Aichi Cancer Center Research Institute

Scientific Report 2000-2001

Chikusa-ku, Nagoya 464-8681 Japan

(The Cover) An aerial photograph of Aichi Cancer Center campus and buildings, standing in the setting of the luxuriant verdure of the Kano-ko Garden Park on the shore of Lake Neko-ga-hora. Published by Dr. Toshitada Takahashi 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. Tetsuo Kuroishi (Division of Epidemiology and Prevention) Dr. Hirotaka Osada (Division of Molecular Medicine) Dr. Masatoshi Fujita (Division of Virology) Dr. Hiroshi Kumimoto (Central Laboratory & Radiation Biology) Mr. Morio Terashima, Photographer (Central Service Unit) Dr. Malcolm A. Moore, English Editor Printed by Nagoya University COOP 1 Furoucho, Chikusa-ku, Nagoya 464-0814, Japan

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Contents ______________________________________________________________________ Preface Takahashi Toshitada 1 Organization of the Aichi Cancer Center Research Institute 2 SCIENTIFIC REPORTS Division of Epidemiology and Prevention

General summary 5 1. Descriptive epidemiologic studies on cancer incidence and mortality Inoue, M., Kuroishi, T., Hirose, K. Tominaga, S. and Tajima, K. 5 2. HERPACC studies on risk and protective factors for main sites of cancer Hirose K., Saito, T., Inoue, M., Takezaki, T., Hamajima, N., Kuroishi, T., Tajima, K., Miura, S., Kuzuya, K., Sugiura, T., Mitsudomi, T., Okuma, K., Ogawa, H., Nishiwaki, K., Sakai, S., Yanagi, T., Ariyoshi, Y., Stellman, S., Wynder, E. and Aoki, K. 7 3. Development of cancer prevention programs Hamajima, N., Hirose, K., Tajima, K. and Miura S. 10 4. Evaluation of secondary prevention of cancers Kuroishi, T., Hirose,K., Suzuki, T., Tominaga, S., Sagawa, M., Fujimura, S. and The Research Group for Lung Cancer Screening 11 5. Ethnoepidemiological studies on cancer Takezaki, T., Hamajima, N., Inoue, M., Hirose, K., Tajima, K., Gao, C-M., Wu, J-Z., Wang, Y-M., TMo, B-Q., Wang, X-R., Yoo, K-Y., Ahn, Y-O., Kim, J-S., Zhou, Z-Y., Cao, J., Li C., Gao, F-C., Tokudome, Y., Sonoda, S., Yashiki, S., Fujiyosh, T., Li, H-C., Zhao, S-H., Horai, S., Chiba, H., Senoh, H. and Tretli, S. 12

Division of Oncological Pathology

General summary 17 1. Reversibility of Heterotopic Proliferative Glands in Glandular Stomach of Helicobacter pylori-infected Mongolian Gerbils on Eradication Tatematsu, M., Nozaki, K., Shimizu, N., Tsukamoto, T., Inada, K., Cao, X., Ikehara, Y., Kaminishi, M. and Sugiyama, A. 18 2. Real-time observation of micrometastasis formation in the living mouse liver using a GFP gene-tagged rat tongue carcinoma cell line Nakanishi, H., Itoh, H., Ikehara, Y., Kato, T., Nakao, A. and Tatematsu, M. 18 3. Intestine specific homeobox genes, Cdx1 and Cdx2, are key molecules in intestinalization of gastric carcinoma cells Inada, K., Mizoshita, T., Tsukamoto, T., Nakanishi, H. and Tatematsu, M. 19 4. Different susceptibilities of p53 knockout (-/-), (+/-) and (+/+) mice to induction of stomach adenocarcinomas by N-methyl-N-nitrosourea and esophageal squamous cell carcinomas by methyl-n-amylnitrosamine

Tsukamoto, T., Yamamoto, M., Shirai, N., Sakai, H., Iidaka, T.,

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Donehower, L.A. and Tatematsu, M. 19 5. Polymorphisms of two fucosyltransferase genes (Lewis and Secretor genes)

involving type I Lewis antigens are associated with the presence of anti- Helicobacter pylori IgG antibodies

Ikehara,Y., Nishihara, S., Yasutomi, H., Kitamura, T., Matsuo, K., Shimizu, N., Inada, K., Kodera, Y., Yamamura, Y., Narimatsu, H., Hamajima, N. and Tatematsu, M. 20

Division of Molecular Oncology General summary 23 1. Multi-faceted analyses of a highly metastatic human lung cancer cell line

NCI-H460-LNM35 suggest mimicry of inflammatory cells in metastasis Kozaki, K., Koshikawa, K., Osada, H., Konishi, H., Tatematsu, Y., Miyaishi, O., Saito, H., Hida, T., Mitsudomi, T. and Takahashi, Ta. 23 2. Gene silencing by aberrant DNA methylation and abnormalities in chromatin

configuration in human lung cancer cells Osada, H., Tatematsu, Y., Yatabe, Y., Masuda, A., Konishi, H., Harano, T.,

Nakagawa, T., Saito, T., Sugiyama, M., Yanagisawa, K., Takada, M. and Takahashi, Ta. 24

3. Persistent increase in chromosome instability in lung cancers Haruki, N., Masuda, A., Harano, T., Kiyono, T., Takahashi, Takao Tatematsu, Y., Shimizu, S., Mitsudomi, T., Konishi, H., Osada, H., Fujii, Y. and Takahashi, Ta. 25 4. Identification of frequent G2 checkpoint impairment and a homozygous deletion of 14-3-3ε at 17p13.3 in small cell lung cancers Konishi, H., Nakagawa, T., Harano, T., Mizuno, K., Saito, H., Masuda, A.,

Osada, H. and Takahashi, Ta. 25 5. In vitro molecular analysis of carcinogenesis of human lung adenocarcinomas

with the aim of clinical applications Masuda, A., Konishi, H., Yatabe, Y., Hida, H., Saito, T. and Takahashi, Ta. 26

Division of Molecular Medicine

General summary 29 1. Search for MALT1-associated proteins using yeast two-hybrid strategy Hosokawa, Y., Suzuki, H. and Seto, M. 29 2. Detection of API2-MALT1 chimeric transcripts involved in mucosa- associated lymphoid tissue lymphomas by a single touchdown multiplex polymerase chain reaction Suguro, M., Suzuki, R., Nakamura, T., Suzuki, H., Hosokawa, Y., Nakamura, S. and Seto, M. 30 3. Detection of cyclin D1 overexpression by real-time reverse-transcriptase mediated quantitative polymerase chain reaction for the diagnosis of mantle cell lymphoma Suzuki, R., Takemura, K., Tsutsumi, M., Nakamura, S., Hamajima, N. and Seto, M. 30 4. Molecular Cytogenetic Analysis of the Breakpoint Region at 6q21-22 in T-Cell Lymphoma/Leukemia Cell Lines Tagawa, H., Miura, I., Suzuki, R., Hosokawa, Y. and Seto, M. 31

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Division of Immunology General summary 33 1. Production of a single chain variable fragment (scFv) antibody against type III mutant EGFR Yoshikawa, K., Nakayashiki, N., 2, Takasu, S., 2, Okamoto, K., Nakamura, K., Hanai, N., Okamoto, S., Mizuno, M., Wakabayashi, T., Saga, S., Yoshida, J. and Takahashi, To. 33 2. Immunogenic gene products in cancer patients Obata, Y., Takahashi, To., Tamaki, H., Tajima, K., Yoshida, M., Miura, S., Iwase, T., Iwata, H, Mitsudomi, T., Takahashi, M., Sakamoto, J., Chen, Y.-T., Stockert, E. and Old, L.J. 34 3. Targeted cloning of cytotoxic T cells specific for minor histocompatibility

an-tigens restricted by HLA class I molecules of interest Akatsuka, Y., Kondo, E., Nishida, T., Taji, H., Morishima, Y., Obata, Y., Kodera Y. and Takahashi, To. 34 4. Binding of thymus leukemia (TL) anti-gen tetramers to normal intestinal

intra-epithelial lymphocytes and thymocytes Tsujimura, K., Obata, Y., Matsudaira, Y., Ozeki, S., Yoshikawa, K., Saga, S. and Takahashi, To. 35

Division of Virology

General summary 37 1. The Epstein-Barr Virus Pol catalytic subunit physically interacts with the

BBLF4/BSLF1/BBLF2/3 Complex. Fujii, K., Yokoyama, N., Kiyono, T., Kuzushima, K., Fujita, M. and Tsurumi, T. 37 2. Purification of the product of the Epstein-Barr virus BZLF1 gene Nakasu, S. and Tsurumi, T. 38 3. Mechanisms by which Cdc2 kinase inhibits re-replication during late S-G2-M

phase in mammalian cells Fujita, M. and Tsurumi, T. 38 4. Immortalization of Human Cells by HPV Kiyono, T. and Tsurumi, T. 39 5. Longitudinal dynamics of Epstein-Barr Virus-specific cytotoxic T lymphocytes

in the posttransplant lymphoproliferative disorder Kuzushima, K., Kimura, H., Hoshino, Y., Yoshimi, A., Tsuge, I., Horibe, K.,

Morishima, T., Kojima, S. and Tsurumi, T. 39 6. Identification of HLA A*2402-restricted cytomegalovirus-specific CD8+ T cell epitopes by a computer algorithm and an enzyme-linked immunospot assay Kuzushima, K., Hayashi, N., Kimura, H. and Tsurumi, T. 40

Division of Molecular Pathology

General summary 43 1. Study of ligand specificity of three selectin family cell adhesion molecules, E-, P- and L-selectins, using genetically engineered cells Kanamori, A., Ohmori, K, Goto, Y., Uchimura, K., Muramatsu, T., Kiso, M.,

Tamatani, T. and Kannagi, R. 43 2. Expression of sialyl 6-sulfo Lewis X, a new ligand for cell adhesion molecules

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of the selectin family, in human colon and cultured colon cancer cells Izawa, M., Kumamoto, K., Kanamori, A., Kanda, K., Goto, Y., Ishida, H.,

Nakamura, S. and Kannagi, R. 44 3. Regulatory mechanisms for expression of functional carbohydrate determinants on malignant and non-malignant cells:

3-1. Roles of sugar nucleotide transporters in the enhanced expression of carbohydrate ligands for selectins, sialyl Lewis X and sialyl Lewis A,

on solid tumors Kumamoto, K., Goto, Y., Ishida, N., Kawakita, M. and Kannagi, R. 45 3. Regulatory mechanisms for expression of functional carbohydrate determinants on malignant and non-malignant cells:

3-2. A T-box transcriptional factor that synergizes with HTLV-1 Tax in transactivating the selectin-ligand synthesizing enzyme, fucosyltrans-

ferase VII Hiraiwa, N., and Kannagi, R. 46 4. A murine model of tumor suppression by vaccination with MUC1 DNA and

dendritic cells Kontani, K. and Taguchi, O. 46

Division of Biochemistry

General summary 49 1. Aurora B and Rho-kinase regulate cleavage furrow-specific vimentin

phosphorylation in the cytokinetic process Yasui, Y., Goto, H., Kawajiri, A., Nigg, E.A., Terada, Y., Tatsuka, M., Matsui, S., Manser, E., Lim, L., Nagata, K. and Inagaki, M. 49 2. Aurora B phosphorylation of histone H3 at serine28 prior to the mitotic

chromosome condensation Goto, H., Yasui, Y., Nigg, E.A. and Inagaki, M. 50 3. Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD Inada, H., Izawa, I., Nishizawa, M., Fujita, E., Kiyono, T., Takahashi, T., Momoi, T. and Inagaki, M. 50 4. ERBIN associates with p0071, an armadillo protein, at cell-cell junctions of epithelial cells Izawa, I., Nishizawa, M., Tomono, Y., Ohtakara, K., Takahashi, T. and Inagaki, M. 51 5. Characterization of a mammalian septin MSF-A Nagata, K., Kawajiri, A., Saito, N., Togashi,H., Takagishi, M., Matsui, S., Hotani, H. and Inagaki, M. 51 6. Functional analysis of DREF using transgenic flies Hirose, F., Ohshima, N., Kwon, E-J., Yoshida, H., Inoue, Y.H., Matsukage, A. and Yamaguchi, M. 52 7. Functional analysis of BEAF32A using transgenic flies Yamaguchi, M., Yoshida, H., Hirose, F., Inoue, Y.H., Hayashi, Y., Yamagishi, M., Nishi Y., Tamai, K., Sakaguchi, K. and Matsukage, A. 52

Division of Central Laboratory & Radiation Biology

General summary 55 1. Analysis of a candidate tumor suppresor gene, LATS2, on 13q11-12 in

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esophageal squamous cell carcinoma Ishizaki, K., Fujimoto, J., Kumimoto, H., Nishimoto, Y., Shimada, Y., Shinoda, M. and Yamamoto, T. 55 2. Different susceptibility of each L-myc genotype to risk factors for esophageal cancer or lung cancer Kumimoto, H., Nishimoto , Y., Hamajima, N., Matsuo, K. and Ishizaki, K. 56 3. Establishment of immortal normal and ataxia telangiectasia fibroblast cell lines by introduction of the hTERT gene Nakamura, H., Fukami, H., Kiyono, T. and Ishizaki, K. 57

Central Service Unit

General summary Tanabe, K., Nakamura, H., Terashima, M., Minoura, Y., Yamamoto, M. and

Hagino, M. 59 Librarians

Adachi, K. Mori, S., Teratani, M. and Yasuda, T. 60 Researches Supported by Special Project Programme

1. Identification of tumor-associated antigens recognized by T cells infiltrating Epstein-Barr virus-positive gastric carcinomas

Kuzushima, K., Nakamura, S., Nakamura, T., Yamamura, Y., Hayashi, N. and Tsurumi, T. 61

Publications

1 Journals 63 2 Reviews and books 78 3. Abstract for international conferences 81

Records of seminars 85 Records of symposia 87 Author index for research reports and publications 95

From left to right The first row; Dr. S. Tominaga (Director, until March 2001, President, as of April 2001) and Dr. To.

Takahashi (Director, as of April 2001) The second row; Mrs. M. Hosokawa (Adachi) and Ms. H. Tamaki.

Preface _______________________________________________________________________________________ First of all, let me introduce myself to you. My name is Toshitada Takahashi, promoted to be the Director of the Institute on April, 2001, as the former director, Dr. Suketami Tominaga, M.D., M.P. H., became President of the Aichi Cancer Center. It is my pleasure to share with you the 17th Scientific Report (2000-2001) of the Aichi Cancer Center Research Institute. Since the establishment of the Research Institute in 1965, the Scientific Reports have been published biennially to document major research activities and highlight progress in and contributions to cancer research at the Institute. As illustrated on the following page, the organization of the Research Institute was remodeled to be 9 Divisions, consisting of three study groups, i.e. cancer prevention/epidemiology, preclinical/ experimental therapy, and carcinogenesis/ molecular biology. A total of 59 full-time staff members, 35 researchers and 24 research assistants, as well as 5 research residents, are now conducting a wide range of cancer studies, together with 2 of graduate school students affiliated with Nagoya University School of Medicine, Nagoya, and approximately 30 visiting research fellows. The major areas being pursued are as follows: - descriptive and analytical epidemiology of cancers - primary and secondary prevention of cancer - molecular pathogenesis of gastrointestinal cancers - molecular oncology of lung cancer - molecular biology of translocation-junction genes of hematopoietic tumors - basic studies for cancer immunotherapy - oncogenicity and molecular biology 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 clinical applications in cancer diagnosis, treatment and prevention. Finally, I would like to express my deep appreciation to the Aichi Prefectural Government, not only for the continuous support received, but also for the new research building (approximately 7100m2), for which construction was completed in January, 2002. Granting support from the Ministry of Education, Science, Sports, Culture and Technology, the Ministry of Health, Labor, and Welfare, and other related organizations is also gratefully acknowledged.

April, 2002

Toshitada Takahashi, M.D., D.M.Sci. Director

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0)rganization ofthe Aichi Cancer Center Research institute

Cher Administrator

H.lto

(un宙lMaに h,2000)

J.Yamada

(as of Apr‖,2000,un付 l March,2001)

Ko Mottta

(as of Ap高 l,2001)

President

M.Ogawa

(un宙l March,2001)

S.Tominaga

(as of Ap克!,2001)

Director

R.Ohno

(aS Of July9 2000)

Director

S.Tominaga

(un廿l March,2001)

Tb.Takahashi

(as of April,2001)

一　

一　

一　

一　

一　

一　

一

一　

一　

一　

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(Chief′ Head)

Division of Epidemiology and Prevention(K.Taiima,M.D.)

