helicobacter pylori- genase-2 in gastric mucosa of Mongo- infected carcinogen-treated Mongolian lian gerbils by a high-salt diet and gerbils Helicobacter pylori infection Tsukamoto, T., Cao, X., Seki, T.*1, Tanaka, H., Toyoda, T., Tsukamoto, T., Hirano, N., Mizoshita, T., Morimura, S.*1, Cao, L., Mizoshita, T., Ban, H., Toyoda, Kato, S., Takasu, S., Ban, H. and Tatematsu, M. T., Maeda, H.*2 and Tatematsu, M. Intake of salt and salty food is known as a risk Oxidative stress is linked to gastric carcinogene- factor for gastric cancer. We have previously dem- sis because of its ability to damage DNA. Here we onstrated that a high-salt diet dose-dependently en- examined antioxidative and anti-inflammatory ef- hances Helicobacter pylori (H. pylori)-associated fects of 4-vinyl-2,6-dimethoxyphenol (canolol), a gastritis and stomach carcinogenesis in Mongolian recently identified potent antioxidative compound gerbils. In this study, we focused on the influence obtained from crude canola oil, on Helicobacter of excessive salt intake on the expression of in- (H.) pylori-induced gastritis and gastric carcino- flammatory mediators involved in progression of H. genesis using a Mongolian gerbil model. The ani- pylori-induced chronic gastritis. mals were allocated to H. pylori-infection alone (12 A total of 45 stomach samples from Mongolian weeks) or H. pylori + N-methyl-N-nitrosourea gerbils were evaluated by immunohistochemistry. (MNU) administration (52 weeks). After oral in- The animals were infected with H. pylori and fed oculation of H. pylori, they were fed for 10 and 44 basal (0.32%) or a high-salt (10%) diet, and sacri- weeks with or without 0.1% canolol. H. py- ficed after 40 weeks. Proliferative activity and ex- lori-induced gastritis, 5'-bromo-2'-deoxyuridine pression of cyclooxygenase-2 (COX-2) in gastric (BrdU) labeling and scores for cyclooxygenase-2 mucosa were significantly increased in H. py- (COX-2) and inducible nitric oxide synthase lori-infected gerbils. The additional high-salt diet (iNOS) immunohistochemistry were attenuated in significantly up-regulated the expression of induc- the canolol-treated groups. Expression of inter- ible nitric oxide synthase (iNOS) and COX-2 in H. leukin-1beta (IL-1beta), tumor necrosis factor-alpha pylori-infected groups (P < 0.01 and P < 0.05, re- (TNF-alpha), COX-2 and iNOS mRNA in the gas- spectively), while no significant effects were noted tric mucosa, and serum 8-hydroxy-2'-deoxy- in non-infected animals. There was significant syn- guanosine (8-OHdG), anti-H. pylori IgG and gastrin ergistic interaction between H. pylori infection and levels were also significantly lower in the ca- 10% NaCl diet on the expression of iNOS (P < nolol-treated groups. Furthermore, the incidence of 0.05) and also a tendency for enhanced COX-2 ex- gastric adenocarcinomas was markedly reduced in pression (P = 0.0599). The present results suggest the H. pylori + MNU + canolol-treated animals that a high-salt diet works synergistically with H. [15.0% (6/40)] compared to the control group pylori infection to enhance iNOS and COX-2 ex- [39.4% (13/33)] (p < 0.05). These data indicate ca- pression in the gastric mucosa of Mongolian gerbils, nolol to be effective for suppressing inflammation, and support the hypothesis that excessive salt intake

17 may be associated with progression of H. py- 6. A new diagnostic and therapeutic strat- lori-induced gastritis. egy targeting micrometastasis for prevention of peritoneal recurrence in gas- 5. Role of IKK and oscillatory NFkappaB tric cancer patients kinetics in MMP-9 gene expression and Nakanishi, H., Ito, S., *1 Kodera, Y., *2 and Tatematsu, M. chemoresistance to 5-fluorouracil in Peritoneal metastasis is a major cause of gastric RKO colorectal cancer cells cancer death. Development of new diagnostic and *1 *1 *1 *1 Fukuyama, R. , Ng, K.P. , Cicek, M. , Kelleher, C. , therapeutic methods for prevention of peritoneal *1 *1 *1 Niculaita, R. , Casey, G. , Sizemore, N. recurrence is therefore an extremely urgent issue to Nuclear factor kappa B (NFkappaB) is a central be addressed in gastric cancer research. Genetic di- participant in the metastasis and chemoresistance of agnosis to detect micrometastasis in the peritoneal colorectal cancer (CRC). However, it is not fully cavity with CEA qRT-PCR developed in our labo- understood to what extent NFkappaB contributes to ratory in 1999 is now widely recognized to be a induction of the metastasis-associated matrix met- highly sensitive method for free cancer cells and alloprotease-9 (MMP-9) gene and sensitivity to the clinical utility for prediction of peritoneal relapse commonly used chemotherapeutic 5-fluorouracil after curative surgery was validated in a prospective (5-Fu) in CRC. Using the RKO human CRC cell study. A phase II clinical trial of adjuvant chemo- line and two NFkappaB signaling deficient RKO therapy for qRT-PCR positive patients showed re- mutants, we investigated NFkappaB's role in the duction of peritoneal recurrence after curative sur- induction of MMP-9 and 5-Fu sensitivity in RKO gery with marginal significance. New genetic diag- CRC cells. Tumor necrosis factor alpha (TNFalpha) nostics such as a custom DNA microarray with an failed to induce MMP-9 in either of the NFkappaB algorithm (SVM) using 59 genes for peritoneal signaling mutants. RKO cells exhibited a robust, washes has been developed for prediction of peri- oscillatory NFkappaB activity in response to toneal metastasis. Furthermore, 6 new genetic TNFalpha not seen in either of the NFkappaB mu- markers including a stem cell marker, CD133, were tant cell lines, which instead demonstrated dimin- also found by genome-wide microarray analysis and ished, nonoscillatory NFkappaB activation. Analy- the resultant multiple qRT-PCR method exhibited sis of TNFalpha-induced phosphorylation and even more diagnostic accuracy. Preclinical study MMP-9 promoter recruitment of the p65 NFkappaB using a GFP-tagged peritoneal micrometastasis subunit revealed a significant reduction in p65 model showed that micrometastases in the perito- phosphorylation as well as reduced and altered re- neal cavity are highly chemo-sensitive, especially to cruitment of p65 to the MMP-9 gene promoter in intraperitoneal chemotherapy (IP) with paclitaxel. A the mutants compared to the parental RKO cell line. phase I clinical trial of IP for far-advanced gastric 5-Fu only activated NFkappaB in the parental RKO cancer patients was initiated. High intraperitoneal cells through induction of IkappaB-kinase (IKK) paclitaxel concentrations were maintained follow- activity and increased sensitivity to 5-Fu was ob- ing IP administration and ascites diminished during served in both NFkappaB mutant lines. Our results the course of the treatment. These results suggest suggest that TNFalpha-dependent induction of that intraperitoneal chemotherapy tailored to pa- MMP-9 gene expression is tightly regulated by os- tients at high-risk for peritoneal relapse would be a cillatory/cumulative activation of NFkappaB and new potential therapeutic modality for advanced that 5-Fu stimulates NFkappaB and RKO CRC cell gastric cancer patients. survival through induction of IKK activity. *1 Department of Gatroenterological Surgery, Aichi *1 Department of Cancer Biology, Cleveland Clinic, Cancer Center Central Hospital, Nagoya, Japan *2 Cleveland, Ohio 44195, USA Department of Surgery II, Nagoya University School of Medicine, Nagoya, Japan

From left to right First row: Ms. Ikuko Tomimatsu, Dr. Keiko Shinjo, Ms. Mari Kizuki, and Ms. Eri Nishikawa Second row: Dr. Yutaro Suzuki, Dr. Taijiro Ozawa, Dr. Yutaka Kondo, Dr. Byonggu An, Dr. Koji Kawaguchi, Dr. Yoshitaka Sekido, Dr. Hideki Murakami, Dr. Hirotaka Osada, Dr. Makiko Fujii, Mr. Yoshio Tatematsu and Dr. Motokazu Ito.

19 ______Division of Molecular Oncology

Yoshitaka Sekido M.D., Ph.D., Chief Hirotaka Osada, M.D., Ph.D., Section Head Yutaka Kondo, M.D., Ph.D., Section Head (as of April 2007) Hideki Murkami, M.D., Ph.D., Senior Researcher (as of April 2006) Makiko Fujii, D.D.S., PhD., Senior Researcher (as of July 2007) Yoshio Tatematsu, B.S., Research Assistant Ikuko Tomimatsu, Semi-regular Employee (as of April 2006) Mari Kizuki, Semi-regular Employee (as of June 2007) Research Resident Hideo Sakamoto, M.D., Nagoya City University School of Medicine (until March 2007) Byonggu An, M.D., Shiga University of Medical Science (as of April 2007) Visiting Trainees Wentao Gao, M.D., Research fellow of the 29th Japan-China Sasagawa (until March 2007) Toshihiko Yokoyama, M.D., Nagoya University School of Medicine (until March 2007) Naohito Sato, M.D., Nagoya University School of Medicine (until March 2006) Tetsuo Taniguchi, M.D., Nagoya University School of Medicine (until September 2007) Yasuhiro Goto, M.D., Nagoya University School of Medicine (until September 2007) Koji Kawaguchi, M.D., Nagoya University School of Medicine (as of April 2006) Yutaro Suzuki, M.D., Nagoya University School of Medicine (as of July 2006) Motokazu Ito, M.D., Nagoya University School of Medicine (as of October 2006) Keiko Shinjo, M.D., Nagoya University School of Medicine (as of August 2007) Taijiro Ozawa, M.D., Nagoya City University School of Medicine (as of August 2007) Eri Nishikawa, Nagoya University School of Medicine (as of August 2007)

General Summary Our goal is to determine genetic lesions giving rise to human solid cancers and use this information for prevention, diagnosis, and treatment of disease. Currently, we are focusing on lung cancer, malignant mesothelioma, colon cancer, hepatoma, and head and neck cancer. Our studies also provide opportunities to dissect biochemical and pathological pathways of malignant phenotypes including dysregulated cell growth, differentiation, invasion and metastasis. Human cancers arise because of genetic mutations in oncogenes and tumor suppressor genes and our approach is to study candidate genes, with a systematic molecular analysis of biochemical pathways and global profiling of gene expression by microarrays, along with comparative genomic hybridization to detect chromosomal abnormalities. Epigenetic changes with DNA methylation and histone modification may also be identified as important mechanisms of inactivation of tumor suppressor genes. We also functionally 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 expression using RNA interference (RNAi) in either tumor or normal cells and then study the resulting phenotype. Understand- ing the functions of mutated genes and disrupted signaling pathways should provide a foundation for translational research for human malignancies, from bench to bedside.

1. Genomic profiling of malignant pleural teration regions including JUN amplifi- mesotheliomas with array-based com- cation on 1p32 parative genomic hybridization shows Taniguchi, T., Karnan, S.*1, Fukui, T.*2, Yokoyama, T., frequent non-random chromosomal al- Tagawa, H.*1, Yokoi, K.*3, Ueda, Y.*3, Mitsudomi,

20 T.*2, Horio, Y.*4, Hida, T.*4, Yatabe,Y.*5, Seto, M.*1, line, and demonstrated up-regulation of a novel Sekido, Y. gene, CLCP1 (CUB, LCCL-homology, coagulation factor V/VIII homology domains protein), also Genome-wide array-based comparative genomic called ESDN/DCBL2, in LNM35 and lung cancer hybridization analysis of malignant pleural meso- specimens. We have subsequently focused on the theliomas (MPMs) was carried out to identify re- potential roles of the gene in cancer metastasis. gions that display DNA copy number alterations. First, we established stable LNM35 RNAi clones, Seventeen primary tumors and nine cell lines de- in which CLCP1 expression was suppressed by rived from 22 individuals were studied, some of RNAi, and found that their motility was signifi- them originating from the same patients. Regions of cantly reduced, though growth rates were not genomic aberrations observed in >20% of individu- changed. Next, in vitro selection of a phage display als were 1q, 5p, 7p, 8q24 and 20p with gains, and library demonstrated that a phage clone displaying a 1p36.33, 1p36.1, 1p21.3, 3p21.3, 4q22, 4q34-qter, peptide similar to a sequence within the Sema do- 6q25, 9p21.3, 10p, 13q33.2, 14q32.13, 18q and 22q main of semaphorin 4B (SEMA4B) interacted with with losses. Two regions at 1p32.1 and 11q22 LNM35. Immunoprecipitation experiments con- showed a high copy gain. The 1p32.1 region confirmed interaction of CLCP1 with SEMA4B. Fi- tains a protooncogene, JUN, and we could further nally, SEMA4B down-regulated the expression demonstrate overexpression of JUN with real-time level of CLCP1 protein through induction of ubiq- polymerase chain reaction analysis. As MPM cell uitination and proteasome degradation. These re- lines did not overexpress JUN, our findings sug- sults are the first to indicate that CLCP1 plays a gested that induction of JUN expression might be role in cell motility, while also showing that at least involved in the development of MPMs in vivo, with one of the ligands is SEMA4B and that their inter- gene amplification in a subset of tumors. However, action mediates proteasome degradation by CLCP1. the most frequent alteration was 9p21.3 deletion, Although the physiological role of the interaction which includes the p16(INK4a)/p14(ARF) locus. between CLCP1 and SEMA4B remains to be inves- With polymerase chain reaction analysis, we deter- tigated, this novel gene may become a target of mined the extent of the homozygous deletion re- therapy to inhibit metastasis of lung cancers. gions of the p16(INK4a)/p14(ARF) locus in MPM cell lines and found the deletion regions to vary *1Division of Molecular Carcinogenesis, Center for with the line. Our results with array comparative Neurological Diseases and Cancer, Nagoya Univer- genomic hybridization analysis have provided new sity Graduate School of Medicine insights into the genetic background of MPM, and *2Division of Surgical Oncology, Department of Surgery, hopefully will give clues to development of new Nagoya University Graduate School of Medicine molecular target therapies for this neoplasm. *3 Department of Pulmonary Medicine, Aichi Cancer *1 Center Hospital Division of Molecular Medicine, Aichi Cancer Center Research Institute *2Division of Thoracic Oncology, Aichi Cancer Center 3. Variable DNA Methylation Patterns Asso- Hospital ciated with Progression of Disease in *3Division of General Thoracic Surgery, Nagoya Univer- Hepatocellular Carcinomas sity School of Medicine Gao, W., Kondo, Y., Shimizu, Y.*1, Sano, T.*1, Yamao, *4 Department of Internal Medicine, Aichi Cancer Center K.*2, Goto, Y., Ito, M., Murakami, H., Osada, H., Sekido, Hospital Y. *5Division of Pathology and Molecular Diagnostics, Ai- Hepatocellular carcinoma (HCC) most com- chi Cancer Center Hospital monly arises with a background of chronic inflam-

mation due to viral infection, and features both ge- 2. CLCP1 interacts with semaphorin 4B and netic and epigenetic abnormalities. However, a regulates motility of lung cancer cells global picture of epigenetic changes in HCC has Osada, H., Nagai, H.*1, Sugito, N.*1, Matsubara H*1, hitherto been lacking. To address this question, we Tatematsu Y, Hida T*3, Sekido Y, Nagino M*2, Nimura conducted genome-wide screening for aberrantly Y*2, Takahashi Ta*1. methylated CpG islands in cancerous tissue and corresponding adjacent non-cancerous tissue We previously established a highly metastatic (chronic hepatitis, CH, or liver cirrhosis, LC) from subline, LNM35, from the NCI-H460 lung cancer

21 HCC patients using the methylated CpG island am- tion in normal tissue and adjacent tissue but high plification-microarray (MCAM) technique with levels in HCC. Methylation in the type IV genes is 6,458 CpG islands. MCAM data were confirmed by characteristic of moderately/poorly differentiated bisulfite pyrosequencing for 56 genes. MCAM cancer. Our extensive analyses revealed that CpG identified 719 prominent targets of hypermethyla- island promoters become methylated in different tion in HCCs, which represent 11% of all CpG is- patterns during progression of the disease, suggest- lands. HCCs arising from LC had very much more ing different mechanisms for acquisition of epige- methylation than those arising from CH (1,249 netic changes. These data support the notion that genes or 19% vs. 444 genes or 7%). There were molecular pathways linked to aberrant DNA me- four patterns of aberrant methylation: Type I (4%, thylation dictate the clinical features of HCCs in e.g. MMP14) represents methylation that develops multiple steps of tumorigenesis. In addition we pro- substantially at the transition between normal tissue vide hundreds of markers that could be of diagnos- and adjacent tissue and does not increase further in tic utility in HCCs and pre-neoplastic lesions. cancer. Type II (55%, e.g. RASSF1A) shows pro- gressively increasing methylation from adjacent *1Department of Gastroenterological Surgery, Aichi tissue to HCC. Type III (4%, e.g. GNA14) shows Cancer Center Hospital decreased methylation in adjacent tissue but either *2Department of Gastroenterology, Aichi Cancer Center similar or increased methylation in HCC. Type IV Hospital (37%, e.g. CDKN2A) shows low levels of methyla-

First row (from left to right): Dr. T. Miyata, Dr. M. Seto, Dr. S. Tsuzuki, Ms. Y. Komai. Second row (from left to right): Dr. K. Karube, Ms. Sato, Dr. K. Honma, Mr. S. Karnan, Dr. M. Nakagawa.

23 ______Division of Molecular Medicine

Masao Seto, M.D., Ph.D. Chief Shinobu Tsuzuki, M.D., Ph.D. Section Chief Masao Nakagawa, M.D., Ph.D. Senior Researcher Kennosuke Karube, M.D., Ph.D. Senior Researcher Hiroyuki Tagawa, M.D., Ph.D. Senior Researcher (Until December, 2006) Hiroko Suzuki, B.P. Senior Research Assistant (Until March, 2007) Yumiko Kasugai, B.S. Research Assistant Keiichiro Honma, M.D., Ph.D. Research Resident Tomoko Miyata, M.D., Ph.D. Research Resident

Visiting Trainees Karnan Sivasundaram, Graduate School of Medical Sciences, Nagoya City University Aya Nakagawa (Oshiro), M.D., Ph.D. Second Department of Internal Medicine, University Hospital, Univer- sity of the Ryukyus (Until January, 2007) Noriko Hukuhara, M.D. Department of Rheumatology and Hematology, Tohoku University School of Medi- cine (Until May, 2007) Guo Ying, M.D., Ph.D. Department of Pathology, Fourth Military Medical University of The People's Republic of China (Until June, 2007)

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 De- partment of Hematology and Cell Therapy (Chief, Dr. Yasuo Morishima) and the Department of Endoscopy (Chief, Dr. Tsuneya Nakamura), Aichi Cancer Center Hospital. Research on hematologic malignancies offers 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 disease development or clinicopathological manifestation. During the last two years, we have studied the issues detailed below.

The function of API2-MALT1 associated with 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 and can scan the entire genome in 1.4 MB segments on average was applied for various hematopoietic malignancies. TEL-AML1 leukemia frequently found in childhood was thereby found to have a characteristic genome profile and oncogenic function of the TEL-AML1 chimeric gene was analyzed in a bone marrow transplantation system. The significance of the AML1 isoform was also analyzed. Mucosa-associated lymphoid tissue (MALT) lymphoma is unique in its clinicopathologic features. A single institution analysis on reactivity to anti-H. pylori therapy was conducted and it was found that a half of non-reactive group (15% of total cases) had API2-MALT chimeric gene. The remain- ing half of the cases with for gastric MALT lymphomas were found to have copy number changes. Spe- cific deletion at 6q23.3 for ocular adnexal MALT lymphoma was analyzed in detail with contig array CGH and it was found that the target gene was TNFAIP3, indicating that NF-kB plays a pivotal role in development of the MALT lymphoma. Peripheral T-cell lymphomas, unspecified (PTCL-U), were also analyzed by array CGH and examples with genomic alterations showed similarity to lymphoma type adult T-cell leukemias/lymphomas (ATLLs).

24 1. Genetic abnormalities involved in t(12; used a retrovirus-based approach to transduce 21) TEL-AML1 acute lymphoblastic leu- AML1a into primitive haematopoietic cells isolated kemia: analysis by means of array- from a mouse and observed that enforced AML1a based comparative genomic hybridiza- expression increased the competitive engraftment tion potential of murine long-term reconstituting stem cells. Thus the proportion of AML1a-expressing Tsuzuki, S., Karnan S. and Seto, M. cells became elevated over time in both primary and The TEL (ETV6)-AML1 (RUNX1) chimeric secondary recipients. Furthermore, AML1a expres- gene fusion is the most common genetic abnormal- sion dramatically increased primitive and commit- ity in childhood acute lymphoblastic leukemias. ted progenitor activity in engrafted animals as as- Evidence suggests that this chimeric gene fusion sessed by long-term culture, cobblestone formation, constitutes an initiating mutation that is necessary and colony assays. In contrast, expression of the but insufficient for the development of leukemia. In full-length isoform AML1b abrogated engraftment a search for additional genetic events that could potential. In vitro, AML1b promoted differentiation play a role, we applied a genome-wide ar- while AML1a enhanced proliferation of progenitors ray-comparative genomic hybridization technique capable of short-term lymphomyeloid engraftment. to 24 TEL-AML1 leukemia samples and two cell Consistent with these findings, the relative abun- lines. It was found that at least two chromosomal dance of AML1a was highest in the primitive imbalances were involved in all samples. Recurrent stem/progenitor compartment of human cord blood, regions of chromosomal imbalance (>10% of cases) and forced expression of AML1a in these cells ele- and representative involved genes were gain of vated maintenance of primitive potential both in vi- chromosomes 10 (17%) and 21q (25%; RUNX1) tro and in vivo. CONCLUSIONS: Our data demon- and loss of 12p13.2 (87%; TEL), 9p21.3 (29%; strate that the "a" isoform of AML1 has the capac- p16INK4a/ARF), 9p13.2 (25%; PAX5), 12q21.3 ity to potentiate stem and progenitor cell engraft- (25%; BTG1), 3p21 (21%; LIMD1), 6q21 (17%; ment, required for successful clinical transplanta- AIM1 and BLIMP1), 4q31.23 (17%; NR3C2), tion. This activity is consistent with its expression 11q22-q23 (13%; ATM) and 19q13.11-q13.12 pattern in both normal and leukaemic cells. Ma- (13%; PDCD5). Enforced expression of TEL and to nipulating the balance of AML1 isoform expression a lesser extent BTG1, both single genes known to may offer novel therapeutic strategies, exploitable be located in their respective minimum common in the contexts of leukaemia and also cord blood region of loss, inhibited proliferation of the transplantation in adults, in whom stem and pro- TEL-AML1 cell line Reh. Together, these findings genitor cell numbers are often limiting. suggest that some of the genes identified as lost by array-comparative genomic hybridization may *1 Division of Epidemiology and Prevention, Aichi partly account for the development of leukemia. Cancer Center Research Institute , Japan

2. Isoform-specific potentiation of stem 3. Chromosomal imbalances are associ- and progenitor cell engraftment by ated with outcome of Helicobacter py- AML1/RUNX1 lori eradication in t(11;18)(q21;q21) Tsuzuki, S., Matsuo K. *1 and Seto, M. negative gastric MALT lymphomas Fukuhara, N. *1,2, Nakamura, T. *3, Nakagawa, M., BACKGROUND: AML1/RUNX1, the most Tagawa, H., Takeuchi, I. *4, Yatabe, Y. *5, Morishima, Y. frequently mutated gene in leukaemia, is central to *6, Nakamura, S. *7 and Seto, M. the normal biology of hematopoietic stem and progenitor cells. However, the role of different AML1 Approximately 70% of gastric mu- isoforms within these primitive compartments is cosa-associated lymphoid tissue (MALT) lympho- unclear. We therefore investigated whether altering mas can be successfully treated with H. pylori relative expression of AML1 isoforms might impact eradication. The translocation t(11;18)(q21;q21) on the balance between cell self-renewal and dif- characteristic of MALT lymphomas is recognized ferentiation in vitro and in vivo. as a marker for H. pylori independence, but this METHODS AND FINDINGS: The human marker is found in only half of the cases resistant to AML1a isoform encodes a truncated molecule with eradication of the bacterium. To address this anom- DNA-binding but no transactivation capacity. We aly, we performed array-based comparative ge-

25 nomic hybridization (array-CGH) for 29 gastric RT-PCR. A correlation between genomic loss MALT lymphomas treated with H. pylori eradica- and expression level was found only with TNFAIP3 tion. These comprised ten cases of t(11;18) positive and we thus concluded that this is the target gene MALT, nine cases of t(11;18) negative MALT with for the deleted region. TNFAIP3 is an inhibitor of H. pylori dependence and ten cases of t(11;18) NF-kB signaling so that its loss may result constitu- negative MALT with H. pylori independence. Ar- tive activation of NF-kB, which has also been im- ray-CGH analysis demonstrated no significant ge- plicated in generation of MALT lymphomas with netic alterations in t(11;18) positive MALT lym- t(11;18)(q21;q21) or t(14;18)(q32;q21). Thus, phomas, but numerous genomic alterations were TNFAIP3 may act as a tumor suppressor gene for detected in t(11;18) negative cases. This indicated ocular adnexal marginal zone B cell lymphoma. that genomic imbalance is associated with H. pylori The function of TNFAIP3 in oncogenesis remains independence in t(11;18) negative gastric MALT unclear so studies aimed at its elucidation are now lymphomas. Gene alterations may thus play an im- underway. portant role in the development of H. pylori inde- *1 Department of Internal Medicine, Samsung Medical pendence. Center, Seoul, Korea *1 Division of Molecular Medicine, Aichi Cancer Cen- *2 Department of Pathology, Samsung Medical Center, ter Research Institute, Nagoya, Japan Seoul, Korea *2 Department of Rheumatology and Hematology, To- hoku University Graduate School of Medicine, Sen- 5. Array CGH analysis of PTCL-U for iden- dai, Japan tification of a distinct subgroup *3 Department of Gastroenterology, Aichi Cancer Cen- Nakagawa, M., Nakagawa-Oshiro, A., Karnan, S., ter Hospital, Nagoya, Japan Nakamura, S.*1, Takeuchi, I.*2, Ohshima, K.*3 and Seto, *4 Division of Computer Science, Mie University M. Graduate School of Engineering, Tsu, Japan *5 Department of Pathology and Molecular Diagnostics, Peripheral T-cell lymphoma unspecified *6 Department of Hematology and Cell Therapy, Aichi (PTCL-U) comprises histopathologically and clini- Cancer Center Hospital, Japan cally heterogeneous groups. Although clarification *7 Pathology/Clinical Laboratories, Nagoya University of the subgroups in PTCL-U is therefore of major Hospital, Nagoya; All the above institutions are lo- importance, the molecular basis of the clinical het- cated in Japan. erogeneity is still poorly understood. In this study, we used array CGH for high-resolution analysis of 4. Identification of the target gene at 51 PTCL-U patients and identified 32 regions with chromosome deletion 6q23.3-24.1 in frequent genomic imbalance, one region with ocular adnexal mucosa associated high-copy number gain at 14q32.2 and one with lymphoid tissue lymphomas homozygous loss at 9p21.3. Gains of 7p and 7q, and loss of 9p21.3 showed significant associations Honma, K., Kim, W-S. *1, Ko, Y-H.*2 and Seto, M. with a poor prognosis. A detailed analysis disclosed Our recent study showed that ocular adnexal that PTCL-U cases with genomic alteration show MALT lymphomas feature recurrent genomic dele- distinct histopathological and prognostic features. tion including homozygous loss in the 2.9 Mb re- Interestingly, they manifest remarkable genetic, gion at chromosome band 6q23.3-24.1. As dele- histopathologic and prognostic resemblance to tion 6q23.3-24.1 is the most common genomic al- lymphoma-type adult T-cell leukemia/lymphoma teration, we assumed that this is a crucial genetic (ATLL), supporting the notion that PTCL-U cases alteration for lymphomagenesis of this specific type featuring genomic alteration might comprise a dis- of MALT lymphoma. In an attempt to identify the tinct subgroup. target gene in this region, we applied contig array CGH analysis to nine ocular MALT lymphoma *1 Pathology/Clinical Laboratories, Nagoya University samples and three lymphoma cell lines with Hospital, Japan 6q23.3-24.1 loss. We narrowed the minimal *2 Division of Information Engineering, Graduate common region down to a length of 586kb region School of Engineering, Mie University, Japan where two genes and four expression sequence tags *3 Department of Pathology, School of Medicine, Ku- (ESTs) exist. All of these genes and ESTs were rume University, Japan subjected to RT-PCR and real-time quantitative

From left to right First row: Dr. H. Torikai, Dr. Y. Akatsuka, Dr. K. Kuzushima, Dr. Y. Watanabe Second row: Ms. K. Shiraishi, Ms. T. Tsuboi, Dr. S. Morishima, Ms. K. Nishida Insets: Dr. M. Kamei, Dr. A. Demachi-Okamura, Ms. M. Tanimoto, Mr. T. Nagao, Dr. K. Tsujimura

27 ______Division of Immunology

Kiyotaka Kuzushima, M.D. Chief Yoshiki Akatsuka, M.D. Section Head Kunio Tsujimura, M.D. Section Head (until March 2007) Yoshinori Ito, M.D. Senior Researcher (until March 2006) Yukiko Watanabe, M.D. Senior Researcher (as of April 2007) Hiroki Torikai, M.D. Senior Researcher (as of July 2007) Ayako Demachi-Okamura, Ph.D. Research Resident (until March 2007) Michi Kamei, M.D. Research Resident (as of April 2007) Satoko Morishima, M.D. Research Resident (as of April 2007) Yasue Matsudaira, B.S. Senior Research Assistant (until March 2006) Keiko Nishida, B.P. Senior Research Assistant Fumiyo Ando, Semi-regular Employee (until October 2006) Michiyo Nakayama, Semi-regular Employee (until January 2006) Yumi Nakao-Ohashi, Semi-regular Employee (until March 2007) Keiko Shiraishi, Semi-regular Employee Miyoko Tanimoto, Semi-regular Employee Tomiko Tsuboi, Semi-regular Employee

Visiting Trainees Ayako Demachi-Okamura, Ph.D. Research Resident of Foundation for Promotion of Cancer Research (Ja- pan) (as of April 2007) Takakazu Kawase, M.D. Cancer Genetics, Nagoya University Graduate School of Medicine (until March 2007) Satoko Morishima, M.D. Cancer Genetics, Nagoya University Graduate School of Medicine (until March 2007) Hiroki Torikai, M.D. Third Department of Internal Medicine, National Defense Medical College (until June 2007) Masataka Haneda, M.D. Department of Immunology, Nagoya University Graduate School of Medicine (as of May 2007, until September 2007) Takashi Nagao, M.D. Department of Pediatrics, Okayama University School of Medicine (as of May 2007, until January 2008) Mayumi Narita, Okayama University School of Medicine (as of September 2007, until November 2007) Kazue Watanabe, M.S. Medical & Biological Laboratories Co., Ltd. (until January 2006)

General Summary The object of our research is to characterize and understand T lymphocyte responses to antigens expressed on cancers and virus-infected cells. The major projects undertaken over the past two years are summarized below. In the field of transplantation immunology, three projects are in progress focusing on epitope identification and fine characterization of the epitope processing, to be recognized by minor histocompatibility (H) antigen-specific cytotoxic T lymphocytes (CTLs). Firstly, HLA-A*3101 and -A*3303-restricted CTL epitopes were determined looking at the Cathepsin H molecule. This was found to be relatively ubiquitously expressed at the protein level, but CTL clones predominantly lysed targets of hematopoietic cell origin. Although the mechanisms involved in the differential susceptibility remain to be determined, these data suggest that the minor H antigens could be targets for graft-versus-leukemia effects. Secondly, a novel HLA-B44–restricted minor H antigen was identified by means of cDNA expression cloning studies. The CTL epitope was encoded by a novel allelic splice variant of the HMSD (Histocompatibility Minor, Serpin Domain containing) gene. In- terestingly, the immunogenicity is generated by a novel mechanism, namely differential protein ex-

28 pression due to alternative splicing controlled by an intronic single-nucleotide polymorphism located in the consensus 5' splice site adjacent to an exon. Thirdly, we have determined that the nonameric peptide VLHDDLLEA, the HLA-A*0201-restricted HA-1H, could be also presented by HLA-A*0206. The finding points to expansion of the patient population who can benefit from HA-1H-based immunotherapy. Another field of our research is to characterize the T cell responses to EBV-related malignancies. To this end, several CD4+ T cell clones specific to Epstein-Barr virus (EBV) nuclear antigen (EBNA) 1 have been established and well characterized. Most importantly, one example proved capable of killing EBV-carrying NK and T cell lines derived from patients with EBV-associated disease, suggesting that EBNA1-specific CD4+ T cells may be useful for immunotherapy targeting EBV carrying NK and T cell malignancies. The clones were used as probes to verify the structure for targeting EBNA1 to the lysosomal/endosomal pathway in mRNA-transfected antigen presenting cells. Among those tested, constructs consisting of the signal sequence of heat shock protein gp96, EBNA1 and lysosome-associated membrane protein 1 proved most effective.