Division of Oncological Pathology(M.Tatematsu,M.D。 )

Division of Molecular Oncology tTaコTakahashi,M.D.)

Division of Molecular Medicine(M,SetO,MttD.)

Division ofimmunology(K.Kuzushima,M,D.)

Division of Virology (Y TSurumi,M.D.)

Division of Molecular Pathology(R.Kannagi,MED。 )

Division of Blochemistry(M口 inagaki,M口 D.)

C e n t r a l L a b o r a t o r y & R a d i a t t o n B i o l o g y t t t s h i Z a k i , P h . D . )

Central Service Unl(H.Nakamura,Ph.D.)

Animal Faci:ity(M.Tatematsu,M.D.)

Laboratory of Transiational Research

SCIENTIFIC REPORTS

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Photo 1 (from left to right), frst row: Dr. T. Kuroishi, Dr. T. Takezaki, Dr. K. Tajima, Dr. N. Hamajima, Dr. M. Inoue; second row: Ms. Y. Yamauchi, Ms. M. Nakano, Ms. S. Hiraiwa, Ms. K. Mizutani, Ms. K. Asai, Ms. K. Sugimoto, Dr. C. Yang and Ms. Y. Hamajima.

Photo 2 (from left to right), Dr. H. Ito, Ms. H. Fujikura, Ms. K. Sanji, Ms. K. Fukaya and Dr. K. Hirose. Photo 3 (from left to right), first row: Ms. M. Kato, Ms. M. Tani, Ms. T. Saito; second row: Dr. K. Matsuo, Dr. A. Hishida and Ms. N. Takeuchi. Photo 4; Dr. C. Nozaki and Dr. X-E Huang. Photo 5; Ms. C. Adachi and Ms. M. Oobuchi. Photo 6; Mr. S. Hanamura. Photo 7; Dr. H. Yuasa.

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2

76

5

4

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Division of Epidemiology and Prevention ________________________________________________________________________________ Kazuo Tajima, M.D. Ph.D. M.P.H. Chief Tetsuo Kuroishi, M.S. Ph.D. Section Head (until March 2002) Nobuyuki Hamajima, M.D. Ph.D. M.P.H. Section Head Toshiro Takezaki, M.D. D.M.Sc. Section Head (as of April 2001) Manami Inoue, M.D. Ph.D. S.M. Senior Researcher Kaoru Hirose, B.P., Ph.D. Senior Research Assistant Toshiko Saito, Research Assistant Visiting Trainees Keitaro Matsuo, M.D. Nagoya University Graduate School of Medicine (until July, 2002) Xinen Huang, M.D. Nagoya City University Medical School Hidemichi Yuasa, D.D.S. Central Hospital of Tokai Medical Institute Kosuke Amano, M.Ed. Showa University School of Medicine Hidemi Ito, M.D. Nagoya City University Medical School (as of April 2001) Asahi Hishida, M.D. Nagoya University Graduate Medical of School (as of October 2001) Chika Nozaki, M.D., Nagoya University Graduate Medical of School (as of April 2001) Lucy Sayuri Ito, M.D. Santa Cruse Hospital, Sao Pauro, Brazil (July 2000-June 2001) Chuanxia Yang, M.D. West China University of Medical School, Chengdu, China (April 2001-March 2002) General Summary The current research activities of the Laboratory of Epidemiology and Prevention cover the following five subjects: 1) descriptive epidemiological studies of cancer incidence and mortality, with special reference to improvement of Aichi Prefectural Cancer Registry; 2) development of the hospital-based epidemiologic re-search program at Aichi Cancer Center (HERPACC) to determine risk and protective factors for main sites of cancer, with special reference to molecular epidemiologic studies on environmental and host-specific fac-tors; 3) development of a cancer prevention program for the general populace according to "Healthy People in Japan 21st, Aichi"; 4) evaluation studies of mass screening programs for main sites of cancer in Japan; 5) ethnoepidemiologic studies in the Asian-Pacific area. Descriptive epidemiologic studies include data from a nation-wide vital statistics, “Vital Statistics Japan”. At the end of 2000 a large-scale HERPACC study was completed and a more advanced version, HER-PACC-II for molecular epidemiology, was started to clarify gene-environment interactions for modification of carcinogenicity. For prevention measures against cancers, primary prevention trials were conducted for control of the smoking habit and obesity. Secondary prevention programs were evaluated by orthodox epi-demiological methods. To promote cancer control programs in the three Northeast Asian countries, a unique international col-laborative study, KOJACH (Korea, Japan and China), was planned in 2000 and a common nutritional and molecular epidemiologic study of gastro-intestinal cancers is now running in Nagoya, Seoul, Nanjing, Chongqin and Benxi. Furthermore, ethnoepidemiological studies on tumor viruses (HTLV and HBV) among Mongoloids were continued for Tibetans in China and Sahme in Northland. Cancer prevention is the final goal of epidemiological studies. Recently we are concentrating attention on molecular epidemiology to clar-ify interaction between host-specific characters and lifestyle exposure to risk factors, with regard to actual function of metabolic and detoxifying enzymes associated with genetic polymorphisms. 1. Descriptive epidemiologic studies on

cancer incidence and mortality Inoue, M., Kuroishi, T., Hirose, K. Tominaga, S.*1 and Tajima, K.

Probability of developing cancer over the whole life span of a Japanese: To obtain a relevant

index of the impact of cancer on the Japanese population, considering curable cases as well as mortalities, the probability of developing cancer in the entire life span of a Japanese was estimated. Data sources were the estimates of cancer incidence in Japan for 1994 by the research group for Popula-tion-based Cancer Registration in Japan, and death

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statistics of all sites and major sites of cancer for 1994 and 1996 obtained from the vital statistics of Japan published by the Ministry of Health and Welfare. Two methods were employed for estima-tion, one based on incidence / death (I/D) ratios of cancer and the other on the cumulative risk. The I/D ratio method gave a lifetime probability of devel-oping cancer in any site of 58 % for males and 51 % for females in 1996. With the cumulative risk, the values up to 85+ years of age were 52 % in males and 31 % in females, and for the average life expectancy of Japanese, 77 years old for males and 84 years old for females, 32 % and 26 % respectively. The estimated probabilities provide reasonable and practical indices of the impact of cancer today, allowing for the limitations of both methods. This approach can be also applied to local estimation if population-based cancer registry data are available. Estimation of the cancer incidence in Aichi prefecture: Use of a model area with good qual-ity registry data: In Japan, population-based can-cer registries are organized by local government and the quality of the registration remains modest, mainly due to the voluntary-based operations with-out legal restrictions and the insufficient budget. The population of Aichi prefecture is estimated to be seven million, and therefore the registry of the

prefecture covers a relatively large population. However, its quality has not reached the level re-quired internationally, for the above-stated reasons, and the derived incidences tend to be underesti-mates. On the other hand, there is a geographically continuous area, “Central Aichi”, with a good qual-ity of registry data, covering a sufficient population, including both urban and rural areas. The present study was aimed at trying to estimate the total can-cer incidence of Aichi Prefecture, using this good quality area as a model. The materials were data on cancer incidence and deaths in 1990-1998 in this central area of Aichi prefecture, with a population of approximately one million, under the jurisdiction of four public health centers covering nine municipalities. The DCN (%) for all sites was around 14%, which was lower than that of other representative registries in Japan, even with the same population size. The Incidence / Death ratio was around 1.9 in Central Aichi. Esti-mated age-standardized incidences were found to be around 270 (per 100,000) for males and 180 for fe-males, these values being high compared with those estimated using data for the whole area of the pre-fecture, but quite close to incidences for other areas with the highest quality of data (Figure 1). Our model area has typical demographic features of Ai-chi prefecture. Therefore, it is suggested that the

Figure 1. Age-standardized cancer incidence rates per 100,000 population in Central Aichi, Aichi and all-Japan (stan-

dard population: world population).

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cancer incidence in the prefecture is indeed being underestimated and that the actual figures may be closer to the present estimates. Trends in cancer mortality in Japan: Cancer mortality statistics in Japan (1950-1999) were cal-culated from the 'Vital Statistics, Japan' series. The age-standardized death rate for all sites of cancer has gradually been increasing in males in recent years, and gradually decreasing in females (Figure 2). The mortality rate from stomach cancer has been decreasing in males and females, while that from lung cancer has been increasing. In 1999 the num-ber of deaths from lung cancer was 37,934 for men, accounting for 21.6% of all male cancer deaths, and 14,243 for women, accounting for 12.4 % of all fe-male cancer deaths, and the total number of these deaths in both sexes ranked top among all sites of cancer. The average age at death from all sites of cancer in males rose 11.1 years over the last 45 years (70.7 years old in 1995 vs. 59.6 years in 1950), and 13.7 years in females (71.4 vs. 57.7). *1 Director

2. HERPACC studies on risk and protec-tive factors for main sites of cancer

Hirose, K., Saito, T., Inoue, M., Takezaki, T., Hamajima, N., Kuroishi, T., Tajima, K., Miura, S.*1, Kuzuya, K.*2 , Sugiura, T.*3, Mitsudomi, T.*4, Okuma, K. *5, Ogawa, H.*6, Nishiwaki, K.*7, Sakai, S.*8, Yanagi, T.*9, Ariyoshi, Y.*10, Stellman, S.*11, Wynder, E.*12 and Aoki, K.*13

Chronic atrophic gastritis and subsequent gastric cancer: Gastric cancer is still one of the most common cancers in Japan. Chronic atrophic gastritis is regarded as an important factor, associ-ated with Helicobacter pylori infection. The pur-pose of the present study was to elucidate chrono-logical change in cumulative risk of gastric cancer occurrence with various degrees of chronic atrophic gastritis by long-term follow-up of a large-scale cohort. A total of 5,373 subjects without cancer who underwent gastroscopic examination and com-pleted a life-style questionnaire at Aichi Cancer Center between 1985-1989 were prospectively fol-lowed until December 1999, by mail survey, hospi-tal record, and hospital- and population-based can-cer registry. Relative risks of gastric cancer associ-ated with baseline endoscopic findings were esti-

Figure 2. Trends in the age-standardized death rates per 100,000 population from cancer for selected sites in Japan, 1950-1999 (standard population: Japanese population in 1985).

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mated using hazard ratios and their 95% confidence intervals with the Cox proportional hazard model, adjusting for gender, age and gastric cancer family history. After an average of 10 years of follow-up, 117 gastric cancer cases were identified. The risk was greatest among the subjects with moderate at-rophy at baseline (hazard ratio=2.2) (Figure 3), es-pecially after 4-6 years of follow-up (hazard ra-tio=4.6-5.0). After this time point, risk attenuation with the length of follow-up period was observed. Our study suggests the possibility that incomplete chronic atrophic gastritis and the processes occur-ring in atrophy are associated with development of gastric cancer. Risk factors for gastric cancer among Japa-nese postmenopausal women: analysis by subsite and histological subtype: To clarify whether re-productive factors have an impact on gastric cancer in Japanese females, a case-control study was con-ducted using data from the HERPACC. The study subjects comprised 365 postmenopausal women with gastric cancer and 1,825 age-class frequency- matched non-cancer outpatients presenting at Aichi Cancer Center in 1988-1998. Cases were further di-vided with regard to the anatomic subsites (upper third, middle third, lower third) and histological subtypes (differentiated, non-differentiated), and associations were evaluated using odds ratios (ORs) estimated by the logistic regression model, adjust-ing for potential confounding factors. A high body weight and corresponding body mass index at age 20 moderately increased the risk of gastric cancer, especially for middle third and non-differentiated cancers. Risk fluctuation with early or late age at menarche and menopause and total duration of fer-tility was not consistent. Individuals with a high age at first parity tended to show a decreased risk of

cancer, irrespective of the subsite or histological subtype. ORs were decreased with a short average period of breastfeeding, especially for upper third and non-differentiated cancers. From these results, however, it appears that height, weight, menstrual and reproductive factors have less impact on gastric cancer than environmental factors such as smoking and dietary habits or a family history of gastric cancer. Protective and risk factors for hormone re-lated cancer in women: To confirm the protective effects of regular exercise on female hormone re-lated cancers, we undertook a case-referent com-parative study using HERPACC data. The case group consisted of 2,367, 222 and 149 women who had first been diagnosed as having breast, endo-metrial and ovarian cancers. The referents were 24,620 female first-visit outpatients who had not previously been diagnosed with any type of cancer. The odds ratios (ORs) and their 95% confidence intervals (95%CI) were estimated using an uncon-ditional logistic regression model. From the cross analysis between frequency of exercise and other selected factors among noncan-cer referents, more frequent exercise was associated with eating more fruits and vegetables and with less cigarette smoking. This result indicated that persons continuing regular exercise for health are much concerned about their health maintenance and they usually contrive to improve their life style. To clar-ify the independent effects of regular exercise for health on the risk reduction, we adjusted for the in-fluence of other related factors. Regular exercise showed a negative association with breast cancer. When women without regular exercise were refer-enced, the adjusted ORs were 0.89 (95%CI: 0.75-1.05) for women exercising 3-4 times per

Figure 3. Hazard ratio for gastric cancer by baseline

endoscopic findings (n=5,373)

Figure 4. Adjusted ORs for hormone related can-

cers according to level of exercise

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month and 0.71 (95%CI: 0.62-0.81) for twice a week or more. The downward trend in the risk of breast cancer with regular exercise was statistically significant (P<0.0001). The risk reduction was most prominent with endometrial cancer, whose figures were 0.40 (95%CI:0.18-0.85) for 3-4 times per month and 0.81 (0.56-1.17) for twice a week or more (Figure 4). The present study thus provided clear evidence that risk of female hormone related cancers is decreased by regular exercise for health. In terms of measuring physical activity, we need to develop techniques to assess optimal intensity and duration of exercise. Comparative epidemiological study on risk and protective factors for lung cancer between the US and Japan: Lung cancer is the leading cau-se of cancer deaths in the US and Japan, but the age-adjusted mortality rate in Japan is still only two thirds of that in the US. To estimate smok-ing-specific relative risks for lung cancer in men, we carried out a multicentric case-control study in New York and Aichi from 1992 to 1998. A total of 371 cases and 373 age-matched controls were inter-viewed in United States hospitals and 410 cases and 252 hospital controls in Japanese hospitals; 411 Japanese healthy controls were also randomly se-lected from electoral rolls. The odds ratio (OR) for lung cancer in current United States smokers rela-tive to nonsmokers was 40.4, which was >10 times higher than the OR of 3.5 for current smokers in Japanese relative to hospital controls and six times higher than in Japanese relative to community con-trols (OR = 6.3). There were no substantial differ-

ences in the mean number of years of smoking or average daily number of cigarettes smoked between United States and Japanese cases or between United States and Japanese controls, but American cases began smoking on average 2.5 years earlier than their Japanese counterparts. To investigate risk modification for lung cancer with diet in Japanese, we also conducted a case-control study and evaluated variation in influ-ence with the histological type at Aichi Cancer Center Hospital. We recruited 367 male and 240 female cases with adenocarcinomas, 381 male and 57 female cases with squamous cell and small cell carcinomas, and 2964 male and 1189 female can-cer-free outpatients as controls. We found de-creased ORs for adenocarcinomas in both males (OR=0.51) and females (OR=0.48) who consumed cooked/raw fish (Figure 5), but not dried/salted fish at the highest quartile frequency, compared with the lowest. Decreased ORs for squamous cell and small cell carcinomas were observed in males with fre-quent consumption of raw and green vegetables, fruit and milk. This study suggests cooked/raw fish consumption lowers the risk of adenocarcinoma of the lung in Japanese. These findings partially support our hypothesis that smoking and dietary habits contribute to dif-ferences in lung cancer mortalities between the two countries. Gene-environmental interactions for cancers: Genetic polymorphisms may modify the effects of environmental risk factors on cancer occurrence. We launched a comprehensive epidemiologic pro-ject, HERPACC-II, including both lifestyle and polymorphism data, following HERPACC-I which solely concentrated on lifestyle. As of December 2001, about 5,300 samples of DNA were stored to conduct case-control studies. Genotyping of about 60 polymorphisms was conducted at the Division of Epidemiology and Prevention. Significant find-ings were found for: 1) gene-environment interac-tion for esophageal cancer between heavy drinking and aldehyde dehydrogenase 2 (ALDH2); 2) inter-action for cancers of the esophagus and lung be-tween smoking and polymorphisms of L-myc and NQO1; 3) altered malignant lymphoma risk with methylenetetrahydrofalate reductase (MTHFR) and methionine synthase (MS); 4) altered cancer risk for breast and colorectum with B2- and B3-adrenoceptors; 5) interactions between smoking and two polymorphisms of interleukin 1B (IL-1B) and myeloperoxidase (MPO) for Helicobacter py-lori infection; and 6) smoking habits with dopa-

Figure 5. Consumption of cooked/raw fish and the odds ratio (OR) for lung cancer by sex, with reference to the histological type: adenocarcinomas (AD), and squamous cell and small cell carcinomas (SQ & SCC).

10

mine receptor D2 (DRD2) and IL-1B. Further stud-ies on the interactions with polymorphisms are continuing to be conducted, using larger sample sizes. The rapid progress in these polymorphism stud-ies has facilitated by a newly developed PCR method, PCR-CTPP (polymerase chain reaction with confronting two-pair primers) in our laboratory. As shown in Figure 6, two-pair primers produce al-lele specific bands for single nucleotide polymor-phisms, which allows electrophoresis directly after PCR. This technique requires half of the cost and time for genotyping by PCR-RFLP. *1 Department of Breast Surgery, Aichi Cancer Center

Hospital *2 Department of Gynecology, Aichi Cancer Center

Hospital *3 Department of Respiratory Diseases *4 Department of Thoracic Surgery *5 Department of Hospital Laboratory *6 Division of Human Science, Aichi Mizuho Univer-

sity *7 Department of Respiratory Diseases, Nagoya Na-

tional Hospital *8 Department of Respiratory Diseases, Red Cross Na-

goya First Hospital *9 Vice Director, Red Cross Nagoya Second Hospital *10 Director, Aichi Prefectural Hospital *11 Division of Epidemiology, American Health Founda-

tion, New York, US *12 Previous President, American Health Foundation

(deceased), New York, US *13 Emeritus President 3. Development of cancer prevention pro-

grams Hamajima, N., Hirose, K., Tajima, K. and Miura, S.*1

Smoking cessation for hospital patients: In order to measure smoking cessation rate among those who visit medical facilities in Japan, a large-scale follow-up study was conducted. Sub-jects were self-reported smokers who visited a can-cer hospital, a general hospital, or one of four health checkup facilities in 1997-98. Their smoking habits were followed by two postal surveys. The first was two months after the visit to hospital or attendance at a health checkup screening, and the second was one year thereafter. In total, 3,552 smokers partici-pated in the present study; 1,131 first visit outpa-tients at a cancer hospital, 214 first visit outpatients at a general hospital, and 2,207 examinees at four health checkup facilities. The response rate for the first follow-up varied from 57.3% to 80.2% of the eligible participants in the six facilities, and that for the second from 50.0 to 67.1%. When non-respondents were classified as non-quitters, the cessation rate two months after their participation was 11.7% (95% confidence interval, 7.4-16.0%)

Figure 6. Logic of the polymerase chain reaction with confronting two-pair primers. At the 3’ ends of the inner primers R1 and F2, the base specific to each allele is included. The difference between a-bp and b-bp should be large enough to be distinguishable by electrophoresis.

11

for the general hospital and 2.7% (2.1-3.5%) for the four health checkup facilities, and that for one year after was 9.8% (6.2-14.6%) and 6.0% (5.1-7.1%), respectively. In the cancer hospital, the rate for self-reported cancer patients was 74.6% (68.5- 80.0%) after two months and 51.3% (44.7-57.9%) after one year. The smoking cessation rate was found to be smaller in the health checkup exami-nees than in the patients. Outpatients seemed to be more amenable to smoking cessation, being a more appropriate target for cessation programs. Based on these baseline cessation rates, intervention studies are now on going. Obesity control trial for hospital patients: In Japan, the mortality rate from female breast cancer began to increase in 1965 and the increasing trend has become more remarkable in recent years. Future estimations of cancer mortality and incidence pre-dict that breast cancer will become the leading can-cer in Japanese women in the 21st century. A num-ber of risk factors for breast cancer related to re-productive events have been established, e.g, early menarche, nulliparity, late age at first birth and late natural menopause. A family history of breast can-cer is associated with an overall increment of risk around twofold and a high Body Mass Index (BMI) increases the risk of breast cancer after menopause. A case-referent study using HERPACC data pro-vided clear evidence that the risk of breast cancer in post-menopausal Japanese women is markedly in-creased by obesity. With the change of nutrient intake after the

World War II, obesity is becoming one of the most serious health problems in Japan, therefore, we planned an intervention trial for obese women. Af-ter obtaining informed consent, we recruited pa-tients over 30 years old with a BMI of 24 or more, a total of 40 being randomly assigned into study groups A (28) and B (12). Group A started the pre-vention program at the entry and group B started three months thereafter, according to the protocol. At baseline, three, six and twelve months, partici-pants were checked for body size, dietary intake and serum chemistry. It was stressed that they should record their daily food intake and physical activity for 3 months. Every weekend they returned their diaries by mail and we gave them appropriate comments by telephone or by mail after reviewing them. This trial was designed to evaluate effective-ness of the intervention trial in the group A during the first 3 month by comparing with group B. After follow up for 3 months, we observed significant improvement in BMI and waist size. For group A the average energy intake per day was about 2,000 Kcal at baseline and, then, 1,600 Kcal after three months. Changes in key biomarkers (serum triglyc-eride, total cholesterol and HDL cholesterol) related to the reduction of BMI in three months were not remarkable in the present interim analysis (Figure 7). *1 Department of Breast Surgery, Aichi Cancer Center

Hospital

Figure 7. Comparison of the change between group A and B during first 3 months.

12

4. Evaluation of Secondary Prevention of Cancers

Kuroishi, T., Hirose,K., Suzuki, T*1., Tominaga, S*2, Sagawa, M.*3, Fujimura, S.*4 and The Research Group of Lung Cancer Screening

In Japan the mortality rate from lung cancer has been increasing in recent years. In 1999 lung cancer was the commonest cause of cancer death in both sexes. Lung cancer screening has been conducted in Japan mainly by chest X-ray examination plus spu-tum cytology, the standard method for lung cancer screening according to the Law of Health Services for the Elderly. The purpose of this study was to evaluate the effectiveness of mass screening for lung cancer in Japan. We calculated the average coverage-rates for lung cancer screening per year from 1986 to 1995 for persons aged 40-69 years for all of the 3,255 municipalities in Japan, selecting "high coverage-rate" municipalities with average coverage-rates of 50%, 60%, 70%, 80% or more. Two municipalities were selected as "controls" for each high coverage-rate municipality, and were matched for population, national health insurance

rate, and the age-adjusted death rate from cancer of the lung in the period 1986-90. We compared the change in the age-adjusted death rate from 1986-90 to 1991-95 of the high coverage-rate municipalities and the comparable controls. The percent reduc-tion in the average age-adjusted death rate from cancer of the lung for those aged 40-69 years, target age-groups of lung cancer screening, in the high coverage-rate municipalities with an average cov-erage-rate of 70%, 80% or more was contrasted with the increase in control municipalities. In the case of the high coverage-rate municipalities with average coverage-rate of 70 % and over, the reduc-tion was statistically significantly greater than those in the controls (Figure 8). These results suggest that mass screening for lung cancer, mainly by chest X-ray examination plus sputum cytology, can con-tribute to a reduction in mortality from lung cancer. *1 Department of Cancer Control and Statistics, Osaka

Medical Center for Cancer and Cardiovascular Dis-eases

*2 Director *3 Department of Thoracic Surgery, Kanazawa Medical

College, Kanazawa, Japan, *4 Tohoku Kosei Nenkin Hospital, Sendai, Japan 5. Ethnoepidemiological studies on can-

cer Takezaki, T., Hamajima, N., Inoue, M., Hirose, K., Tajima, K., Gao, C-M.*1,Wu, J-Z.*1, Wang, Y-M.*2, Mo, B-Q.*2, Wang, X-R.*2, Yoo, K-Y.*3, Ahn, Y-O.*3, Kim, J-S.*4, Zhou, Z-Y.*5, Cao, J.*5, Li, C.*6, Gao, F-C.*6, Tokudome, Y.*7, Sonoda, S.*8, Yashiki, S.*8, Fujiyoshi, T.*8, Li, H-C.*8, Zhao, S-H.*9, Horai, S.*10, Chiba, H.*11, Senoh, H.*12 and Tretli, S.*13

Comparative epidemiological study on GI-tract cancers focusing on Korea, Japan and China (KOJACH study): China is one of the highest risk areas for esophageal and gastric cancer in the world. To clarify the environmental and host factors associated with risk of esophageal and stomach cancers, we have been conducting a com-parative epidemiological study in Jiangsu Province, China, since 1996, focusing high and low risk-areas for these cancers. We found frequent consumption of garlic, in addition to other anticancer foods, to lower the risk of these cancers in a case-control study of a low-risk area, Pizhou, concordant with our previous ecological findings for the general population in high- and low-risk areas, and a case-control study of a high-risk area. Frequent

Figure 8. Percentage change in the age-adjusted death rate of cancer of the lung from 1986-90 to 1991-95 for 127 high coverage-rate munici-palities with an average coverage-rate of 70% and over and for 254 comparable control mu-nicipalities.

13

consumption of these foods thus appears to be a factor in low mortality from esophageal and stom-ach cancer. To investigate the gene and environmental in-teraction for risk of esophageal and stomach can-cers, we conducted a case-control study in a high-risk area, Huaian, recruiting 191 cases and 196 population-based controls. We selected gene poly-morphisms for cytochrome P-450 2E1 (CYP2E1), hOGG1, GSTM1 and GSTT1, because CYP2E1 is involved in metabolic activation of environmental chemical carcinogens; hOGG1 encodes an enzyme which repairs 8-hydroxyguanine adducts produced by oxidative stress; and the GSTs are primarily re-sponsible for detoxication of xenobiotics. We found a significant positive interaction between heterozy-

gous and homozygous RsaI rare alleles for CYP2E1 and ever-smoking in the odds ratio (OR) for stom-ach cancer. A frequent drinking habit and pickled vegetable consumption elevated the OR for stomach cancers in individuals with the Cys/Cys genotype of hOGG1, as compared to Ser/Ser and Ser/Cys carri-ers (Figure 9). The GSTM1 null genotype was asso-ciated with an increased OR for esophageal cancer, but not for stomach cancer. A combined effect was also observed between smoking and the GSTM1 null genotype with regard to esophageal risk. These findings suggest that the polymorphisms are in-volved in determining susceptibility to esophageal and stomach cancer development. To establish cancer prevention measurement in northeastern countries bearing a historically com-mon cultural background, we started a case-referent study on colorectal cancer, based on a standardized epidemiological approach, in Korea (Seoul), Japan (Nagoya) and China (Nanjing, Chongqing and Benxi), the so called KOJACH Study, in 2000. First we developed a semi-quantitative food frequency questionnaire (SQFFQ) for the five cities according to the Tokudome’s method (Tokudome et al, Jpn J Clin Oncol 28: 679, 1998), and evaluated their va-lidity and reproducibility for further study. We started collecting lifestyle data by standardized questionnaire and blood samples for plasma and DNA after obtaining informed consent from colo-rectal cancer cases, hospital referents and popula-tion-based referents. The intent is to analyze risk and protective factors for colorectal cancer using

Figure 9. Drinking habit and the odds ratio (OR)

for stomach cancer, with reference to the hOGG1 Ser326Cys polymorphism.

Figure 10. Worldwide distribution of HBV genotypes among various ethnic groups. Seventeen ethnic groups

(Usuda S, et al., 1999; Bowyer SM and Sim JGM. 2000; Nakamo T, et al., 2001; Bowyer SM, et al., 1997; Arauz-Ruiz P, et al., 1997; Blitz L, et al., 1998; Norder H, et al., 1993) were used in the compari-son, including the Tibetans.

14

data for a total of 1,000 cases and 2,000 referents. Immunogenetic study on Mongoloid popula-tions: Human T-cell leukemia virus type 1 (HTLV-1), the main cause of adult T-cell leuke-mia/lymphoma, spread throughout the world but microgeographical clusters of hyperendemicity. Epidemiologic studies among Mongoloids showed that HTLV-1 is highly endemic in South Japan (one million carriers) and in the Andes district of South America. On the other hand HTLV-II (also a risk factor for adult T-cell leukemia/lymphoma) is broadly distributed in the whole of South America, except the Andes line. We now know that there are no other HTLV-I/II clusters in the Asian Pacific except among Aborigines in north Australia and Melanesians in Papua New Guinea. Hepatitis B virus (HBV), distributed throughout the world, is classified into seven geographically separated genotypes designated A to G. Since the prevalence of HBV infection in isolated ethnic Ti-betan populations in China, and the HBV genotypes involved have hitherto remained unclear, we col-lected 262 blood samples from four isolated vil-lages in east and west regions of Tibet (Photo). The prevalence of HBV infection was estimated by EIA for HBV Ag and HBV Ab, and the HBV genotypes were determined by a PCR-microwell plate hy-bridization method using plasma DNA. The preva-lence of HBV Ag and HBV Ab positives was 19.1% and 29.0%, respectively (Figure 10). We de-tected only the C genotype, known to be a pre-

dominant among Mongoloid populations in Asia. The evidence including ethnoepidemiologic find-ings for HTLV-I opens doors to a new paradigm for virus anthropology. *1 Division of Epidemiology, Cancer Institute of Ji-

angsu Province, Nanjing, China *2 Nanjing Medical University, Nanjing, China *3 Department of Preventive Medicine, College of

Medicine, Seoul National University, Seoul, Korea *4 Department of Food and Nutrition in Oriental Medi-

cine, Semyung University, Semyung, Korea *5 Laboratory of Molecular Toxicology, Third Military

Medical University, Chongqing, China *6 Bengan General Hospital, Benxi, China *7 Department of Life Science, Nagoya Bunri College *8 Department of Virology, Faculty of Medicine, Ka-

goshima University, Kagoshima

*9 Department of Blood Transfusion, Southwest Hospi-tal, Chongqing, P.R.China

*10 Department of Biosystems Sciences, The Graduate University for Advanced Studies, Kanagawa

*11 Department of Laboratory Medicine, Hokkaido Uni-versity School of Medicine, Sapporo

*12 Department of Anatomy, Akita University School of Medicine, Akita, Japan,

*13 Institute of Population-based Cancer Research, Oslo, Norway

Photograph: Dr. Liang is collecting blood samples from local people in Eastern Tibet.

15

Dr. Parry J. Guilford, from University of Otago, New Zealand, giving us the lecture entitled "E-cadherin Germline Mutations in Familial Gastric Cancer" in the 8th Aichi Cancer Center International Symposium held on February 16, 2002 (see p. 88).

16

From left to right First row; Mr. H. Tanaka , Dr. K. Inada, Dr. M. Tatematsu, Dr. H. Nakanishi, Dr. T. Tsukamoto and Dr. A. Tanaka. Second row; Dr. M. Goto, Dr. X. Cao, Dr. T. Mizoshita, Dr. N. Ogasawara, Ms. C. Tomita, Ms. N. Yamada and Ms. M.

Yamamoto. Third row; Dr Y. Tsukamoto, Dr. K. Matsumoto, Ms. S. Tokumasu, Ms. H. Ban and Ms. H. Maejima. Inset; Dr. K. Nozaki, Dr. Y. Ikehara, Dr. N. Shirai, Dr. T. Iidaka, Ms. R. Haruta and Dr. A. Hirata.