1. The human cathepsin H gene encodes volved in GVHD and anti-leukemic/tumour re- two novel minor histocompatibility an- sponses. Interestingly, however, CTL clones pre- tigen epitopes restricted by HLA-A*3101 dominantly lysed targets of hematopoietic cell ori- and -A*3303 gin, which could not be explained in terms of the immunoproteasome system. Although the unique Torikai, H., Akatsuka, Y., Miyazaki, M.*1, Tsujimura, mechanisms involved in the differential susceptibil- A.*2, Yatabe, Y.*3, Kawase, T., Nakao, Y., Tsujimura, ity remain to be determined, these data suggest that K.*4, Motoyoshi, K.*5, Morishima Y.*6, Kodera Y.*2, CTSH-encoded minor H antigens could be targets Kuzushima, K., and Takahashi, To*7. for GVL effects. Minor histocompatibility (H) antigens play crucial roles in the induction of graft-versus-host dis- *1Department of Internal Medicine & Molecular Science, ease (GVHD) and/or graft-versus-leukemia (GVL) Nagoya City University Graduate School of Medical effects following HLA-identical hematopoietic stem Sciences cell transplantation (HSCT). We identified a novel *2Department of Hematology, Japanese Red Cross Na- minor H antigen epitope using two HLA-A*3101 goya First Hospital and -A*3303-restricted cytotoxic T lymphocyte *3Department of Pathology and Molecular Diagnostics, (CTL) clones derived from different HSCT recipi- Aichi Cancer Center Central Hospital ents with high-risk acute leukemia who both main- *4Department of Infectious Diseases, Hamamatsu Uni- tained durable remission more than 2 years after versity School of Medicine well-controlled acute/chronic GVHD. The epitopes *5Third Department of Internal Medicine, National De- identified by cDNA expression cloning were en- fense Medical College coded by the same nonsynonymous single nucleo- *6Department of Hematology and Cell Therapy, Aichi tide polymorphism (SNP) in the leader sequence of Cancer Center Central Hospital the Cathepsin H gene. The polymorphic amino acid *7Aichi Health Plaza residue controlling the antigenicity was located at the C terminus of identified epitopes which is the 2. Identification of a novel minor histo- major anchor motif (e.g. Arg or Lys) for both compatibility antigen generated by al- HLA-A*3101 and -A*3303. Fine epitope mapping ternative splicing due to an intronic revealed that the nonameric sequence ATLPLL- SNP in HMSD CAR was defined as an HLA-A*3101-restricted Kawase, T., Akatsuka, Y., Torikai, H., Morishima, S., epitope (CTSHR/A31), while a decameric peptide Oka, A.*1, Tsujimura, A.*2, Miyazaki, M.*3, Tsujimura, featuring a one N-terminal amino acid extension, K.*4, Miyamura, K.*2, Ogawa, S.*5,6, Inoko, H.*1, WATLPLLCAR, was presented by HLA-A*3303 Morishima, Y.*7, Kodera, Y.*2, Kuzushima, K., (CTSHR/A33). HLA peptide binding assays clearly Takahashi, To.*8 revealed that the immunogenicity of both epitopes was totally dependent on the differential HLA Minor histocompatibility (H) antigens are MHC binding capacity. CTSH is relatively ubiquitously (human leukocyte antigen, HLA in hu- expressed at the protein level and thus may be in- man)-associated peptides originating mainly from

29 polymorphisms in the genome that trigger T cell *3Department of Internal Medicine & Molecular Science, responses between MHC identical allogeneic indi- Nagoya City University Graduate School of Medical viduals. Graft-versus-host disease and graft-versus- Sciences leukemia/lymphoma (GVL) effects in hematopoi- *4Department of Infectious Diseases, Hamamatsu Uni- etic stem cell transplant recipients are initiated by versity School of Medicine donor T cell recognition of minor H antigens on re- *5Department of Regeneration Medicine for Hematopoi- cipient cells. Identification of suitable target anti- esis, Graduate School of Medicine, University of gens for selective GVL effects is warranted. Tokyo We have identified a novel HLA-B44–restricted *6Core Research for Evolutional Science and Technol- minor H antigen (designated as ACC-6) with ex- ogy (CREST) of Japan, Science and Technology pression limited to hematopoietic cells using a cy- Corporation (JST) totoxic T lymphocyte (CTL) clone isolated from a *7Department of Hematology and Cell Therapy, Aichi post-transplant patient with acute myeloid leukemia. Cancer Center Central Hospital cDNA expression cloning studies demonstrated that the CTL epitope of interest was encoded by a novel allelic splice variant of the HMSD (Histocompati- 3. The HLA-A*0201-restricted minor his- H bility Minor, Serpin Domain containing) gene, tocompatibility antigen HA-1 peptide hereafter designated as HMSD-v. The immuno- can also be presented by another genicity of the epitope was generated by differential HLA-A2 subtype, A*0206 protein expression due to alternative splicing, which Torikai H., Akatsuka, Y., Miyauchi, H., Terakura, S.*1, was completely controlled by 1 intronic sin- Onizuka, M.*1, Tsujimura, K.*2, Miyamura K.*1, gle-nucleotide polymorphism located in the con- Morishima Y.*3, Kodera Y.*1, Kuzushima, K., and sensus 5' splice site adjacent to an exon. Both Takahashi, To*4. HMSD-v and HMSD transcripts were selectively HA-1H is one of the most attractive minor histo- expressed at higher levels in mature dendritic cells, compatibility (H) antigens as a target for immuno- primary leukemia cells, especially those of myeloid therapy of hematopoietic malignancies, but lineage, and primary myeloma cells. Engraftment of HLA-A*0201 and -B60 molecules capable of pre- myeloid leukemia stem cells that were positive for senting HA-1H-derived peptides are less common in the ACC-6 minor H antigen into nonobese dia- eastern Asian when compared with Caucasian betic/severe combined immunodeficient populations. Therefore, an attempt was made to (NOD/SCID)/γnull mice was completely inhibited by search for novel epitopes presented by HLA alleles in vitro preincubation with an ACC-6-specific CTL other than those previously reported by generating clone, suggesting that this minor H antigen epitope CTL lines from patients undergoing HLA-identical, is expressed on leukemic stem cells. The patient HA-1 disparate hematopoietic stem cell transplanta- from whom the CTL clone was isolated demon- tion (HSCT) by stimulation with a 29-mer HA-1H strated a significant increase of ACC-6-specific T peptide spanning a central polymorphic histidine cells in post-transplant peripheral blood, whereas (His). Two CTL clones established were found to ACC-6-specific T cells were undetectable in be restricted by HLA-A*0206, which is the second pre-transplant peripheral blood and in peripheral or third most common HLA-A2 subtype worldwide. blood from his donor. These findings suggest that Epitope mapping revealed that the clones recog- the HMSD–v–encoded minor H antigen, designated nized the same nonameric peptide as ACC-6, could serve as a target antigen for immu- A*0201-restricted HA-1H, VLHDDLLEA. This notherapy against hematologic malignancies. The epitope was unexpected, since it does not contain clinical significance of the ACC-6 minor H antigen any preferred anchor motifs for HLA-A*0206. will be evaluated in a vaccination study in However, HLA peptide binding assays revealed post-transplant patients with high risk of malignan- stronger binding of this peptide to A*0206 than to cies. A*0201. We next examined the in vivo immuno-

genicity of HLA-A*0206-restricted, HA-1H-specific *1Department of Genetic Information, Division of Mo- T cells by means of tetramers. Peripheral blood lecular Life Science, Tokai University School of mononucleocytes (PBMCs) were obtained after Medicine HSCT from a patient who was positive for both *2Department of Hematology, Japanese Red Cross Na- HLA-A*0201 and -A*0206 and received an HA-1 goya First Hospital. disparate marrow transplant. After HA-1H peptide

30 pulsed antigen presenting cell stimulation, individ- *1Department of Pediatrics, Nagoya University Graduate ual tetramers detected tetramer+ CD8+ cells. Thus, School of Medicine A*0201 and A*0206 restricted HA-1H might even *2Department of Infectious Diseases, Hamamatsu Uni- be immunogenic. This finding points to expansion versity School of Medicine of the patient population who might benefit from *3Department of Cell Therapy, Aichi Cancer Center Cen- HA-1H-based immunotherapy. tral Hospital *4Aichi Health Plaza *1Department of Hematology, Japanese Red Cross Na- goya First Hospital 2 5. Effective antigen presentation to * Department of Infectious Diseases, Hamamatsu Uni- EBNA1-specific CD4+ T lymphocytes versity School of Medicine Watanabe, Y., Demachi-Okamura, A., and Kuzushima, *3Department of Hematology and Cell Therapy, Aichi K. Cancer Center Central Hospital *4Aichi Health Plaza Induction of potent and sustained antitumor immunity is dependent on efficient and longitudinal activation of cytotoxic T lymphocytes (CTLs) and 4. Epstein-Barr virus (EBV) nuclear anti- + gen 1-specific CD4+ T cells directly kill CD4 T lymphocytes. Antigens expressed in EBV-carrying NK and T cells mRNA-transfected antigen presentation cells (APCs) are mainly processed in the MHC class I Demachi-Okamura, A., Ito, Y.*1, Akatsuka, Y., pathway. Targeting antigens to the ly- Tsujimura, K.*2, Morishima Y.*3, Takahashi, To.*4, and sosomal/endosomal pathway in APCs can enhance Kuzushima, K. presentation of MHC class II-restricted epitopes de- Epstein-Barr virus (EBV) nuclear antigen rived from tumor and viral antigens. We therefore (EBNA) 1 is expressed in every EBV-infected cell, explored structures for effective antigen presenta- regardless of the state of EBV infection. Although tion to CD4+ T lymphocytes. Essentially three con- EBNA1 is thought to be a promising antigen for structs, lysosome-associated membrane protein 1 immunotherapy of all EBV-associated malignancies, (LAMP1), invariant chain and autophagy-related it is less clear whether EBNA1-specific CD4+ T light chain 3, were compared for their ability to in- cells can act as direct effectors. Here, we investi- troduce EBNA1 to the class II pathway. As signal gated the ability of CD4+ T cell clones induced with sequences, we used both of LAMP1 and the heat overlapping peptides covering the C-terminal re- shock protein gp96. Autologous CD40-activated B gion of EBNA1, and identified minimal epitopes cells transfected with mRNA encoding EBNA1 and their restricted MHC class II molecules. Of with the targeting signals to class II were used to these, a novel epitope, EYHQEGGPD, was found to stimulate an EBNA1-specific CD4+ T clone. IFN-γ be presented by DRB1*0401, 0403, and 0406. Five production from the clone was evaluated by en- CD4+ T cell clones produced IFN-γ upon stimula- zyme-linked immunospot assays. As a result, gene tion with autologous EBV-transformed lym- products from gp96-EBNA1-LAMP1 were most phoblastoid cell lines (LCLs) recognizing epitopes effectively presented among those tested. produced through autophagic pathways, as con- CD40-activated B cells transfected with the same firmed by RNA interference (RNAi) of an auto- structural mRNA were able to stimulate an phagy-related gene. One example proved capable of EBNA1-specific CTL clone, indicating that the killing EBV-carrying NK and T cell lines derived class I pathway is also enhanced by the modifica- from patients with chronic active EBV infection tion. Currently, simultaneous induction of (CAEBV), presenting an epitope through autopha- EBNA1-specific CTL and CD4+ T lymphocytes gic pathways, suggesting that EBNA1-specific using the APCs is ongoing. Our findings should CD4+ T cells may be helpful for immunotherapy prove useful not only for identification of novel targeting EBV carrying NK and T cell malignan- epitopes but also for monitoring T-cell responses cies. during immunotherapy.

From left to right First row: Mr. S. Toyama, Dr. S. Iwahori, Dr. T. Tsurumi, Dr. S. Nakayama, Dr. A. Kudoh, and Mr. Y. Nishikawa. Second row: Dr. S. Nakasu, Dr. T. Murata, Dr. H. Isomura, and Mr. Y. Sato.

32 Division of Virology ______

Tatsuya Tsurumi, M.D. Chief Sho Nakasu, PhD. Senior Researcher Hiroki Isomura, M.D. Senior Researcher Takayuki Murata, Vet. M.D. Researcher (as of September 2007) Satoko Iwahori, Ph.D. Research Resident Sanae Nakayama, Ph.D. Research Resident Yasuhiro Nishikawa, Research Assistant Ayumi Kudoh, M.D. Research Fellow of the Japanese Society for the Promotion of Science

Visiting Trainees Yoshitaka Sato, 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 Epstein-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 2006-2007, our research interest was concentrated on the following issues: 1) phosphorylation of MCM4 at sites inactivating DNA helicase activity of the MCM4-6-7 complex during Epstein-Barr Virus productive replication; 2) expression of Epstein-Barr Virus BZLF1 immediate-early protein induces p53 degradation independent of MDM2, leading to repression of p53-mediated transcription; 3) enhanced phosphorylation of transcription factor Sp1 in response to herpes simplex virus type 1 infection is dependent on the ataxia telangiectasia-mutated protein; 4) purification of the product of the Epstein-Barr virus BZLF1 gene; 5) a cis-element between the TATA Box and the transcription start site of the Major Immediate-Early (MIE) promoter of human cytomegalovirus determines efficiency of viral replication.

1. Phosphorylation of MCM4 at sites inac- EBV-PK with the same loss of helicase activity as tivating DNA helicase activity of the with CDK2/cyclin A. Introducing mutations in the MCM4-6-7 complex during Epstein-Barr N-terminal six Ser and Thr residues of MCM4 re- virus productive replication duced the inhibition by CDK2/cyclin A, while EBV-PK inhibited the helicase activity of both the Kudoh, A. and Tsurumi, T. wild type and the mutant MCM4-6-7 hexamer. Induction of Epstein-Barr virus (EBV) lytic rep- Therefore, phosphorylation of MCM4 by the con- lication blocks chromosomal DNA replication not- secutive actions of CDK2 kinase and EBV-PK dur- withstanding an S-phase like cellular environment ing lytic replication might provide one mechanism with high CDK2 activity. We report here that the to block chromosomal DNA replication in infected phosphorylated form of MCM4, a subunit of the cells, through inactivation of DNA unwinding by MCM complex essential for chromosomal DNA the MCM4-6-7 complex. replication, increases with progression of lytic replication, Thr-19 and Thr-110 being CDK2 targets 2. Expression of Epstein-Barr virus BZLF1 whose phosphorylation inactivates MCM4-6-7 immediate-early protein induces p53 complex-associated DNA helicase. Expression of degradation independent of MDM2, EBV-encoded protein kinase (PK) in HeLa cells leading to repression of p53-mediated causes phosphorylation of these sites on MCM4. In transcription vitro, the sites of MCM4 in the MCM4-6-7 Sato, Y. and Tsurumi, T. hexamer were confirmed to be phosphorylated by

33 Induction of the Epstein-Barr virus (EBV) lytic transcription activity of Sp1 is regulated in part by program elicits ATM-dependent DNA damage re- post-translational modifications, which include sponses, with activation of the ATM-Chk2-p53 phosphorylation, glycosylation, acetylation and signal transduction pathway, resulting in phos- sumoylation. It has been previously reported that phorylation of p53 at Ser15. This prevents interac- herpes simplex virus type 1 (HSV-1) infection in- tion with its negative regulator MDM2. However, duces activation of protein kinase activity of ATM p53-downstream signaling is blocked and the mo- and hyper-phosphorylation of transcription factor, lecular mechanism involved in this process remains Sp1. to be clarified. In this study, we found that during In the present study, we showed that ATM is in- lytic infection p53 is actively degraded in a protea- timately involved in Sp1 hyper-phosphorylation some-dependent manner even with a reduced level during HSV-1 infection, rather than individual of MDM2. Turnover of p53 is stimulated in the HSV-1 encoded protein kinases. In ATM-deficient lytic phase compared with that in the latent phase, cells or cells silenced for ATM expression by suggesting that EBV regulates p53 protein-levels shRNA targeting, hyper-phosphorylation of Sp1 also after induction of lytic replication. was prevented even as HSV-1 infection progressed. Co-immunoprecipitation analysis revealed that the Mutational analysis of putative ATM phosphoryla- EBV BZLF1 immediate-early protein interacts with tion sites on Sp1 and immunoblot analysis with the DNA-binding domain near the N-terminus of phosphopeptide-specific Sp1 antibodies clarified p53. It was confirmed that BZLF1 protein elicited that at least Ser-56 and Ser-101 residues on Sp1 proteasome-dependent degradation of p53 and re- become phosphorylated upon HSV-1 infection. pressed the p53-mediated transactivation in SaOS-2 Serine to alanine mutations at both sites on Sp1 cells. Degradation of p53 was observed even in the considerably abolished hyper-phosphorylation of presence of Nutlin-3, an inhibitor of p53-MDM2 Sp1 upon infection. Although ATM phosphorylated interaction, and also in mouse embryo fibroblasts Ser-101 but not Ser-56 on Sp1 in vitro, phosphory- lacking the mdm2 gene, indicating the BZLF1 pro- lation of Sp1 at both sites was not detected at all tein-induced degradation of p53 to be independent upon infection in ATM-deficient cells, suggesting of MDM2. Furthermore, Nutlin-3 increased the that some cellular kinase(s) activated by ATM level of p53 in the latent phase of EBV infection could be involved in phosphorylation at Ser-56. but not in the lytic phase. Thus, although the p53 Upon viral infection Sp1-dependent transcription in level is regulated by MDM2 in the latent phase, it ATM expression-silenced cells was almost the same might be mediated by the BZLF1 protein-associated as that in ATM-intact cells, suggesting that E3 ubiquitin ligase in the lytic phase. These find- ATM-dependent phosphorylation of Sp1 might ings provide insight into how EBV regulates the have only limited effects on transcriptional activity cellular environment advantageous for lytic replica- during HSV-1 infection. ATM-dependent Sp1 tion through degradation of p53, leading to inhibi- phosphorylation appears to be a global response to tion of p53 downstream signaling. various DNA damage stresses including viral DNA replication. 3. Enhanced phosphorylation of transcription factor Sp1 in response to herpes 4. Purification of the product of Epstein- simplex virus type 1 Infection is de- Barr virus BZLF1 Gene pendent on the ataxia telangiecta- Nakasu, S. and Tsurumi, T. sia-mutated protein The product of the BZLF1 gene (pBZLF1) of Iwahori, S. and Tsurumi, T. the Epstein-Barr virus (EBV) is a nuclear protein The ATM protein, a member of the related which activates the lytic cycle in cells latently in- PI-3-like kinase family encoded by a gene respon- fected with EBV. pBZLF1 has been suggested to sible for the human genetic disorder ataxia te- activate genes required for the lytic cycle and to langiectasia, regulates cellular responses to DNA induce viral DNA replication as a DNA binding damage and viral infection. Transcription factor protein specific for the viral lytic origin of DNA Sp1 functions as a trans-activator of gene expres- replication (ori lyt). sion and its recognition elements are distributed In order to understand the role of pBZLF1 in widely in various promoters of cellular and viral induction of the lytic cycle, we have focused on pu- genes. As with many other transcription factors, the rification and characterization of its biochemical

34 features. We first tried to purify pBZLF1 from in- 5. A cis-element between the TATA Box sect cells infected with baculoviruses which over- and the transcription start site of the produced pBZLF1. However, partially purified major immediate-early (MIE) promoter pBZLF1 from insect cells tended to form aggre- of human cytomegalovirus determines gates and was eluted with a wide range of salt con- efficiency of viral replication centrations from ion exchange chromatography Isomura, H. and Tsurumi, T. columns. The results suggested an altered confor- mation of the pBZLF1 produced in the insect cells. The promoter of the major immediate-early Therefore we next tried to purify pBZLF1 from (MIE) genes of human cytomegalovirus (HCMV), B95-8 cells, a cell line latently infected with EBV. also referred to as the CMV promoter, possesses a The lytic cycle was induced by chemicals and cis-acting element positioned downstream of the pBZLF1 could be extracted from the induced cells TATA box between -14 and -1 relative to the tran- with high salt buffer (0.6 to 1 M NaCl) and purified scription start site (+1). We determined the role of more than 100 fold with hydrophobic chromatog- the cis-acting element in viral replication by com- raphy, DEAE Sephacel chromatography, and phos- paring recombinant viruses with the cis-acting ele- pho (P) cellulose chromatography. Judging from ment replaced with other sequences. Like the sucrose gradient centrifugation, most pBZLF1 was wild-type virus, recombinant virus with the simian present in complexes with other proteins in low salt CMV counterpart replicated efficiently in human buffer (< 0.12 M NaCl) during these steps and be- foreskin fibroblasts. In contrast, replacement with haved as a monomer protein in high salt buffer (e.g. the murine CMV counterpart caused inefficient 1.5 M NaCl). Thus the steps were necessary to pu- MIE gene transcription, RNA splicing, MIE and rify pBZLF1 under high salt conditions. We found early viral gene expression, and viral DNA replica- pBZLF1 to flow through hydroxyapatite in the tion. To determine which nucleotides in the presence of phosphate and become absorbed to hy- cis-acting element are required for efficient MIE droxyapatite in the absence of phosphate under high gene transcription and splicing, we constructed mu- salt conditions. After hydroxyapatite chromatogra- tations within the cis-acting element in the context phy, a sample was absorbed to heparin sepharose in of a recombinant virus. While mutations in the 0.3 M NaCl buffer and pBZLF1 was eluted with 0.7 cis-acting element had only a minor effect on in vi- M NaCl buffer. Starting from 3 l culture, pBZLF1 tro transcription, effects on viral replication were and several other proteins were visible on silver major. The nucleotides at -10 and -9 in the staining of the SDS page of the final fraction (0.4 cis-acting element relative to the transcription start ml) but were only faint with CBB staining. There- site (+1) affected efficient MIE gene transcription fore a larger scale purification procedure is now and splicing at early times after infection. The required for further investigation. cis-acting element also acts as a cis-repression sequence when the viral IE86 protein accumulates in infected cells. We were thus able to demonstrate that the cis-acting element has an essential role in viral replication.

From left to right First row: Dr. Zenta Yasukawa, Dr. Osamu Taguchi, Dr. Reiji Kannagi, Dr. Mamoru Kyogashima and Dr. Akiko Kanamori. Second row: Ms. Keiko Miyazaki, Ms. Akiko Nishioka, Ms. Mineko Izawa, Dr. Akiko Yusa, Dr. Keiko Tamiya-Koizumi, Dr. Lim Khe-Ti, Ms. Yoshiko Goto, Ms. Sachiko Kondo, and Mr. Kouji Tanaka. Third row: Dr. Jun Yin, Dr. Hirokazu Yagi, Dr. Masahiro Fujii, Dr. Keiichiro Sakuma Inset: Ms. Naoko Kimura, Dr. Guo-Yun Chen, Ms. Sasako Eguchi, Ms. Kumiko Oseto, Ms. Kazumi Hagiwara, Ms. Sayaka Sobue, Ms. Sayaka Miyagi and Ms. Risa Umeyama.

______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 Akiko Kanamori, Ph.D., Senior Researcher Keiichiro Sakuma, M.D., D.M.Sc., Senior Researcher (as of April, 2007) Zenta Yasukawa, Ph.D., Research Resident Akiko Yusa, Ph.D., Research Resident (as of April 2007) Mineko Izawa, B.A., Research Assistant (until March, 2007) 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 Jun Yin, Ph.D. Foundation for Promotion of Cancer Research (as of April, 2006) Khe-Ti Lim, Ph.D., National Institute of Biomedical Innovation Guo-Yun Chen, M.D., D.M.Sc., Japan Science and Technology Agency (until March, 2007) Keiko Miyazaki, M.T., Japan Science and Technology Agency Mineko Izawa, B.A., Japan Science and Technology Agency (as of April, 2007) Takashi Murate, M.D., D.M.Sc., Nagoya University School of Health Sciences Keiko Tamiya-Koizumi, Ph.D., Nagoya University School of Health Sciences

Visiting Trainees Naoko Kimura, B.Ph., Nagoya City University Masahiro Fujii, M.D. Nagoya University School of Medicine (as of June, 2006) Kazumi Hagiwara, M.T., Nagoya University School of Health Sciences Sayaka Sobue, M.T., Nagoya University School of Health Sciences Hirokazu Yagi, M.P. Nagoya City University Sachiko Kondo, M.E. Nagoya City University Akiko Yusa, Ph.D., Aichi University of Education (until March, 2007) Kouji Tanaka, B.Ph., Shinshu University School of Medicine Risa Umeyama, Sugiyama Jogakuen University (until December, 2006) Sayaka Miyagi, Sugiyama Jogakuen University (as of February, 2007) Kumiko Oseto, Sugiyama Jogakuen University (as of February, 2007) Tetsufumi Koike, M.D., Fukushima University School of Medicine (until March, 2006) Atsushi Akutagawa, M.D., Nagoya University School of Medicine (until March, 2006)

General Summary Cell adhesion mediated by selectins and their carbohydrate ligands, sialyl Lewis X and sialyl Lewis A, plays an important role in cancer metastasis and tumor angiogenesis. Expression of these carbohydrate determinants is markedly enhanced in cancer cells compared to non-malignant epithelial cells. Our recent studies indicated the presence of further-modified complex forms of sialyl Lewis X/A in non-malignant epithelial cells, which have additional sulfation or sialylation. Such complex determinants serve as specific ligands for another family of carbohydrate-recognizing molecules, siglecs, and maintain immunological homeostasis in normal mucous membranes. The impairment of these additional modifications in cancer cells, which we term "incomplete synthesis," leads to considerable accumulation of sialyl Lewis X/A in cancer cells at early stages of malignant transformation. Epigenetic gene silencing through DNA methylation and/or histone deacetylation is proposed to confer the incomplete synthesis.