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Division of Oncological Pathology ________________________________________________________________________________ Tatematsu Masae, M.D. Chief Hayao Nakanishi, M.D. Section Head Ken-ichi Inada, M.D. Senior Researcher Tetsuya Tsukamoto, M.D. Senior Researcher Yuzuru Ikehara, M.D. Researcher Sachiko Tokumasu, B.D., Research Assistant Masami Yamamoto, D.V.M., Research Assistant Harunari Tanaka, B.P., Research Assistant Michiyo Tominaga, Semi-regular Employee Nami Yamada, Semi-regular Employee Hisayo Ban, Semi-regular Employee Visiting Scientists Malcolm A. Moore, Ph.D. Asian Pacific Organization for Cancer Prevention Kato Kazuo, M.D., Fujita Health University School of Medicine Visiting Trainees Koji Nozaki, M.D., Research Resident Hirofumi Yuasa, D.V.M., Tanabe Seiyaku Co., Ltd. Takasuke Yamachika, M.D., Kita Hospital Kiyoshi Kobayashi, D.V.M., Mitsubishi-Tokyo Pharmaceuticals, Inc. Hiroki Sakai, D.V.M., Gifu University Norimitsu Shirai, D.V.M., Pfizer Pharmaceuticals Inc. Azusa Tanaka, D.D.S., School of Dentistry, Aichi-Gakuin University Tsutomu Mizoshita, M.D., School of Medicine, Nagoya City University Seiji Ito, M.D., Department Surgery II, Nagoya University School of Medicine Yoshinari Mochizuki, M.D., Department Surgery II, Nagoya University School of Medicine Takeshi Iidaka, D.V.M., Pfizer Pharmaceuticals Inc. Yoshitaka Tsukamoto, D.D.S., School of Dentistry, Aichi-Gakuin University Xueyuan Cao, M.D., Faculty of Medicine, University of Tokyo General Summary The responsibilities of the Division of Oncological Pathology include both autopsy and research activities. From its establishment in 1965 up through the end of 2001, our laboratory performed a total of 2,477 autop-sies. Postmortem examinations are a source of valuable information on the behavior of neoplasms and their response to therapy. Research activities in our laboratory are divided into two main areas. The first deals with the molecular basis of chemical carcinogenesis in the gastrointestinal tract of man, the rat, mouse and Mongolian gerbils, along with the mechanisms regulating differentiation of stomach epithelium, especially intestinal metaplasia. During 2000-2001, the research focused on various issues: a) heterotopic proliferative glands (HPGs) related to Helicobacter pylori (Hp) infection, which frequently develop in the glandular stomach of infected gerbils with slightly dysplastic change - an eradication experiment revealed the reversibility of this lesion, implying that it is an entity distinct from a true neoplasm; b) Hp attachment to the gastric mucosa through adhesin, which binds to Lewis b (Le(b)) or H type I carbohydrate structures - polymorphisms of the secretor (Se) and Lewis (Le) genes, both involved in type I Le antigen synthesis, were found to alter the risk of Hp infection; c) susceptibility of p53 nullizygote (–/–), heterozygote (+/–), and wild type (+/+) mice to N-methyl- N-nitrosourea (MNU) gastric carcinogenesis and to methyl-n-amylnitrosamine (MNAN) esophageal car-cinogenesis - p53 may not be a direct target of MNU but rather play an important role as a gatekeeper in mouse stomach carcinogenesis, whereas p53 mutations may be involved in the development of esophageal SCCs induced by MNAN; d) intestine specific homeobox genes, Cdx1 and Cdx2, candidate genes for di-recting intestinal development and differentiation of intestinal phenotypes in the gastrointestinal tract epithe-lium - human gastric cancer tissues were examined and a positive correlation was demonstrated between

19

may reside in their capacity to attach stably to the vessel wall rather than their potential for initial cell arrest or subsequent growth. The system used in the present study provides a powerful tool for analyzing targets of various anti-metastatic agents in the se-quential process of metastasis. *1 Department of Gastroenterological Surgery, Aichi

Cancer Center Hospital *2 Department of Surgery II, Nagoya University of

Medicine 3. Intestine specific homeobox genes,

Cdx1 and Cdx2, are key molecules in intestinalization of gastric carcinoma cells

Inada, K., Mizoshita, T.*1, Tsukamoto, T., Nakanishi, H. and Tatematsu, M.

Caudal-type homeobox genes Cdx1 and Cdx2 are candidates for directing intestinal development, differentiation, and maintenance of the intestinal phenotype in the human and mouse gastrointestinal tract. The aim of this study was to assess relation-ships among expression of Cdx1 and Cdx2 mRNAs, histological classification and phenotypic expres-sion of carcinoma cells. Fresh human gastric cancer tissues were collected from surgically resected specimens from 70 patients after obtaining in-formed consent. Northern hybridization was per-formed against Cdx1 and Cdx2 mRNAs after ex-tracting RNAs. In addition, the carcinoma tissues were evaluated both histologically and phenotypi-cally using mucin histochemical and immunohisto-chemical methods. Neither Cdx1 nor Cdx2 expres-sion had any relation with the Lauren’s histological classification (“intestinal” types: n=32, “diffuse” types: n=38). On the other hand, Cdx1 was appar-ently associated with intestinal phenotypic differen-tiation both in the “intestinal” and “diffuse” type {gastric phenotype: n=15, gastric and intesti-nal-mixed phenotype: n=18, intestinal phenotype: n=17, unclassified (null) type: n=20, p< 0.05}. Cdx2 was also related with the phenotypically in-testinal gastric cancers only in intestinal type of Lauren’s histological classification. The results suggest that both Cdx1 and Cdx2 might play im-portant roles in expression of the intestinal pheno-type, not only in the normal intestine but also in gastric neoplasms. *1 Department of Internal Medicine, School of Medicine,

Nagoya City University

4. Different susceptibilities of p53 knock-out (–/–), (+/–) and (+/+) mice to induc-tion of stomach adenocarcinomas by N-methyl-N-nitrosourea and esophageal squamous cell carcinomas by methyl-n-amylnitrosamine

Tsukamoto, T., Yamamoto, M., Shirai, N.*2, Sakai, H.*1, Iidaka, T.*2, Donehower, L.A.*3 and Tatematsu, M.

Mutations of the p53 tumor suppressor gene constitute one of the most frequent molecular changes in a wide variety of human cancers. Mice deficient in p53 have recently attracted attention for their potential to identify chemical genotoxins. In this study we investigated the susceptibility of p53 nullizygote (–/–) , heterozygote (+/–), and wild type (+/+) mice to N-methyl-N-nitrosourea (MNU) gas-tric carcinogenesis and to methyl-n-amylnitrosa- mine (MNAN), which specifically induces eso-phageal tumors in mice. In the first experiment, p53 knockout mice were treated with 30 ppm MNU in drinking water one week on and one week off and killed after 5 weeks. The numbers of pepsi-nogen altered pyloric glands (PAPG), putative pre-neoplastic lesions, were 1.8, 1.7 and 22.6 in p53 (+/+), (+/–), and (–/–) mice, respectively. In a 15-week experiment, adenomas were found in 0 of 19 (+/+) (0%), 2 of 21 (+/–) (9.5%), and 6 of 10 (–/–) (60.0%) animals. Also one well differenti-ated adenocarcinoma was observed in a p53 (–/–) mouse. After forty weeks treatment with 120 or 30 ppm MNU, there was no significant difference in the incidence of gastric tumors between p53 (+/+) and (+/–) mice, but mortality from carcino-gen-induced lymphomas, leukemias and sarcomas was very much greater in the latter group. Homo-zygous KO animals could not be maintained long-term. PCR-single strand conformation polymorphism analysis of exons 5-8 of the p53 gene demonstrated no mutations, in DNA extracts from 68 gastric tumors from 16 and 20 p53 (+/+) and (+/–) mice receiving 30 ppm, respectively, and 14 and 18 given 120 ppm. For esophageal car-cinogenesis, the p53 (+/–), and (+/+) mice were treated with 5 or 15 p.p.m. MNAN in their drinking water for 8 weeks then maintained without further treatment for an additional 7 or 17 weeks, being killed at experimental weeks 15 or 25. An addi-tional group of p53 (–/–) mice were given 5p.p.m. MNAN for 8 weeks and killed at week 15. In the 5p.p.m. groups, squamous cell carcinomas (SCCs) were observed in 10/12 (83.3%) p53 (–/–) and 1/15 (6.7%) p53 (+/–) mice, but in none of the p53 (+/+) mice. With 15 p.p.m., 2/14 (14.3%) p53 (+/–) and

18

Cdx1 expression and intestinal phenotypic differentiation. Our second research area involves the molecular basis of cancer metastasis and applications in diagnosis and treatment, especially for micrometastases. Several micrometastasis models featuring tagged with green fluorescence protein (GFP) gene were established, including a rat tongue cancer cell line, human gastric and colonic cancer cell lines with different metastatic potentials. Sequential steps in hematogenous, peritoneal and lymphogenous metastasis in living mouse were then documented by an intravital videomicroscopy tech-nique. In addition, a rapid quantitative method for detection of micrometastases in peritoneal washes of pa-tients with gastric carcinoma was developed using real-time RT-PCR and prognostic potential demonstrated. 1. Reversibility of heterotopic proliferative

glands in glandular stomach of Helico-bacter pylori-infected mongolian gerbils on eradication

Tatematsu, M., Nozaki, K., Shimizu, N., Tsukamoto, T., Inada, K., Cao, X., Ikehara, Y., Kaminishi, M. *1 and Sugiyama, A. *2

Helicobacter pylori (Hp) infection is an impor-tant factor in human gastric disorders. Mongolian gerbils can be easily infected with Hp and provide an excellent experimental model for clarifying the role of the bacterium in chronic active gastritis, peptic ulceration, intestinal metaplasia, and gastric carcinoma development. We have proved the en-hancing effects of Hp infection on all histological types of gastric cancers in Mongolian gerbils ex-posed to chemical carcinogens. Heterotopic pro-liferative glands (HPGs) also frequently develop with Hp infection in the glandular stomach of in-fected gerbils, with a slightly dysplastic change of constituent cells. Distinguishing reversible inflam-matory lesions from true neoplasms upon eradica-tion is necessary for further biological or histo-chemical investigations using this model. For this purpose, we employed an experimental model of long-term Hp infection and eradication in gerbils. HPGs finally developed with a phenotypic shift of intestinalization, including generation of Paneth cells. After eradication, HPGs were obviously re-duced, and gastric lesions in mucosa also improved with few remnants of the former injury. This shows that reversible HPGs are frequently induced solely by Hp infection in this animal species, and are re-lated to severe gastritis, rather than being malignant in character. Thus, distinction of reversible lesions from true neoplasms is necessary to elucidate rela-tionship between Hp infection and gastric carcino-genesis in this animal model. *1 Department of Gastrointestinal Surgery, Postgraduate

School of Medicine, The University of Tokyo *2 First Department of Surgery, Shinshu University,

School of Medicine

2. Real-time observation of micrometasta-sis formation in the living mouse liver using a GFP gene-tagged rat tongue carcinoma cell line

Nakanishi, H., Itoh, H.*1, Ikehara, Y., Kato, T.*1, Nakao, A.*2 and Tatematsu, M.

Initial arrest, attachment, extravasation, and subsequent extravascular growth of tumor cells in secondary organs are believed to be crucial events for hematogenous metastasis, but the actual proc-esses in living animals remains unclear. For the present study, we established green fluorescent protein (GFP)-expressing rat tongue carcinoma cell lines permiting real-time analysis of micrometasta-sis formation in combination with intravital video microscopy (IVVM). GFP expressing metastatic (LM-EGFP) and non-metastatic (E2-EGFP) cell lines could be visualized at the cellular level in live mice for more than one month. Real-time IVVM analysis of liver metastases after intraportal injec-tion of the cells via mesenteric vein revealed that both LM-EGFP and E2-EGFP tumor cells arrest similarly in sinusoidal vessels near terminal portal venules within 0.4 sec, during which time no evi-dence of “rolling” -like movement along endothelial cell surface is observed. Quantitative analysis of GFP positive foci showed that E2-EGFP cells were completely sheared from the liver sinusoid within 3 days, with no solitary dormant cells, whereas a sub-stantial number of LM-EGFP cells remained in the liver, probably due to stable attachment to the si-nusoidal wall. Confocal laser scanning microscopy (CLSM), in combination with laminin immunohis-tochemistry, revealed that only LM-EGFP cells started growth at 3 to 4 days after inoculation and that most of the growing foci were surrounded by a subsinusoidal basement membrane (BM). Our re-sults suggest that micrometastasis formation by LM-EGFP cells consists of initial tumor cell arrest due to size constraints of the vessel, stable attach-ment to subsinusoidal BM and subsequent in-travascular growth before extravasation. The dif-ference in metastatic potential between the 2 lines

20

1/11 (9.1%) p53 (+/+) mice developed SCCs. At 25 weeks, the incidences of SCCs were 7/16 (43.8%) and 8/14 (57.1%) in p53 (+/–) mice and 1/13 (7.7%) and 2/10 (20.0%) in p53 (+/+) mice receiving 5 and 15 p.p.m., respectively. Of the SCCs examined by PCR-single strand conformation polymorphism analysis, 61% (14/23) from p53 (+/–) and 50% (6/12) from p53 (+/+) mice demon-strated mutations in the p53 gene (exons 5-8). These results suggest that p53 may not be a direct target of MNU but rather play an important role as a gatekeeper in mouse stomach carcinogenesis in-duced by this direct acting agent. However, they indicate the order of susceptibility to MNAN-induced esophageal tumorigenesis to be as follows: nullizygotes (–/–) > heterozygotes (+/–) > wild type (+/+), and provide strong evidence of in-volvement of p53 mutations in the development of esophageal SCCs. *1 Department of Veterinary Pathology, Gifu University *2 Nagoya Laboratories, Pfizer Global Research & De-

velopment *3 Department of Molecular Virology and Microbiology,

Baylor College of Medicine, Houston 5. Polymorphisms of two fucosyltrans-

ferase genes (Lewis and Secretor genes) involving type I Lewis antigens are associated with the presence of anti-Helicobacter pylori IgG antibodies

Ikehara,Y., Nishihara, S.*1, Yasutomi, H., Kitamura, T., Matsuo, K., Shimizu, N., Inada, K., Kodera, Y.*2, Yamamura, Y.*2, Narimatsu, H.*1, Hamajima, N.*3 and Tatematsu, M.

Helicobacter pylori attach to the gastric mucosa though adhesin, which binds to Lewis b (Le(b)) or H type I carbohydrate structures. The products of the Secretor (Se) and Lewis (Le) gene are involved in type I Le antigen synthesis. The present study was therefore performed to investigate the possibil-ity that Se and Le gene polymorphisms alter the risk of H. pylori infection. Two hundred thirty-nine par-ticipants were genotyped for Se and Le and tested for the presence of anti-H. pylori IgG antibodies. Using the normal gastric mucosa from 60 gastric cancer patients, we then assessed immunohisto-chemically whether type I Le antigen expression depended on the Se and Le genotype. The H pylori infection rate was positively associated with the number of Se alleles (se/se group, 45.1%; Se/se group, 64.6%; and Se/Se group, 73.3%) and nega-

tively associated with the number of Le alleles (le/le group, 76.4%; Le/le group, 68.3%; and Le/Le group, 55.6%). When the subjects were classified into three groups [low risk, (se/se, Le/Le) genotype; high risk, (Se/Se, le/le), (Se/Se, Le/le), and (Se/se, le/le) genotypes; moderate risk, other than low- or high-risk group], the odds ratio relative to the low-risk group was 3.30 (95% confidence interval, 1.40-7.78) for the moderate-risk group and 10.33 (95% confidence interval, 3.16-33.8) for the high-risk group. Immunohistochemical analysis supported the finding that Se and Le genotypes af-fected the expression of H. pylori adhesin ligands. We conclude that Se and Le genotypes strongly af-fect susceptibility to H. pylori infection.

*1 Division of Cell Biology, Institute of Life Science, Soka University

*2 Division of Gastroenterological Surgery, Aichi Can-cer Center Hospital

*3 Epidemiology and Prevention, Aichi Cancer Center Research Institute

21

Professor Youlin Qiao, from Cancer Institute and Hospital Chinese Academy of Medical Sciences and Peking Union Medical College giving us the lecture entitled “ Helicobacter pylori Seropositivity and Cardia Stomach Cancer: Positive Association in a Prospective, Nested Case-cohort Study from Linxian, China“ in the 8th Aichi Cancer Center International Symposium held on February 16, 2002 (see p. 89).

22

From left to right First row: Dr. T. Nakagawa, Mr. Y. Tatematsu, Dr. H. Nagai and Dr. S. Tomida. Second row: Dr. A. Masuda, Dr. T. Takeuchi, Dr. H. Osada, Dr. K. Koshikawa, Dr. H. Konishi, Dr. Ta.

Takahashi, Dr. K. Mizuno, Ms. T. Harano and Dr. H. Saito.

23

Division of Molecular Oncology ________________________________________________________________________________ Takashi Takahashi, M.D., Ph.D., Chief Hirotaka Osada, M.D., Ph.D., Section Head Akira Masuda, Ph.D., Senior Researcher Ken-ichi Kozaki, D.D.S., Ph.D., Senior Researcher (until Dec. 2000) Hiroyuki Konishi, M.D., Ph.D., Senior Researcher (as of Jul. 2001) Hiroko Saito, Ph.D., Research Assistant Tomoko Harano, B.S., Research Assistant Yoshio Tatematsu, B.S., Research Assistant Postdoctoral Fellows Hiroyuki Konishi, M.D., Ph.D. (until Jun. 2001) Toshiyuki Takeuchi, Ph.D. (as of Jul. 2001) Visiting Trainees Kiyoshi Yanagisawa, M.D., Nagoya University School of Medicine (until Nov. 2000) Nobuhiro Haruki, M.D., Nagoya City University School of Medicine (until Oct. 2000) Katsumi Koshikawa, M.D., Nagoya University School of Medicine (as of May 2000) Taku Nakagawa, M.D., Nagoya University School of Medicine (as of Apr. 2000) Kotaro Mizuno, M.D., Nagoya City University School of Medicine (as of Sept. 2000) General Summary Lung cancer is soon expected to become the leading cause of cancer death in Japan, currently claiming nearly 50,000 lives annually. Our goal is to understand the molecular pathogenesis of this fatal disease, as a basis for designing novel strategies for better diagnosis, treatment and prevention. Accumulating evidence indicates that lung cancer is a disease caused by accumulation of multiple genetic defects and our previous studies identified multiple tumor suppressor genes and dominant oncogenes in-volved in its pathogenesis. In addition, aiming at an in vitro model system suitable for analysis of various aspects of lung carcinogenesis, we have established and extensively characterized peripheral lung epithelial cell lines, termed HPL1A to E, which are, to our knowledge, the first immortalized human peripheral airway cells. Metastasis is a major cause of cancer-related deaths and we are also devoting ourselves to investigate the underlying mechanisms with the goal of control of this life-threatening process. We are continuing to focus on molecular pathogenesis of lung cancer, employing a variety of strategies for its better understanding in line with realization of our major objective, "from bench top to bedside". 1. Multi-faceted analyses of a highly me-

tastatic human lung cancer cell line NCI-H460-LNM35 suggest mimicry of in-flammatory cells in metastasis

Kozaki, K., Koshikawa, K., Osada, H., Konishi, H., Tatematsu, Y., Miyaishi, O.*1, Saito, H., Hida, T.*2, Mitsudomi, T.*3 and Takahashi Ta.

Despite considerable advances in the under-standing of the molecular pathogenesis of lung cancer, the majority of patients eventually die be-cause of widespread metastases. To date, various molecules have been suggested as playing a role in the underlying processes, but it is also evident that we still need to learn more about how cancer cells metastasize to distant organs. To propel studies on the molecular mechanisms of lung cancer metasta-

sis, we have established a highly metastatic human lung cancer cell line, NCI-H460-LNM35 (hereafter referred to as LNM35), which is capable of sponta-neous metastasis, not only via hematogenous but also lymphogenous routes, with a 100% incidence. To gain insight into the molecular mechanisms of the highly metastatic capability of LNM35, ge-nome-wide screening for genes differentially ex-pressed in LNM35 and a representative low metas-tatic clone (NCI-H460-N15, hereafter referred to as N15) of NCI-H460, the parental line for LNM35, was performed with the aid of a microarray con-taining 9,844 cDNA fragments. This search con-sequently identified 13 genes with a more than 2.5-fold up-regulation in LNM35, whereas a more than 2.5-fold down-regulation was detectable for 15 genes. Our previous study showed increased

24

COX-2 expression in LNM35 and significant inhi-bition, although in vitro, of motility and invasion as a result of treatment with nimesulide, and we here noted up-regulation of various other genes known to be related to inflammation, including a proin-flamatory cytokine IL-1α, two C-X-C chemokines (i.e., ENA-78 and NAP-2), complement component 3 and a complement-activating factor, CD55 in the present microarray analysis. In addition, we cloned and characterized a novel gene, CLCP1, whose corresponding EST was identified in our ex-pression profiling analysis. CLCP1 expression was confirmed by Northern blot analysis to be up-regulated during in vivo selection of LNM35, and increase was also identified in a significant fraction of human lung cancer cases in vivo, espe-cially in lymphogenous metastatic sites. In fact, our results indicate increased CLCP1 expression to be present in 36% of primary sites, in 58% of lymph node metastases and in 33% of distant me-tastases. Taken together, these findings indicate that future studies with our unique model system are warranted in order to improve understanding of the molecular mechanisms of lung cancer metasta-sis, in the hope that this may ultimately provide clues as to how to reduce the present, extremely large number of lung cancer deaths. *1 Department of Basic Gerontology, National Institute

for Longevity Sciences *2 Department of Internal Medicine, Aichi Cancer Cen-

ter Hospital *3 Department of Thoracic Surgery, Aichi Cancer Cen-

ter Hospital 2. Gene silencing by aberrant DNA me-

thylation and abnormalities in chroma-tin configuration in human lung cancer cells

Osada, H., Tatematsu, Y., Yatabe, Y.*1, Masuda, A., Konishi, H., Harano, T., Nakagawa, T., Saito, T.*2, Sugiyama, M.*2, Yanagisawa, K., Takada, M.*3 and Takahashi, Ta.

TGF-β strongly inhibits epithelial cell prolifera-tion and alterations of its signaling are thought to play a role in tumorigenesis. We have found that most lung cancer cell lines demonstrated loss of the growth-inhibitory responses to TGF-β, and the af-fected tumors being divided into two major groups, TGFβRII (+)/Smad7(+) and TGFβRII (-)/Smad7(-), suggesting heterogeneity in mechanisms underlying TGF-β response. The mechanism of the loss of

TGFβRII expression in the latter group was further studied, identifying aberrant DNA methylation of the promoter region in a limited fraction of cell lines. Interestingly, we found that alteration of chromatin structure because of histone deacetyla-tion may also be involved, showing a good correla-tion with loss of TGFβRII expression. This notion was supported by the findings of a restriction en-zyme accessibility assay, of a chromatin immuno-precipitation assay with anti-acetyl histone anti-bodies, and of in vivo induction of TGFβRII ex-pression by histone deacetylase inhibitors including trichostatin A (TSA) and sodium butyrate. In vitro induction of TGFβRII promoter reporter activity by TSA was also detected and found to require a CCAAT box within the –127/-75 region. This is the first study to demonstrate that, in addition to the TSA-responsive region in the TGFβRII promoter, the alteration of histone deacetylation may be in-volved in loss of TGFβRII expression in lung can-cer cell lines. We also identified another target for epigenetic alteration in lung cancers, i.e, 14-3-3σ, one isoform of the 14-3-3 family, which plays a role in the G2 checkpoint by sequestering Cdc2-cyclinB1 in the cytoplasm. Loss of expression has been suggested to cause a G2 checkpoint defect, resulting in chro-mosomal aberrations. Our recent study on DNA methylation status and expression level of the 14-3-3σ gene in lung cancer cell lines and primary lung tumor specimens revealed small cell lung can-cer (SCLC) cell lines to frequently feature DNA hypermethylation (~70%) and silencing of the 14-3-3σ gene. Among non-small cell lung cancers (NSCLC), only large cell lung cancer cell lines showed frequent hypermethylation and silencing of 14-3-3σ (~60%), whereas hypermethylation oc-curred very rarely (6%) in other types including squamous cell carcinomas and adenocarcinomas. All eight primary SCLC specimens examined also showed loss or significant reduction of 14-3-3σ ex-pression in vivo, while this was very rare in primary NSCLC specimens (5%). This is the first descrip-tion that indicates lung cancers frequently show significant inactivation of the 14-3-3σ gene, mainly due to DNA hypermethylation, in SCLC, but rarely in NSCLC, suggesting involvement of the 14-3-3σ gene in lung tumorigenesis in a histological type-specific manner. These epigenetic changes in lung cancers may be of considerable clinical inter-est, because their potential reversible nature may be regarded as a rational target for development of novel therapeutic measures.

25

*1 Department of Pathology and Molecular Diagnostics,

Aichi Cancer Center Hospital. *2 Laboratory of Ultrastructural Research (until March

1999) *3 Pulmonary Medicine, Rinku General Medical Center. 3. Persistent increase in chromosome in-

stability in lung cancers Haruki, N., Masuda, A., Harano, T., Kiyono, T.*1, Takahashi, Takao*2, Tatematsu, Y., Shimizu, S.*3, Mitsudomi, T.*3, Konishi, H., Osada, H., Fujii, Y. *4 and Takahashi, Ta.

Lung cancer cells have been shown to exhibit frequent chromosomal abnormalities, from both numerical (i.e. aneupoidy) and structural points of view. These are believed to contribute to tumor de-velopment and progression by facilitating loss of heterozygosity and inactivation of tumor suppressor genes, as well as favoring polysomy of chromo-somes that habor growth-promoting genes. How-ever, it has not been directly established whether aneuploidy is in fact associated with a persistent in-crease in the rate of chromosomal losses and gains (i.e. chromosomal instability or CIN). To clarify whether CIN is a common feature in lung cancer cell lines in association with the pres-ence of significant aneuploidy, we examined the rates of divergence of chromosome numbers in 10 lung cancer cell lines during cultivation by means of fluorescence in situ hybridization with specific centromere probes. Then, we examined the corre-lation of degree of CIN with various other molecu-lar and cellular parameters in lung cancer cell lines, such as the presence of mutations in p53, degree of aneuploidy, morphology of centrosomes, and integ-rity of the mitotic spindle checkpoint. As results, we found that lung cancer cell lines exhibiting sig-nificant aneuploidy showed a persistent CIN phe-notype. In addition, the CIN phenotype correlated well with the presence of p53 mutations, and mod-erately with centrosome abnormalities. However, human papilloma virus 16-E6-directed inactivation of p53 in a representative non-CIN lung cancer cell line did not result in the induction of CIN, at least up to the 25th generation, suggesting that inactiva-tion of this tumor suppressor gene is itself unlikely to directly induce CIN in lung cancer cells. Inter-estingly, however, significant CIN could be induced in p53-inactivated cells in conjunction with the generation of aneuploid populations, when mitotic spindle formation was transiently abrogated with a

microtubule interfering reagent. In the present study, we provided clear evidence for the first time that the majority of human lung cancer cell lines, which exhibit significant ane-uploidy, show persistent CIN. The results suggest that inactivation of p53 may allow lung cancer cells to go through an inappropriate second division cy-cle under certain forms of mitotic stress, which would result in the induction of the CIN phenotype in conjunction with generation of aneuploidy. Fur-ther studies are now needed to identify and clarify the underlying mechanisms directly responsible for the induction of CIN. Their clarification should provide important clues to the development of new therapeutic approaches for this fatal cancer. *1 Laboratory of Viral Oncology *2 Laboratory of Ultrastructural Research (until March

1999) *3 Department of Thoracic Surgery, Aichi Cancer Cen-

ter Hospital *4 Department of Surgery II, Nagoya City University

School of Medicine

4. Identification of frequent G2 checkpoint

impairment and a homozygous deletion of 14-3-3ε at 17p13.3 in small cell lung cancers

Konishi, H., Nakagawa, T., Harano, T., Mizuno, K., Saito, H., Masuda, A., Osada, H. and Takahashi Ta.

It has been shown that the short arm of chromo-some 17 (17p) is one of the most frequently af-fected chromosomal regions in lung cancers, as well as in a variety of other human neoplasias. Although the p53 gene at 17p13.1 is well accepted as a genu-ine molecular target in the frequent 17p deletions, we previously performed a detailed LOH analysis for 17p using 100 cases of primary lung cancers representing all four major histologic subtypes, and suggested that, in addition to the p53 gene, an as yet unidentified tumor suppressor gene(s) residing at 17p13.3 might also play a role in lung carcinogene-sis. We therefore screened 65 lung cancer cell lines with the aid of nine markers mapped within the commonly deleted region of lung cancers at 17p13.3. A single STS marker corresponding to the 14-3-3ε gene was consequently found to yield no amplification products in either of two SCLC cell lines on duplex PCR analysis. The two SCLC cell lines, ACC-LC-48 and ACC-LC-52, had been established from distinct metastases of the same pa-

26

tient after different treatment periods, suggesting the occurrence of homozygous deletions before metastasis in vivo. Northern blot analysis showed complete absence of 14-3-3ε expression in the two SCLC cell lines. 14-3-3ε is one of the seven 14-3-3 isoforms thus far identified, which form a complex with a variety of molecules. Among their binding partners, Cdc25C has been shown to be involved in the G2 checkpoint response, and to bind mainly to 14-3-3ε among the seven 14-3-3 isoforms, in Xenopus egg extract. Although 14-3-3ε also forms a complex with Cdc25C in human cells, it is not clear which 14-3-3 isoform actually plays a significant role in the G2 checkpoint response. We therefore exam-ined the function of the G2 checkpoint using a 14-3-3ε-null ACC-LC-48 cell line. The mitotic index in ACC-LC-48 after one Gy irradiation re-mained at over 50% of the non-irradiated value, suggesting an inefficient G2 arrest and G2 check-point impairment. In addition, the G2 checkpoint response could be restored to a significant extent by both transient and stable introduction of exogenous 14-3-3ε into ACC-LC-48. These observations indi-cate that the homozygous loss of 14-3-3ε perturbs the G2 checkpoint response to x-ray-irradiation in ACC-LC-48. Next we examined mitotic indices af-ter exposure to one Gy irradiation in an additional seven SCLC cell lines, and found the G2 check-point response to be frequently impaired to various degrees. The G2 checkpoint is one of the most highly conserved mechanisms that regulate the cell cycle by preventing damaged cells from progression through the cell cycle. Our finding that a significant fraction of SCLCs, which are very sensitive to irra-diation and chemotherapy and at the same time the most aggressive type of lung cancers, exhibit an abnormal G2 checkpoint response is thus of great interest not only from a biological point of view but also in terms of clinical implications. 5. In vitro molecular analysis of carcino-

genesis of human lung adenocarcino-mas with the aim of clinical applications

Masuda, A., Konishi, H., Yatabe, Y.*1, Hida, H. *2, Saito, T. *3 and Takahashi, Ta.

It is well known that oncogenes such as K-ras, c-myc and c-jun are often activated or overex-pressed in human lung cancers. However, the pre-cise roles of such oncogene activation in the devel-

opment and progression of human lung cancers are not yet fully understood. In the present study, we examined effect of modulation of signaling using a newly established immortalized human peripheral lung epithelial cell line (HPL1D) and an immortal-ized lung airway epithelial cell line (BEAS2B), with the aim of identifying molecular targets for future therapeutic modalities. We observed that a transformed cell-like mor-phology could be induced in HPL1D in response to EGF under the anchorage-dependent conditions and that apoptosis with anchorage-independence was markedly reduced, consequently resulting in colony formation in soft agar. Interestingly, although an-chorage-dependent proliferation of HPL1D was markedly enhanced with HGF, which is known to stimulate cell proliferation via signal transduction pathways similar to those elicited with EGF, HGF did not induce anchorage-independent cell growth at all. We are currently investigating the mecha-nisms underlying these differential responses. In addition, the biological effects of oncogene activation were examined by establishing stable transfectants of BEAS2B, in which oncogenes were placed under the regulation by tetracycline-off sys-tem. Addition of either EGF or FCS failed to in-duce endogenous c-jun under anchor-age-independent conditions and did not lead to formation of soft agar colonies. In contrast, ex-pression of exogenously introduced c-jun consid-erably stimulated anchorage-independent colony formation. Interestingly, eight of 18 human lung cancer cell lines exhibited loss of the anchor-age-dependence of c-jun induction. Furthermore, we could show that introduction of activated K-ras alone was insufficient to induce anchor-age-independent colony formation, but that an-chorage-independent cell growth could be stimu-lated in conjunction with the forced expression of c-jun, suggesting cooperative roles in transforma-tion of lung epithelial cells. Taken together, these results point to the utility of immortalized normal human lung epithelial cells for understanding tumorigenesis in the lung. *1 Department of Pathology and Molecular Diagnostics,

Aichi Cancer Center Hospital. *2 Department of Internal Medicine, Aichi Cancer Cen-

ter Hospital *3 Laboratory of Ultrastructural Research (until March

1999)

27

Dr. Roderich E. Schwarz, from University of Medicine and Dentistry of New Jersey Robert Wood John-son Medical School, U.S.A, giving us the lecture entitled “Surgery and Adjuvant Therapy for Gatsric Carcinoma in the U.S.A.“ in the 8th Aichi Cancer Center International Symposium held on February 16, 2002 (see p. 93).