In later stages of cancer progression, increased transcription of several glycogenes responsible for the synthesis of sialyl Lewis X/A determinants by hypoxia-inducible factors further accelerates expression of these determinants on cancer cells. Hematogenous metastasis mediated by sialyl Lewis X/A-selectin interactions determines the prognostic outcome of patients with advanced cancers. Cell adhesion molecules equipped with specific carbohydrate-recognition domains figure prominently in the biological behavior of cancer cells as well as in cell-cell interactions in normal mucosal membranes. During the period 2006-2007, we have made progress in elucidating the mechanisms involved in induction of cancer-associated carbohydrate determinants including sialyl Lewis X, sialyl Lewis A and N-glycolyl GM2 in human cancers. At the same time, we have also studied the role of CD44 in cancer progression, and identified several important molecules involved in immune recognition.

1. Significance of interconversion tion in immune cells by recruiting tyrosine between carbohydrate ligands for phosphatases SHP-1 and SHP-2. On trans- selectins and siglecs expressed fection into human macrophage cell lines, on normal colonic epithelium and Siglec-7/-9 suppressed LPS-induced COX2 cancer cells and IL-12 production. These results imply that normal glycans of colonic epithelial cells Miyazaki, K. Izawa, M., Ohmori, K.*1, Hashimoto, exert a suppressive effect on tissue macro- Y.*2 and Kannagi, R. phage COX2 expression in colonic mucosa, Cancer-associated carbohydrate determi- thus maintaining immunological homeostasis nants, such as sialyl Lewis A and sialyl in normal mucosal membranes. The results Lewis X, are known to serve as ligands for also suggest that abnormal glycosylation selectins, and mediate cancer cell adhesion to during early carcinogenesis abrogates this vascular beds in the course of hematogenous suppressive effect. metastasis. Expression of these determinants is markedly increased in cancer cells *1 Department of Laboratory Medicine, Kyoto compared to normal epithelial cells. We University, School of Medicine. have recently shown that non-malignant *2 Glyco-chain Functions Laboratory, Frontier epithelial cells express carbohydrate deter- Research System, RIKEN Institute. minants, such as disialyl Lewis A or sialyl 6-sulfo Lewis X, having more complex struc- 2. Tumor hypoxia induces expres- tures than sialyl Lewis A/X. Their expression of gangliosides containing sion is lost at an early stage of colon car- abnormal sialic acid on cancer cinogenesis due to the epigenetic silencing of cells glycogenes involved in their synthesis. Yin, J., Izawa, M., Miyazaki, K., Yasukawa, Z. Loss of these normal carbohydrate determi- Kitajima, K.*1 and Kannagi, R. nants then leads to accumulation in cancers of less-complex determinants such as sialyl Tumor hypoxia plays important roles in Lewis A and sialyl Lewis X. Disialyl Lewis malignant progression by altering intracellu- A on normal epithelial cells serves as a lar glucose metabolism and inducing angio- ligand for siglec-7 and siglec-9, but sialyl genic factor production, thus selecting and Lewis A on cancer cells has no binding activ- expanding more aggressive hypoxia-resistant ity. Binding assays at the cellular level in- cancer cell clones. Little is known, however, dicated that sialyl 6-sulfo Lewis X also regarding hypoxia-induced changes in cell serves as a ligand for siglec-7, but sialyl surface molecules, which can serve as mark- Lewis X does not. It is noteworthy that ers of hypoxia-resistant cancer cells. only the carbohydrate determinants on nor- We therefore have investigated expression mal epithelial cells serve as ligands for of NeuGc-GM2, a cancer-associated gan- siglecs, while cancer-associated determinants glioside GM2 containing a non-human sialic do not. Siglec-7/-9 are known to have acid, N-glycolyl sialic acid (NeuGc), in hu- ITIM-motifs which inhibit signal transduc- man cancers. Tissues prepared from breast

which is not endogenously synthesized through CMP-NeuAc hydroxylase, since humans lack a gene for the synthetic enzyme. As hypoxia is associated with diminished response to radio- and chemotherapy, NeuGc-GM2 is a potential therapeutic target for hypoxic cancer cells. Expression of many other carbohydrate determinants is also induced by tumor hypoxia. One must bear in mind here that hypoxia-induced change is not limited to malignant cells, and that normal cells respond to hypoxia as well. If cell surface determinants induced simply by hypoxia are chosen as a target of therapeutic attack, such determinants can be induced on the surfaces of hypoxic normal cells, which will also be open to attack. In this context, it is inter- esting to note that the GM2 ganglioside, even that with normal sialic acid, is known to be preferentially expressed on cancer cells, and is regarded as a good target for immune Fig. 1. Preferential expression of NeuGc- therapy of cancers. NeuGc-GM2 is therefore GM2 in human cancer tissues. Expression of expected to be a better target for therapy of NeuGc-GM2 in human cancer tissues; immuno- hypoxia-resistant highly-aggressive cancers histochemical staining with the anti-NeuGc- than GM2 having normal sialic acid. GM2 antibody (MK2-34) of normal colonic epithelial cells (panel A) and cancer cells (panel B) in tissue sections prepared from a patient *1 Bioscience and Biotechnology Center, Nagoya with colon cancer. Bar, 100 µm. Adopted University,. from Cancer Res., 66: 2937-2945, 2006. and colon cancers frequently expressed 3. Studies on cell adhesion activity NeuGc-GM2, while it was virtually absent in of CD44 non-malignant mammary and colonic epithe- 3.1. Roles of carbohydrate side chains of the lia. Studies on cultured cancer cells have CD44 molecule indicated that non-human sialic acid can be Lim, K.T., Miyazaki, K., Kimura, N. Murakami, incorporated from culture medium. Hy- Y.*1 and Kannagi, R. poxic culture markedly induced mRNA for a sialic acid transporter, sialin, and this was Cell adhesion molecules such as CD44 are accompanied by enhanced incorporation of modified by a variety of carbohydrate chains NeuGc. Transfection of cells with the including N-glycans, O-glycans and glyco- Sialin gene conferred accelerated sialic acid saminoglycans. Using human cancer cells transport and induced cell surface expression we have studied functional roles of the car- of NeuGc-GM2. bohydrate side chains of CD44, known to From these results we propose that the specifically bind to a carbohydrate ligand, preferential expression of NeuGc-GM2 in hyaluronate. Sialylation of CD44 is known cancers is closely associated with tumor hy- to suppress, while 6-sulfation facilitates this poxia. This latter induces expression of the binding to hyaluronate. Western blotting sialic acid transporter, and enhances incor- analyses of CD44 in cultured human colon poration of non-human sialic acid from the cancer cells indicated that it carried the sialyl external milieu. A consequence of this is Lewis X determinant, which is known as a acquisition of cancer-associated cell-surface ligand for E-selectin. CD44 in colon cancer gangliosides, typically GM2, containing ab- cells were also found to express sialyl Lewis normal non-human sialic acid (NeuGc), A and sialyl 6-sulfo Lewis X determinants.

We previously showed that the sialyl Lewis exclusively mediated by the CD44-HA inter- A determinant serves as a ligand for action, because it was inhibited by HA, but E-selectin as well as sialyl Lewis X, and that not IαI, and was completely abolished by the sialyl 6-sulfo Lewis X determinant is a pretreating the cells with anti-CD44 antibod- specific ligand for L-selectin. These car- ies. SHAP appears to potentiate the interac- bohydrate determinants are carried mainly by tion by increasing the avidity of HA for the splicing variants of CD44 (CD44v) in CD44 and altering their distribution on cell cancer cells. Introduction of a gene for the surfaces. Large amounts of the SHAP-HA sialyltransferase that transfers sialic acid to complex accumulate in the hyperplastic N-acetylglucosamine residues induced ex- synovium of rheumatoid arthritis patients. pression of the disialyl Lewis A determinant, Infiltrating leukocytes are strongly positive which we recently showed to serve as a for HA, SHAP, and CD44 on their surfaces, ligand for siglec-7, a member of the immu- suggesting a role for the adhesion-enhancing nosuppressive receptor family recognizing effect of SHAP in pathogenesis. sialic acid. These results suggest that carbohydrate side chains of CD44v molecules in *1 Institute for Molecular Science of Medicine, colon cancers not only regulate the binding to Aichi Medical University. hyaluronate, but also function as ligands for *2 Nagoya University, School of Medicine. multiple cell adhesion molecules, and that expression of functional carbohydrate side 4. Diversity of sulfatide molecular chains is highly dependent on presence of the species from biological materials splicing variants. by MALDI-TOF MS

Kyogashima, M., Tadano-Aritomi, K.*1, Murate, *1 Institute of Medical Science, Tokyo Univer- T., Tamiya-Koizumi, K., Hara, A.*2, Aoyama, T.*2 sity and Kannagi, R.

3.2. Potentiation of CD44-mediated leukocyte By combining the partition method for adhesion to the hyaluronan substratum enrichment of sulfatides without any chro- by SHAP matographic procedures and a preparation method for lysosulfatides, we have succeeded Zhuo, L.S.*1, Kanamori, A., Kannagi, R. Itano, in analyzing sulfated glycosphingolipids N.*1, Wu, J.W.*1, Hamaguchi, M.*2, Ishiguro, N.*2, from biological materials by MALDI-TOF and Kimata, K.*1 MS within a single day. We found SM4s CD44-hyaluronan (HA) interaction is in- (galactosylsulfatide) to be composed of dif- volved in diverse physiological and patho- ferent species. While the exact composition logical processes. Regulation of interaction depended on the source material, SM4s al- avidity is well studied for CD44 but less is ways contained hydroxy fatty acids to vari- known regarding HA. We discovered a ous degrees. In addition to the common unique covalent modification of HA by a sphingoid 4-sphingenine (d18:1), uncom- protein, SHAP, that corresponds to the heavy mon/unusual sphingoids phytosphingosine chains of inter-α-trypsin inhibitor family (t18:0), 4-eicosasphinganine (d20:0), molecules circulating in blood. Formation 4-eicosasphingenine (d20:1), and sphingadi- of the SHAP-HA complex is often associated enine (d18:2) were readily detected. Finally, with inflammation, a process well known to in addition to SM4s, sulfatide SM3 (sulfated involve CD44-HA interaction. We there- lactosylceramide) and SM2 (sulfated gan- fore examined the effect of SHAP on the gliotriaosylceramide) were clearly present in CD44-HA interaction-mediated lymphocyte renal tubule cells. The major SM4s were adhesion. Under both static and flowing found to comprise ceramides possessing conditions, Hut78 cells (CD44-positive) and d18:1 with C22 hydroxy fatty acids (C22:0h), CD44-transfected Jurkat cells (originally C23:0h, and C24:0h, whereas the major CD44-negative) adhered preferentially to SM3/SM2 forms were composed of cera- immobilized SHAP-HA complexes rather mides possessing t18:0 with C22 normal fatty than to HA. The enhanced adhesion was acids (C22:0), C23:0, C24:0. Namely, in

these two series of sulfatides, either fatty ac- recognize GlcA-GlcNAc, IdoA2S-GlcNAc, ids or sphingoids were hydroxylated, and and IdoA-GlcNAc, respectively. Several hu- chain lengths of these components were ex- man colon, squamous cell, lung, and breast actly the same, consequently resulting in a cancer cell lines as well as breast cancer tis- similar polarity of ceramide moieties. sues were then examined by flow-cytometry These results demonstrate diversity of sulfa- and immunohistochemistry using these anti- tide molecular species, not only with respect bodies. While NAH46 intensively stained all to sugar- but also to ceramide moieties, cell lines, staining patterns with 10E4 and which is probably important for specific ef- JM403 were variable, suggesting that the de- fective functions in particular microenviron- gree of HSPG sulfation differed depending ments, such as lipid membrane microdo- on the cancer cell. AS22 and ACH55 stained mains. some colon cancer cell lines and breast cancer tissues, indicating that HSPGs having the *1 Department of Biochemistry, Teikyo Univer- IdoA2S-GlcNAc and/or IdoA-GlcNAc sity School of Medicine. structures may also be expressed in human *2 Department of Metabolic Regulation, Institute cancers. on Aging and Adaptation, Shinshu University Graduate School of Medicine. *1 Central Research Laboratories, Seikagaku Corporation. *2 5. Molecular profiling of heparan Natural Products Research Institute, College of Pharmacy, Seoul National University. sulfate glycosaminoglycans in *3 human cancers using monoclonal Institute for Molecular Science of Medicine, antibodies Aichi Medical University *4 Department of Breast Oncology, Aichi Cancer Yusa, A., Fujii, M., Goto, Y., Suzuki, K.*1, Ishi- Center Hospital. maru , T.*1, Yamamoto, K.*1, Kim, Y.*2, Kimata,

K.*3, Iwata, H.*4, Kyogashima, M., and Kannagi, R. 6. Studies on molecules involved in immune regulation Cancer cell surface heparan sulfate pro- teoglycans (HSPGs) play important roles in 6.1. Interaction of GATA-3/T-bet transcription cell proliferation, invasion and metastasis. factors regulates expression of selectin Information, however, on sugar chain struc- ligands in human leukemic cells. tures of the glycosaminoglycans (GAGs) on Chen, G.Y., Osada, H.*1, Santamaria-Babi, L.F.,*2 malignant cells has been more limited, as and Kannagi, R. compared to the core proteins, because of Selectin-mediated cell adhesion is in- their extreme complexity. In order to address volved in hematogenous metastasis of cancer this issue, we have profiled cancer HSPGs by cells and tissue infiltration by leukemic cells. flow-cytometry and immunohistochemistry It is also implicated in extravasation of leu- using various monoclonal anti-HS antibodies, kocytes in inflammatory diseases as well as including 10E4 recognizing GlcNS and in routine homing of lymphocytes. Se- GlcNAc, JM403 recognizing GlcNH2, and lectin-mediated cell adhesion is regulated by newly generated antibodies, NAH46, AS22 levels of selectins and their carbohydrate and ACH55. NAH46 was generated by im- ligand, sialyl Lewis X, whose expression on munizing mice with KLH-coupled bacterial cell surfaces depends on transcriptional polysaccharide K5 heparosan. AS22 and regulation of the fucosyltransferase VII gene ACH55 were generated with KLH-coupled (FUT7), encoding the rate-limiting enzyme acharan sulfate prepared from the giant Afri- for sialyl Lewis X synthesis. We have pre- can snail, and its desulfated form acharan, viously shown that enhanced sialyl Lewis X respectively. Specificities of these antibodies expression on adult T cell leukemic cells is were tested using a panel of GAGs, including due to induction of FUT7 transcription by the chondroitin sulfate, keratan sulfate, hyalu- Tax protein, an oncogenic transcription acti- ronic acid, heparin and HS. NAH46, AS22 vator encoded by the etiological virus and ACH55 were confirmed to specifically HTLV-1. The induction was mediated by

Fig. 2. Transcription factors involved in regulation of the gene for fucosyltransferase VII (FUT7), which is the rate-limiting step enzyme in the synthesis of the specific selectin ligand, sialyl Lewis x. The factors indicated in dark grey (GATA-3 and HDACs) exert suppressing effects, whereas others in light grey promote Fuc-T VII transcription. Adopted from Proc. Natl. Acad. Sci. U.S.A., 103:16894-16899, 2006. phosphorylation-independent interaction of cer Center. CREB/ATF-family transcription factors with *2 Department of Dermatology, Hospital del Mar, the Tax protein. IMAS, Barcelona, Spain. We have now further investigated transcription factors involved in regulation of the 6.2. Identification of cutaneous lymphocyte FUT7 gene. The 5'-regulatory region of antigen as sialyl 6-sulfo-Lewis X, a se- FUT7 turns out to be equipped with binding lectin ligand expressed on a subset of sites for multiple transcription factors, in- skin-homing helper memory T cells cluding Sp1, MZF-1, CREB/ATF, GATA-3 Ohmori, K.*1, Sakuma, K., Kiso, M.*2, Imai, T.*3, and T-bet. Among these factors, only Yoshie, O.*4, Hasegawa, H.*5, Matsushima, K.*6 GATA-3 repressed, while the other promoted, and Kannagi, R. FUT7 transcription. All formed transcription factor complexes on the FUT7 promoter; We previously identified the carbohydrate GATA-3 interacted with HDAC-3/-5, T-bet determinant sialyl 6-sulfo Lewis X as the and CREB/ATF, and T-bet interacted with major L-selectin ligand on high endothelial GATA-3 CBP/P300 and Sp1. The GATA- venules of peripheral lymph nodes. In recent 3/ T-bet interaction most dynamically regu- studies, we examined the distribution of the lated composition of the transcriptional com- sialyl 6-sulfo Lewis X determinant among plex on the FUT7 promoter. GATA-3 re- peripheral lymphocytes. It was thereby found pressed FUT7 transcription through phos- to be expressed on a subset of helper memory phorylation-dependent interaction with T and NK cells. Helper memory T cells ex- HDAC-3 and -5, while T-bet promoted its pressing sialyl 6-sulfo Lewis X were bright+ high+ + + transcription through recruiting CBP/P-300. CD45RO PSGL-1 CCR4 L-selectin + T-bet interfered with binding of GATA-3, CCR7 but lacked α4β7 integrin and CCR9, which in turn interfered with binding of T-bet indicating that they were the skin-homing to the FUT7 promoter as evidenced by chro- population of central memory T cells. The matin immunoprecipitation assays. Since T-cell subset significantly expressed mRNAs GATA-3 and T-bet are two opposing factors for 6-sulfotransferase HECGlcNAc6ST and in T-helper-1 (Th1) and T-helper-2 (Th2) fucosyltransferase Fuc-T VII, responsible for development, this well explains the preferen- the synthesis of sialyl 6-sulfo Lewis X. tial expression of sialyl Lewis X in Th1 Characteristics of the T-cell population were compared to Th2 cells. In addition, we have similar to those previously described for cu- generated evidence implying that HIF (hy- taneous lymphocyte-associated antigen poxia inducible factor) also binds to the (CLA)–positive T cells defined by the FUT7 promoter. This would explain the HECA-452 or 2F3 antibodies. Binding of the hypoxia-induced expression of sialyl Lewis T-cell subset with the specific anti–sialyl X in cancer cells, as described in our previ- 6-sulfo Lewis X antibody G152 was almost ous report. completely abrogated by HECA-452 or 2F3. Binding of recombinant E-, P-, and *1 Division of Molecular Oncology, Aichi Can- L-selectins to the T-cell subset was also sig-

nificantly inhibited by G152 and by HECA-452 antibodies. We propose that CLA, which is expressed without any activation stimuli on peripheral skin-homing helper memory T cells in healthy persons, is at least partly the sialyl 6-sulfo Lewis X determinant.

*1 Kyoto University, School of Medicine. *2 Gifu University, School of Agriculture. *3 Kan Research Institute. *4 Kinki University, School of Medicine. *5 Ehime University, School of Medicine. *6 Tokyo University, School of Medicine.

6.3. Identification of α2-6 sialylated 6-sulfo- LacNAc as a preferred ligand for CD22/siglec-2 on human B-lymphocytes *1 Kimura, N., Ohmori, K. , Miyazaki, K., Izawa, *2 *2 M., Matsuzaki, Y. , Yasuda, Y. , Takematsu, Fig. 3. Specific inhibition of CD22/Siglec-2 H.*3, Kozutsumi, Y.*3, Moriyama, A.*4, and Kan- binding to human peripheral B-lymphocytes nagi, R. by newly generated antibody specific to α2→6-sialylated 6-sulfo-LacNAc determinant. CD22/Siglec-2, an important inhibitory Note that binding of recombinant CD22-Fc to co-receptor on B-lymphocytes, is known to B-lymphocytes is significantly reduced by the recognize α2-6-sialylated glycans as a spe- KN343 antibody that is specific to α2→6-sialy- cific ligand. Here we propose that the lated 6-sulfo-LacNAc determinant, but not by the GL7 antibody, that is directed to non-sulfated α2-6-sialylated 6-GlcNAc-sulfated determi- α2→6-sialylated LacNAc determinant, corrobo- nant serves as a preferred ligand for CD22, rating importance of 6-sulfation in the binding of because binding of a human B-cell line to CD22/Siglec-2. Adopted from J Biol Chem., CD22 was almost completely abrogated after 282: 32200-32207,2007. incubation with NaClO , an inhibitor of cel- 3 CD22/Siglec-2 functions in humans. lular sulfate metabolism, and also significantly inhibited with a newly generated *1 Kyoto University, School of Medicine. monoclonal antibody specific to the *2 Central Research Laboratories, Seikagaku α2-6-sialylated 6-sulfo-LacNAc determinant Corporation. (KN343, murine IgM). The α2-6-sialylated *3 Kyoto University, School of Pharmaceutical 6-sulfo-LacNAc determinant defined by the Sciences. antibody was significantly expressed on a *4 Nagoya City University, School of Natural majority of normal human peripheral Science. B-lymphocytes as well as follicular B-lymphocytes in peripheral lymph nodes. 6.4. Alpha-internexin as a corneal autoanti- The determinant was also expressed by en- gen in a spontaneous auto-immune dothelial cells of high-endothelial venules of keratitis mouse model secondary lymphoid tissues including lymph Hattori, T.*1, Takeuchi, M.*1, Usui, M.*1 and nodes, tonsils and gut-associated lymphoid Taguchi, O. tissues, more strongly than on B-lymphocytes, suggesting a role for CD22 in B cell interac- The purpose of this study was to identify tion with blood vessels and trafficking. target antigens of autoimmune keratitis in a These results indicate that the α2-6-sialylated disease-prone mouse model. BALB/c nude 6-sulfo-LacNAc determinant serves as an mice grafted with embryonic rat thymi (TG endogenous ligand for human CD22, and nude mice) develop various organ-localized provide evidence that 6-GlcNAc sulfation as autoimmune lesions, including keratitis. A well as α2-6 sialylation may regulate hybridoma producing a monoclonal antibody

(OT-20), specific for corneal epithelium was these mice on stimulation with al- established using spleen cells from this pha-internexin exhibited proliferation re- mouse model of keratitis, and the target of sponses and produced IL-2, IFN-gamma and OT-20 was identified by immunoblot analy- TNF-alpha, but not IL-4 or IL-5. These re- sis. T-cell proliferation and cytokine produc- sults suggest that alpha-internexin is one of tion by TG nude mice with keratitis were the corneal antigens associated with keratitis then examined. Immunoblot analysis re- developing spontaneously in TG-nude mice, vealed alpha-internexin to be the target anti- with a probable pathogenic role. gen of OT-20 that specifically recognizes corneal epithelium. Sera from TG nude mice *1 Department of Ophthalmology, Tokyo Medical with keratitis reacted with alpha-internexin University, Tokyo. on Western blot analysis, and the T cells of

From left to right First row: Dr. M. Ibi, Ms. Y. Takada, Dr. Y. Ohmuro, Ms. T. Yuhara Second row: Dr. T. Shiromizu, Dr. Y. Ikegami, Dr. Y. Hayashi, Dr. H. Goto Third row: Dr. M. Inagaki, Dr. K. Kasahara, Mr. M. Enomoto, Dr. I. Izawa, Dr. A. Inoko Inset: Dr. A. Kawajiri, Dr. P. Zou, Dr. T. Yamaguchi, Dr. T. Oguri

45 Division of Biochemistry ______

Masaki Inagaki, M.D. Chief Ichiro Izawa, M.D. Section Head Hidemasa Goto, M.D. Section Head Akihito Inoko, M.D. Senior Researcher Kousuke Kasahara, Ph.D. Researcher (as of April 2007) Yuko Hayashi, Ph.D. Research Assistant Peng Zou, M.D. Research Resident (until March 2007) Takashi Siromizu, Ph.D. Research Resident (as of April 2006) Miho Ibi, D.D.S. Research Resident (as of April 2007)

Visiting Trainees Yuki Ohmuro, Ph.D. Research Resident of Foundation for Promotion of Cancer Research (as of April 2007) Masato Enomoto, M.S. Department of Cellular Oncology, Graduate School of Medicine, Nagoya University (as of April 2006) Yousuke Ikegami, M.D. Department of Nephro-Urology, Nagoya City University Graduate School of Medical Sciences (as of April 2006) Aie Kawajiri, Ph.D. Department of Pathology, Nagoya University School of Medicine (until March 2006) Takashi Oguri, M.S. Department of Cancer Genetics, Nagoya University School of Medicine (until March 2006) Takashi Shiromizu, M.S. Department of Cancer Genetics, Nagoya University School of Medicine(until March 2006) Tomoya Yamaguchi, M.S. Department of Cancer Genetics, Nagoya University School of Medicine (until March 2007)

General Summary Cells need to respond to environmental signals to proliferate in a coordinated fashion during development and differentiation. In epithelia, the architecture is also intimately involved in control of cell cycle machinery. Mutations in genes functioning in cell cycle control and the maintenance of tissue architecture lead to uncontrolled proliferation, genetic instability, and cell invasion (metastasis) in cancer cells. However, the precise mechanisms of carcinogenesis remain largely unknown. Our research aim is to elucidate mechanisms by which the cell cycle (including cell cycle checkpoints) and tissue architecture (including the 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 checkpoints; (2) Regulation of cytoskeletal proteins (especially intermediate filaments) and associated elements active in cell adhesion and determination of cell polarity.

1. Chk1 phosphorylation by cyclin- tated- and Rad3-related (ATR) and thereby acti- dependent kinase 1 controls mitotic vated. This prevents premature entry into mitosis. entry through cytoplasmic sequestra- However, Chk1 dynamics in mitosis remain to a tion of Chk1 great extent unknown. We recently reported Chk1 to be phosphorylated Enomoto, M., Goto, H., Kasahara, K., Ikegami, Y., at Ser286 and Ser301 by cyclin-dependent kinase Yamaguchi, T., Shiromizu, T., Tomono, Y.*1, Tsujimura, (Cdk) 1 during mitosis. In order to elucidate func- K.*2, Kiyono, T.*3 and Inagaki, M. tional consequences, we produced a rat monoclonal Chk1 is one of the most important players in antibody which specifically recognizes Chk1 phos- genetic stability, disorders of which are often ob- phorylated at Ser301. Immunocytochemical analy- served in cancer cells. In response to stalled replica- ses using this antibody revealed Chk1-Ser301 tion and genotoxic stresses, Chk1 is phosphorylated phosphorylation from entry into mitosis (prophase). at Ser317 and Ser345 by ataxia-telangiectasia mu- The level of phosphorylation appeared to peak at

46 metaphase and to gradually decrease after anaphase. lation state-specific antibodies for Ser296, Ser301 In prophase when chromosome condensation occurs (a Cdk1 site), or Ser345 (an ATR site) on Chk1. without nuclear envelope breakdown, we observed Hydroxyurea (HU) treatment or ultraviolet (UV) ir- two phosphorylation patterns. At earlier stages, the radiation were confirmed to induce Ser296 and phophorylation tended to be observed mainly in the Ser301 phosphorylation, together with Ser345 nucleus. However, at later stages of prophase, Chk1 phosphorylation, by ATR. Treatment with UCN-01, phosphorylated at Ser301 appeared to exist mainly a Chk1-specific inhibitor, reduced the phosphoryla- in the cytoplasm. This translocation was inhibited tion at Ser296 but not at Ser345, suggesting that by treatment with leptomycin B, which blocks Ser296 may be an autophosphorylation site on Crm1-mediated nuclear export. To further address Chk1. Interestingly, in response to HU treatment or the role of mitotic Chk1 phosphorylation, we estab- UV irradiation, Chk1 phosphorylated at Ser296 or lished HeLa cells in which Chk1 wild type (WT) or Ser345 formed foci in the nucleus, but the foci Chk1 mutated to Ala at Ser286 and Ser301 didn’t completely overlap with each other. These (S286A/S301A) were expressed in a tetracy- results imply that Chk1 function may be spatially cline-dependent manner. Introduction of regulated by phosphorylation at different sites in the S286A/S301A caused significant delay in mitotic checkpoint response. Now, we are further analyzing entry, compared with the WT case. In the specific roles of each site phosphorylation in S286A/S301A mutant prophase cells, staining was checkpoint responses. observed mainly in the nucleus although in WT cells location was also found in the cytoplasm. *1 Division of Molecular and Cell Biology, Shigei GST-pull down assays revealed that Chk1 could Medical Research Institute bind to 14-3-3 proteins in a Ser301- phosphoryla- *2 Department of Infectious Diseases, Hamamatsu Uni- tion-dependent manner. These results suggested that versity School of Medicine Cdk1 controls the cytoplasmic sequestration of *3 Virology Division, National Cancer Center Research Chk1 likely through binding to 14-3-3 proteins. We Institute propose that this pathway is a key control mechanism for maintaining the G2/M transition. 3. Palmitoylation of ERBIN is required for its plasma membrane localization *1 Division of Molecular and Cell Biology, Shigei Izawa, I., Nishizawa, M., Hayashi, Y. and Inagaki, M. Medical Research Institute *2 Department of Infectious Diseases, Hamamatsu Uni- LAP (leucine-rich repeats (LRR) and versity School of Medicine PSD-95/Dlg/ZO-1 (PDZ)) family proteins, includ- *3 Virology Division, National Cancer Center Research ing Scribble, LET-413, ERBIN, Densin-180, and Institute Lano, are involved in the regulation of cell polarity. Drosophila Scribble is a cell-junction localized 2. Spatial regulation of Chk1 phosphoryla- protein implicated in control of epithelial cell polar- tion in checkpoint response ity and growth. In addition, it has been shown that Scribble mutants cooperate with oncogenic Ras, Kasahara, K., Ikegami, Y., Goto, H., Enomoto, M., Raf, or Notch to cause invasive and metastatic neo- Tomono, Y.*1, Tsujimura, K.*2, Kiyono, T.*3 and plasia in Drosophila larvae. The LRR domains of Inagaki, M. LAP proteins are reported to mediate basolateral Precise chromosome duplication is critical to membrane localization and to be essential for their cellular functions, and errors in the process can re- function. To further dissect the mechanisms of sult in aneuploidy, which is often observed in can- plasma membrane localization of ERBIN, we in- cer cells. Cells respond to DNA damage and repli- troduced various mutants of ERBIN into cultured cation blocks by activating a checkpoint network, a cells and observed the intracellular localization. system which arrests the cell cycle and facilitates When a LRR domain mutant lacking amino acid DNA repair. Chk1, one of the key players in the residues 1-32 at the amino (N) terminal region was checkpoint system, is controlled by several protein overexpressed in cells, localization was in the cyto- kinases such as ATR and Cdk1. However, the roles plasm, rather than at the plasma membrane. We of each phosphorylation site on Chk1 are not fully found that cysteines 14 and 16 in the N-terminal re- understood. gion of ERBIN are palmitoylated and that this For analysis, we produced site- and phosphory- palmitoylation is required for its plasma membrane