28

From left to right Front row: Dr. M. Suguro-Katayama, Ms. Y. Kasugai, Ms. H. Suzuki, Dr. M. Seto and Dr. Y. Hosokawa Back row: Dr. R. Suzuki, Dr. H. Tagawa, Dr. Dr. K. Mayama, Dr. K. Izumiyama, Dr. S. Tsuzuki and Mr.

S. Karnan

29

Division of Molecular Medicine ________________________________________________________________________________ Masao Seto, M.D., Dr.M.Sc. Chief Yoshitaka Hosokawa, M.D., Dr.M.Sc. Section Chief Ryoji Ishida, Ph.D. Senior Researcher Ritsuro Suzuki, M.D., Dr.M.Sc., Senior Researcher Shinobu Tsuzuki, M.D., Ph.D., Senior Researcher (As of October, 2001) Keiko Nishida, B.P. Senior Research Assistant (Until March, 2000) Hiroko Suzuki, B.P. Senior Research Assistant Yumiko Maeda, B.S. Research Assistant Hiroyuki Tagawa, M.D., Ph.D. Research Resident (As of April, 2000) Visiting Trainees Yoshitoyo Kagami, M.D., Department of Hematology and Chemotherapy, Aichi Cancer Center Hospital Hidenobu Takahashi, M.D., The First Department of Internal Medicine, Niigata University School of Medicine (Until September, 2000) Masakatsu Yonezumi, M.D., The Third Department of Internal Medicine, Hokkaido University School of Medicine (Until March, 2001) Ko Izumiyama, M.D., The Third Department of Internal Medicine, Hokkaido University School of Medicine (As of April, 2001) Toshihiro Nakanishi, B.P., Gifu Pharmaceutical University (Until March, 2000) Karnan Sivasundaram, Nagoya University Graduate School of Science (As of June, 2001) Miyuki Suguro-Katayama, M.D., The second Department of Internal Medicine, Mie University School of Medicine (As of July, 2001) Dr. Ko Mayama, M.D., The First Department of Internal Medicine, Hirosaki University School of Medicine (As of October 2001) 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 researchers of the De-partment of Hematology and Chemotherapy (Chief. Dr. Yasuo Morishima), and the Pathology and Clinical Laboratories (Dr. Shigeo Nakamura). Hematologic malignancies have several advantages for studying the molecular bases of neoplasia. Chromosomal abnormalities have been analyzed by a large number of re-searchers and the observed strong association between specific chromosome changes and specific hemato-poietic tumors provides direct evidence that the resultant gene alterations play a pivotal role in the disease development. Over the last two years we have concentrated attention on the 18q2l translocation (associated with the mucosa-associated lymphoid tissue lymphoma), the 3q27 translocation (associated with diffuse lar-ge cell lymphomas), and the 11q13 (associated with mantle cell lymphomas). The second advantage of studying hematopoietic malignancies is that the various leukemias and lymphomas have been classified in detail with respect to cell surface markers, developmental lineages and stages, so that they can be used for studying important factors and signals for cell differentiation and proliferation. Indeed, we could identify subsets of diffuse large B-cell lymphoma with cell surface markers. Finally, we are also trying to analyze T-cell malignancies, most of whose chromosome aberrations are not well understood. We could identify the TCBA1 gene, a candidate that may be involved in chromosome 6q aberrations. 1. Search for MALT1-associated proteins

using yeast two-hybrid strategy Hosokawa, Y., Suzuki, H., and Seto, M.

The category of mucosa-associated lymphoid tissue (MALT) lymphoma was first proposed by

Isaacson et al. and it is now clearly recognized and categorized as extranodal marginal zone lymphoma of MALT type in REAL classification. It often originates from chronic inflammation, such as H. pylori gastritis or autoimmune disease, tends to re-main localized for a long time and mostly exhibits

30

an indolent clinical course. The t(11;18) translo-cation has been reported as a characteristic chro-mosomal abnormality in this lymphoma, and recent studies including ours have demonstrated that this translocation results in the fusion of two genes, API2 at 11q21 and a novel gene, MALT1, at 18q21. API2 is a member of the IAP (inhibitor of apop-tosis) gene family, whereas the precise function of MALT1 remain obscure. To identify possible regulators and clarify signal transduction pathway of MALT1, the yeast two-hybrid strategy was em-ployed. Using the two domains of MALT1, namely the Ig-like domain and the caspase-like domain, as baits, cDNAs were isolated from human bone marrow and testis pretransformed cDNA li-braries. Two of cDNAs most frequently isolated from these libraries were found to encode metal-lothionein 2A and galectin-1. Metallothionein (MT) is a zinc-binding proteins that play an impor-tant regulatory role in zinc uptake, distribution, storage, and release. It has been demonstrated that MT2A is involved in the interaction with the NF-kB DNA-binding domain, suggesting a poten-tial role for NF-kB in mediating the anti-apoptotic effects of MT2A. It has also been demonstrated that galectin-1, a β-galactoside-binding protein, plays a crucial role in apoptosis. Thus, further studies of two MALT1-associated proteins should highlight not only a novel signal transduction pathway of MALT1 but also a role of API2-MALT1 chimeric protein in the molecular pathogenesis of MALT lymphoma. 2. Detection of API2-MALT1 chimeric

transcripts involved in mu-cosa-associated lymphoid tissue lym-phomas by a single touchdown multi-plex polymerase chain reaction

Suguro, M., Suzuki, R., Nakamura, T.*1, Suzuki, H., Hosokawa, Y., Nakamura, S.*2 and Seto, M.

T(11;18)(q21;q21), which results in a chimeric transcript between API2 at 11q21 and MALT1 at 18q21, is a characteristic chromosome aberration in extranodal marginal zone B-cell lymphomas occur-ring in (MALT). API2-MALT1 chimeric tran-scripts are present in 20-30 % of gastric MALT lymphomas and prognostic of lymphoma resistance to antibiotic eradication of Helicobactor pylori. Detection of API2-MALT1 fusion transcripts is al-so important for MALT lymphomas originating in tissues other than the stomach because it represents direct evidence of clonal expansion of lymphoma

cells. The reverse transcriptase-polymerase chain reaction is a major tool for the detection of API2-MALT1. However, multiple PCR reactions are occasionally necessary to cover all possible chimeric transcripts, because at least four different fusion-points in API2 and MALT1 mRNAs may be involved. We have therefore established a touch-down multiplex PCR to detect API2-MALT1 chi-meric transcripts in a single reaction. All five variants from patient samples with t(11;18) (q21;q21) showed specific amplification with this approach and detection of 100 copies of API2-MALT1 transcripts was verified. We could also demonstrate specific amplification with a pe-ripheral blood sample of a MALT patient, indicat-ing that the established multiplex touchdown in the present study PCR assay is sensitive and specific. *1 Department of Gastroenterology *2 Department of Pathology and Clinical Laboratories

3. Detection of cyclin D1 overexpression

by real-time reverse-transcriptase me-diated quantitative polymerase chain reaction for the diagnosis of mantle cell lymphoma

Suzuki, R., Takemura, K.*1, Tsutsumi, M.*1, Nakamura, S.*2, Hamajima, N.*3 and Seto, M.

The diagnosis of mantle cell lymphoma (MCL) is particularly important for clinical management because of the remarkable prognostic difference between this and other types of B-cell lymphoma. In addition to immunohistochemical analysis, we have established a 5’ exonuclease-based real-time reverse-transcriptase mediated quantitative poly-merase chain reaction (RQ-PCR) method to detect cyclin D1 overexpression for diagnostic. The RQ-PCR could detect cyclin D1 overexpression in all 9 MCL cases, in contrast to genomic PCR, which detected t(11;14) in only 2 of the 9 cases. With RQ-PCR the expression of G6PDH was sig-nificantly higher in myeloid leukemias than in B-cell lymphomas (p=0.018). As a result, the cy-clin D1/G6PDH ratio ranged from 0.78 to 12.4 (mean: 1.83) in MCL, very much higher than those for other B-cell lymphomas (0.00009~0.16) and myeloid leukemias (0.00011~ 0.085). Elevated expression of cyclin D1 in certain myeloid leuke-mias was found to reflect their proliferative activity and not to represent oncogenic overexpression. The 95% confidence interval of the cyclin D1/G6PDH ratio was 0.29~11.1 for MCL,

31

0.014~0.25 for other B-cell lymphomas and 0.000014~0.083 for myeloid leukemias, suggesting that the cutoff value can be set at 0.25. RQ-PCR for cyclin D1 is convenient and particularly useful for the diagnosis of MCL. *1 SRL Inc., Tokyo, Japan. *2 Department of Pathology and Genetics

*3 Division of Epidemiology and Prevention, Aichi Cancer Center, Nagoya

4. Molecular cytogenetic analysis of the breakpoint region at 6q21-22 in T-Cell lymphoma/leukemia cell lines

Tagawa, H., Miura, I.*1, Suzuki, R., Hosokawa, Y. and Seto, M.

Chromosome band 6q21 is reported to be one of the most frequent target regions in T-cell lympho-mas for both translocations and deletions. In order to explore whether the breakpoint clustering in T-cell malignancies indicates the presence of a common breakpoint region in 6q, we employed fluorescence in situ hybridization analysis using various YAC, BAC, and PAC clones aligned at 6q21-22. We identified two T-cell lym-phoma/leukemia cell lines with different differen-tiation stages that had breakpoints within the same novel gene, TCBA1 (T-cell lymphoma breakpoint associated target 1). In a T-cell lymphoblastic lym-phoma cell line, HT-1, the TCBA1 fused to SUSP1 (SUMO-1-specific protease) creating a SUSP1-TCBA1 chimeric gene. However, in an adult T-cell leukemia cell line, ATN-1, no chimeric gene was detected although aberrant TCBA1 transcripts were produced. It can be strongly inferred that TCBA1 is a target of 6q21 aberrations in T-cell ma-lignancies because two independent T-cell lym-phomas feature breakpoints in this gene. Alterna-tively, TCBA1 may be a fragile site at 6q in T-cell lymphomas. It is also possible that TCBA1 gene translocation causes deregulation of juxtaposed genes, like immunoglobulin translocations in B-cell malignancies. *1 Third Department of Internal Medicine, Akita Uni-

versity School of Medicine

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From left to right First row: Ms. Y. Matsudaira, Dr. K. Kuzushima and Ms. Y. Nakao. Second row: Dr. E. Kondo, Dr. K. Tsujimura, Dr.

To. Takahashi, Ms. H. Tamaki, Dr. K. Tajima, Ms. K. Nishida, Dr. M. Miyazaki, Dr. Y. Akatsuka and Dr. T. Nishida.

Insets: Dr. Y. Obata and Mr. S. Iwase.

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Division of Immunology ________________________________________________________________________________ Toshitada Takahashi, M.D. Chief Yuichi Obata, Ph.D. Section Head (until March 2001) Kunio Tsujimura, M.D. Senior Researcher Yoshiki Akatsuka, M.D. Senior Researcher (as of July 2000) Yasue Matsudaira, B.S. Senior Research Assistant Keiko Nishida, B.P. Senior Research Assistant (as of April 2001) Satoshi Ozeki, D.V.M. Research Assistant (until March 2001) Visiting Scientists Kazuhiro Yoshikawa, B.M.T., M.D. Second Department of Pathology, Aichi Medical University Yuichi Obata, Ph.D. Head, Department of Biological Systems, RIKEN BioResource Center (as of April 2001) Visiting Trainees Shigeru Iwase, B.P. Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Nagoya City University Keigo Mizutani, M.D. Department of Pediatrics, Nagoya City University School of Medicine Eisei Kondo, M.D. Second Department of Internal Medicine, Okayama University School of Medicine Kouhei Tajima, M.D. First Department of Surgery, Gunma University School of Medicine Masahiro Yoshida, M.D. Department of Orthopedic Surgery, Nagoya University School of Medicine Mikinori Miyazaki, M.D. Second Department of Internal Medicine, Nagoya City University School of Medicine Tetsuya Nishida, M.D. First Department of Internal Medicine, Nagoya University School of Medicine

General Summary The object of our research is to characterize the biological nature of cancer cells, with emphasis on im-munogenetic analysis of cell-surface molecules of both human and experimental tumors. The major projects undertaken over the past two years are summarized below. In the field of human tumor antigens, three projects are in progress. Firstly, production of single chain an-tibodies (scFv) reactive with a truncated type of the epidermal growth factor receptor (EGFR) expressed on glioblastomas was achieved and their application for tumor imaging has been investigated. Secondly, we have carried out an antigen analysis of stomach, breast, and prostate cancers using the expression cloning method (SEREX). Fifty to 100 cDNA clones encoding antigens detected by autologous IgG antibodies were selected for each type of cancer and are now being analyzed. Thirdly, an attempt has been made to detect minor histocompatibility antigens by generating cytotoxic T cells (CTL) from HLA identical bone marrow transplantation patients. In addition, an efficient in vitro CTL generation method was established using retro-virally transduced B cells as antigen-presenting cells. In the second area of interest, studies on mouse tumor antigens have been conducted with the thy-mus-leukemia (TL) antigen as a model. Immunization with dendritic cells engineered to express TL is able to induce rejection of TL positive lymphoma cells. In addition, in order to monitor TL-specific CTL, TL tetramers were prepared. Unexpectedly, these proved reactive not only with TL-specific CTL, but also with normal intestinal intra epithelial lymphocytes and thymocytes.

1. Production of a single chain variable

fragment (scFv) antibody against type III mutant EGFR

Yoshikawa, K.*1, Nakayashiki, N.*1, 2, Takasu, S.*1, 2, Okamoto, K.*1, 2, Nakamura K.*3, Hanai, N.*3, Okamoto, S.*2, Mizuno, M.*2, Wakabayashi, T.*2, Saga, S.*1, Yoshida, J.*2 and Takahashi, To.

The type III deletion-mutant of the epidermal growth factor receptor (EGFR) is a potential target

in diagnostic and therapeutic approaches for glio-blastomas characterized by its expression. In this study, a single chain variable fragment (scFv) antibody was produced, based on the mouse mono-clonal antibody, 3C10 (IgG2b) specifically recog-nizing this mutant EGFR. (Nakayashiki, et al., Jpn. J. Cancer Res., 91, 1035-1043, 2000). Partial de-termination of its N-terminal amino acid sequence and preparation of adequate primers for VH and VL genes were assembled with a linker, (Gly4Ser)3 and

34

ligated into a bacterial expression vector to express the scFv as cytoplasmic inclusion bodies. After ap-propriate refolding, the scFv were purified using a mutant peptide-conjugated column. On Biacore analysis, the affinity (KA) of the parental 3C10 for the mutant peptide was 9.7 x 107 M-1, while that of 3C10 scFv was 2.45-2.48 x 107 M-1, approximately 4 fold weaker. On ELISA, 3C10 scFv showed se-lective reactivity with the mutant peptide, similarly to the parental 3C10 antibody. Immunostaining analysis revealed scFv staining in a glioblastoma case with type III mutant EGFR, the biodistribution of 99mTc-labeled 3C10 scFv being evaluated in athymic mice bearing transformants expressing the mutant EGFR. 99mTc-labeled 3C10 scFv accumu-lated in tumors, with a tumor/blood radio of 3-5, which was, however found to be lower than that with the parental antibody (12-18). The results al-together suggest that the scFv antibody still main-tains the antibody structure to detect a conforma-tional epitope, similarly to the parental antibody. *1 Second Department of Pathology, Aichi Medical

University *2 Department of Neurosurgery, Nagoya University

School of Medicine *3 Tokyo Research Laboratories, Kyowa Hakko Kogyo

Co. Ltd. 2. Immunogenic gene products in cancer

patients Obata, Y.*1, Takahashi, To., Tamaki, H., Tajima, K., Yoshida, M., Miura, S.*2, Iwase, T.*2, Iwata, H*2, Mitsudomi, T.*3, Takahashi, M.*4, Sakamoto, J.*5, Chen, Y.-T.*6, Stockert, E.*6 and Old, L.J.*6

Molecular characterization of cancer antigens recognized by cytotoxic T cells by Dr. Boon's group has opened a new era of cancer immunology. The search for cancer antigens that can be used for im-munodiagnosis and immunotherapy of cancer is still at a very early stage. For their identification, several methods using cytotoxic T cells have been developed. In addition, a molecular technique using antibodies produced in cancer patients was also de-vised by Dr. Pfreundschuh and his colleagues. This technique, named SEREX (serological analysis of cancer antigens by recombinant cDNA expression cloning), identifies protein antigens that have elic-ited high titer IgG responses in cancer patients. Ex-pression cDNA phage libraries constructed using mRNA from tumor specimens are screened with autologous or allogeneic sera from cancer patients.