47 localization. The overexpressed 1-196 amino acids cells, respectively, revealed that keratins promote of ERBIN, without the latter half of LRR, were the accumulation of associated trichoplein on palmitoylated but did not localize at the plasma themselves. Furthermore, we found that trichoplein membrane, suggesting that both palmitoylation and localizes even on desmosomes in the small intestine LRR are required for the localization. Overexpres- and T84, colon cancer, cells. Experiments are now sion of wild-type ERBIN in Neuro2a cells resulted ongoing to clarify the function of trichoplein at in formation of cellular processes, lacking in the cell-cell borders with trichoplein stable knockdown mutant in which cysteines 14 and 16 were changed T84 cells. to serines. These results indicate that the palmitoy- With Albatross, localization is not only on kera- lation of ERBIN is critical for its biological func- tin filaments but also in the vicinity of the apical tion. junctional complex (AJC), a cell-cell adhesive ap- paratus composed of tight junctions, adherens junc- 4. Trichoplein and Albatross: unexpected tions and desmosomes. Knockdown in A549, lung passengers among keratin filaments, adenocarcinoma, cells revealed that Albatross centrioles and cell-cell contacts regulates the formation of AJC and lateral domains in cells. In addition, we found that keratin promotes Inoko, A., Zou, P., Ohmuro-Matsuyama, Y., Shiromizu, this function, using keratin-rescued SW13 cells. T., Ibi, M., Hayashi, Y., Yonemura, S.*1, Nakayama, Furthermore, we found that Albatross also lo- M.*2, Kaibuchi, K.*2, Kiyono, T.*3, Izawa, I. and Inagaki, calizes even on centrioles and clarification of func- M. tions in this site is now being targeted with knock- To reveal the function of cytoskeleton elements down experiments. particular to epithelial cells, we have searched for Finally, we found that both trichoplein and Al- binding partners with keratin 8/18 filaments and batross have a similar domain designated as the recently identified two novel keratin fila- trichohyalin/plectin homology domain (TPHD), ment-binding proteins: trichoplein and Albatross. which binds to keratins. Screening is ongoing to Trichoplein was found to localize not only on kera- find other TPHD proteins. tin filaments but also on mother centrioles. Knock- down in HeLa cells revealed that trichoplein is es- *1 RIKEN Center for Developmental Biology sential for anchoring of microtubules and ninein on *2 Department of Cell Pharmacology, Graduate School appendages of mother centrioles. In addition, over- of Medicine, Nagoya University expression and knockdown of keratin in SW13 of *3 Virology Division, National Cancer Center Research keratin-deficient cells and T24 bladder carcinoma Institute

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

49 Division of Central Laboratory & Radiation Biology ______

Kanji Ishizaki, Ph.D. Chief Hiroshi Kumimoto, Ph.D. Researcher Hideaki Nakamura, Ph.D. Research Resident Shoichiro Kitajima, D.D.S. Research Resident (as of April 2007) Hiroki Furue, D.D.S. Research Resident (until March 2007) Yoshihiro Yasui, Ph.D. Research Assistant Noriko Saito, Research Assistant

Visiting Trainees Kei Ijichi, M.D. School of Medicine, Nagoya City University Shoichiro Kitajima, D.D.S. Aichigakuin University School of Dentistry (until March 2007) Hiroki Furue, D.D.S. School of Medicine, Nagoya University (as of April 2007) Makoto Adachi, D.D.S. Asahi University School of Dentistry (until March 2007)

General Summary One of our main research projects is molecular genetic analysis of human esophageal and oral tumors. An especial focus is on mutations in mitochondrial DNA and we have found an increased mutation frequency in Japanese esophageal tumors. To ascertain the mechanisms responsible for mitochondrial mutations in tumor cells, we are also studying X-ray induced mutations. So far we have obtained results suggesting that tumor cells with p53 mutations exhibit high frequencies of induced mutations in mitochondrial DNA. Another research project is the study of genetic effects of low-dose-rate radiation (LDR) on human cells. For this purpose we have established immortal cell lines derived from normal individuals and patients with ataxia telangiectasia (AT), NBS, or Artemis deficiency. The Artemis and NBS genes are known to function downstream of the AT gene. Using these cell lines we have revealed that cytotoxic effects and mutation induction by LDR in normal cells is much lower than with high-dose-rate radiation (HDR) but that AT cell lines exhibit similar radiation sensitivity independent of the dose-rate. In contrast, NBS and Artemis deficient cells show higher resistance to LDR than to HDR while exhibiting the same sensitivity to HDR as AT cells. Using phosphorylated H2AX foci as indicators of DNA double strand breaks (DSBs), we have demonstrated that AT cells exhibit a partial but significant defect in the repair of DSBs, while NBS and Artemis deficient cells feature only a minor repair deficiency after LDR.

1. Mitochondria DNA instability due to ion- by mutagenic agents such as X-ray, we screened for izing radiation mtDNA mutations in 4 esophageal cancer cell lines, KYSE-30, 110, 150 and 410, and a normal cell line, Kumimoto, H., Saito, N. and Ishizaki, K. SuSa/T-n, after X-ray irradiation. These cell lines Previously we analyzed mutations in the D-loop were irradiated with X-ray at the doses giving sur- region of mitochondrial DNA (mtDNA) in eso- vival rate of 0.01 in each cell line. After sin- phageal tumors and found frequent somatic muta- gle-cell colonies were picked up, DNA was ex- tions (in 34 % of tumors) (Kumimoto et al. Int J tracted. Since there are up to 103 copies of Cancer, 108, 228-231, 2004). We also determined mtDNA in each cell and the same mutations may nuclear genomic instability, but did not find any not occur in all mtDNA, evaluation was conducted correlation between somatic mtDNA mutations and by assessing the ratio of mutated mtDNA to the to- nuclear genomic instability, suggesting that insta- tal mtDNA. Since the D310 region which has a 7 bility of mtDNA in esophageal cancer might be in- continuous C stretch in mtDNA frequently showed dependent of nuclear genomic instability. To 1 base insertion or deletion mutations, we analyzed know whether mtDNA mutations could be induced this region by GeneScan. Colonies with more than

Fig. 1. Frequencies of colonies which showed more than 0.3 of D310 insertion/deletion index were calculated for each cell line with or without X-ray irradiation

0.3 for the proportion of mutated mtDNA were de- 2. Role of ATM in repair of the double- fined as mutant colonies (Fig. 1). In SuSa/T-n, no strand break induced by low-dose-rate change was observed in D310 length after X-ray irradiation radiation, and two esophageal cancer cell lines, Nakamura, Hid., Yasui, Y., Kitajima S., Saito, N. and KYSE-150 and 410, also did not exhibit change al- Ishizaki, K. teration. On the other hand, 11.8% and 31.8% of colonies from irradiated cells of the esophageal Effects of low-dose-rate radiation (LDR) on cancer cell lines, KYSE-30 and 110, exhibited cells derived from normal humans (Normal) length changes in the D310 region. Since the p53 and patients with ataxia-telagiectasia (AT), gene was mutated in these two mtDNA-mutation NBS (NBS1, NBS2 and NBS3) and Artemis positive cell lines but not in the mtDNA-mutation (Artemis1 and Artemis2) deficiency were in- negative cell lines, the p53 gene might play an im- vestigated. Non-proliferating cells were irradi- portant role in mtDNA stability. ated with LDR (0.3 mGy/min) continuously for up to 2 weeks or were exposed to high-

Fig. 2. Survival curves of Artemis-deficient Fig. 3. Survival curves of NBS-deficient cells after HDR or LDR radiation. cells after HDR or LDR radiation.

51 dose-rate radiation (HDR; 1 Gy/min). AT cells showed hypersensitivity to HDR radiation compared with normal cells, a property shared by NBS and Artemis cells. While the survival of normal cells after LDR radiation was significantly higher than that after HDR radiation, AT cells showed essentially similar survival in both cases. In contrast, survival of Artemis deficient cells after LDR radiation was more resistant (Fig. 2). Surprisingly, the survival of NBS cells after LDR radiation was to that of normal cells (Fig. 3). Although only a few γH2AX foci were observed in LDR-irradiated normal cells, significant numbers remained in AT cells. In Artemis deficient cells, numbers of γH2AX foci after LDR irradiation were about half those in AT cells. Only a few foci were observed in the case of LDR irradiated NBS Fig. 4. RBE-LET curves for normal, AT and cells. These results suggest that ATM plays an AT-heterozygote cells after exposure to heavy ion important role in repairing the double-strand beams. breaks (DSBs) induced by LDR radiation. Moreover, they indicate that ATM might activate not only Artemis but also other factors in was determined by colony formation assay, and the DSB-repair pathway. On the other hand, relative biological effectiveness (RBE) was ob- functions of NBS in cells irradiated with LDR tained (Fig.4). To calculate the RBE, we used the might differ from those in HDR-irradiated radiation dose which gave a survival rate of 0.1 cells. (D10) after X-rays and heavy ions, and the D10 for X-rays was divided by that for the heavy-ion beams with various LETs. In a normal cell line, RBE was 3. Lethal effects of a heavy-ion beam on flat from 24-40 keV/µm but increased LET depen- AT-heterozygote cells dently above 40 keV/µm. In contrast, although Kitajima, S., Nakamura, Hid., Yasui, Y. and Ishizaki, K. RBEs of AT-heterozygote cell lines at 24 keV/µm It is well recognized the heavy-ion radiotherapy were similar to that of normal cells, increase is effective for cancer. However, it is not clear what LET-dependently was noted above 24 keV/µm. In effects beam exposure might have on an AT cell line, the RBE increased from 24-40 AT-heterozygotes, which are thought to account for keV/µm. However, above 40 keV/µm, a plateau about 1% of the population. In this study, we ana- was evident. These results suggest that ATM het- lyzed effects of carbon and iron-ion beams with erozygosity could influence cellular survival after various energy levels (expressed as LET) on heavy-ion beam therapy. We now need to determine AT-heterozygote cells. LETs of the heavy-ion beam the mechanisms underlying the heavy-ion sensitiv- used were 24, 40, 50, 60 and 200 keV/µm. Survival ity of AT-heterozygotes.

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

53 Central Service Unit ______

Hiromu Nakamura, D.M.Sc. Section Head Miwako Nishizawa, B.P. Senior Research Assistant Masami Yamamoto, Ph.D. Senior Research Assistant Harunari Tanaka, B.P. Research Assistant (as of April 2007)

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 include a DNA sequencer (ABI 3100), two flow-cytometers (Bec- ton-Dickinson FACS Calibur), imaging analyzers (Fujix BAS-2500Mac, Amersham-Pharmacia ImageMaster-CL and FluorImager-595), two X-ray machines (Hitachi MBR-1520R3), a scanning electron microscope (Hitachi SEM), two real-time PCR set-ups (ABI 7500 Fast Real-Time PCR System and Roche Light Cycler), seven ultracentrifuges (Beckman-Coulter and Hitachi), a mi- croplate reader (TECAN GENios) and computer systems for image analysis (Windows and Macin- tosh systems). Furthermore, we maintain and manage the radioisotope experimental facilities, SPF and conventional animal rooms, laboratories for translational research, a hazardous chemical storehouse, ultra-low temperature freezers, cold rooms, a liquid nitrogen storage room, security systems, air-conditioning, and water purifying and waste water treatment systems, as well as a carbon diox- ide gas supply, thereby contributing to many other of the Institute’s functions. During the last two years we replaced several instruments such as a confocal laser microscope (Carl-Zeiss LSM510 META), and an X-ray machine for SPF animals (Hitachi MBR-1520R3) and have held technical seminars on advanced biotechnology. Our activities thus provide essential background support for all of the research carried out by the Research Institute.

54 ______Librarians

From Left to right First row: Ms. T. Yasuda, Ms. T. Shibata Second row: Ms. K. Adachi, Ms. K. Horayama, supporting scientific and medical information.

55 ______Researches Supported by Special Project Programme

1. Roles of mitotic kinases in carcino- antibody which specifically recognizes Chk1 phos- genesis phorylated at Ser301. Immunocytochemical analyses using this antibody revealed Chk1-Ser301 Goto, H., Enomoto, M., Kasahara, K., Ikegami, Y., phosphorylation from entry into mitosis (prophase). Yamaguchi, T., Shiromizu, T., Tomono, Y.*1, Tsujimura, The level of phosphorylation appeared to peak at K.*2, Kiyono, T.*3 Kawajiri, A., Hayashi, Y., Urano, T.*4, metaphase and to gradually decrease after anaphase. Furukawa, K.*4, Nigg, E.A.*5, Hayashi, N.*6 and Inagaki, In prophase when chromosome condensation occurs M. without nuclear envelope breakdown, we observed Appropriate chromosome duplication cycles are two phosphorylation patterns. At earlier stages, the critical to cellular function, and mitosis is the final phophorylation tended to be observed mainly in the step in which duplicated chromosomes are divided nucleus. However, at later stages of prophase, Chk1 into two daughter cells correctly. Errors in mitosis phosphorylated at Ser301 appeared to exist mainly can result in aneuploidy, which is often observed in in the cytoplasm. This translocation was inhibited cancer cells. Mitotic chromosomal dynamics are by treatment with leptomycin B, which blocks regulated by the coordinated activities of many mi- Crm1-mediated nuclear export. To further address totic kinases, such as cyclin-dependent kinase 1 the role of mitotic Chk1 phosphorylation, we estab- (Cdk1), Aurora-B or Polo-like kinase 1 (Plk1), but lished HeLa cells in which Chk1 wild type (WT) or mechanisms of coordination remain largely un- Chk1 mutated to Ala at Ser286 and Ser301 known. We have found two novel examples of sig- (S286A/S301A) were expressed in a tetracy- naling crosstalk among mitotic kinases. cline-dependent manner. Introduction of S286A/S301A caused significant delay in mitotic (1) Complex Formation of Plk1 and INCENP entry, compared with the WT case. In Required for Metaphase-Anaphase Transi- S286A/S301A mutant prophase cells, staining was tion observed mainly in the nucleus although in WT We have established that Cdk1 phosphorylates cells location was also found in the cytoplasm. Thr59 and Thr388 on the inner centromere protein GST-pull down assays revealed that Chk1 could (INCENP), which regulates localization and kinase bind to 14-3-3 proteins in a Ser301-phosphoryla- activity of Aurora-B in the transition from prophase tion-dependent manner. These results suggested that to metaphase. INCENP depletion disrupts Plk1 lo- Cdk1 controls the cytoplasmic sequestration of calization specifically at the kinetochore. This phe- Chk1 likely through binding to 14-3-3 proteins. We notype is rescued by the exogenous expression of propose that this pathway is a key control mecha- INCENP wild type (WT) and INCENP mutated at nism for maintaining the G2/M transition. Thr59 to Ala (T59A), but not at Thr388 to Ala (T388A). Replacement of endogenous INCENP *1 Division of Molecular and Cell Biology, Shigei Medi- with T388A results in delay of progression from cal Research Institute metaphase to anaphase. We propose that INCENP *2 Department of Infectious Diseases, Hamamatsu Uni- phosphorylation by Cdk1 is necessary for recruit- versity School of Medicine ment of Plk1 to the kinetochore, and that complex *3 Virology Division, National Cancer Center Research formation of Plk1 and Aurora-B on INCENP may Institute play critical roles in the regulation of chromosomal *4 Department of Biochemistry II, Nagoya University dynamics. Graduate School of Medicine *5 Department of Cell Biology, Max-Planck Institute for (2) Chk1 phosphorylation by cyclin-dependent Biochemistry, Germany kinase 1 controls mitotic entry through cyto- *6 Department of Urology, Aichi Cancer Center Hospital plasmic sequestration of Chk1 We recently reported Chk1 to be phosphorylated at Ser286 and Ser301 by cyclin-dependent kinase (Cdk) 1 during mitosis. In order to elucidate functional consequences, we produced a rat monoclonal

56 2. Array CGH analysis of malignant lym- we examined whether genomic copy number gains phomas and its application for molecu- and losses detected in this way could be used to lar diagnosis differentiate lymphoma diseases or subtypes. Copy Tagawa, H. number gains and losses in 46 DLBCLs and 29 mantle cell lymphoma (MCL) were assessed by ar- Array comparative genomic hybridization (array ray CGH and gene expression in the DLBCL cases CGH) analysis has identified frequent 13q31 ampli- was profiled. Hierarchical clustering revealed 28 to fication in diffuse large B-cell lymphomas be of activated B-cell (ABC) type while 18 were of (DLBCLs) and Burkitt's lymphomas. We found the germinal center-B-cell (GCB) type. A computer al- C13orf25/miR-17 cluster to include the responsible gorithm was developed to classify lymphoma dis- gene in this region and could show that C13orf25 eases or subtypes on the basis of copy number gains contains the microRNA-17-18-19-20-92 polycistron. and losses whose application allowed correct classi- We further demonstrated that MYC and the miR-17 fication of the DLBCL and MCL subtypes in 89% cluster synergistically contribute to cancer devel- of cases, and of ABC and GCB subtypes in 83%. opment from rat fibroblast (Rat-1) transfectants These results demonstrate that copy number gains through agarose colony formation assays as well as and losses detected by array CGH can be used for in nude mice. In addition, we identified TGFβRII as classifying lymphomas into biologically and clini- the true target for the miR-17 cluster. In an attempt cally distinct diseases or subtypes. to apply array CGH data for molecular diagnosis,

57 Publications ______

Shimoyama, Y., Yoshino, T., Morishima, Y. and Journals Nakamura, S.: Cytotoxic molecule expression is

predictive of prognosis in Hodgkin’s-like anaplastic J001. Adachi, M., Inoko, A., Hata, M., Furuse, large cell lymphoma. Histopathology, 50: 705-715, K., Umeda, K., Itoh, M. and Tsukita, S.: Normal 2007. (PMID: 17493234) establishment of epithelial tight junctions in mice J008. Biswas, MH., Hasegawa, HH., Rahman, and cultured cells lacking expression of ZO-3, a MA., Huang, P., Mon, NN., Ruhul Amin, AR., tight-junction MAGUK protein. Mol Cell Biol, 26: Senga, T., Kannagi, R. and Hamaguchi, M.: 9003-9015, 2006. (PMID: 17000770) SHP-2-Erk signaling regulates Concanavalin J002. Akatsuka, Y., Torikai, H., Inamoto, Y., A-dependent production of TIMP-2. Biochem Tsujimura, K., Morishima, Y., Kodera, Y., Biophys Res Commun, 348: 1145-1149, 2006. Kuzushima, K. and Takahashi, To.: Bone marrow (PMID: 16904070) may be a reservoir of long-lived memory T cells J009. Boumber, YA., Kondo, Y., Chen, X., Shen, specific for minor histocompatibility antigen. Brit J L., Gharibyan, V., Konishi, K., Estey, E., Haematol, 135: 413-414, 2006. (PMID: 16978218) Kantarjian, H., Garcia-Manero, G. and Issa, JP.: J003. Akutagawa, A., Fukami, K., Banno, Y., RIL, a LIM gene on 5q31, is silenced by Takenawa, T., Kannagi, R., Yokoyama, Y., Oda, methylation in cancer and sensitizes cancer cells to K., Nagino, M., Nimura, Y., Yoshida, S. and apoptosis. Cancer Res, 67: 1997-2005, 2007. Tamiya-Koizumi, K.: Disruption of phospholipase (PMID: 17332327) Cδ4 gene modulates the liver regeneration in J010. Cao, X., Tsukamoto, T., Seki, T., Tanaka, cooperation with nuclear protein kinase C. J H., Morimura, S., Cao, L., Mizoshita, T., Ban, H., Biochem, 140: 619-625, 2006. (PMID: 16998201) Toyoda, T., Maeda, H. and Tatematsu, M.: J004. Aoki, K., Aoki, M., Sugai, M., Harada, N., 4-Vinyl-2,6-dimethoxyphenol (Canolol) suppresses Miyoshi, H., Tsukamoto, T., Mizoshita, T., oxidative stress and gastric carcinogenesis in Tatematsu, M., Seno, H., Chiba, T., Oshima, M., Helicobacter pylori-infected carcinogen-treated Hsieh, CL. and Taketo, MM.: Chromosomal Mongolian gerbils. Int J Cancer, in press. (PMID: instability by beta-catenin/TCF transcription in 18059022) APC or beta-catenin mutant cells.Chromosomal J011. Chen, G.Y., Osada, H., Santamaria-Babi, instability by beta-catenin/TCF transcription in LF. and Kannagi, R.: Interaction of GATA-3/T-bet APC or beta-catenin mutant cells. Oncogene, 26: transcription factors regulates expression of sialyl 3511-3520, 2007. (PMID: 17160019) Lewis X homing receptors on Th1/Th2 J005. Aoyama, J., Akazawa, Y., Kasahara, K., lymphocytes. Proc Natl Acad Sci USA, 103: Higashiyama, Y., Kikuchi, I., Fukumoto, Y., 16894-16899, 2006. (PMID: 17075044) Saburi, S., Nakayama, Y., Fukuda, MN. and J012. Daikoku, T., Kudoh, A., Sugaya, Y., Yamaguchi, N.: Nuclear localization of magphinins, Iwahori, S., Shirata, N., Isomura, H. and Tsurumi, alternative splicing products of the human trophinin T.: Postreplicative mismatch repair factors are gene. J Cell Biochem, in press. (PMID: 17559068) recruited to Epstein-Barr Virus replication J006. Asano, N., Ohshiro, A., Matsuo, K., compartments. J Biol Chem, 281: 11422-11430, Kagami, Y., Ishida, F., Suzuki, R., Kinoshita, T., 2006. (PMID: 16510450) Shimoyama, Y., Tamaru, JI., Yoshino, T., J013. Demachi-Okamura, A., Ito, Y., Akatsuka, Kitamura, K., Fukutani, H., Morhishima, Y. and Y., Tsujimura, K., Morishima, Y., Takahashi, To. Nakamura, S.: Prognostic significance of T-cell or and Kuzushima, K.: Epstein-Barr virus (EBV) cytotoxic molecules phenotype in classical latent membrane protein-1-specific cytotoxic T Hodgkin's lymphoma: a clinicopathologic study. J lymphocytes targeting EBV-carrying natural killer Clin Oncol, 24: 4626-4633, 2006. (PMID: cell malignancies. Eur J Immunol, 36: 593-602, 16954517) 2006. (PMID: 16479544) J007. Asano, N., Suzuki, R., Matsuo, K., Kagami, J014. Ebi, H., Matsuo, K., Sugito, N., Suzuki, M., Y., Ishida, F., Tamaru, JI., Jin, GS., Sato, Y.,

58 Osada, H., Tajima, K., Ueda, R. and Takahashi, Kasugai, Y., Karnan, S., Kameoka, J., Sasaki, T., Ta.: Novel NBS1 heterozygous germ line mutation Morishima, Y., Nakamura, S. and Seto, M.: causing MRE11-binding domain loss predisposes to Characterization of target genes at the 2p15-16 common types of cancer. Cancer Res, 67: amplicon in diffuse large B-cell lymphoma. Cancer 11158-11165, 2007. (PMID: 18056440) Sci, 97: 499-504, 2006. (PMID: 16734728) J015. Enomoto, M., Sawano, Y., Kosuge, S., J022. Fukushima, A., Yamaguchi, T., Sumi, T., Yamano, Y., Kuroki, K. and Ohtsuki, K.: High Fukuda, K., Kumagai, N., Nishida, T., Takeuchi, phosphorylation of HBV core protein by two M., Taguchi, O. and Ueno, H.: Roles of alpha-type CK2-activated cAMP-dependent protein CD4+CD25+ T cells in the development of kinases in vitro. FEBS Lett, 580: 894-899, 2006. experimental murine allergic conjunctivitis. Graefes (PMID: 16430890) Arch Clin Exp Ophthalmol, 245: 705-714, 2007. (PMID: 17024440) J016. Enomoto, S., Maekita, T., Tsukamoto, T., Nakajima, T., Nakazawa, K., Tatematsu, M., J023. Furue, H., Kumimoto, H., Matsuo, K., Ichinose, M. and Ushijima, T.: Lack of association Suzuki, T., Hasegawa, Y., Shinoda, M., Sugimura, between CpG island methylator phenotype in T., Mitsudo, K., Tohnai, I., Ueda, M., Tajima, K. human gastric cancers and methylation in their and Ishizaki, K.: Opposite impact of NKG2D background non-cancerous gastric mucosae. Cancer genotype by lifestyle exposure to risk of Sci, 98: 1853-1861, 2007. (PMID: 17900260) aerodigestive tract cancer among Japanese. Int J Cancer, in press. J017. Esaki, M., Sano, T., Shimada, K., Sakamoto, Y., Wakai, K and, Kosuge, T.: J024. Furue, H., Matsuo, K., Kumimoto, H, Randomized controlled clinical trial of hepatectomy Hiraki, A., Suzuki, T., Yatabe, Y., Komori, K., using intermittent pedicle occlusion; ischemic Kanemitsu, Y., Hirai, T., Kato, T., Ueda, M., intervals of 15 versus 30 minutes. Brit J Surg, 93: Ishizaki, K. and Tajima, K.: Decreased risk of 944-951, 2006. (PMID: 16823832) colorectal cancer with the high Natural-Killer (NK)-cell activity NKG2D genotype in Japanese. J018. Fujii, N., Hiraki, A., Aoe, K., Murakami, Carcinogenesis, in press. (PMID: 18174257) T., Ikeda, K., Masuda, K., Matsuo, K., Shinagawa, K., Ishimaru, F., Sugi, K., Darzynkiewicz, Z. and J025. Go, S., Sato, C., Yin, J., Kannagi, R. and Tanimoto, M.: Serum cytokine concentrations and Kitajima, K.: Hypoxia-enhanced expression of free acute graft-versus-host disease after allogeneic deaminoneuraminic acid in human cancer cells. peripheral blood stem cell transplantation: Biochem Biophys Res Commun, 357: 537-542, Concurrent measurement of ten cytokines and their 2007. (PMID: 13434449) respective ratios using cytometric bead array. Int J J026. Goto, C., Tokudome, Y., Imaeda, N., Mol Med, 17: 881-885, 2006. (PMID: 16596275) Takekuma, K., Kuriki, K., Igarashi, F., Ikeda, M. J019. Fujiwara, Y., Kiura, K., Toyooka, S., and Tokudome, S.: Validation study of fatty acid Takigawa, N., Tokumo, M., Hotta, K., Aoe, M., consumption assessed with a short food frequency Tabata, M., Matsuo, K., Date, H. and Tanimoto, questionnaire against plasma concentration in M.: Relationship between epidermal growth factor middle-aged Japanese people. Scandinavian J Food receptor gene mutations and the severity of adverse Nutr, 50: 77-82, 2006. events by gefitinib in patients with advanced J027. Goto, H. and Inagaki, M.: Production of a non-small cell lung cancer. Lung Cancer, 52: site- and phosphorylation state-specific antibody. 99-103, 2006. (PMID: 16503086) Nature Protocols, 2: 2574-2581, 2007. (PMID: J020. Fukuhara, N., Nakamura, T., Nakagawa, 17948000) M., Tagawa, H., Takeuchi, I.. Yatabe, Y., J028. Goto, H., Kiyono, T., Tomono, Y., Morishima, Y., Nakamura, S. and Seto, M.: Kawajiri, A., Urano, T., Furukawa, K., Nigg, EA. Chromosomal imbalances are associated with and Inagaki, M.: Complex formation of Plk1 and outcome of Helicobacter pylori eradication in INCENP required for Metaphase-anaphse transition. t(11;18)(q21;q21) negative gastric malt lymphomas. Nature Cell Biol, 8: 180-187, 2006. (PMID: Gene Chromosome Cancer, 46: 784-790, 2007. 16378098) (PMID: 17492756) J029. Hara, M., Hirai, T., Nakanishi, H., J021. Fukuhara, N., Tagawa, H., Kameoka, Y., Kanemitsu, K., Komori, K., Tatematsu, M. and

59 Kato, T.: Isolated tumor cell in lateral lymph node development of histiocytic sarcomas in p53 has no influences on the prognosis of rectal cancer knockout mice treated with patients. Int J Colorectal Dis, 22: 911-917, 2007. N-bis(2-hydroxypropyl)nitrosamine. Oncol Rep, (PMID: 17318555) 18: 755-761, 2007. (PMID: 17786332) J030. Hara, M., Nakanishi, H. (Corresponding J037. Hirata, A., Tsukamoto, T., Yamamoto, M., author), Jun, Q., Hirai, T., Kanemitsu, K., Ito, S., Takasu, S., Sakai, H., Ban, H., Yanai, T., Masegi, Mochizuki, Y., Kodera, Y., Tatematsu, M., T., Donehower, LA. and Tatematsu, M.: Yamamura, Y. and Kato, T.: Comparative analysis Organ-dependent susceptibility of p53 knockout of intraperitoneal minimal free cancer cells between mice to 2-amino-3-methylimidazo[4,5-f]quinoline colorectal and gastric cancer patients using (IQ). Cancer Sci, 98: 1164-1173, 2007. (PMID: quantitative RT-PCR. Possible reason for rare 17532756) peritoneal recurrence in colorectal cancer. Clin Exp J038. Hirose, K., Matsuo, K., Iwata, H. and Metastas, 24: 179-189, 2007. (PMID: 17487561) Tajima, K.: Dietary patterns and the risk of breast J031. Hattori, T., Takeuchi, M., Ohno, K., cancer in Japanese women. Cancer Sci, 98: Ogawara, M., Asatani, T., Usui, Y., Muramatsu, 1431-1438, 2007. (PMID: 17627618) R., Inagaki, M., Usui, M. and Taguchi, O.: J039. Hirose, K., Niwa, Y., Wakai, K., Matsuo, Alpha-internexin as a corneal autoantigen in K., Ito, H., Nakanishi, T. and Tajima, K.: Coffee spontaneous autoimmune keratitis mouse model. consumption lowers the risk of endometrial cancer : Invest Ophth Vis Sci, 47: 249-255, 2006. (PMID: a case-control study on women’s hormone related 16384970) cancer in Japan. Cancer Sci, 98: 411-415, 2007. J032. Hiraki, A., Matsuo, K., Wakai, K., Suzuki, (PMID: 17270030) T., Hasegawa, Y. and Tajima, K.: Gene-gene and J040. Hiyama, T., Yoshihara, M., Matsuo, K., gene-environment interactions between alcohol Kusunoki, H., Kamada, T., Ito, M., Tanaka, S., drinking habit and polymorphisms in Nishi, N., Chayama, K. and Haruma, K.: alcohol-metabolizing enzyme genes and the risk of Meta-analysis of the effects of prokinetic agents in head and neck cancer in Japan. Cancer Sci, 98: patients with functional dyspepsia. J Gastroen 1087-1091, 2007. (PMID: 17489985) Hepatol, 22: 304-310, 2007. (PMID: 17295758) J033. Hiraki, A., Murakami, T., Aoe, K., Sueoka, J041. Hiyama, T., Yoshihara, M., Matsuo, K., E., Sueoka, N., Taguchi, K., Kamei, T., Sugi, K., Kusunoki, H., Kamada, T., Ito, M., Tanaka, S., Ueoka, H. and Kishimoto, T.: Heterogeneous Nishi, N., Chayama, K. and Haruma, K.: nuclear ribonucleoprotein B1 expression in Treatment of functional dyspepsia with serotonin malignant mesothelioma. Cancer Sci, 97: agonist:a meta-analysis of randomized controlled 1175-1181, 2006. (PMID: 16939492) trials. J Gastroen Hepatol, 22: 1566-1570, 2007. J034. Hirano, N., Tsukamoto, T., Mizoshita, T., (PMID: 17845684) Koriyama, C., Akiba, S., Campos, F., Carrasquilla, J042. Hotta, K., Fujiwara, Y., Matsuo, K., G., Carrascal, E., Cao, X., Toyoda, T., Ban, H., Suzuki, T., Kiura, K., Tabata, M., Takigawa, N., Miki, K. and Tatematsu, M.: Down regulation of Ueoka, H. and Tanimoto, M.: Recent improvement gastric and intestinal phenotypic expression in in survival of patients with advanced non-small-cell Epstein-Barr virus-associated stomach cancers. lung cancer enrolled into phase III trials of first-line Histol Histopathol, 22: 641-649, 2007. (PMID: systemic chemotherapy. Cancer, 109: 939-948, 17357094) 2007. (PMID: 17285602) J035. Hirata, A., Tsukamoto, T., Yamamoto, M., J043. Hu, R., Li, G., Kamijo, Y., Aoyama, T., Sakai, H., Yanai, T., Masegi, T., Donehower, LA. Nakajima, T., Inoue, T., Node, K., Kannagi, R., and Tatematsu, M.: Organ-specific susceptibility of Kyogashima, M. and Hara, A.: Serum sulfatides as p53 knockout mice to N-bis(2-hydroxypropyl) a novel biomarker for cardiovascular disease in nitrosamine carcinogenesis. Cancer Lett, 238: 271 patients with end-stage renal failure. Glycoconj J, 283, 2006. (PMID: 16150539) 24:565-571, 2007. (PMID: 17657603) J036. Hirata, A., Tsukamoto, T., Yamamoto, M., J044. Hyuga, S., Hyuga, M., Nakanishi, H., Ito, Takasu, S., Sakai, H., Ban, H., Yanai, T., Masegi, H., Watanabe, K., Oikawa, T. and Hanawa, T.: T., Donehower, LA. and Tatematsu, M.: Early Maoto, Kampomedicine, suppresses the metastatic

60 potential of highly metastatic osteosarcoma cells. J M., Murata, T., Shimotohno, K. and Maruyama, Tradi Med, 24: 51-58, 2007. T.: 5'-O-Masked 2'-deoxyadenosine analogues as lead compounds for hepatitis C virus (HCV) J045. Ibi, M., Ishisaki, A., Yamamoto, M., Wada, therapeutic agents. Bioorg Med Chem, 15: S., Kozakai, T., Nakashima, A., Iida, J., Takao, S., 6882-6892. 2007. (PMID: 17766124) Izumi, Y., Yokoyama, A. and Tamura, M.: Establishment of cell lines that exhibit pluripotency J053. Isaka, T., Nakamura, T., Tajika, M., from miniature swine periodontal ligaments. Arch Kawai, H., Imaoka, H., Okamoto, Y., Aoki, M., Oral Biol, 52: 1002-1008, 2007. (PMID: 17543882) Inoue, H., Takahashi, K., Mizuno, N., Sawaki, A., Yamao, K., Seto, M., Yokoi, T., Yatabe, Y. and J046. Ichihara, K., Shimizu, H., Taguchi, O., Nakamura, S.: API2-MALT1 chimeric Yamaguchi, M. and Inoue, Y.H.: A Drosophila transcript-positive gastroduodenal MALT orthologue of larp protein family is required for lymphoma with subsequent development of multiple processes in male meiosis. Cell Struct adenocarcinoma as a collision tumour over a Funct, 32: 89-100, 2007. (PMID: 17951964) clinical course of 7 years. Histopathology, 51: J047. Ijichi, K., Adachi, M., Hasegawa, Y., 119-123, 2007. (PMID: 17542995) Ogawa, T., Nakamura, H., Kudoh, A., Yasui, Y., J054. Ishii, A., Ikeda, T., Hitoshi, S., Fujimoto, Murakami, S. and Ishizaki, K.: Pretreatment with I., Torii, T., Sakuma, K., Nakakita, S., Hase, S. 5-FU enhances cisplatin cytotoxicity in head and and Ikenaka, K.: Developmental changes in the neck squamous cell carcinoma cells. Cancer expression of glycogenes and the content of Chemother Pharmacol, in press. (PMID: 18075740) N-glycans in the mouse cerebral cortex. J048. Ikehara, S., Ikehara, Y., Matsuo, K., Glycobiology, 17: 261-276, 2007. (PMID: Hirose, K., Niwa, T., Ito, H., Ito, S., Kodera, Y., 17172259) Yamamura, Y., Nakanishi, H., Tatematsu, M. and J055. Iso, H., Date, C., Wakai, K., Fukui, M. Tajima, K.: A polymorphism of C to T substitution and Tamakoshi, A.: The relationship between at -31 IL1B is associated with the risk of advanced green tea and total caffeine intake and risk for gastric adenocarcinoma in a Japanese population. J self-reported type 2 diabetes among Japanese adults. Hum Genet, 51: 927-933, 2006. (PMID: 17006606) Ann Intern Med, 144: 554-562, 2006. (PMID: J049. Ikehara, Y., Niwa, T., Biao, L., Ikehara, S., 16618952) Ohashi, N., Kobayashi, T., Shimizu, Y., Kojima, N. J056. Isomura, H., Stinski, MF., Kudoh, A., and Nakanishi, H.: A carbohydrate Murata, T., Nakayama, S., Sato, Y., Iwahori, S. recognition-based drug delivery and controlled and Tsurumi, T.: Noncanonical TATA Sequence in release system using intraperitoneal macrophages as the UL44 Late Promoter of Human a cellular vehicle. Cancer Res, 66: 8740-8748, 2006. Cytomegalovirus is required for the accumulation (PMID: 16951190) of late viral transcripts. J Virol, 82:1638-1646. 2008. J050. Ikehara, Y., Sato, T., Niwa, T., Nakamura, (PMID: 18057245) S., Gotoh, M., Ikehara, SK., Kiyohara, K., Aoki, C., J057. Isomura, H., Stinski, MF., Kudoh, A., Iwai, T., Nakanishi, H., Hirabayashi, J., Nakayama, S., Iwahori, S., Sato, Y. and Tsurumi, Tatematsu, M. and Narimatsu, H.: Apical Golgi T.: The late promoter of the human localization of N,N'-diacetyllactosediamine cytomegalovirus viral DNA polymerase synthase, β4GalNAc-T3, is responsible for processivity factor has an impact on delayed early LacdiNAc expression on gastric mucosa. and late viral gene products but not on viral DNA Glycobiology, 16: 777-785, 2006. (PMID: synthesis. J Virol, 81: 6241-6247, 2007. (PMID: 16728562) 17409154) J051. Ikehara, Y., Shiuchi, N., Ikehara, S., J058. Isomura, H., Stinski, MF., Kudoh, A., Nakanishi, H., Yokoyama, N., Takagi, H., Nakayama, S., Murata, T., Sato, Y., Iwahori, S. Takahashi, T., Tsujimura, K. and Kojima, N.: and Tsurumi, T.: A cis element between the TATA Effective induction of anti-tumor immune Box and the transcription start site of the major responses with oligomannose-coated liposome immediate-early promoter of human targeting to intraperitoneal phagocytic cells. cytomegalovirus determines efficiency of viral Cancer Letters, in press. (PMID: 18077084) replication. J Virol, 82: 849-858, 2008. (PMID: J052. Ikejiri, M., Ohshima, T., Kato, K., Toyama, 17989180)

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64 Tanaka, R. and Tajima, K.: Meat, milk, saturated mice. J Neurol Sci, 264: 77-86, 2008. (PMID: fatty acids, the Pro12Ala and C161T 17854834) polymorphisms of the PPARgamma gene and J108. Lim, K.T., Miyazaki, K., Kimura, N., colorectal cancer risk in Japanese. Cancer Sci, 97: Izawa, M. and Kannagi, R.: Clinical application of 1226-1235, 2006. (PMID: 16965392) functional glycoproteomics ─ Dissection of J101. Kuriki, K., Hirose, K., Wakai, K., Matsuo, glycotopes carried by soluble CD44-variants in sera K., Ito, H., Suzuki, T., Hiraki, A., Saito, T., Iwata, of patients with cancers. Proteomics, in press. H., Tatematsu, M. and Tajima, K.: Breast cancer J109. Lu, JW., Gao, CM., Wu, JZ., Cao, HX., risk and erythrocyte compositions of n-3 highly Tajima, K. and Feng, JF.: Polymorphism in the unsaturated fatty acids in Japanese. Int J Cancer, 3'-unsatulated region of the thymidylate synthase 121: 377-385, 2007. (PMID: 17354239) gene and sensitivity of stomach cancer to J102. Kuriki, K., Tajima, K. and Tokudome, S.: fluoropyrimidine-based chemotherapy. J Hum Accelerated solvent extraction for quantitative Genet, 51: 155-160, 2006. (PMID: 16424979) measurement of fatty acids in plasma and J110. Maeda, O., Kondo, M., Fujita, T., Usami, erythrocytes. Lipids, 41: 605-614, 2006. (PMID: N., Fukui, T., Shimokata, K., Ando, T., Goto, H. 16981438) and Sekido, Y.: Enhancement of J103. Kuriki, K., Wakai, K., Hirose, K., Matsuo, GLI1-transcriptional activity by b-catenin in human K., Ito, H., Suzuki, T., Saito, T., Kanemitsu, Y., cancer cells. Oncol Rep, 16: 91-96, 2006. (PMID: Hirai, T., Kato, T., Tatematsu, M. and Tajima, K.: 16786128) Risk of colorectal cancer is linked to erythrocyte J111. Maekita, T., Nakazawa, K., Mihara, M., compositions of fatty acids as biomarkers for Nakajima, T., Yanaoka, K., Iguchi, M., Arii, K., dietary intakes of fish, fat and fatty acids. Cancer Kaneda, A., Tsukamoto, T., Tatematsu, M., Epidemiol Biomarker Prev, 15: 1791-1798, 2006. Tamura, G., Saito, D., Sugimura, T., Ichinose, M. (PMID: 17035384) and Ushijima, T.: High levels of aberrant DNA J104. Kyogashima, M., Tamiya-Koizumi, K., methylation in Helicobacter pylori-infected gastric Ehara, T., Li, G.., Hu, R., Hara, A., Aoyama, T. mucosae and its possible association with gastric and Kannagi, R.: Rapid demonstration of diversity cancer risk. Clin Cancer Res, 12: 989-995, 2006. of sulfatide molecular species from biological (PMID: 16467114) materials by MALDI-TOF MS. Glycobiology, 16: J112. Maeno, K., Masuda, A., Yanagisawa, K., 719-728, 2006. (PMID: 16670104) Konishi, H., Osada, H., Saito, T., Ueda, R. and J105. Li, G., Hu, R., Kamijo, Y., Nakajima, T., Takahashi, Ta.: Altered regulation of c-jun and its Aoyama, T., Inoue, T., Node, K., Kannagi, R., involvement in anchorage-independent growth of Kyogashima, M. and Hara, A.: Establishment of a human lung cancers. Oncogene, 25: 271-277, 2006. quantitative, qualitative, and high-throughput (PMID: 16158054) analysis of sulfatides from small amounts of sera by J113. Masuda, K., Hiraki, A., Fujii, N., matrix-assisted laser desorption ionization-time of Watanabe, T., Tanaka, M., Matsue, K., Ogama, Y., flight mass spectrometry. Anal Biochem, 362: 1-7, Ouchida, M., Shimizu, K., Ikeda, K. and Tanimoto, 2007. (PMID: 17254536) M.: Loss or down-regulation of HLA class I J106. Li, GQ., Xia, HHX., Chen, MH., expression at the allelic level in freshly isolated Tsukamoto, T., Tatematsu, M., Gu, Q., Qiao, L., leukemic blasts. Cancer Sci, 98: 102-108, 2007. Cho, CH., So, WHL., Yuen, MF., Hu, PJ., Liang, (PMID: 17083564) YJ., Lin, HL., Chan, AOO. and Wong, BCY.: J114. Mathonnet, G., Fabian, MR., Svitkin, YV., Effects of aspirin on the development of Parsyan, A., Huck, L., Murata, T., Biffo, S., Helicobacter pylori-induced gastric inflammation Merrick, WC., Darzynkiewicz, E., Pillai, RS., and heterotopic proliferative glands in Mongolian Filipowicz, W., Duchaine, TF. and Sonenberg, N.: gerbils. Helicobacter, in press. MicroRNA inhibition of translation initiation in J107. Li, ZF., Wu, X., Jiang, Y., Liu, J., Wu, C., vitro by targeting the cap-binding complex eIF4F. Inagaki, M., Izawa, I., Mizisin, AP., Engvall, E. Science, 317: 1764-1767. 2007. (PMID: 17656684) and Shelton, GD.: Non-pathogenic protein J115. Matsubara, H., Takeuchi, T., Nishikawa, aggregates in skeletal muscle in MLF1 transgenic E., Yanagisawa, K., Hayashita, Y., Ebi, H.,

65 Yamada, H., Suzuki, M., Nagino, M., Nimura, Y., maternal and neonatal outcomes. Acta Obster Gyn Osada, H. and Takahashi, Ta.: Apoptosis Scand, 86: 191-197, 2007. (PMID: 17364282) induction by antisense oligonucleotides against J123. Miyazawa, M., Utsunomiya, H., Inada, K., miR-17-5p and miR-20a in lung cancers Yamada, T., Okuno, Y., Tanaka, H. and overexpressing miR-17-92. Oncogene, 26: Tatematsu, M.: Inhibition of Helicobacter pylori 6099-6105, 2007. (PMID: 17384677) motility by (+)-Syringaresinol from unripe Japanese J116. Matsumoto, M., Yaginuma, K., Igarashi, apricot. Biol Pharm Bull, 29: 172-173, 2006. A., Imura, M., Hasegawa, M., Iwabuchi, K., Date, (PMID: 16394533) T., Mori, T., Ishizaki, K., Yamashita, K., Inobe, M. J124. Mizoshita, T., Tsukamoto, T., Inada, KI., and Matsunaga, T.: Perturbed GAP-filling Hirano, N., Tajika, M., Nakamura, T., Ban, H. synthesis in nucleotide excision repair causes and Tatematsu, M.: Loss of MUC2 expression histone H2AX phosphorylation in human quiescent correlates with progression along the cells. J Cell Science, 120:1104-1112, 2007. (PMID: adenoma-carcinoma sequence pathway as well as 17327276) de novo carcinogenesis in the colon. Histol J117. Matsuo, K., Hiraki, A., Hirose, K., Wakai, Histopathol, 22: 251-260, 2007. (PMID: 17163399) K., Ito, H., Saito, T. and Tajima, K.: Impact of J125. Mizoshita, T., Tsukamoto, T., Takenaka, Alcohol Dehydrogenease (ADH) 2 and ADH3 Y., Cao, X., Kato, S., Kaminishi, M. and polymorphisms on drinking behavior in Japanese. Tatematsu, M.: Gastric and intestinal phenotypes Hum Mutat, 28: 506-510, 2007. (PMID: 17285601) and histogenesis of advanced glandular stomach J118. Matsuo, K., Ito, H., Wakai, K., Hirose, K., cancers in carcinogen-treated, Helicobacter Saito, T., Suzuki, T., Kato, T., Hirai, T., pylori-infected Mongolian gerbils. Cancer Sci, 97: Kanemitsu, Y., Hamajima, N. and Tajima, K.: A 38-44, 2006. (PMID: 16367919) gene-gene interaction between ALDH2 Glu487Lys J126. Mizoshita, T., Tsukamoto, T., Toyoda, T., and ADH2 His47Arg polymorphisms regarding the Ban, H., Nozaki, K. and Tatematsu, M.: Intestinal risk of colorectal cancer in Japan. Carcinogenesis, phenotypes of stomach cancers arising after 27: 1018-1023, 2006. (PMID: 16332725) helicobacter pylori eradication in J119. Matsuo, K., Ito, H., Yatabe, Y., Hiraki, A., carcinogen-treated mongolian gerbils. Asian Pac J Wakai, K., Hirose, K., Kosaka, K., Suzuki, T., Cancer Prev, 8: 267-271, 2007. (PMID: 17696744) Tajima, K. and Mitsudomi, T.: Risk factors differ J127. Mizukoshi, E., Nakamoto, Y., Marukawa, for non-small-cell lung cancers with and without Y., Arai, K., Yamashita, T., Tsuji, H., Kuzushima, EGFR mutation: assessment of smoking and sex by K., Takiguchi, M. and Kaneko, S.: Cytotoxic T cell a case-control study in Japanese. Cancer Sci, 98: responses to human telomerase reverse transcriptase 96-101, 2007. (PMID: 17054433) in patients with hepatocellular carcinoma. J120. Matsuo, K., Wakai, K., Hirose, K., Ito, H., Hepatology, 43:1284-1294, 2006. (PMID: Saito, T. and Tajima, K.: The ADH2 His47Arg 16729333) polymorphism influences drinking habits J128. Mori, K., Suzuki, T., Uozaki, H., independently of the ALDH2 Glu487Lys Nakanishi, H., Ueda, T., Matsuo, Y., Kodera, Y., polymorphism: Analysis of 2,299 Japanese Subjects. Sakamoto, H., Yamamoto, N., Sasako, M., Cancer Epidemiol Biomarker Prev, 15: 1009-1013, Kaminishi, M. and Sasaki, H.: Detection of 2006. (PMID: 16702384) minimal gastric cancer cells in peritoneal washings J121. Mihara, M., Yoshida, Y., Tsukamoto, T., by focused microarray analysis eith multiple Inada, K., Nakanishi, Y., Yagi, Y., Imai, K., markers: Clinical implications. Ann Surg Oncol, Sugimura, T., Tatematsu, M. and Ushijima, T.: 14: 1694-1702, 2007. (PMID: 17294072) Methylation of multiple genes in gastric glands with J129. Morishima, S., Akatsuka, Y., Nawa, A., intestinal metaplasia: A disorder with polyclonal Kondo, E., Kiyono, T., Torikai, H., Nakanishi, T., origins. Am J Pathol, 169: 1643-1651, 2006. Ito, Y., Tsujimura, K., Iwata, K., Ito, K., Kodera, (PMID: 17071588) Y., Morishima, Y., Kuzushima, K. and Takahashi, J122. Miyazaki, K., Furuhashi, M., Matsuo, K., To.: Identification of an HLA-A24-restricted Minami, K., Yoshida, K., Kuno, N. and Ishikawa, cytotoxic T lymphocyte epitope from human K.: Impact of subclinical chorioamnionitis on papillomavirus type-16 E6: The combined effects

66 of bortezomib and interferon-gamma on the 6449: 644910-1-9, 2007. presentation of a cryptic epitope. Int J Cancer, 120: J137. Naota, H., Miyahara, Y., Okumura, S., 594-604, 2007. (PMID: 17096336) Kuzushima, K., Akatsuka, Y., Hiasa, A., Kitano, S., J130. Morishima, Y., Yabe, T., Matsuo, K., Takahashi, To., Yuta, A., Majima, Y. and Shiku, Kashiwase, K., Inoko, H., Saji, H., Yamamoto, K., H.: Generation of peptide-specific CD8+ T cells by Maruya, E., Akatsuka, Y., Onizuka, M., Sakamaki, phytohemagglutinin-stimulated antigen-mRNA- H., Sao, H., Ogawa, S., Kato, S., Juji, T., Sasazuki, transduced CD4+ T cells. J Immunol Methods, 314: T. and Kodera, Y.: Effects of HLA allele and killer 54-66, 2006. (PMID: 16828790) immunoglobulin-like receptor ligand matching on J138. Narimatsu, H., Ota, Y., Kami, M., clinical outcome in leukemia patients undergoing Takeuchi, K., Suzuki, R., Matsuo, K., Matsumura, transplantation with T-cell-replete marrow from an T., Yuji, K., Kishi, Y., Hamaki, T., Sawada, U., unrelated donor. Biol Blood Marrow Transplant, Miyata, S., Sasaki, T., Tobinai, K., Kawabata, M., 13: 315-328, 2007. (PMID: 17317585) Atsuta, Y., Tanaka, Y., Ueda, R. and Nakamura, J131. Munesue, S., Yoshitomi, Y., Kusano, Y., S.: Clinicopathological features of Koyama, Y., Nishiyama, A., Nakanishi, H., pyothorax-associated lymphoma; a retrospective Miyazaki, K., Ishimaru, T., Miyaura, S., Okayama, survey involving 98 patients. Ann Oncol, 18: M. and Oguri, K.: A novel function of syndecan-2, 122-128, 2007. (PMID: 17043091) suppression of MMP-2 activation, which causes J139. Narimatsu, H., Terakura, S., Matsuo, K., suppression of metastasis. J Biol Chem, 282: Oba, T., Uchida, T., Iida, H., Hamaguchi, M., 28164-28174, 2007. (PMID: 17623663 ) Watanabe, M., Kohno, A., Murata, M., Sawa, M., J132. Murata, T. and Shimotohno, K.: Ubi- Miyamura, K. and Morishita, Y.: Short-term quitination and proteasome-dependent degradation methotrexate could reduce early immune reactions of human eukaryotic translation initiation factor 4E. and improve outcomes in umbilical cord blood J Biol Chem, 281: 20788-20800, 2006. (PMID: transplantation for adults. Bone Marrow Transpl, 16720573) 39: 31-39, 2007. (PMID: 17115066) J133. Nagai, H., Sugito, N., Matsubara, H., J140. Narimatsu, Y., Ikehara, Y., Iwasaki, H., Tatematsu, Y., Hida, T., Sekido, Y., Nagino, M., Nonomura, C., Sato, T., Nakanishi, H. and Nimura, Y., Takahashi, Ta. and Osada, H.: Narimatsu, H.: Immunohistochemical analysis of CLCP1 interacts with semaphorin 4B and regulates intarcellular dynamics of C1GalTassociated with motility of lung cancer cells. Oncogene, 26: molecular chaperone, Cosmc. Biochem Biophys 4025-4031, 2007. (PMID: 17213806) Res Commun, 366: 199-205, 2008. (PMID: 18061573) J134. Nakagawa, M., Hashino, S., Takahata, M., Kawamura, T., Fujisawa, F., Kahata, K., Kondo, J141. Niibe, Y., Tsunoda, S., Jobo, T., Imai, M., T., Imamura, M., Ando, S. and Asaka, M.: Arai, M., Arai, T., Kawaguchi, M., Futai, M., Successful reduced-intensity stem cell Hamada, Y., Yago, K., Matsuo, K., Matsunaga, K., transplantation with cord blood for a Unno, N. and Hayakawa, K.: Phase II study of poor-prognosis adult with refractory chronic active radiation therapy combined with weekly nedaplatin epstein-barr virus infection. Int J Hematol, 85: in locally advanced uterine cervical carcinoma: 443-445, 2007. (PMID: 17562623) Kitasato Gynecologic Radiation Oncology Group (KGROG0501)-Initial analysis. Eur J J135. Nakamura, H., Yasui, Y., Saito, N., Gynaecoloncol, in press. Tachibana, A., Komatsu, K. and Ishizaki, K.: DNA repair defect in AT cells and their hypersensitivity J142. Nishimura, T., Yamaguchi, T., Tokunaga, to low-dose-rate radiation. Radiat Res, 165: A., Hara, A., Hamaguchi, T., Kato, K., Iwamatsu, 277-282, 2006. (PMID: 16494515) A., Okano, H. and Kaibuchi, K.: Role of Numb in Dendritic Spine Development with a Cdc42 GEF J136. Nakanishi, H., Hara, H., Ikehara, Y. and Intersectin and EphB2. Mol Biol Cell, 17: Tatematsu, M.: Non-invasive and real-time 1273-1285, 2006. (PMID: 16394100) monitoring of molecular targeting therapy for lymph node and peritoneal metastasis in nude mice J143. Nishino, Y., Wakai, K., Kondo, T., Seki, N., bearing xenografts of human colorectal cancer cells Ito, Y., Suzuki, K., Ozasa, K., Watanabe, Y., tagged with GFP and DsRed. Proceeding of SPIE, Aondo, M., Tsubono, Y., Tsuji, I. and Tamakoshi,

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R010. Kannagi, R., Miyazaki, K., Kimura, N. and Reviews and Books Yin, J.: Selectin-mediated metastasis of tumor cells: Alteration of carbohydrate-mediated cell-cell interactions in cancers induced by epigenetic R001. Akatsuka, Y., Morishima, Y., Kuzushima, K., Kodera, Y. and Takahashi, To.: Minor silencing of glycogenes, in Sansom C, Markman O. (eds), Glycobiology, Bloxham, Oxfordshire, UK, histocompatibility antigens as targets for Scion Publishing Ltd. pp274-287, 2007. immunotherapy using allogeneic immune reactions. Cancer Sci, 98: 1139-1146, 2007. (PMID: R011. Kannagi, R., Ohmori, K. and Kimura, N.: 17521316) Anti-oligosaccharide antibodies as tools for studying sulfated sialoglycoconjugate ligands for R002. Akatsuka, Y., Torikai, H., Inamoto, Y., Tsujimura, K., Morishima, Y., Kodera, Y., siglecs and selectins. Glycoconj J, in press. Kuzushima, K. and Takahashi, To.: Bone marrow R012. Kannagi, R., Yin, J., Miyazaki, K. and may be a reservoir of long-lived memory T cells Izawa, M.: Current relevance of incomplete specific for minor histocompatibility antigen. Brit J synthesis and neo-synthesis for cancer-associated Haematol, 135: 408-417, 2006. (PMID: 16978218) alteration of carbohydrate determinants. Biochim Biophys Acta, in press. (PMID: 17980710) R003. Cao, X., Tsukamoto, T., Nozaki, K., Tanaka, H., Cao, L., Toyoda, T., Takasu, S., Ban, R013. Kannagi, R.: Carbohydrate antigen sialyl H., Kumagai, T. and Tatematsu, M.: Severity of Lewis a - its pathophysiological significance and gastritis determines glandular stomach induction mechanism in cancer progression. Chang carcinogenesis in Helicobacter pylori-infected Gung Med J, 30: 189-209, 2007. (PMID: Mongolian gerbils. Cancer Sci, 98: 478-483, 2007. 17760270) (PMID: 17284248) R014. Kudoh, A. and Tsurumi, T.: Classification R004. Daikoku, T.: Latent infection and and structure of Gammaherpesvirus. Nippon Rinsho, Reactivation of Epstein-Barr virus. Nippon Rinsho, 64: 525-530, 2006. (PMID: 16615528) 64: 584-588, 2006. (PMID: 16615539) R015. Kuriki, K. and Tajima, K.: The increasing R005. Hino, O., Wakabayashi, K., Tatematsu, M. incidence of colorectal cancer and the preventive and Tajima, K.: On environmental carcinogens: strategy in Japan. Asian Pac J Cancer Prev, 7: from an era of risk evaluation to an era of risk 495-501, 2006. (PMID: 17059355) management. Cancer Sci, 98: 617-619, 2007. R016. Matsuo, K., Kiura, K., Tabata, M., Uchida, (PMID: 17284256) A., Hotta, K., Niiya, D., Kubonishi, S., Ogino, A., R006. Hotta, K., Matsuo, K., Kiura, K., Ueoka, H. Fujiwara, Y., Nakajima, H., Shinagawa, K., and Tanimoto, M.: Advances in our understanding Ishimaru, F., Ueoka, H. and Tanimoto, M.: of postoperative adjuvant chemotherapy in Clustered Incidence of Acute Promyelocytic resectable non-small-cell lung cancer. Curr Opin Leukemia During Gefitinib Treatment for Oncol, 18: 144-150, 2006. (PMID: 16462183) Non-Small Cell Lung Cancer: Experience at Single Institution. Am J Hematol, 81: 349-354, 2006. R007. Hotta, K., Matsuo, K., Kiura, K., Ueoka, H. (PMID: 16628731) and Tanimoto, M.: Long-standing debate on which regimen is superior, cisplatin-based chemotherapy R017. Matsuo, K., Suzuki, T. and Tajima, K.: or carboplatin-based chemotherapy in the treatment CagA status and successful eradication of of advanced non-small cell lung cancer. J Thorac Helicobacter pylori: authors’ replya. Aliment Oncol, 2: 96, 2007. (PMID: 17410021) Pharm Therap, 24: 1263, 2006. (PMID: 17014588) R008. Isomura, H. and Tsurumi, T.: Tissue R018. Mizoue, T., Tanaka, K., Tsuji, I., Wakai, tropism for herpesviruses. Nippon Rinsho, 64: K., Nagata, C., Otani, T., Inoue, M., Tsugane S. 24-28, 2006. (PMID: 16615435) for the Research Group for the Development and Evaluation of Cancer Prevention Strategies in R009. Izawa, I. and Inagaki, M.: Regulatory Japan: Alcohol drinking and colorectal cancer risk: mechanisms and functions of intermediate filaments: A study using site- and phosphorylation an evaluation based on a systematic review of epidemiologic evidence among the Japanese state-specific antibodies. Cancer Sci, 97: 167-174, population. Jpn J Clin Oncol, 36: 582-597, 2006. 2006. (PMID: 16542212) (PMID: 16870695)