Positive clones are isolated and sequenced for iden-tification of genes encoding antigens. One of the advantages of the SEREX method in identification of cancer antigens is the lack of any requirement for establishing cultured cell lines from tumor speci-mens, a process that is very difficult for most epithelial cancers. Another advantage is direct iden-tification of genes encoding antigens through DNA sequencing. Since the introduction of the method, SEREX analysis targeting various cancer types has defined several categories of antigens with potential use in the clinical setting: cancer-testis (CT) anti-gens, mutational antigens, fusion antigens, differen-tiation antigens, over-expressed antigens, spliced-variant antigens and viral antigens. In our own SEREX study, we have surveyed so far five gastric, three prostate, two breast, one lung and one colon cancer libraries. The immune sys-tems of the cancer patients were found to be sur-prising extremely active, with antibodies production against diverse sets of gene products. Nearly 500 antigens were identified. To select examples useful for diagnosis and therapy, the genes and their prod-ucts were analyzed for; (1) “DNA alterations” such as mutation, splicing abnormality and translocation; (2) “cancer-restricted expression” by examining the presence of transcripts in cancer and normal tissues; and (3) “cancer-restricted immune recognition” by examining the frequency of antibody production in cancer patients and control individuals. Characteri-zation of these and future SEREX-defined antigens promises identification of potential targets for im-munodiagnosis and immunotherapy of cancer, as well as genes that are significant in cancer biology. *1 RIKEN BioResource Center *2 Department of Breast Surgery *3 Department of Thoracic Surgery *4 Department of Orthopedic Surgery *5 Aichi Hospital *6 Ludwig Institute for Cancer Research 3. Targeted cloning of cytotoxic T cells

specific for minor histocompatibility an-tigens restricted by HLA class I mole-cules of interest

Akatsuka, Y., Kondo, E., Nishida, T., Taji, H.*1, Morishima, Y.*1, Obata, Y.*2, Kodera Y.*3 and Takahashi, To.

Minor histocompatibility antigens (mHAs) are MHC (HLA in human)-associated peptides origi-nating from polymorphisms in the genome that

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trigger T cell responses between MHC identical al-logeneic individuals. Graft-versus-host disease (GVHD) and graft-versus-tumor (GVT) effects in hematopoietic stem cell transplant (HCT) recipients are initiated by donor T cell recognition of mHAs on recipient cells, and it has been suggested that particular mHAs may be selectively involved in GVHD or GVT reactions. Thus, identification of novel mHAs, especially restricted by HLA-A24 which is common in Japanese, is important to spec-ify recipients at risk of severe GVHD and antigenic targets for immunotherapy to augment GVT re-sponses. We have developed a novel approach to isolate T cell clones restricted by HLA class I alleles of in-terest directly from cytotoxic T lymphocyte (CTL) cell lines using interferon (IFN)-γ-based techniques. CTL lines were successfully generated from 3 of 4 patients entered in this study. Enzyme-linked im-munospot (ELISPOT) assays were first performed to identify the HLA alleles presenting mHAs to the CTL lines using panels of B-LCL that do not share any HLA alleles with the recipients and engineered to express each one of the recipient HLA-A, B and C alleles. ELISPOT assays conducted on 2 CTL lines generated from peripheral blood specimens of 2 patients early after HCT demonstrated almost all T cells in these CTL lines to be restricted by HLA-B44 and HLA-A24, respectively. Multiple HLA alleles were used as restriction molecules in the other 3 CTL lines generated relatively long after HCT. IFN-γ secreting T cells were then positively selected in response to stimulation with the B-LCL expressing the HLA alleles selected based on the ELISPOT results, and then directly cloned. We have successfully cloned 2 distinct HLA-B44-restricted CTL clones and 4 distinct HLA-A24-restricted CTL clones. Of note is that HLA-A24-restricted CTL clones were obtained from all of 3 HLA-A24 positive patients with this approach. All CTL clones showed hematopoietic lineage-specific cytotoxicity, so it can be expected that they will recognize mHAs useful for immuno-therapy of hematopoietic malignancies. We are currently identifying genes encoding mHAs recog-nized by these CTL clones. This new method could potentially be applied to isolate T cell clones that recognize any antigen in the context of a specific HLA allele of interest. *1 Department of Hematology and Chemotherapy *2 RIKEN BioResource Center *3 Department of Hematology, Japanese Red Cross

Nagoya First Hospital 4. Binding of thymus leukemia (TL) anti-

gen tetramers to normal intestinal intraepithelial lymphocytes and thymo-cytes

Tsujimura, K., Obata, Y.*1, Matsudaira, Y., Ozeki, S., Yoshikawa, K.*2, Saga, S.*2 and Takahashi, To.

Thymus leukemia (TL) antigens belong to the family of nonclassical MHC class I antigens and can be recognized by both TCRαβ and TCRγδ CTL with TL- but not H-2-restriction. We previously reported that the CTL epitope is TAP-independent, but antigenic molecules presented by TL have yet to be determined. In the present study, TL tetramers were prepared with T3b-TL and murine β2-microglobulin not including antigenic peptides, to determine binding specificity. CTL clones against TL antigens were stained with the T3b-TL tetramer, and the binding shown to be CD3- and CD8-dependent. Normal lymphocytes from various origins were also studied. Surprisingly, most CD8+ intraepithelial lymphocytes (IEL) derived from the small intestines (iIEL), as well as CD8+ and CD4+CD8+ thymocytes, were stained, while only very minor populations of CD8+ cells derived from other peripheral lymphoid tissues such as spleen and lymph nodes were positive. The binding of T3b-TL tetramers to CD8+ iIEL and thymocytes was CD8-dependent but CD3-independent, in contrast to that to TL-restricted CTL. These results alto-gether show that TL-restricted CTL can be moni-tored by CD3-dependent binding of T3b-TL tetram-ers. In addition, CD3-independent T3b-TL tetramer binding to iIEL and thymocytes may imply that TL expressed on intestinal epithelium and cortical thymocytes has a physiological function, interacting with these tetramer+CD8+ T lymphocytes. *1 RIKEN BioResource Center *2 Second Department of Pathology, Aichi Medical

University

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From left to right First row: Dr. T. Kiyono, Dr. T. Tsurumi, Dr. H. Nakamura, and Dr. K. Kuzushima. Second row: Dr. M. Fujita, Ms. A. Kudoh, Ms. N. Hayashi, Dr. S. Nakasu, Ms. T. Yoshida, Dr. Y.

Sugaya, and Mr. Y. Nishikawa.

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Division of Virology ________________________________________________________________________________ Tatsuya Tsurumi, M.D. Chief Tohru Kiyono, M.D. Section Head (until March 2002) Kiyotaka Kuzushima, M.D. Section Head (until March 2002) Hiromu Nakamura , PhD. Section Head (from April 2000 to March 2002) Masatoshi Fujita, M.D. Senior Researcher Shou Nakasu, PhD. Senior Researcher (as of April 2000) Naoaki Yokoyama, Vet. M.D. Researcher (until September 2000) Yutaka Sugaya, PhD. Research Resident (as of April 2001) Toyoko Yoshida. Research Assistant Yasuhiro Nishikawa. Research Assistant (as of April 2000) Visiting Trainees Yo Hoshino. Department of Pediatrics, Nagoya University School of Medicine (until March 2002) Ken Fujii. Division of Biological Science, Nagoya University Graduate School of Science (until March 2001) Ayumi Kudoh. Graduate School of Science and Technology, Faculty of Science, Kumamoto University Naomi Hayashi. Department of Pediatrics, Nagoya University School of Medicine (until March 2002) Yoriko Yamashita. First Department of Pathology, Nagoya University School of Medicine (From March 2000 to March 2002) 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) and human papillomavirus (HPV) are the objects 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 lym-phomas, some gastric cancers, and B cell lymphomas in immunosuppressed patients. HPV is causally linked to cervical cancers and probably to other anogenital and also to some skin and oropharyngeal cancers. Our research aims are to elucidate the molecular mechanisms of viral DNA replication and oncogenesis of EBV and HPV as part of the world-wide effort to combat virus-infected cancers and to characterize cellu-lar immunity against EBV-associated tumors in order to contribute to clinical diagnosis and therapy. During the period 2000-2001, our research interest was concentrated on the following issues: 1) protein-protein in-teractions among EBV replication proteins; 2) purification and characterization of EBV replication pro-teins; 3) mechanisms of inhibiting re-replication during late S-G2-M phase in mammalian cells ; 4) im-mortalization of human cells by HPV; 5) determination of Epstein-Barr virus-specific CD8+ T cell fre-quencies by flow cytometry and related clinical applications; 6) identification of HLA A*2402-restricted EBV or CMV-specific CD8+ T cell epitopes by a computer algorithm and an enzyme-linked immunospot assay. 1. The Epstein-Barr Virus Pol catalytic

subunit physically interacts with the BBLF4/BSLF1/BBLF2/3 complex

Fujii, K., Yokoyama, N. and Tsurumi, T.

At a replication fork, the following enzymatic reactions play very important roles for the rapid and accurate duplication of the genetic information: 1) a DNA helicase unwinds the parental template; 2) a primase manufactures RNA primers for Okazaki fragment synthesis; 3) a DNA polymerase synthe-sizes the nascent leading and lagging strands. Dur-ing these reactions, various protein-protein interac-

tions between the replication proteins have been observed. For example, bacteriophage T7 gene 5 DNA polymerase interacts with the gene 4 heli-case/primase, while the catalytic subunit of the HSV-1 DNA polymerase interacts with the car-boxyl terminus of the UL8 protein of the HSV-1 helicase/primase heterotrimeric complex. In the case of EBV, the EBV DNA polymerase holoenzyme consists of the BALF5 protein (Pol catalytic subunit) and the BMRF1 protein (Pol ac-cessory subunit). Although enzymatic activities of the EBV BBLF4/BSLF1/BBLF2/3 heterotrimeric complex have yet to be demonstrated, we assume

38

that it may act as a helicase and primase like the HSV-1 UL5/UL52/UL8 complex. In the present study, we revealed by immunoprecipitation analy-ses that the EBV DNA Pol catalytic subunit inter-acts with the BBLF4/BSLF1/BBLF2/3 complex. The same approach using anti-BSLF1 or anti-BBLF2/3 antibodies with clarified lysates of B95-8 cells in a viral productive cycle suggested that the EBV Pol holoenzyme interacts with the BBLF4/BSLF1/BBLF2/3 complex. By experi-ments utilizing lysates from insect cells superin-fected with combinations of recombinant baculovi-ruses capable of expressing each of the viral repli-cation proteins, it was shown that not the BMRF1 Pol accessory subunit but rather the BALF5 Pol catalytic subunit directly interacts with the BBLF4/BSLF1/BBLF2/3 complex. Furthermore, double infection with pairs of recombinant viruses revealed that each component of the BBLF4/ BSLF1/BBLF2/3 complex makes contact with the BALF5 Pol catalytic subunit. The interactions of the EBV DNA polymerase with the EBV putative helicase-primase complex warrant particular atten-tion because they are thought to coordinate leading and lagging strand DNA synthesis at the replication fork. 2. Purification of the product of the Ep-

stein-Barr virus BZLF1 gene Nakasu, S. and Tsurumi, T.

The product of the BZLF1 gene (pBZLF1) of Epstein-Barr virus (EBV) encodes a nuclear protein which is activators of the lytic cycle in cells latently infected with EBV. pBZLF1 is suggested to acti-vate the genes required for the lytic cycle and in-duction of viral DNA replication as a DNA binding protein specific for the viral lytic origin of DNA replication (ori lyt). In order to understand the role of pBZLF1 in induc-tion of the lytic cycle, we have been trying to purify the protein and to characterize its biochemical fea-tures. This was first attempted with insect cells in-fected with baculoviruses overproducing pBZLF1, but the partially purified protein tended to form ag-gregates and was eluted with a wide range of the salt concentrations on ionic chromatography. The results suggested the conformation of the pBZLF1 produced in the insect cells would not reflect the in vivo case. Therefore, we next tried to purify pBZLF1 from B95-8 cells, the cell line latently in-fected with EBV. The lytic cycle was induced with chemical agents such as TPA, sodium n-butyrate,

and calcium ionophore, and pBZLF1 could be ex-tracted with high salt buffer(0.6 to 1 M NaCl). The pBZLF1 and purified more than 50 fold with the hydrophobic column chromatography, DEAE sephacel chromatography, phosphocellulose chro-matography and Heparin agarose chromatography. However, the protein was not the main component in the final fraction and further purification proce-dures are therefore required. 3. Mechanisms by which Cdc2 kinase in-

hibits re-replication during the late S-G2-M phase in mammalian cells

Fujita, M. and Tsurumi, T.

Genomic DNA needs to be replicated com-pletely and only once during a single cell cycle and inhibition of re-replication is one of most important aspects of cell cycle control to maintain genome integrity. We have demonstrated that, in mam-malian cells, Cdc2 kinase governs the inhibition of re-replication during late S-G2-M phase through prohibition of re-binding of the MCM hetero-hexameric complex, an essential DNA replication initiation factor, to chromatin. MCM complexes are believed to be loaded onto chromatin by an ori-gin recognition complex (ORC) and CDC6 protein. We have suggested that phosphorylation of the MCM complex and CDC6 protein by Cdc2 kinase may be one of mechanisms underlying prohibition of re-binding. Recently, the Cdt1 protein has been identified as another MCM-loading factor, its func-tion being proposed to be suppressed through bind-ing by a counteractive protein, geminin, thus pro-hibiting MCM re-binding in the mitotic phase of the Xenopus egg cell cycle. Interestingly, this gem-inin-mediated regulation of Cdt1 function appears to be independent of mitotic Cdc2 kinase activity. However, considering our previous experimental data, it is quite possible that the Cdt1 function is regulated by Cdc2 kinase during the late phase of the cell cycle in mammalian somatic cells (see be-low). Therefore, we have been more precisely analyzing mechanisms by which Cdc2 kinase inhib-its re-replication during the late phase of the mam-malian cell cycle, especially focusing on interrela-tionships among Cdt1, geminin and Cdc2 kinase. Using Cdc2 temperature-sensitive mutant mur-ine FT210 cells, we previously demonstrated that inactivation of Cdc2 kinase leads to re-binding of MCM complexes to chromatin during the G2/M phase. We first investigated intranuclear behavior of Cdt1 and geminin proteins when Cdc2 is inacti-

39

vated in FT210 cells. In G2/M phase-enriched FT210 cells, MCM, Cdt1 and geminin were present as soluble nucleoplasmic proteins. When Cdc2 kinase was inactivated, re-binding of MCM proteins was observed, as we reported previously. Under such conditions, most Cdt1 proteins also became associated with chromatin, while geminin remained soluble. It has been reported that the latter can physically interact with Cdt1. One simple explana-tion for the finding, therefore, might be that Cdc2 kinase activity enhances the Cdt1-geminin interac-tion, and when Cdc2 is inactivated, Cdt1 becomes free from geminin binding, then associating with chromatin and functioning to load MCM. We found that the Cdc2/cyclinA complex, but not Cdc2/cyclin B complex, binds to Cdt1 through its Cy motif in 293T human kidney cells, this not interfering with geminin binding to Cdt1. We also found that when 293T cells are treated with purvalanol A, a very specific Cdk2 and Cdc2 inhibitor, both Cdt1-geminin and Cdt1-Cdc2/cyclin A interactions are diminished. From these results, our current working hypothesis is that Cdc2 inhibits Cdt1 re-loading MCM through (1) enhancing geminin binding to Cdt1 by their phosphorylation and (2) directly binding as a Cdc2/cyclin A complex to Cdt1. For confirmation, we are now preparing re-combinant Cdt1, geminin and Cdk/cyclin com-plexes to establish in vitro binding assay. We are also examining whether wild-type or Cy-mutated Cdt1 can induce re-replication in 293T cells. 4. Immortalization of human cells by HPV Kiyono, T. and Tsurumi, T.