R019. Mizuoe, T., Inoue, M., Tanaka, K., Tsuji, R028. Osada, H. and Takahashi, Ta.: Inclusion I., Wakai, K., Nagata, C. and Tsugane, S.: of ASH1 gene governing neuroendocrine Tobacco smoking and colorectal cancer risk: an differentiation of lung epithelium as an additional evaluation based on a systematic review of prototypic “lineage-survival oncogene”. Nat Rev epidemiologic evidence among the Japanese Cancer, 7: doi:10.1038/nrc1947-c1, 2007. population. Jpn J Clin Oncol, 36: 25-39, 2006. R029. Sihag, RK., Inagaki, M., Yamaguchi, T., (PMID: 16423841) Shea, TB. and Pant, HC.: Role of phosphorylation R020. Moore, M. and Tajima, K.: Establishment on the structural dynamics and function of types III of the UICC Asia Regional Office and Direction for and IV intermediate filaments. Exp Cell Res, 313: Collaborative Efforts in the Future. Asian Pac J 2098-2109, 2007. (PMID: 17498690) Cancer Preve, 7: 345-347, 2006. (PMID: 17059322) R030. Tajima, K.: Virus related cancers in Asian R021. Moore, M. and Tajima, K.: Practical Pacific with special reference to adult T-cell cancer prevention-Where is it going to occure and leukemia/lymphoma. Developments in cancer who is going to be responsible? Asian Pac J Cancer Epidemiology-prospects for cancer control in the Prev, 7: 1-2, 2006. (PMID: 16629537) Asian Pasific, 79-84, 2006. R022. Nagata, C., Mizoue, T., Tanaka, K., Tsuji, R031. Tanaka, K., Tsuji, I., Wakai, K., Nagata, I., Wakai, K., Inoue, M., Tsugane, S. Research C., Mizoue, T., Inoue, M., Tsugane, S. for the Group for the Development and Evaluation of Research Group for the Development and Cancer Prevention Strategies in Japan: Tobacco Evaluation of Cancer Prevention Strategies in smoking and breast cancer risk: an evaluation based Japan.: Cigarette smoking and liver cancer risk: an on a systematic review of epidemiologic evidence evaluation based on a systematic review of among Japanese population. Jpn J Clin Oncol, 36: epidemiologic evidence among Japanese. Jpn J Clin 387-394, 2006. (PMID: 16766567) Oncol, 36: 445-456, 2006. (PMID: 16782973) R023. Nakagawa, M., Seto, M. and Hosokawa, R032. Tatematsu, M., Tsukamoto, T. and Y.: Molecular pathogenesis of MALT lymphoma: Mizoshita, T.: Significant factors on gastric two signaling pathways underlying the carcinogenesis revealed by experimenta animal antiapoptotic effect of API2-MALT1 fusion protein. models. J Toxicol Pathol, 19: 75-86, 2006. Leukemia, 20: 929-936, 2006, (PMID: 16572204) R033. Tatematsu, M., Tsukamoto, T. and Toyoda, R024. Nakamura, H.: hTERT-Immortalized Cells T.: Effects of eradication of Helicobacter pylori on Useful for Analyzing Effects of Low-Dose-Rate gastric carcinogenesis in experimental models. J Radiation on Human Cells. J Radiat Res, in press. Gastroenterol, 42 Suppl 17: 7-9, 2007. (PMID: 17238018) R025. Nakanishi, H., Ito, S., Mochizuki, Y., Ohashi, N., Ikehara, Y., Yamamura, Y., Kodera, R034. Tokudome, S., Kojima, M., Suzuki, S., Y.: Peritoneal Carcinomatosis: A new Diagnostic Ichikawa, H., Ichikawa, Y., Miyata, M., Maeda, K., and Therapeutic Strategy. In Research Focus on Marumoto, M., Agawa, H., Arakawa, K., Tanaka, Gastric Cancer, Edited by Dorothy C. Cardinni, T., Ando, R., Hattori, N., Goto, C., Tokudome, Y., 2007. Imaeda, N., Kuriki, K., Okuyama, H. and Moore, MA.: Marine n-3 fatty acids and colorectal cancer: R026. Nishino, Y., Inoue, M., Tsuji, I., Wakai, K., is there a real link? Cancer Epidemiol Biomarker Nagata, C., Mizoue. T., Tanaka, K. and Tsugane, Prev, 15: 406-407, 2006. (PMID: 16492940) S. for the Research Group for the Development and Evaluation of Cancer Prevention Strategies R035. Tsukamoto, T., Mizoshita, T. and in Japan. Tobacco smoking and gastric cancer risk: Tatematsu, M.: Animal models of stomach an evaluation based on a systematic review of carcinogenesis. Toxicol Pathol, 35: 636-648, 2007. epidemiologic evidence among the Japanese (PMID: 17654405) population. Jpn J Clin Oncol, 36: 800-807, 2006. R036. Tsukamoto, T., Mizoshita, T. and (PMID: 17210611) Tatematsu, M.: Gastric Cancer Stem Cells: A R027. Osada, H. and Takahashi, Ta.: Hypothesis In: Sunitha M. Current Research in MicroRNAs in biological processes and Pathologyed. Kerala, India: Research Media, in carcinogenesis. Carcinogenesis, 28: 2-12, 2007. press. (PMID: 17028302)

R037. Tsukamoto, T., Mizoshita, T. and Abstracts for international conferences Tatematsu, M.: Gastric-and-intestinal mixed-type intestinal metaplasia: aberrant expression of transcription factors and stem cell intestinalization. A001. Akatsuka, Y., Torikai, H., Kawase, T., Gastric Cancer, 9: 156-166, 2006. (PMID: Tsujimura, K., Miyamura, K., Kodera, Y., 16952033) Morishima, Y., Kuzushima, K. and Takahashi, To.: Immunotherapy of hematological malignancies R038. Tsukamoto, T., Mizoshita, T., Cao, X. and targeting hematopoietic cell-specific minor Tatematsu, M.: Helicobacter pylori infection and histocompatibility antigens. Abstract (Workshop modifying factors in gastric carcinogenesis in 2-3) for The 11th Congress of The Asia Pacific Mongolian gerbils. In: Tanaka T. Cancer: Disease Blood and Marrow Transplantation. Nagoya, 2006. Progression and Chemopreventioned. Kerala: Research Signpost, 2007:15-25. A002. Chen, G.Y., Osada, H., Santamaria-Babi, LF. and Kannagi, R.: Interaction of GATA-3/T-bet R039. Tsurumi, T. and Kudoh, A.: Epstein-Barr transcription factors regulates expression of ligands virus: Lytic Genes, Shindan to Chiryosha, in press. for selectin-family cell adhesion molecules in R040. Tsurumi, T. and Kudoh, A.: Replication of human leukemic cells. 7th AACR-JCA Joint DNA viruses and host DNA damage responses. International Conference "In the Forefront of Basic Tanpakushitsu Kakusan Koso, 52: 1155-1160, 2007. and Translational Cancer Research" (Chaired by (PMID: 17824233) LM Matrisian and K Imai), Hawaii, 2007. R041. Varki, A., Kannagi, R. and Toole, BP.: A003. Chen, G.Y., Osada, H., Santamaria-Babi, Glycosylation changes in cancer. In A. Varki, R. D. LF. and Kannagi, R.: Interaction of GATA-3/T-bet Cummings, J. D. Esko, H. H. Freeze, G. W. Hart, transcription factors regulates homing receptor and J. D. Marth (eds.), Essentials of Glycobiology, expression on Th1/Th2 lymphocytes. Experimental 2 ed. New York: Cold Spring Harbor Laboratory Biology 2007, Washington DC, USA, 2007. Press, in press. A004. Chen, G.Y., Osada, H., Santamaria-Babi, R042. Wakai, K. and Matsuo, K.: The JICA LF. and Kannagi, R.: Transcriptional regulation of Training Course, Community-based selectin ligand expression in helper T-lymphocytes CancerPrevention for the Asian and Pan-pacific through chromatin modulation. Hakomori Countries, Fiscal Year 2006 (Epidemiological Commemorative Forum "Glycobiology and Approach). Asian Pac J Cancer Prev, 8: 150-154, Sphingobiology 2007" (Chaired by Roger A. Laine), 2007. (PMID: 17696724) Tokushima, 2007. R043. Wakai, K., Inoue, M., Mizoue, T., Tanaka, A005. Fukuhara, N., Tagawa, H., Nakamura, T., K., Tsuji, I., Nagata, C., Tsugane, S. for the Inagaki, H., Morishima, Y., Nakamura, S., Seto, Research Group for the Development and M.: Genomic imbalances are associated with Evaluation of Cancer Prevention Strategies in outcome of helicobacter pylori eradication in Japan: Tobacco smoking and lung cancer risk: an t(11;18)(q21;q21)-negative gastric MALT evaluation based on a systematic review of lymphomas. The American Society of Hematology epidemiological evidence among the Japanese 48th Annual Meeting and Exposition, Orlando, population. Jpn J Clin Oncol, 36: 309-324, 2006. 2006. (PMID: 16735374) A006. Goto, Y., Kondo, Y., Gao, D., Toyota, M., R044. Wakai, K.: The JICA training course, Suzuki, H., Ito, M., Yokoyama, T., Taniguchi, T., community-based cancer prevention for the Asian Osada, H., Murakami, H., Tokino, T., Imai, K., Pacific countries, fiscal years 2004 and 2005 Hasegawa, Y., Shimokata, K. and Sekido, Y.: (epidemiological approach). Asian Pac J Cancer Genome wide analyses for aberrant DNA Prev, 7: 5-10, 2006. (PMID: 16629508) methylation observed in malignant pleural mesothelioma. 98th American Association for R045. Yanada, M., Matsuo, K. and Naoe, T.: Cancer Research Annual Meeting, Los Angeles, Author reply. Cancer, 109: 304, 2007. (PMID: 2007. 17154186) A007. Goto, Y., Maeda, H., Suzuki, K., Ishimaru, T., Yamamoto, K., Miyaura, S., Kim, YS., Kimata, K., Kyogashima, M. and Kannagi, R.: Profiling of

78 heparan sulfate glycosaminoglycans on the human A015. Izawa, M., Kimura, N., Miyazaki, K., Yusa, cancer cell surfaces by various monoclonal A. and Kannagi, R.: Epigenetic silencing of antibodies. Satellite Symposium of IUBMB 2006 DTDST, a sulfate transporter gene, is involved in "Extracellular glycomatrix in health and disease," the induction of tumor-associated carbohydrate Hyogo, 2006. determinant sialyl Lewis X in colon cancers. 7th AACR-JCA Joint International Conference "In the A008. Honma, K., Tsuzuki, S., Nakagawa, M., Forefront of Basic and Translational Cancer Karnan, S., Kim, W-S., Ko, Y-H. and Seto, M.: Research" (Chaired by LM Matrisian and K Imai), Ocular adnexal marginal zone B cell lymphoma has Waikoloa, Hawaii, 2007. characteristic deletion at chromosome band 6q23.3-24.1 whose target is TNFAIP3. The A016. Kamiyama, S., Sasaki, N., Goda, E., American Society of Hematology 49th Annual Ui-Tei, K., Saigo, K., Narimatsu, H., Jigami, Y., Meeting and Exposition, Atlanta, 2007. Kannagi, R., Irimura, T. and Nishihara, S.: Identification and characterization of A009. Inagaki, M.: Phosphorylation by Cdk1 3'-phosphoadenosine 5'-phosphosulfate transporter induces Plk1-mediated vimentin phosphorylation genes. Satellite Symposium of IUBMB 2006 during mitosis. Gordon Conference on Intermediate "Extracellular glycomatrix in health and disease," Filaments, 2006. Hyogo, 2006. A010. Inoko, A., Sugimoto, M., Zou, P., Hayashi, A017. Kannagi, R., Chen, G.Y., Sakuma, K., Y., Izawa, I., Mikio, S., Uji, Y., Kiyono, T. and Osada, H., Santamaria-Babi, LF. and Ohmori, K.: Inagaki, M.: Fbf-1, a novel keratin Regulation of selectin ligand expression in human filament-binding protein, is needed to form apical T-lymphocyte subsets. 19th International junctions and apical-basal cell polarity. Gordon Symposium on Glycoconjugates (Glyco19), Cairns, Conference on Intermediate Filaments, 2006. Australia, 2007. A011. Isomura, H., Stinski, M.F., Kudoh, A., A018. Kannagi, R.: Cell-Cell interactions Iwahori, S., Shirata, N., Daikoku, T. and Tsurumi, mediated by sialoconjugates. [Invited Speaker] NIH T.: A cis-element downstream of TATA box in the Workshop "Frontiers in Glycomics: Bioinformatics HCMV major IE promoter stabilizes the and Biomarkers in Disease", (Organized by Pamela TBP-TFIIB-DNA complex for efficient RNA Marino) NIH, Bethesda, Maryland, USA, 2006. transcription and splicing. The 31st International Herpesvirus Workshop. 1.20. Seattle, Washington, A019. Kannagi, R.: Current relevance of USA, 2006. "incomplete synthesis" and "neo-synthesis" for cancer-associated alteration of carbohydrate A012. Isomura, H., Stinski, M.F., Kudoh, A., determinants. [Invited Speaker] Hakomori Nakayama, S., Iwahori, S., Sato, Y. and Tsurumi, Comemorative Forum "Glycobiology and T.: The late promoter of the human Sphingobiology 2007" (Chaired by Roger A. Laine), Cytomegalovirus viral DNA polymerase Tokushima, 2007. processibity factor has an impact on delayed early and late viral gene products but not on viral DNA A020. Kannagi, R.: Functional roles of synthesis. The 32nd International Herpesvirus carbohydrate determinants in cancer progression. Workshop. 1.50. Asheville, North Carolina, USA, International Symposium on Molecular 2007. Immunology of Complex Carbohdyrate-3 (MICC-3), Taipei, 2007. A013. Iwata, S., Kawakami, N., Sato, C., Ando, H., Kiso, M., Kannagi, R. and Kitajima, K.: A021. Kannagi, R.: Hypoxia-induced expression Studies on acid instability of O-glycosides of cyclic of cancer-associated glycoconjugates. The 27th sialic acid. Sialoglycoscience 2006, 5th. Sapporo Cancer Seminar International Symposium International Conference, Mishima, 2006. "Glycomics: new perspectives in cancer cell behavior", Sapporo, 2007. A014. Iwata, S., Sato, C., Ando, H., Kiso, M., Kannagi, R. and Kitajima, K.: Quantitative A022. Kannagi, R.: Lymphocyte homing: Role of detection of cyclic sialic acid in mammalian cells. carbohydrate ligands for selectins. [Invited 20th IUBMB International Congress of Speaker] Osaka University 21st Century Center of Biochemistry and Molecular Biology and 11th Excellence Program International Symposium, FAOBMB Congress. Kyoto, 2006. "Integrated Functional Analyses of

Disease-Associated Sugar Chains and Proteins" A030. Kim, W., Karnan, S., Tagawa, H., Seto, M., Osaka, 2006. Ko Yonug H.: Genome-wide array-based comparative genomic hybridization of ocular A023. Kannagi, R.: Selectin-mediated cell marginal zone B-cell lymphoma: Comparison with adhesion in cancer metastasis. 19th FAOBMB pulmonary and nodal marginal zone B-cell Conference, Symposium "Cell adhesion and lymphoma. The 9th Korean-Japanese migration", Seoul, 2007. Lymphoreticular Workshop, Jeju Island, 2006. A024. Kannagi, R.: Sialic acid recognition in the A031. Kitajima, K., Go, S., Yin, J. and Kannagi, skin and gut. [Invited Speaker] Sialoglycoscience R.: De novo synthesis of free deaminoneuraminic 2006, 5th. International Conference, Mishima, acid (Kdn) is regulated by network of metabolisms 2006. of sialic acids and mannose 6-phosphate in normal A025. Kannagi, R.: Tumor hypoxia induces and tumor cells. 19th International Symposium on expression of carbohydrate determinants involved Glycoconjugates (Glyco19), Cairns, Australia, in adhesion of circulating cancer cells to vascular 2007. endothelium. [Invited Speaker] The 11th A032. Kondo, S., Yagi, H., Lim, K.T., Miyazaki, International Congress of the Metastasis Research K., Takahashi, N., Kato, K. and Kannagi, R.: Society, Tokushima, 2006. Structural analysis of N-glycans carrying disialyl A026. Kawamura, YI., Adachi, Y., Kannagi, R., Lewis a determinant. Hakomori Commemorative Mizutani, N., Toyama-Sorimachi, N., Curiel, DT., Forum "Glycobiology and Sphingobiology 2007" Nishimoto, N. and Dohi, T.: Therapeutic gene (Chaired by Roger A. Laine), Tokushima, 2007. transfer of a glycosyltransferase efficientlyprevents A033. Kondo, Y., Sakamoto, H., Osada, H., gastric cancer metastasis. 20th IUBMB Murakami, H., Ueda, R. and Sekido, Y.: RARβ2 International Congress of Biochemistry and gene silencing by histone H3 lysine 27 Molecular Biology and 11th FAOBMB Congress, tri-methylation independent of promoter DNA Kyoto, 2006. methylation. 98th American Association for Cancer A027. Kawamura, YI., Adachi, Y., Nishimoto, N., Research Annual Meeting, Los Angeles, 2007. Curiel, DT., Kannagi, R. and Dohi, T.: A034. Kondo, Y., Shen, L., Ahmed, S., Boumber, Therapeutic potential of gene transfer of a Y., Haddad, BR., Murakami, H., Osada, H., glycosyltransferase using adenoviral vector to Sekido, Y. and Issa, JPJ.: Down regulation of prevent cancer metastasis. 5th International histone H3 lysine 9 methyltransferase G9 induces Symposium on Glycosyltransferases (GlycoT2006), centrosome disruption in cancer cells. 98th Ibaraki, 2006. American Association for Cancer Research Annual A028. Kawamura, YI., Toyota, M., Kawashima, Meeting, Los Angeles, 2007. R., Hagiwara, T., Miyake, O., Takeshita, E., Saito, A035. Koyama, H., Isogai, Z., Yoneda, M., Y., Kawamura, YJ., Konishi, F., Kannagi, R. and Fujimori, M., Amano, J., Kannagi, R., Kimata, K., Dohi, T.: Epigenetic mechanism for cancer- Taniguchi, S. and Itano, N.: Hyperproduction of associated silencing of a gene encoding a hyaluronan in neu-induced mammary tumor β1,4N-aceytylgalactosaminyltransferase, which is accelerates angiogenesis through stromal cell exclusively expressed in normal gastrointestinal recruitment: Possible involvement of mucosa. 19th International Symposium on PG-M/versican. The 11th International Congress of Glycoconjugates (Glyco19), Cairns, Australia, the Metastasis Research Society, Tokushima, 2006. 2007. A036. Koyama, H., Isogai, Z., Yoneda, M., A029. Kawase, T., Akatsuka, Y., Torikai, H., Fujimori, M., Kannagi, R., Kimata, K., Taniguchi, Morishima, S., Kodera, Y., Morishima, Y., S. and Itano, N.: Hyaluronan accelrtates tumor Kuzushima, K. and Takahashi, To.: Alternative angiogenesis through stromal cell recruitment. 20th Splicing due to an Intronic SNP defines a Novel IUBMB International Congress of Biochemistry HLA-B44-Restricted-Hemato-poiesis-Restricted and Molecular Biology and 11th FAOBMB Minor Histocompatibility Antigen. Abstract Congress (Chair T Honjo). Kyoto, 2006. (#3232) for The 48th American Society of Hematology Annual Meeting, Orlando, Florida, A037. Kudoh, A. and Tsurumi, T.: 2006. Phosphorylation of MCM4 at Sites Inactivating

DNA Helicase Activity of the MCM4-6-7 Complex and Tajima, K.: Risk factors differ for during Epstein-Barr virus Productive Replication. non-small-cell lung cancers with and without EGFR The 12th Symposium of the International mutation: an epidemiological evaluation in Japan. Association for Researh on Epstein-Barr Virus and AACR Annual Meeting 2006, Washington D.C., Associated Diseases. p148. Boston, USA, 2006. 2006. A038. Kumimoto, H., Saito, N. and Ishizaki, K.: A046. Miyazaki, K., Izawa, M., Ohmori, K., Induction of Mitochondrial-DNA Mutation in Mitsuki, M., Yamaji, T., Hashimoto, Y., Suzuki, A. Esophageal Cancer Cells by X-ray Irradiation. In and Kannagi, R.: Biological significance of the Forefront of Basic and Translational Cancer interconversion and overlap between carbohydrate Research AACR/JCA 7th Joint Conference. HI, ligands for selectins and siglecs expressed on USA, 2007. normal colonic epithelium and cancer cells. 19th International Symposium on Glycoconjugates A039. Kumimoto, H., Saito, N. and Ishizaki, K.: (Glyco19), Cairns, Australia, 2007. Mitochondria DNA instability by ionizing radiation. 20th IUBMB International Congress of A047. Miyazaki, K., Izawa, M., Ohmori, K., Biochemistry and Molecular Biology and 11th Mitsuki, M., Yamaji, T., Hashimoto, Y., Suzuki, A. FAOBMB Congress. Kyoto, Japan, 2006. and Kannagi, R.: Cancer-associated "incomplete synthesis" of carbohydrate determinants enhances A040. Kuriki, K., Hirose, K., Wakai, K., Matsuo, mucosal macrophage COX2 expression in colonic K., Ito, H., Suzuki, T., Hiraki, A., Saito, T., Iwata, carcinogenesis. Hakomori Commemorative Forum H., Tatematsu, M. and Tajima, K.: Breast cancer "Glycobiology and Sphingobiology 2007" (Chaired risk and erythrocyte compositions of n-3 by Roger A. Laine), Tokushima, 2007. polyunsaturated fatty acids in Japanese. The 7th Joint Conference of AACR and JCA, Hawaii, 2007. A048. Miyazaki, K., Izawa, M., Ohmori, K., Mitsuki, M., Yamaji, T., Hashimoto, Y., Suzuki, A. A041. Kyogashima, M., Tadano-Aritomi, K., and Kannagi, R.: Implication of cancer-associated Murate, T., Tamiya-Koizumi, K., Aoyama, T., abnormal glycosylation for enhanced COX2 Kannagi, R. and Hara, A.: Diversities and their expression in mucosal macrophages during colonic apoptotic activities of hydroxy-ceramides carcinogenesis. 7th AACR-JCA Joint International containing long-chain bases with unusual alkyl Conference "In the Forefront of Basic and chain lengths. 19th International Symposium on Translational Cancer Research" (Chaired by LM Glycoconjugates (Glyco19), Cairns, Australia, Matrisian and K Imai), Waikoloa, Hawaii, 2007. 2007. A049. Murakawa, A., Itano, N., Kimata, K., A042. Kyogashima, M., Tadano-Aritomi, K., Kannagi, R., Mori, T. and Okahata, Y.: Tamiya-Koizumi, K., Aoyama, T., Hara, A. and Preperation of hyaluronan synthase 2 by Kannagi, R.: Demonstration of diversities of novel baculovirus-infected insect cells expression system. free hydroxyceramides molecular species Satellite Symposium of IUBMB 2006 containing unusual sphingoid bases. Hakomori "Extracellular glycomatrix in health and disease," Commemorative Forum "Glycobiology and Hyogo, 2006. Sphingobiology 2007" (Chaired by Roger A. Laine), Tokushima, 2007. A050. Murata, T. and Tsurumi, T.: TORC Promotes EBV Reactivation from Latency by A043. Kyogashima, M.: Sulfated glycoconjugates Interacting with BZLF1. The ASM Conference on in diseases. Annaul Conference of Korea Manipulation of Nuclear Processes by DNA Glycosciene Society, Seoul, 2007. Viruses, Charleston, South Carolina, USA, 2008. A044. Lim, K.T., Miyazaki, K., Kimura, N. and A051. Nakagawa, M., Nakagawa-Oshiro, A., Kannagi, R.: Roles of carbohydrate side chains of Tagawa, H., Karnan, S., Tsuzuki, S., Ohshima, K., immunoglobulin superfamily cell adhesion Nakamura, S. and Seto, M.: Array CGH analysis molecules (IgCAMs). 55th Fujihara Seminar, of peripheral T-cell lymphoma-unspecified revealed Comprehensive understanding of the physiological common genomic alterations with adult T-cell and pathological significance of cell to cell leukemia/lymphoma. An AACR special conference adhesion by TSLC1/IGSF4. Tomakomai, 2006. in cancer reseach; oncogenomics, Arizona, 2007. A045. Matsuo, K., Ito, H., Yatabe, Y., Hirose, K., A052. Nakagawa, M., Nakagawa-Oshiro, A., Wakai, K., Kosaka, K., Suzuki, T., Mitsudomi, T.

Karnan, S., Tagawa, H., Nakamura, S., Itakeuchi, A059. Sato, Y. and Tsurumi, T.: Expression of I., Ohshima, K. and Seto, M.: Array CGH analysis Epstein-Barr Virus BZLF1 Immediate-Early Protein for PTCL-U revealed a distinct subgroup with Induces p53 Degradation Independent of MDM2, pathological and clinical relevance. The American Leading to Repression of p53-mediated Society of Hematology 49th Annual Meeting and Transcription. The ASM Conference on Exposition, Atlanta, 2007. Manipulation of Nuclear Processes by DNA Viruses, Charleston, South Carolina, USA, 2008. A053. Nakagawa, M., Oshiro, A., Tagawa, H., Karnan, S., Tsuzuki, S., Ohshima, K., Nakamura, A060. Suzuki, T., Matsuo, K., Hiraki, A., Saito, S., Seto, M.: Array CGH analysis for PTCL-U T., Sato, S., Ueda, R., Hasegawa, Y. and Tajima, revealed two genetically distinct subgroups. The K.: One-carbon metabolism related gene American Society of Hematology 48th Annual polymorphisms and risk of head and neck Meeting and Exposition, Orlando, 2006. squamous cell carcinoma. AACR Annual Meeting 2007, Los Angeles, 2007. A054. Nannya, Y., Onizuka, M., Kashiwase, K., Sanada, M., Akatsuka, Y., Satake, M., Chiba, S., A061. Tagawa, H., Karube, K., Tsuzuki, S., Kurokawa, M., Saji, H., Maruya, E., Inoko, H., Ohshima, K., Seto, M.: The synergistic action of Morishima, Y., Kodera, Y. and Ogawa, S.: the microRNA-17 cluster with c-MYC Exploring genetic basis of GVHD by inAggressive cancer development. The American whole-genome association studies in a large series Society of Hematology 48th Annual Meeting and from the Japan Marrow Donation Program (JMDP). Exposition, Orlando, 2006. Abstract (#3229) for The 49th American Society of A062. Tajima, K., Sonoda, S., Senoh, H. and Hematology Annual Meeting, Atlanta, Georgia, Chiba, H.: Anthropo-genetic approach on 2007. Mongoloids HTLV dispersal in Pan-Pacific regions. A055. Osada, H., Hayashita, Y., Tatematsu, Y., 13th Int Conference on Human Retrovirology, Yamada, H., Yanagisawa, K., Tomida, S., Yatabe, Hakone, 2007. Y., Kawahara, K., Sekido, Y. and Takahashi, Ta.: A063. Tajima, K.: Asian cancer etiology and A polycistronic miRNA cluster, miR-17-92, is prevention, Proceeding of Asian Medical Forum overexpressed in human lung cancers and enhances The Lancer 2007, Singapore, 2007. cell proliferation. 20th IUBMB International Congress of Biochemistry and Molecular Biology A064. Taniguchi, T., Kaman, S., Fukui, T., and 11th FAOBMB Congress, Kyoto, 2006. Yokoyama, T., Tagawa, H., Yokoi, K., Ueda, Y., Mitsudomi, T., Horio, Y., Hida, T., Yatabe, Y., A056. Osada, H., Nagai, H., Matsubara, H., Seto, M. and Sekido, Y.: Genomic profiling of Sugito, N., Tatematsu, Y., Hida, T., Sekido, Y., malignant pleural mesothelioma with array-based Nagino, M., Nimura, Y. and Takahashi, Ta.: comparative genomic hybridization shows frequent CLCP1 interacts with semaphorin 4B and regulates nonrandom chromosomal alteration regions motility of lung cancer cells. 98th American including JUN amplification on 1p32. 98th Association for Cancer Research Annual Meeting, American Association for Cancer Research Annual Los Angeles, 2007. Meeting, Los Angeles, 2007. A057. Osada, H.: The ASH1 Gene may be a A065. Taniguchi, T., Kaman, S., Fukui, T., Prototypic “Lineage-Survival Oncogene” and a Yokoyama, T., Tagawa, H., Yokoi, K., Ueda, Y., Specific the Rapeutic Target for Lung Cancers with Mitsudomi, T., Horio, Y., Hida, T., Yatabe, Y., Neuroendocrine Features. First JCA-AACR Special Seto, M. and Sekido, Y.: Genomic profiling of Joint Conference: The Latest Advances in Lung malignant pleural mesothelioma with array-based Cancer Research: From Basic Science to comparative genomic hybridization. 12TH World Therapeutics, Nagoya, 2007. Conference on Lung Cancer, Seoul, 2007. A058. Oshiro, A., Tagawa, H., Nakagawa M., A066. Tsurumi, T. and Iwahori, S.: Enhanced Oshima, K., Utsunomiya, A., Nakamura, S., Phosphorylation of Transcription Factor Sp1 Morishima, Y., Seto, M.: Genomic alterations in dependent on Ataxia Telangiectasia-Mutated aggressive adult T-cell leukemia/ lymphoma and (ATM) in response to Herpes Sinplex Virus Type 1 PTCL-U. The 9th Korean-Japanese Lymphoreticular Infection. The 7th Awaji International Forum on Workshop, Jeju Island, 2006. Infection and Immunity. p74. Awaji, Hyogo, Japan,

2007. Trichoplein, a keratin filament-binding protein, is a functional component of centrosomes. Gordon A067. Tsurumi, T., Kudoh, A. and Daikoku, T.: Conference on Intermediate Filaments, 2006. Postreplicative Mismatch Repair Factors are recuired to Epstein-Barr Virus replication Compartment. The 12th Symposium of the

International Association for Researh on Epstein-Barr Virus and Associated Diseases. p146. Boston, USA, 2006. A068. Yagi, H., Yamada, K., Yamaguchi, Y., Takahashi, N., Oka, S., Kawasaki, T., Uchimura, K., Kannagi, R. and Kato, K.: Application of the multi-dimensional hplc mapping method to the branch specificties of sulfotransferases and glycosyltransferases. 5th International Symposium on Glycosyltransferases (GlycoT2006), Ibaraki, 2006. A069. Yin, J., Izawa, M., Miyazaki, K., Chen, G.Y., Cheng, FL., Lin, CH. and Kannagi, R.: N-glycolyl GM2, a cancer-associated ganglioside on human breast and colon cancers induced by tumor hypoxia. 19th International Symposium on Glycoconjugates (Glyco19), Cairns, Australia, 2007. A070. Yin, J., Izawa, M., Miyazaki, K., Chen, G.Y., Cheng, FL., Lin, CH. and Kannagi, R.: Tumor hypoxia induces expression of N-glycolyl GM2, a cancer-associated ganglioside on human cancer cells through "neosynthesis" mechanism. Hakomori Commemorative Forum "Glycobiology and Sphingobiology 2007" (Chaired by Roger A. Laine), Tokushima, 2007. A071. Yin, J., Izawa, M., Miyazaki, K., Chen, G.Y., Cheng, FL., Lin, CH. and Kannagi, R.: Tumor hypoxia induces expression of Sialin, a sialic acid transporter, and cancer-associated gangliosides containing abnormal sialic acid on human cancer cells. 7th AACR-JCA Joint International Conference "In the Forefront of Basic and Translational Cancer Research" (Chaired by LM Matrisian and K Imai), Waikoloa, Hawaii, 2007. A072. Yusa, A., Fujii, M., Goto, Y., Suzuki, K., Ishimaru, T., Yamamoto, K., Kim, YS., Kimata, K., Kyogashima, M. and Kannagi, R.: Profiling of heparan sulfate glycosaminoglycans of the human cancers by monoclonal antibodies. 19th International Symposium on Glycoconjugates (Glyco19), Cairns, Australia, 2007. A073. Zou, P., Inoko, A., Kiyono, T., Hayashi, Y., Izawa, I., Hirotsune, S. and Inagaki, M. ：

83 ______Record of Seminars Invited Speakers

2006 Jan. 25 Hiraki, A. (University of Texas, San Antonio): Role of osteopontine in breast cancer metastasis.

Jun. 09 Hirota, T. (The JFCR Cancer Institute Dept. Experimental Pathology): How Aurora kinases control mitotic chromosome assembly.

Jun. 30 Urano, T. (Dept. of Molecular Biochemistry, Nagoya University Graduate School of Medicine): Ca2+/calmodulin regulates Aurora-A kinase activity at anaphase.

July 24 Oshima, M. (Division of Genetics, Cancer Research Institute, Kanazawa University): Gastric tumorigenesis through cooperation of Wnt signaling and COX-2/PGE2 pathway.

July 24 Yamamoto, S. (National Cancer Center, Center for Cancer Control and Information Service, Information System Division): Introduction of methods in clinical research.

Oct. 04 Shen, L. (Department of Leukemia, MD Anderson Cancer Center): CpG island methylation is a poor prognostic factor in myelodysplastic syndrome patients and is reversed by Decitabine therapy-Results of a Phase III randomized study.

Oct. 26 Hirano, N. (Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School): Long-lived CTL generated using artificial antigen-presenting cells: Translational cancer research from bench to bedside.

Oct. 31 Riddell, S. R. (Fred Hutchinson Cancer Research Center, University of Washington): Establishing T cell memory by adoptive transfer of T cell clones.

Nov. 28 Rickinson, A. (Cancer Research UK Institute for Cancer Studies, University of Birmingham): T cell responses to Epstein-Barr virus infection.

Dec. 06 Takenawa, T. (Department of Biochemistry, The Institute of Medical Science, The University of Tokyo): Breakdown of regulation of cell motility and cancer invasion/metastasis.

2007 Mar. 05 Hoffman, R. M. (Department of Surgery, University of California): The use of fluorescent protein imaging to visualize new cellular and subcellular targets for cancer chemotherapy in vivo.

May 09 Ohta, K. (Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo): ADLib system: rapid and flexible design of monoclonal antibodies by the chicken DT40 cell line.

May 17 Tanaka, H. (Department of Cancer Control and Statistics, Osaka Medical Center for Cancer and Cardiovascular Diseases): Epidemiology of liver cancer and national policy for liver cancer control.

84 May 25 Eriksson, J. E. (Dept. of Biology, Abo Akademi University, Turku, Finland and Turku Center for Biotechnology, Univ. of Turku and Abo Akademi University): Intermediate filaments as signaling organizers.

Magin, T. M. (Institute for Physiological Chemistry, Division of Cell Biochemistry and LIMES, University of Bonn): Characterization of a novel multidomain protein involved in cell polarity and tight junction formation.

May 29 Yamaguchi, T. (Center for Experimental Medicine, Institute of Medical Science, University of Tokyo/ Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science And Technology): Establishment of transgenic mice that express SV40 large T antigen by Cre/loxP recombination and applications for immortalized blood/lymphatic endothelial cells.

Jun. 28 Matsuyama, M. (Center for Developmental Biology, RIKEN Kobe): The role of secreted frizzled-related protein (Sfrp) in the Wnt signaling pathway.

Aug. 08 Minami, M. (Division of Hematology-Oncology, Department of Medicine and Moores - UCSD Cancer Center, University of California San Diego School of Medicine, USA): BCR-ABL-transformed leukemia stem cells display innate resistance to imatinib.

Sep. 10 Cavalli, F. (Oncology Institute of Southern Switzerland, and President of UICC): Extranodal lymphomas in Europe/Lugano lymphoma conference; history and future.

Institute Speakers

2006 Jan. 12 Kyogashima, M. (Molecular Pathology): Functions of sulfated glycoconjugates, sulfatides and glycosaminoglycans.

Feb. 16 Yamaguchi, T. (Biochemistry): Phosphorylation by Cdk1 induces Plk1-mediated vimentin phosphorylation during mitosis.

Mar. 09 Kumimoto, H. (Central Laboratory & Radiation Biology): Mitochondria DNA instability by ionizing radiation.

Mar. 23 Nakamura, Hir. (Central Laboratory & Radiation Biology): Summary of my research work.

Jun. 14 Tajima, K.（Epidemiology and Prevention）: Current situation and future prospects of Aichi Cancer Center Research Institute.

July 31 Cao, X. (Oncological Pathology); Chemoprevention of N-methyl-N-nitrosourea induced gastric cancer using Helicobacter pylori infected Mongolian gerbils.

Oct. 26 Tagawa, H. (Molecular Medicine): Genome alterations in malignant lymphoma.

Dec. 25 Isomura, H. (Virology): Transcriptional regulation of human cytomegalovirus.

2007 Jan. 31 Chen, G.-Y. (Molecular Pathology): Transcriptional regulation of sialyl Lewis X expression.

Feb. 15 Peng, Z. and Inoko, A. (Biochemistry): Unexpected correlations among keratins, centrosomes and apical junctional complex - A novel mechanism for cancer growth which is different from EMT?-

Mar. 05 Akatsuka, Y.（Immunology）: From bed to bench to cell processing facility, and finally to bed.

Mar. 22 Ijichi, K. (Central Laboratory & Radiation Biology): Pretreatment with 5-FU enhances cisplatin cytotoxicity in head and neck squamous cell carcinoma cells.

Aug. 08 Nakanishi, H. (Oncological Pathology): Basic research and clinical application of micrometastasis of gastrointestinal malignancy.

Sep. 13 Fujii, M. (Molecular Oncology): Enhancement of c-Myc transcriptional activity by SNIP1 (Smad Nuclear Interacting Protein 1).

Oct. 30 Nakagawa, M. (Molecular Medicine): Subtyping of peripheral T-cell lymphoma, unspecified, by array CGH.

Dec. 26 Takayuki Murata (Virology): Replication and gene expression of hepatitis C virus.

86 ______Record of Symposia

The 12th Aichi Cancer Center International Symposium “Perspective of Oncological Strategy for Gastrointestinal Cancer”

Organizing Committee: Yasuo Morishima (Chairperson), Tetsuya Mitsudomi, Masae Tatematsu, Kenji Yamao, Kei Muro, Yasuhisa Hasegawa, Akira Sawaki, Yoshitsugu Horio, Kazuhito Yamamoto, Naoki Tanaka, Takanori Umeda

January 13, 2007, International Conference Hall, Aichi Cancer Center.

Program of symposium

Opening Remarks: Toshitada Takahashi (Aichi Cancer Center)

Basic Research and Preclinical Study on Cancer Therapeutics Jean-Pierre Issa (MD Anderson Cancer Center, USA) Integrated genetic and epigenetic analysis identifies colon cancer as three different diseases Toshikazu Ushijima (National Cancer Center Research Institute) DNA methylation as a marker for the past and future Hayao Nakanishi (Aichi Cancer Center Research Institute) HER family as potential molecular targets for anti-cancer therapy against gastrointestinal malignancies

Development of Chemotherapy for Pancreatic Cancer Malcolm J. Moore (Princess Margaret Hospital, Canada) Perspectives in pancreatic cancer chemotherapy Takuji Okusaka (National Cancer Center Hospital) Treatment of pancreatic cancer: challenges in Japan Akira Sawaki (Aichi Cancer Center Hospital) The future direction of chemotherapy for pancreatic cancer

Chemoradiotherapy for Head & Neck and Esophageal Cancers Carol R. Bradford (University of Michigan, USA) Current thoughts on the role of chemotherapy and radiation in advanced head and neck cancer Tetsuya Ogawa (Aichi Cancer Center Hospital) Prediction of chemosensitivity for head and neck cancer Satoshi Ishikura (National Cancer Center Hospital) Radiotherapy for esophageal cancer－Current status and future directions－ Kei Muro (Aichi Cancer Center Hospital) Definitive chemoradiotherapy for stage I-III esophageal squamous cell carcinoma: Current results in Japan.

Chemotherapy for Colorectal Cancer Claus-Henning Köhne (Klinikum Oldenburg, Germany) Systemic chemotherapy for colorectal cancer Atsushi Ohtsu (National Cancer Center Hospital East) Systemic chemotherapy for colorectal cancer－Current status and future perspectives in Japan－ Yoshitaka Inaba (Aichi Cancer Center Hospital) Hepatic arterial infusion chemotherapy for liver metastasis from colorectal cancer Concluding Remarks: Yasuo Morishima

87 Abstracts

Integrated genetic and epigenetic analysis DNA methylation as a marker for the past identifies colon cancer as three and future different diseases Toshikazu Ushijima Jean - Pierre Issa Carcinogenesis Division, National Cancer Center M. D. Anderson Cancer Center, Houston, Texas, USA Research Institute Tokyo, Japan Background: Colon cancer affects 6 percent of DNA methylation, a representative epigenetic the population in the US, and is one of the most modification, is stably inherited upon cell common malignances. Colon cancer has been replication, and can ‘permanently’ repress gene viewed as the result of progressive accumulation of transcription. In spite of the deep involvement of genetic and epigenetic abnormalities. However, this aberrant methylation in gastric cancers, its view does not fully reflect the molecular induction mechanisms have been unclear. heterogeneity of the disease. We quantified methylation levels of eight CpG Methods: We have analyzed both genetic islands (CGIs) of seven genes in the gastric (mutations of BRAF, KRAS and p53, as well as mucosae of healthy individuals with and without microsatellite instability) and epigenetic alterations Helicobacter pylori (HP) infection, a potent gastric (DNA methylation of 27 CpG island promoter carcinogen. HP infection was detected by the serum regions) in a group of 97 primary colorectal cancer antibody test and/or the culture method, reflecting patients. Two clustering analyses on the basis of the current or recent infection status. It was found either epigenetic profiling or a combination of that methylation levels were 5- to 50-fold higher in genetic and epigenetic profiling were performed to individuals with current HP infection than those identify subgroups with distinct molecular without. We further isolated 48 promoter CGIs that signatures. can be methylated in gastric cancers, and found that Results: Unsupervised hierarchical clustering specific genes were methylated in individuals with of the DNA methylation data identified 3 distinct HP. It was suggested that a field defect with subgroups of colon cancers named CpG island inactivation of multiple genes was induced by HP methylator phenotype (CIMP) 1, CIMP2 and CIMP infection and that this can be detected by negative. Genetically, these three clusters methylation of specific genes. correspond to very distinct profiles; CIMP1 cases Methylation levels of the initial eight CGIs were are characterized by MSI (80%) and BRAF also analyzed in non-cancerous gastric mucosae of mutations (53%) but rare KRAS and p53 mutations cases with a gastric cancer. Individuals with current (16% and 11%, respectively). CIMP2 is associated HP infection showed high methylation levels with 92% KRAS mutations but rare MSI, BRAF or regardless of the presence or absence of gastric p53 mutations (0%, 4% and 31% respectively). cancers. In contrast, among individuals without CIMP negative cases have a high rate of p53 current HP infection, cases with a gastric cancer mutations (71%) and lower rates of MSI (12%) or had 2- to 20- fold higher methylation levels than mutations of BRAF (2%) or KRAS (33%). healthy individuals. We further showed that the Clustering based on both genetic and epigenetic level of FLNc methylation had an increasing trend parameters also identifies three distinct (and in the order of: healthy individuals, cases with a homogeneous) groups that largely overlap with the single gastric cancer, and cases with multiple previous classification. The three groups are gastric cancers. The risk given by high methylation independent of age, gender or stage, but CIMP 1 levels was independent from that given by the and 2 are more common in proximal tumors. extent of endoscopic atrophy. It was indicated that Conclusion: Integrated genetic and epigenetic methylation levels in gastric mucosae of individuals analysis reveals that colon cancers correspond to without current HP infection can be used to three molecularly distinct diseases. These diseases estimate a future risk of gastric cancers. also differ in terms of histology, precursor lesion It is generally known that, for specific cancers, and clinical course, suggesting truly distinct DNA methylation of some genes in cancer tissues is pathogenesis. associated with responses to chemotherapies and that of other genes is with patient prognosis. Our

88 study here showed DNA methylation in administration. In these GLM cell lines, no non-cancerous tissues is also useful, and might be significant inhibition of Akt phosphorylation and related to methylation patterns in cancer tissues. induction of apoptosis were observed by trastuzumab treatment in vitro, whereas significant HER family as potential molecular targets antibody-dependent cellmediated cytotoxicity for anticancer therapy against (ADCC) was observed by trastuzumab treatment. gastrointestinal malignancies These results suggest that the anti-tumor effects of gefitinib and the anti-metastatic (peritoneal) effects Hayao Nakanishi of trastuzumab against GLM cell lines are mainly Division of Oncological Pathology, Aichi Cancer Center due to the effective inhibition of HER2- driven Research Institute, Nagoya, Japan constitutive activation of phosphatidylinositol- 3-kinase (PI3K)/Akt pathway and induction of HER family, a receptor tyrosine kinase ADCC, respectively. In colorectal cancers, we including HER1 (EGFR), HER2 (neu/erbB2), found that intact EGFR expressing COLM-5 cells, a HER3 (erbB3) and HER4 (erbB4), regulates poorlydifferentiated colorectal cancer cell line with essential cellular functions, such as growth, high metastatic potential established in our differentiation and apoptosis in epithelial cells, and laboratory, showed super-sensitivity to both their activation is associated with carcinogenesis gefitinib and cetuximab in vivo. Primary sc tumor and progression in various cancers including growth, lymph node metastasis and peritoneal gastrointestinal malignancies. The binding of metastasis were markedly inhibited by gefitinib and ligands to the extracellular region of HER family cetuximab monotherapy in some of the treated nude induces receptor homo- or hetero-dimerization and mice cured. However, no apparent apoptosis activation of cytoplasmic tyrosine kinase which in induction and inhibition of Akt and ERK1/2 turn leads to autophosphorylation and initiation of phosphorylation were observed in vitro. Although downstream signaling. Recently, drugs targeting the mechanism of the anti-tumor effect of gefitinib EGFR and HER2 is clinically used for lung and and cetuximab on COLM-5 cells remains unclear, breast cancer patients, and their clinical efficacy has these results suggest the possible presence of a been proved. In gastrointestinal malignancies, a subset of aggressive colorectal cancers with high chimeric EGFR monoclonal antibody (mAb) sensitivity for EGFR targeting drugs. Gastric and (cetuximab) is approved for clinical application in colorectal cancers with HER family overexpression metastatic colorectal cancer, but molecular targeting would therefore be potential targets for molecular therapy for gastric cancer is still unavailable. We therapy with gefitinib, cetuximab, trastuzumab and found that gastric cancer metastasized to the liver possibly other agents. overexpresses HER2 at a significantly higher incidence than primary gastric cancers. We thus developed three new HER2 overexpressing gastric Perspectives in pancreatic cancer cancer cell lines (GLM-1, GLM-2, GLM-4) without chemotherapy EGFR mutations from such liver metastasis, two of Malcolm J. Moore which had HER2 gene amplifications. Interestingly, Department of Medical Oncology & Hematology, all these GLM series of cell lines were highly Princess Margaret Hospital, Toronto, Canada sensitive to gefitinib (Iressa) in vitro, a specific inhibitor of EGFR tyrosine kinase, whereas most of Gemcitabine has represented the gold standard the HER2 low expressing counterparts were not. In for palliative chemotherapy in advanced pancreatic these HER2 overexpressing GLM series, Akt, but cancer for over a decade. This benchmark was not ERK1/2 was constitutively phosphorylated, and established by a phase III trial which randomized gefitinib efficiently inhibited this Akt 126 symptomatic patients with advanced pancreatic phosphorylation, induced strong apoptosis in vitro cancer to either gemcitabine or 5-fluorouracil. and exhibited antitumor activity in subcutaneous Treatment with gemcitabine was associated with (sc) tumor xenografts in nude mice. On the other improvements in one-year survival (18% versus hand, a humanized HER2 mAb (trastuzumab) only 2%) and clinical benefit response (24% versus 5%) weakly inhibited growth of sc tumor xenografts, but over 5-FU. Since this trial was published in 1997, showed a marked anti-tumor effect against many large international phase III trials involving peritoneal metastasis of HER2 overexpressing thousands of patients have been conducted GLM-1 cells after intraperitoneal (ip) comparing gemcitabine alone (as the reference

89 standard) to a gemcitabine combination with either (mTOR), such as RAD001 and temsirolimus, and a cytotoxic or molecularly targeted agent. Almost Src kinase inhibitors. Gemcitabine and erlotinib, an all have failed to meet the standard of improving orally available antagonist of the epidermal growth overall survival. factor receptor (EGFR), was the first combination Gemcitabine and 5-fluorouracil have been the regimen to demonstrate statistically significant most widely used chemotherapy agents in superiority in terms of overall survival over pancreatic cancer. It is rational, therefore, to gemcitabine alone. The hazard ratio was 0.81 which combine these agents in the hopes of achieving translates to an overall 23% improvement in benefit over either drug alone. SAKK/CECOG survival; in absolute terms this translated to an compared GemCap, the combination of gemcitabine average improvement in survival of 5 weeks. and capecitabine, an oral prodrug of 5-FU, to Further analyses suggest there is a subset of patients gemcitabine alone in patients with locally advanced with pancreatic cancer who receive a greater benefit or metastatic cancer. Consistent with the majority although at this time we cannot prospectively of phase III trials in pancreatic cancer before it, this identify these patients. trial by Herrmann et al. failed to reach their primary Phase II trials of novel agents need to continue endpoint; in this case, a 2-month improvement in and more resources need to be devoted to the median overall survival. ECOG 2297, enrolled 327 development of new agents. These trials also patients to standard doses of gemcitabine, given provide the platform for necessary translational weekly 3 weeks out of 4, with or without 5-FU by research to identify predictive biomarkers for these bolus infusion at a dose of 600 mg/ m2 weekly in new agents. By returning to the benchtop from the the same schedule. The median survival for bedside, we might be able to establish an enriched gemcitabine alone was 5.4 months, compared to 6.7 subset of patients who might benefit from any of a months with gemcitabine and 5-FU (p=0.09). number of available combination therapies. To date, Modulating the effect of 5-FU, by the use of such predictive biomarkers have been elusive for 24-hour infusion and leucovorin were not EGFR antagonists and antiangiogenic agents. successful in improving efficacy when combined However, with the use of innovative strategies, such with gemcitabine. A phase III trial of 533 patients as functional imaging, genomics and other related conducted in the UK comparing GemCap to technologies, we may finally move toward truly gemcitabine alone has been reported in abstract individualized and targeted treatment for patients form, suggesting a significant survival advantage with advanced pancreatic cancer. [HR=0.80] to the combination therapy. Similarly studies of gemcitabine with oxaliplatin, irinotecan Treatment of pancreatic cancer: challenges and pemetrexed have not shown benefit. After ten in Japan. years of exhaustive clinical investigations with Takuji Okusaka gemcitabine doublet regimens in advanced pancreatic cancers, the arguments in favor of Hepatobiliary and Pancreatic Oncology Division, combination chemotherapy are modest. The only National Cancer Center Hospital, Tokyo, Japan positive study with gemcitabinecapecitabine is Despite recent advances in imaging modalities, tempered by a similar study that was negative and most patients with pancreatic cancer are surgically several other negative gemcitabine-fluoro- unresectable at the time of their diagnosis and have pyrimidine studies. an extremely poor prognosis. Even for those who A wave of new molecularly targeted agents has undergo resections, the risk of recurrence is been emerging into the clinic and many more agents exceedingly high, and patient outcome remains are in preclinical development. The combination of unsatisfactory. Therefore, to improve the prognosis gemcitabine with molecularly targeted agents is a of pancreatic cancer patients, the development of potentially more fruitful avenue of exploration. effective non-surgical treatments for this disease is Encouraging results in phase II trials have been essential. documented with EGFR antagonists, such as Gemcitabine, a deoxycytidine analogue, showed erlotinib and cetuximab, as well as antiangiogenic a significant impact on survival and clinical benefit agents, such as bevacizumab. Other agents on the response, such as pain alleviation and improved horizon include multitargeted tyrosine kinase performance status, in a randomized trial comparing inhibitors, such as sorafenib and sunitinib, it to 5-FU. However, there is still substantial room inihibitors of the mammalian target of rapamycin for improvement in chemotherapy for pancreatic

90 cancer, because gemcitabine can only confer a internally validated cindex (receiver operating limited survival benefit. S-1, an oral characteristics area under the curve) of this model fluoropyrimidine-based antineoplastic agent was 0.711. Applied to another data set, the consisting of the fluorouracil (5-FU) prodrug externally validated c-index was 0.692. This index tegafur combined with two modulators, gimeracil improved predictive ability in patients with and potassium oxonate, showed a promising result metastatic pancreatic cancer treated with in our early and late phase II studies. Furthermore, gemcitabine. S-1 may be useful in combination with gemcitabine, We describe the preliminary results of because the toxicity of S-1 was generally mild and expression of multiple genes in pancreatic cancer its profile was distinct from that of gemcitabine. with DNA microarray systems. EUS-FNA, which is Currently, we are conducting a multi-institutional established as the preferred method to confirm a phase II study of this combination treatment for diagnosis of pancreatic cancer, can also be used to metastatic pancreatic cancer. provide pancreatic cancer tissue for microarray A pharmacokinetics study for gemcitabine in analysis. Total mRNA was harvested from each Japanese cancer patients showed that carriers of the sample which was amplified to provide adequate cytidine deaminase *3 allele had a reduced mRNA for the analysis. The patient with the highest gemcitabine clearance and a corresponding increase dCMP deaminase or RRM1 resulted in progressive in the neutrophil reduction ratio. Evolving disease (PD) for gemcitabine. DNA microarray- understanding of molecular and genetic biology based gene expression profiling combined with should facilitate research to establish individualized EUSFNA may be a promising tool to predict therapy regimens and to develop novel non-surgical chemoresistance in advanced pancreatic cancer. The treatment for this disease. rapidly evolving understanding of the molecular biology of pancreatic cancer may contribute to the The future direction of chemotherapy for development and use of targeted therapies with pancreatic cancer novel agents for even more effective treatments in the near future. Akira Sawaki Department of Gastroenterology, Aichi Cancer Center Current thoughts on the role of Hospital, Nagoya, Japan chemotherapy and radiation in Pancreatic cancer is a devastating illness that advanced head and neck cancer has the briefest survival of any solid tumor, Carol R. Bradford accounting for approximately 20,000 deaths per University of Michigan Medical School and year in Japan. The efficacy of gemcitabine was Comprehensive Cancer Center, Ann Arbor, Michigan, reported by significantly prolonged overall survival USA demonstrating the existence of a subpopulation of pancreatic cancer patients who benefit from The paradigm for the management of advanced chemotherapy. Prediction of treatment outcome squamous cell carcinoma of the upper aerodigestive helps tailor more effective treatment strategies and tract has shifted from a predominantly is important to avoid over-treatment. We mention surgically-based treatment approach to an organ the possibility of tailor made treatment according to preservation approach that utilizes combinations of the readily available clinical data and molecular chemotherapy and radiation. This approach has processes associated with the development and been shown to be a valid approach in which organ progression of the disease. preservation is feasible and can be attached without We investigated pre-treatment characteristics sacrificing the potential for meaningful survival. and computerized tomography findings in patients, Careful pretreatment clinical, radiographic, and in order to identify the most effective readily endoscopic assessments of these patients are available prognostic factors in predicting survival essential for accurate tumor staging. Treatment for metastatic pancreatic cancer patients. strategies focusing on the integration of Multivariate analysis identified performance status, chemotherapy and radiation will be discussed primary tumor location, and C-reactive protein as including the role of induction chemotherapy and important independent predictive factors. Patients tumor response assessment. One of the critical were divided into three groups according to the issues is the post-treatment surveillance of these score based on coefficients of the Cox model. The patients. The management of advanced nodal

91 disease in this patient population will be described were collected from surgery or biopsy. 1: The including the indications and timing of salvage neck chemosensitivity of surgically respected specimens dissection. The high incidence of postoperative was investigated in vitro using histoculture drug complications with salvage surgery for local- response assay (HDRA) for 5-fluorouracil (5-FU) regional recurrence is mitigated by the use of free and cisplatin, respectively. TS and DPD activities tissue transfer. Careful attention should be paid to were also measured. 2: Biopsy specimens were quality of life and functional outcomes following taken from patients before administration of surgical versus nonsurgical approaches. Targeted induction chemotherapy with 5-FU and cisplatin, treatment using intensity modulated radiation and their clinical responses were estimated. 3: therapy (IMRT) to spare saliva (parotid) function Using specimens collected from both surgery and and the pharyngeal constrictors has resulted in biopsy, we subsequently analyzed the mRNA improved swallowing results. Our present approach expression levels of 13 markers that we thought for advanced cancers of the oropharynx is were likely predictors of response to anti-cancer concomitant carboplatin, docetaxel, and IMRT. Our agents. These mRNA expressions were quantified approach for advanced laryngeal cancers not by real-time reverse transcription polymerase chain amenable to surgical organ preservation is induction reaction (real-time RT-PCR) assay, after which we chemotherapy (cisplatin/5- flurouracil) followed by investigated the associations of these mRNA concomitant cisplatin and radiation therapy in expression levels with chemosensitivity in the responders. Biomarkers such as p53 and Bcl-xL HDRA, TS and DPD activities, and clinical have proven important for predicting response to response, respectively. organ preservation approachs. In oropharynx Results: We found that HER2 mRNA cancers, presence of human papillomavirus expression level was inversely correlated with 5-FU portends an excellent prognosis whereas and cisplatin sensitivity in the HDRA, respectively. overexpression of epidermal growth factor receptor An inverse correlation was also found between portends a poor prognosis. Patients who continue to beta-tubulin expression level and cisplatin smoke (“current smokers”) have a poorer outcome sensitivity in the HDRA. Moreover, associations of than patients who have quit and never smokers. cisplatin sensitivity in the HDRA with MRP1 and Rb1 expression levels were also demonstrated, Prediction of chemosensitivity for head and respectively, albeit just above the level of statistical neck cancer significance. There was a positive correlation between TS and DPD activity, and an inverse Tetsuya Ogawa correlation was detected between TS activity and Department of Head and Neck Surgery, Aichi Cancer 5-FU sensitivity in the HDRA. However, Center Hospital, Nagoya, Japan associations of TS and DPD activity with their mRNA expression levels were not detected. In the Head and neck squamous cell Introduction: patients who were subjected to induction carcinoma (HNSCC) is classified as a tumor with chemotherapy, the overall response rate was 73.5% high sensitivity to anti-cancer agents. From the (complete response, 38.2%; partial response, viewpoint of clinical efficacy, however, 35.3%). However, no significant correlation was chemotherapy for head and neck tumors remains observed between any of the investigated mRNA challenging. Because conventional clinical and expressions and clinical response to chemotherapy. pathologic parameters cannot be used to accurately Further studies are required to predict the response to chemotherapy or disease Conclusion: determine whether HER2 expression will be a outcome for patients with HNSCC, there exists a useful predictive maker for chemosensitivity in great need to identify new markers with which to patients with HNSCC. A study with additional define the subset of patients who will respond to patients to provide more data on the response to chemotherapy. To help establish order-made cancer chemotherapy is currently underway. chemotherapy, we performed multigene analysis to identify predictive markers for response to chemotherapy in patients with HNSCC. Radiotherapy for esophageal cancer Method: Patients undergoing radical treatment -Current status and future directions- for HNSCC at the Department of Head and Neck Satoshi Ishikura Surgery, Aichi Cancer Center Hospital were Clinical Trials and Practice Support Division, Center for included in the present study, and tumor specimens

92 Cancer Control and Information Services, National Background: In Japan, extended radical Cancer Center, Tokyo, Japan esophagectomy is thought to be the only way to obtain the cure of EC. Purpose: The purpose is to Carcinoma of the esophagus has been a evaluate the efficacy and toxicities of CRT for challenging disease. In contrast to Western clinical stage I-III (T1-3N0-1 and M0) esophageal countries where the number of patients with squamous cell carcinoma (ESCC). Methods: For adenocarcinoma has been increasing, most patients stage I, treatment consisted of two 4-week courses still have squamous cell carcinoma (SCC) in Japan. of cisplatin 70 mg/m2 (day 1) and 5FU 700 Recently, the number of patients with stage I mg/m2/day (days 1-4) combined with concurrent disease has been increasing, although most patients radiotherapy of 60 Gy in 30 fractions over 7 weeks are still diagnosed with advanced disease and with one week break. For stage II, III, consisted of dismal prognosis. The standard therapy for patients two 5-week courses of cisplatin 40 mg/m2 (day 1, 8) with resectable disease has been surgery with / and 5FU 400 mg/m2/day (days 1-5, 8-12) combined without adjuvant therapy in Japan. In 80’s, with concurrent radiotherapy of 60 Gy in 30 radiotherapy alone had been indicated in fractions over 8 weeks with 2-week break. For unresectable or medically inoperable patients as a responders, two 4-week courses of chemotherapy of definitive or palliative treatment. In 90’s, cisplatin 80 mg/m2 (day 1) and 5FU 800 mg/m2/day chemoradiotherapy (CRT) became a standard for (days 1-5) were added. Results: From June 1997 to patients who received non-surgical treatment. September 2003, consecutive 158 patients were Recent data suggested that patients who achieved retrospectively analyzed. Patient characteristics complete response (CR) after CRT had a substantial were as follows: median age of 63 (range 34-78), risk of late toxicity such as pericarditis, pleural male/female; 132/26, PS 0/1/2; 84/70/4, stage effusion, heart failure, and radiation pneumonitis, I/IIA/IIB/III; 70/25/18/45, scc/others with scc especially when treated with traditional component; 154/4. Of 70 patients (pts) with stage I, 2-dimensional radiotherapy. The intergroup there were 64 complete responses (CRs) for CR rate randomized study RTOG 9405 / INT 0123, which of 91.4% (95% confidence interval (CI); 85-98%). compared standard dose (50.4 Gy) vs. high dose (64 Regarding stage I, 6 pts with residual tumor Gy) radiotherapy with concurrent chemotherapy, successfully underwent endoscopic mucosal also showed that local failure was still dominant resection (EMR) or esophagectomy, and local even with high dose radiotherapy, and suggested recurrence or new lesion occurred in 11 pts (17%) that the tumor control probability of current CRT of 64 CRs, who successfully underwent EMR in 8 approaches reached a plateau. and esophagectomy in 3 as salvage treatment, In 2003, we changed the treatment scheme respectively. Of 88 pts with stage II, III, there were which intended to prevent late toxicity by reducing 58 CRs for CR rate of 66% (95%CI; 56-75%). the dose to normal tissues without compromising Regarding stage II, III, 28 pts (48%) of 58 CRs the efficacy. This included 3-dimensional showed recurrent disease, and 22 pts (79%) of 28 conformal radiotherapy, radiotherapy dose recurrences underwent EMR or esophagectomy as reduction from 60 Gy to 50.4 Gy, and selected salvage. On the other hand, of 30 non-CRs, there salvage surgery. So far, the treatment outcome were 10 pts (33%) who underwent EMR or seems good, although it is preliminary and esophagectomy as salvage. With a median long-term follow up is necessary. We also expect follow-up duration of 4.5 years, the 1, 3, 5-year that newer cytotoxic drugs and / or molecular survival rates in pts with stage I were 96%, 79%, targeted therapy in combination with radiotherapy and 74%, respectively. With a median follow-up may improve local control and overall survival in duration of 3.4 years, the 1, 2, 3-year survival rates the near future. in pts with stage II, III were 76%, 56%, and 46%,

respectively. These survival data are comparable Definitive chemoradiotherapy for stage I-III with those obtained by ordinary surgery in Japan. esophageal squamous cell carcinoma: Acute toxicities and late radiation morbidities Current results in Japan. increased dependent on the steps of stages, but they Kei Muro were tolerable and manageable. There was one treatment related death due to severe diarrhea in Department of Clinical Oncology, Aichi Cancer Center stage III EC pt. Conclusions: Definitive CRT for Hospital, Nagoya, Japan stage I-III ESCC is effective in CR rate and

93 short-term survival with acceptable toxicities, but agents such as bevacizumab and cetuximab are salvage treatment for locoregional failure after CRT candidates to further increase the response rate and is necessary to improve the prognosis. potential resection rate. Currently available data suggest that cetuximab may be a good candidate as it has single agent efficacy and high response rates Systemic chemotherapy for colorectal have been reported in combination with cancer chemotherapy. Bevacizumab may cause peri- Claus-Henning Köhne operative bleeding complications and thus has to be used with caution in this setting. Future trials will Clinic of Oncology and Haematology, Klinikum be necessary to better define the relative value to Oldenburg,Oldenburg, Germany fthese new agents. Colorectal cancer is a curative disease. About Secondary resection may be considered as a 50% of patients will be cured by the surgeon, paradigmatic shift from palliation to cure in patients however the other half of patients will experience a with metastatic disease and should be considered in relapse. Adjuvant chemotherapy has been any patient with a good response following established as an important treatment option to chemotherapy. increase the cure rate. Twelve cycles of FOLFOX Nowadays, we have to decide whether a are the most efficacious therapy. Long lasting curative approach might be possible in order to peripheral neuropathy are however potential sides initiate the most efficacious regimen that may result effects that may not be tolerable for everyone. Oral is resection of metastases. Hereby, a higher degree fluorpyrimidienes either with capecitabine or with of toxicity may be acceptable to achieve this goal. UFT are potential less toxic options. Adjuvant In a more palliative approach, a low degree of therapy is established for node positive disease toxicity with a maximum effect of tumor control is (UICC stage III), but controversial for stage II the aim and the sequential use of chemotherapy patients. A group of high risk stage II patients has agents probably with a stop and go strategy to been defined; the value of systemic chemotherapy maximise quality of life in patients is a potential is however less well established. The liver is the strategy. main site of metastases in colorectal cancer. If resectable cure is possible in about 30% - 50% of Systemic chemotherapy for colorectal patients. In patients in which liver metastases have cancer -Current status and future been resected the use of adjuvant chemotherapy is perspectives in Japan- again controversial. Unfortunately, only 15-20% of Atsushi Ohtsu patients may have respectable disease and treatment of theses patients is mostly palliative. Systemic Division of Gastrointestinal Oncology/Digestive Endo- chemotherapy has become more efficacious with scopy, National Cancer Center Hospital East, Kashiwa, the use of regimens including infusional 5-FU Japan modulated with folinic acid and combined with Chemotherapy for colorectal cancer is a rapidly either irinotecan and or oxalipaltin. Response rate evolving field. This is largely due to the of 50-60% may be expected especially in patients development of various novel agents, such as with metastases confined to the liver. Phase II irinotecan, oxaliplatin, capecitabine, bevacizumab, studies have been performed with carefully selected cetuximab, and panitumumab, over the past decade. patients that were considered non-resectable by These agents have provided not only survival their surgeon but probably resectable after systemic prolongation in patients with metastatic disease but chemotherapy. Although criteria of nonresectablity also additional cure rate after curative resection in may differ between surgeons about 50-70% of these stage III disease. Although the approval status of previously nonresectable cases became resectable. these agents had been far behind from the Westaern In other phase II studies not selecting patients as countries, the delay is now being improved and carefully the resection rate was 20%. Secondary these advantage will be available very soon for the resection of metastases is reported in phase III Japanese patients. studies mainly as an incidental event in below 10%. There are few ethnic differences in PK / PD of The rate of secondary resection depends on the these new agents between the West and Japanese selection of patients and on the efficacy of the used population, except for oral fluorouracils where preoperative chemotherapy. The new targeted Japanese patients have lower incidence of diarrhea

94 as compared to Caucasians. Nowadays, oxaliplatin Some RCTs comparing 5-FU based systemic or irinotecan in combination with infusional chemotherapy and hepatic arterial infusion fluorouracil (FOLFOX / FOLFIRI) are becoming chemotherapy (HAIC) have not shown the impact popular in most of the Japanese institutions where of HAIC contributes to improving on the survival medical oncologists lead the chemotherapy. The prolongation in patients with liver metastasis from registration trials of molecular targeting agents such colorectal cancer in the last 20 years. In these as bevacizumab, cetuximab, and panitumumab have studies, although HAIC was superior to systemic already completed and these agents will be chemotherapy on the local response, HAIC had low commercially available soon. During these feasibility and high incidence of extra-hepatic foci. developments, there have been no obvious However, there is a great difference in techniques differences in safety and efficacy results as for HAIC between western countries and Japan. compared with those in overseas and we are ready The indwelling catheter for HAIC is placed under to accept global results. According to the recent laparotomy in western countries, while it is change of regulatory guidelines in Japan which performed with percutaneous radiological facilitate to enter into global studies, we are also procedures in Japan. HAIC in Japan actually has planning to participate global IND studies with high feasibility and low invasion. In Japanese phase newly developing (next generation) agent. In case II studies, the median survival time (MST) of HAIC of adjuvant trials, several Japanese institutions have with 5-FU only showed over 20 months. At that participated in a global IND trial comparing time, there were not new agents such as irinotecan FOLFOX4 with FOLFOX4 + bevacizumab or and oxaliplatin. But since HAIC was the regional XELOX + bevacizumab (AVANT study). The therapy, it could not prevent the extra-hepatic number of post-marketing studies is also increasing disease from spreading anyway. and various combination regimens including oral Now systemic chemotherapy for advanced or fluorouracil which seems favorite for Japanese recurrent colorectal cancer has approximately 50% population, and some of them are being of the response rate and over 20 months of the MST, investigated in randomized trials. These studies, but liver metastasis is still one of the most either global or domestic, will resolve the issues important survival limiting factors. In US, whether there are true ethnic differences in terms of combination therapy with hepatic arterial infusion efficacy and safety between Japanese and and systemic irinotecan or oxaliplatin might be non-Japanese populations. more useful recently. HAIC may have a potential Mortality rate from colorectal cancer in Japan role in pushing up the MST of patients with liver has already become similar to those in the West and metastasis even after the failure of systemic it should be an urgent issue for Japanese health. We chemotherapy. have just entered into the global development line HAIC supported by the adequate techniques is of new agents for colorectal cancer and will play an safely administered. HAIC with 5- FU only is important role in this field, which would provide reasonable, while new agents are costly. In more advantage for the patients with colorectal considering the disease condition as well as quality cancer over the world. of life and medical expenses of patients, we are groping the new strategy in which HAIC is Hepatic arterial infusion chemotherapy for performed only in the time when liver metastasis is liver metastasis from colorectal cancer dominant and it is converted into systemic chemotherapy in the time when extrahepatic lesions Yoshitaka Inaba are growing. Department of Diagnostic and Interventional Radiology, Aichi Cancer Center Hospital, Nagoya, Japan

The 12th Aichi Cancer Center International Symposium“Perspective of On- cological Strategy for Gastrointestinal Cancer,” held on January 13, 2007.

96 Author index for research reports and publications ______Adachi, K. 55. Hirano, N. 16, 17, J034, J124, J194, J205, Akatsuka, Y. 29, 30, 31, J002, J013, J061, J062, J206. J078, J129, J130, J137, J200, J203, Hirose, F. J230. J204, J211, R001, R002, A001, Hirose, K. 7, 12, J038, J039, J048, J099, J100, A029, A054. J101, J103, J117, J118, J119, J120, An, B. 19. J144, J177, J179, J180, J181, J182, Aoki, K. J004. J183, J184, J185, J220, A040, Arimura, N. J148. A045. Ban, H. 16, 17, J010, J034, J036, J037, Honma, K. 26, J082, A008. J124, J126, J191, J192, J193, J194, Hoshino, Y. J161. J205, J206, J214, R003. Hosokawa, Y. R023. Cao, L. 17, J010, R003. Horayama, K. 55. Cao, X. 17, J010, J034, J125, J147, J193, Horio, Y. 20, J198, J218, J244, A064, A065. R003, R038. Ibi, M. 48, J045. Chen, G.Y. 41, J011, J239, A002, A003, A004, Ichinose, M. J016, J111. A017, A069, A070, A071. Ikegami, Y. 46, 47, 56. Daikoku, T. J012, J095, J199, J249, R004, Ikehara, S. J048, J049, J051, J240. A011, A067. Ikehara, Y. J048, J049, J050, J051, J136, J140, Demachi-Okamura, A. 31, J013, J061, J062, J211. J228, J240, J241, R025. Enomoto, M. 46, 47, 56, J015, J154. Imai, T. J151, J223. Fujii, M. 41, A072. Inada, K. 16, J121, J123, J147, J206. Fujita, M. J215. Inagaki, N. J148. Fukami, H. J212, J213. Inagaki, M. 46, 47, 48, 56, J027, J028, J031, Fukuhara, N. 25, J020, J021, A005. J107, J148, J167, J207, J215, Fukui, F. J151. R009, R029, A009, A010, A073. Fukui, T. 20, J110, J162, J173, J198, J218, Inoko, A. 48, J001, J148, J167, A010, A073. J242, A064, A065. Inoue, M. R018, R019, R022, R026, R031, Fukuyama, R. 18. R043. Furue, H. J023, J024. Ishida, H. J084, J091. Gao, W. 21, J162. Ishizaki, K. 50, 51, 52, J023, J024, J047, J097, Goto, H. 46, 47, 56, J027, J028, J110, J167, J116, J135, J173, A038, A039. J207. Isomura, H. 35, J012, J056, J057, J058, J063, Goto, Ya. 21, J162, J242, A006. J095, J249, R008, A011, A012. Goto, Yo. 41, A007, A072. Ito, H. 6, 7, 11, J039, J044, J048, J060, Gotoh, M. J050. J100, J101, J103, J117, J118, J119, Hamajima, N. 7, J060, J100, J118, J144, J183, J120, J144, J180, J181, J182, J183, J224. J185, J220, A040, A045. Hara, S. J064. Ito, M. 21, J040, J041, A006. Hasegawa, Y. 7, J023, J032, J047, J153, J177, Ito, S. 18, J030, J048, J085, J086, J149, J185, J242, A006, A060. J229, R025. Hashimoto, A. J239. Ito, Y. J013, J061, J062, J085, J094, J129, Hayashi, Y. 47, 48, 56, J163, A010, A073. J143, J176, J211, J241. Hayashita, Y. J115, A055. Iwahori, S. 34, J012, J056, J057, J058, J063, Hida, T. 20, 21, J133, J178, J198, J218, J095, J249, A011, A012, A066. J244, A056, A064, A065. Iwata, H. 41, J038, J064, J101, J179, A040. Hirai, T. 7, J024, J029, J030, J100, J103, Izawa, I. 47, 48, J107, J148, R009, A010, J118, J220. A073. Hiraki, A. 7, 11, 12, J018, J024, J032, J033, Izawa, M. 38, 43, J083, J108, J239, R012, J074, J075, J101, J113, J117, J119, A015, A046, A047, A048, A069, J169, J177, J178, J179, J184, A070, A071. A040, A060. Joh, T. J147.

97 Kagami, Y. J006, J007, J174. Kuriki, K. 7, 12, J026, J065, J099, J100, J101, Kamei, M. 27. J102, J103, J220, J222, R015, Kameoka, Y. J021. R034, A040. Kanamori, A. 40, J146, J250. Kuzushima, K. 29, 30, 31, J002, J013, J061, J062, Kanemitsu, Y. 7, J024, J100, J103, J118, J220. J078, J127, J129, J137, J203, J204, Kannagi, R. 38, 39, 40, 41, 42, 43, J003, J008, J211, R001, R002, A001, A029. J011, J025, J043, J066, J067, J083, Kyogashima, M. 40, 41, J043, J104, J105, A007, J091, J092, J093, J104, J105, J108, A041, A042, A043, A072. J146, J151, J159, J168, J201, J226, Lim, K.T. 39, J108, A032, A044. J229, J239, J248, J250, R010, Maeno, K. J112, J197. R011, R012, R013, R041, A002, Masuda, A. J112. A004, A007, A013, A014, A015, Matsudaira, Y. J211. A016, A017, A018, A019, A020, Matsuo, K. 6, 7, 10, 11, 12, 25, J006, J007, A021, A022, A023, A024, A025, J014, J018, J019, J023, J024, J032, A026, A027, A028, A031, A032, J038, J039, J040, J041, J042, J048, A035, A036, A041, A042, A044, J060, J064, J074, J075, J079, J081, A046, A047, A048, A049, A068, J087, J098, J100, J101, J103, J117, A069, A070, A071, A072. J118, J119, J120, J122, J130, J138, Karnan, S. 20, 25, 26, J021, J068, J082, J198, J139, J141, J144, J171, J172, J173, J217, A008, A030, A051, A052, J177, J178, J179, J180, J181, J182, A053. J183, J184, J185, J196, J208, J209, Kasahara, K. 46, 47, 56, J005, J070, J071, J072, J210, J216, J219, J220, J225, J227, J073, J096. J233, J234, J235, J236, J243, J244, Kasugai, Y. J021, J217. R006, R007, R016, R017, R042, Kato, K. J052, J142, J217, A032, A068. R045, A040, A045, A060. Kato, S. 10, 17, J076, J077, J079, J125, Minoura, Y. 53. J130, J205. Mitsudomi, T. 20, J119, J178, J195, J197, J198, Kato, T. 7, J024, J029, J030, J100, J103, J202, J208, J209, J244, A045, J118, J149, J220. A064, A065. Kawajiri, A. J028. Miyata, T. 33. Kawase, T. 6, 10, 29, J078, J079, J172, J179, Miyauchi, H. 30, J203. J204, A001, A029. Miyazaki, K. 38, 39, 43, J083, J108, J131, J239, Kawajiri, A. 56, J028. R010, R012, A015, A032, A044, Kimura, N. 39, 43, J083, J108, R010, R011, A046, A047, A048, A069, A070, A015, A044. A071. Kitajima, S. 51, 52. Mizoshita, T. 16, 17, J004, J010, J034, J076, Kiyono, T. J028, J129, J232, A010, A073. J077, J124, J125, J126, J147, J193, Kobayashi, T. J049, J236. J194, J205, J206, R032, R035, Kodera Y. 29, 30. R036, R037, R038. Kondo, E. J062, J129, J199. Mizuno, M. J144, J215. Kondo, M. J110, J162, J218, J242. Mochizuki, Y. J030, J085, J149, J240, R025. Kondo, S. A032. Moore, M. R020, R021. Kondo, Y. 21, J009, J089, J150, J156, J162, Morishima, S. 29, J078, J129, A029. J164, J165, J166, J187, J188, J223, Morishima, Y. 10, 25, 29, 30, J002, J007, J013, A006, A033, A034. J020, J021, J061, J062, J078, J079, Konishi, H. J112. J129, J130, J158, J174, J180, J189, Kontani, K. J090, J091, J201. J200, J203, J204, J227, R001, Koshikawa, K. J202. R002, A001, A005, A029, A054, Kudoh, A. 33, J012, J047, J056, J057, J058, A058. J063, J095, J249, R014, R039, Murakami, H. 21, J156, A006, A033, A034. R040, A011, A012, A037, A067. Murata, T. J052, J056, J058, J114, J132, Kumimoto, H. 50, J023, J060, J097, J173, A038, A050. A039. Murate, T. 40, J168, A041.

98 Nagata, K. J168. A012, A059. Nakagawa, M. 25, 26, J020, J134, J155, R023, Seki, T. 17, J010. A008, A051, A052, A053. Sekido, Y. 20, 21, J089, J110, J133, J156, Nakagawa-Oshiro, A. 26, A051, A052. J162, J197, J198, J218, J223, J224, Nakamura, Hid. 51, 52, J047, J135, R024. A006, A033, A034, A055, A056, Nakamura, Hir. 54. A064, A065. Nakamura, S. J006, J007, J020, J021, J050, J053, Seto, M. 20, 25, 26, J020, J021, J053, J068, J065, J084, J138, J158, J174, J180, J069, J082, J158, J174, J189, J190, J189, J222, A005, A051, A052, J198, J216, J217, J245, R023, A053. A058. A005, A008, A030, A051, A052, Nakamura, T. 16, 25, J020, J053, J124, J161, A053, A058, A061, A064, A065. J181, J182, J183, J194, A005. Shibata, T. 55. Nakanishi, H. 18, J029, J030, J044, J048, J049, Shima, H. J148. J050, J051, J084, J085, J086, J128, Shimizu, N. J169, J210. J131, J136, J140, J149, J153, J228, Shimizu, Y. 21, J049. J240, J241, R025. Shinoda, M. J023. Nakanishi, T. J039, J129, J144. Shirata, N. J012, J063, J095, A011. Nakao, Y. 29, J204. Shiromizu, T. 46, 48, 56, J167. Nakasu, S. 34. Sobue, S. J168. Nakayama, S. J056, J057, J058, J063, A012. Sugaya, Y. J012. Nawa, A. J088, J129, J199, J249. Sugimoto, M. A010. Nimura, Y. 21, J003, J115, J133, J229, A056. Suguro, M. J174. Nishida, K. 27, J061, J211. Suzuki, Hid. 15. Nishida, T. J022, J175, J200. Suzuki, Hir. 24. Nishikawa, Y. 32. Suzuki, T. 7, 11, J023, J024, J032, J042, J100, Nishizawa, M. 47, 54. J101, J103, J118, J119, J128, J177, Niwa, T. J048, J049, J050, J224. J178, J179, J180, J181, J182, J183, Nozaki, K. J126, R003. J184, J185, J220, J233, J234, Obata, Y. J096, J211. R017, A040, A045, A060. Ogasawara, N. J147, J192, J193, J194. Tagawa, H. 20, 25, 57, J020, J021, J068, J069, Oguri, T. J148. J082, J158, J174, J189, J190, J198, Ohashi, N. J049, J085, J149, J241, R025. A005, A030, A051, A052, A053, Ohmuro-Matsuyama, Y. 48, J152. A058, A061, A064, A065. Okuma, K. J060. Taguchi, O. 43, J022, J031, J046, J080, J090, Onishi, J. vi. J091, J163, J175, J201. Osada, H. 21, 41, J011, J014, J089, J112, Tajika, M. 16, J053, J124. J115, J133, J156, J162, J195, J197, Tajima, K. 6, 7, 12, 13, J014, J023, J024, J032, J202, J230, J242, R027, R028, J038, J039, J048, J060, J075, J099, A002, A003, A004, A006, A017, J100, J101, J102, J103, J109, J117, A033, A034, A055, A056, A057. J118, J119, J120, J144, J145, J173, Oshiro, A. J158, A051, A052, A053, A058. J177, J178, J179, J180, J181, J182, Otsuka, T. J194. J183, J184, J185, J220, R005, Saito, N. 50, 51, J135, A038, A039. R015, R017, R020, R021, R030, Saito, T. 7, J060, J100, J101, J103, J112, A040, A045, A060, A062, A063. J117, J118, J120, J177, J178, J180, Takahashi, Ta. 21, J014, J112, J115, J133, J156, J183, A040, A060. J202, J230, R027, R028, A055, Sakamoto, H. J128, A033. A056. Sakai, H. J035, J036, J037, J191, J212, J213, Takahashi, To. 29, 30, 31, J002, J013, J061, J062, J221. J078, J129, J137, J051, J203, J204, Sakuma, K. 42, J054, J160, J161, J186, A017. J211, R001, R002, A029. Sano, T. 21, J017. Takasu, S. 16, 17, J036, J037, J191, J192, Sato, N. J162. J205, J206, R003. Sato, Y. 33, J007, J056, J057, J058, J063, Takenaka, Y. 16, J125, J147, J193, J194, J206,

99 J246. Tsuzuki, S. 25, J068, J190, J216, J217, A008, Takezaki, T. J100. A051, A053, A061. Tamaki, H. vi. Ueda, R. J014, J068, J089, J112, J138, J177, Tamiya-Koizumi, K. 40, J003, J104, A041, A042. J178, J180, J181, J182, J184, J185, Tanaka, Hi. 6. J217, A033, A060. Tanaka, Ha. 16, 17, 54, J010, J077, J123, J206, Wakai, K. 7, 12, J032, J039, J055, J060, J094, R003. J100, J101, J103, J117, J118, J119, Taniguchi, T. 20, J162, J198, J218, A006, A064, J120, J143, J176, J177, J180, J181, A065. J182, J183, J184, J185, J219, J220, Tatematsu, M. 16, 17, 18, J004, J010, J016, J029, J221, R018, R019, R022, R026, J030, J034, J035, J036, J037, J048, R031, R042, R043, R044, A040, J050, J076, J077, J086, J101, J103, A045. J106, J111, J121, J123, J124, J125, Watanabe, Y. 31, J094, J143, J176, J196, J223. J126, J136, J147, J149, J150, J153, Yagi, H. A032, A068. J191, J192, J193, J194, J205, J206, Yamaguchi, M. J046, J163. J212, J213, J214, J228, J231, J240, Yamaguchi, T. 46, 56, J022, J142, J148, J230, J241, J246, R003, R005, R032, R029. R033, R035, R036, R037, R038, Yamamoto, M. 54, J035, J036, J037, J045, J213. A040. Yamamura, Y. J030, J048, J085, J149, J194, Tatematsu, Y. 21, J133, J156, A055, A056. R025. Torikai, H. 29, 30, J002, J078, J129, J203, Yamao, K. 21, J053, J181, J182, J183. J204, R002, A001, A029. Yanagisawa, K. J112, J115, J195, J197, J202, Tomida, S. J156, J195, J197, J202, A055. A055. Toyoda, T. 16, 17, J010, J034, J126, J191, Yasuda, T. 55. J205, J206, R003, R033. Yasui, Y. 51, 52, J047, J135. Tsujimura, K. 29, 30, 31, 46, 47, 56, J002, J013, Yasukawa, Z. J237, J238. J051, J059, J061, J062, J078, J129, Yatabe, Y. 11, 20, 25, 29, J020, J024, J053, J203, J204, J211, R002, A001. J084, J119, J156, J178, J195, J197, Tsukamoto, T. 16, 17, J004, J010, J016, J034, J198, J202, J204, J208, J209, J240, J035, J036, J037, J076, J077, J106, J244, A045, A055, A064, A065. J111, J121, J124, J125, J126, J147, Yin, J. 38, J025, J239, R010, R012, A031, J150, J157, J170, J191, J192, J193, A069, A070, A071. J194, J205, J206, J212, J213, J214, Yokoyama, N. J051. J246, R003, R032, R033, R035, Yokoyama, T. 20, J162, J198, J218, J242, A006, R036, R037, R038. A064, A065. Tsurumi, T. 33, 34, 35, J012, J056, J057, J058, Yokoyama, Y. J003. J063, J088, J095, J199, J232, J249, Yoshikawa, A. J213. R008, R014, R039, R040, A011, Yusa, A. 41, J247, A015, A072. A012, A037, A050, A059, A066, Zou, P. 48, A010, A073. A067.

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