Normal human cells in culture undergo a limited number of divisions and then enter a nondividing state called replicative senescence. Most cancer cells can divide indefinitely by escaping this senes-cence program. The E6 and E7 genes of human papillomavirus can cooperatively immortalize nor-mal human epithelial cells originating from skin or mammal glands. Both inactivation of the RB pathway by E7 and activation of telomerase by E6 are required for the immortalization. In the past two years, we have tried to immortalize many cell types, including: epithelial cells originated from bronchus, small air ways, esophagus, stomach, mammal- and prostate glands, endometrium, surface of the ovary, tooth root, and hair follicle; endothelial cells from umbilical veins, microvessels, and thoracic ducts; mesothelial cells from omentum; mesenchymal stem cells from bone marrow, T- and B lympho-

cytes; and fibroblasts from many tissues. Among these cell types, only skin fibroblasts could be im-mortalized by introduction of TERT, the catalytic subunit of telomerase reverse transcriptase. Such introduction proved sufficient for induction of te-lomerase activity in all the cell types tested, but in-sufficient for immortalization except in the skin fi-broblasts. We are trying to elucidate the underly-ing mechanisms which could explain this anomaly. So far, we found that all the above cell types other than skin fibroblast showed gradual increase in p16Ink4a expression with increasing number of population doublings, which in turn causes de-creased phosphorylation of RB resulting in cell cy-cle arrest. Introduction of E6 and E7 extended the life span of all the cell types tested, and in some cases resulted in immortalization with active te-lomerase. However, some cell types, including mesenchymal stem cells from bone marrow, showed an extended life span with no active telom-erase, and finally stopped growing. These cell types were eventually immortalized by introduction of TERT in addition to E6 and E7. These results support our previous conclusion that both inactiva-tion of the RB pathway and activation of telomerase are required for immortalization to many cell types. However, inactivation of the RB pathway by E7 induced a significantly higher rate of apoptosis as well as extending life span in cell types including mesenchymal stem cells, and those introduced with E7 together with TERT were difficult to propagate, not because of senescence but because of apoptosis. At least, practically, those cells required another gene such as E6 to inhibit apoptosis induced by E7 for immortalization. E6 alone induces telomerase in some cell types including skin keratinocytes and mammary epithelial cells, but not in others. Intro-duction of E7 together with TERT is a good strategy to immortalize normal human cells, but is not ideal for establishing normal human cell lines, because E7 induces chromosomal instability and sometimes apoptosis. We are looking for a better strategy to establish normal human cell lines, which might be useful not only for many research fields but also in clinical areas such as regeneration medicine. 5. Longitudinal dynamics of Epstein-Barr

Virus-specific cytotoxic T lymphocytes in the posttransplant lymphoprolifera-tive disorder

Kuzushima, K, Kimura, H.*1, Hoshino, Y.*1, Yoshimi, A.*1, Tsuge, I.*1, Horibe, K.*1, Morishima, T.*2, Kojima, S.*1 and Tsurumi, T.

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The Epstein-Barr virus (EBV)-associated lym-phoproliferative disorder (LPD) is a serious com-plication after allogeneic bone marrow transplanta-tion (BMT). Dynamics of EBV-specific cytotoxic T lymphocytes (CTL), which are important in con-trolling EBV, during LPD have yet to be fully elu-cidated. A patient with Wiskott-Aldrich syndrome was diagnosed as suffering with the LPD on day 47 after BMT. Fluorescence-activated cell sorter (FACS) analysis for interferon-γ production re-vealed more than 70% of the patient’s CD8+ T cells to be EBV-specific. They were directly cytotoxic to donor-derived EBV+ lymphoblastoid cells, thus be-ing blocked by an anti-class I antibody. EBV-specific CD8+ T cell counts declined in paral-lel with the EBV genome load and full recovery of LPD was obtained with relaxation of immunosup-pressive drugs. The results illustrate longitudinal dynamics of EBV-specific CTL during post-transplant LPD and feature the advantages of FACS analysis for EBV-specific CTL for treatment decision making. *1 Departments of Pediatrics/Developmental Pediatrics

and *2 Health Science, Nagoya University School of Medicine, Nagoya Japan

6. Identification of HLA A*2402-restricted

cytomegalovirus-specific CD8+ T cell epitopes by a computer algorithm and an enzyme-linked immunospot assay

Kuzushima, K., Hayashi, N., Kimura, H.*1 and Tsurumi, T.

Antigenic peptides recognized by virus-specific cytotoxic T lymphocytes (CTLs) are useful tools for studying CTL responses specifically among those

who possess the presenting major histocompatibil-ity (MHC) class I molecules. For widening the ap-plication, an efficient strategy to determine such epitopes in the context of a given MHC is highly desirable. We present here a rapid and effective method for determination of CTL epitopes through multiple screenings, consisting of a com-puter-assisted algorithm, and MHC stabilization and enzyme-linked immunospot assays. A major cytomegalovirus (CMV)-specific CTL epitope, QYDPVAALF in the amino acid sequence of its lower matrix 65 kilo dalton phosphoprotein (pp65), presented by HLA A*2402 molecules was identi-fied from 83 candidate peptides. The results indi-cate that the CMV-specific CTL response is highly focused on pp65 in the context of HLA A*2402. Endogenous processing and presentation was con-firmed using a peptide-specific CD8+ T cell clone as the effector and autologous fibroblast cells in-fected with recombinant vaccinia virus expressing pp65 gene or CMV as the antigen presenting cells. Flow cytometric analysis of intracellular inter-feron-γ production revealed between 0.04 and 0.27 % of CD8+ T cells in peripheral blood of HLA A24-positive and CMV-seropositive donors to be specific for the peptide. The tetrameric MHC-peptide complexes specifically bound to the reactive T cell clone and 0.79% of CD8+ T cells in peripheral blood from a seropositive donor. The peptide could thus be a useful reagent to study CTL responses to CMV among populations positive for HLA A*2402. *1 Departments of Pediatrics/Developmental Pediatrics,

Nagoya University School of Medicine, Nagoya Ja-pan

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Professor C.J.H. van de Velde, from Leiden University Medical Center, Netherlands, giving us the lecture entitled “Ten Yeras Results of Prospective Randomized D1/D2 Gastric Cancer Trial Limited but Definitive Benefits“ in the 8th Aichi Cancer Center International Symposium held on February 16, 2002 (see p. 93).

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From left to right First row: Ms. Keiko Miyazaki, Dr. Akiko Kanamori, Dr. Reiji Kannagi, Ms. Masumi Usui-Nozaki and

Dr. Nozomu Hiraiwa. Second row: Dr. Takaaki Hattori, Mr. Takunori Ogaeri, Dr. Osamu Taguchi, Ms. Mineko Izawa and Dr. Akinari Watanabe.

Insets, Dr. Satoshi Saito, Dr. Kensuke Kumamoto, Ms. Yoshiko Goto, Dr. Kou Tei and Ms. Sasako Eguchi.

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Division of Molecular Pathology ________________________________________________________________________________ Reiji Kannagi, M.D., D.M.Sc., Chief Osamu Taguchi, D.M.Sc., Section Head Nozomu Hiraiwa, M.D., D.M.Sc., Senior Researcher Akiko Kanamori, Ph.D., Researcher Kumamoto Kensuke, Research Resident (as of April, 2001) Mineko Izawa, B.A., Research Assistant Chikako Mitsuoka, M.T., Research Assistant (until March, 2000) Yoshiko Goto, D.V.M., Research Assistant Keiko Miyazaki, M.T., Research Assistant (as of November, 2001) Sasako Eguchi, Semi-regular Employee Masumi Usui-Nozaki, Semi-regular Employee Kayoko Kanda, M.T., Semi-regular Employee (until March, 2000) Visiting Scientists Hiroshi Ikeda, M.D., Aichi Medical University Visiting Trainees Katsuhiro Ohno, M.D., Nagoya University School of Medicine (until March, 2001) Kensuke Kumamoto, M.D., Fukushima University School of Medicine (until March, 2001) Chikako Mitsuoka, M.T., Shiroyama Hospital (as of April 2000, until April, 2001) Kou Tei, M.D., Kyoto Prefectural University School of Medicine Satoshi Saito, M.D., Nagoya University School of Medicine Akinari Watanabe, M.D., Fukushima University School of Medicine (as of March, 2001) General Summary Cell adhesion molecules play important roles in infiltrative growth and distant metastasis of cancers, and expression of functional carbohydrate determinants implicated in cell adhesion is remarkably enhanced upon malignant transformation of cells. Especially, the carbohydrate determinants, sialyl Lewis a and sialyl Lewis x, are frequently expressed on human malignant cells in patients with cancers or leukemia. These determinants serve as ligands for selectins, cell adhesion proteins present on activated human endothelial cells, and intimately involved in the process of hematogenous metastasis. During the period 2000-2001, our research interest was concentrated on the following issues; 1) basic study on the ligand requirements of three members of the selectin family of cell adhesion molecules; 2) expression and functional roles of a newly found sulfated selectin ligand, sialyl 6-sulfo Lewis x, in solid tumors; 3) mechanisms of specific in-duction of sialyl Lewis a and sialyl Lewis x expression upon malignant transformation of cells; and 4) ex-perimental trials for treatment of cancers by vaccination of MUC1 cDNA with dendritic cells. 1. Study of ligand specificity of three se-

lectin family cell adhesion molecules, E-, P- and L-selectins, using genetically engineered cells

Kanamori, A., Ohmori, K*1, Goto, Y., Uchimura, K.*2, Muramatsu, T.*2, Kiso, M.*3, Tamatani, T.*4 and Kannagi, R.

Selectins, a family of cell adhesion molecules with a C-type lectin domain at the outer terminus of each molecule, have been shown to recognize spe-cific carbohydrate ligands such as sialyl Lewis X and sialyl Lewis A. Cell adhesion mediated by

selectin and their carbohydrate ligands is implicated in recruitment of leukocytes in inflammation, he-matogenous metastasis of cancer cells, and tissue infiltration of leukemic cells. Recently we identi-fied sialyl 6-sulfo Lewis x as a major L-selectin ligand on high endothelial venules of human pe-ripheral lymph nodes. We further investigated the ligand activity of sialyl 6-sulfo Lewis x with E- and P-selectins and made a comparison with the binding activity of conventional sialyl Lewis x, using cul-tured human lymphoid cells expressing both car-bohydrate determinants. The results of the recom-binant selectin binding studies and the non-static

44

monolayer cell adhesion assays indicated both sia-lyl 6-sulfo Lewis x and conventional sialyl Lewis x to serve as ligands for E- and P-selectins, while L-selectin appears quite specific for sialyl 6-sulfo Lewis x. Treatment with anti-PSGL-1 antibodies as well as O-sialoglycoprotein endopeptidase al-most completely abrogated the binding of P-selectin but barely affected the binding of E-selectin. This indicates that these carbohydrate determinants car-ried by O-glycans of PSGL-1 selectively serve as ligands for P-selectin, while the ligand for E-selectin is not restricted to PSGL-1 nor to O-sialoglycoprotein endopeptidase-sensitive gly-cans. The binding of L-selectin was markedly re-duced by O-sialoglycoprotein endopeptidase treat-ment but only minimally affected by anti-PSGL-1 antibodies, indicating O-glycans carrying sialyl 6-sulfo Lewis x to be the major L-selectin ligands, while PSGL-1 is only a minor core protein for L-selectin in these cells. These results indicated that each member of the selectin family has a dis-tinct ligand binding specificity. *1 Department of Biochemistry, Nagoya University,

School of Medicine. *2 Department of Laboratory Medicine, Kyoto Univer-

sity, School of Medicine. *3 Department of Applied Bioorganic Chemistry, Gifu

University, School of Agriculture. *4 Research Center for Advanced Science and Tech-

nology, the University of Tokyo. 2. Expression of sialyl 6-sulfo Lewis X, a

new ligand for cell adhesion molecules of the selectin family, in human colon and cultured colon cancer cells

Izawa, M., Kumamoto, K., Kanamori, A., Kanda, K., Goto, Y., Ishida, H.*1, Nakamura, S.*1 and Kannagi, R.

We recently identified sialyl 6-sulfo Lewis X determinant as a major ligand for L-selectin on high endothelial venules of human peripheral lymph nodes. However, its expression is not limited to endothelial cells. From our investigation of its distribution in human colorectal cancer tissues and cultured colon cancer cells, the sialyl 6-sulfo Lewis X determinant is preferentially expressed in nonma-lignant colonic epithelium rather than cancer cells (P < 0.001; n = 23). This is in contrast to the dis-tribution of conventional sialyl Lewis X, which is preferentially expressed in cancer tissues rather than nonmalignant epithelia (P = 0.007; n = 23), indicat-ing that 6-sulfation predominantly occurs in non-

malignant tissues and is suppressed upon malignant transformation (Fig. 1). In confirmation of this, a non-sialylated determinant 6-sulfo Lewis X was also found to be preferentially localized in nonma-lignant epithelium. Significant expression of sia-lyl 6-sulfo Lewis X was observed in only 2 of 13 cultured colon cancer cell lines, whereas 8 were positive for conventional sialyl Lewis X. Trans-fection of cells with fucosyltransferase (Fuc-T) VI induced expression of sialyl 6-sulfo Lewis X, whereas transfection of Fuc-T III did not, suggest-ing that the determinant was synthesized mainly by Fuc-T VI in colonic epithelial cells. Members of the sialic acid cyclase pathway, the de-N-acetyl sialyl 6-sulfo Lewis X and cyclic sialyl 6-sulfo Lewis X determinants, were also found to be pref-erentially expressed in nonmalignant epithelium rather than colonic cancer cells (P < 0.001; n = 23). Stimulation of the sialyl 6-sulfo Lewis X-positive colon cancer cell line with a calcium ionophore ionomycin markedly reduced sialyl 6-sulfo Lewis X and induced cyclic sialyl 6-sulfo Lewis X expres-

Fig. 1. Sialyl 6-sulfo sialyl Lewis X and conventional

sialyl Lewis X in a colon cancer. Note the preferential expression of the 6-sulfo form on non-malignant colonic epithelial cells (upper panel), while non-sulfated sialyl Lewis X is strongly expressed on cancer cells (lower panel).

45

sion. These results suggest that the metabolic conversion of sialyl 6-sulfo Lewis X into cyclic sialyl 6-sulfo Lewis X by a calcium-dependent en-zyme, sialic acid cyclase, as we hypothesized for human leukocytes previously (C. Mitsuoka et al., Proc. Natl. Acad. Sci. USA, 96: 1597–1602, 1999), also occurs in nonmalignant colonic epithelium. *1 Central Clinical Laboratory, Aichi Cancer Center

Hospital. 3. Regulatory mechanisms for expression

of functional carbohydrate determi-nants on malignant and non-malignant cells:

3-1. Roles of sugar nucleotide transporters in the enhanced expression of carbo-hydrate ligands for selectins, sialyl Lewis X and sialyl Lewis A, on solid tumors

Kumamoto, K., Goto, Y., Ishida, N.*1, Kawakita, M.*1 and Kannagi, R.

Extravasation of malignant cells involves inter-action of carbohydrate ligands on their surfaces with selectins, cell adhesion molecules on endothe-lial cells lining the blood vessels. Several mo-lecular species of carbohydrate ligands for selectins are expressed on malignant cells, including sialyl Lewis X and sialyl Lewis A, especially in solid tu-mors. The molecular mechanisms underlying ac-celerated expression of sialyl Lewis X/A in cancers is not well understood. Cancer-associated induction of some glycosyl-transferases has been assumed to influence expres-sion of determinants. Recent studies, however,

have indicated that cancer-associated alteration in sugar transportation and intermediate carbohydrate metabolism also play important roles in the induc-tion of sialyl Lewis X/A expression in cancer. A series of human nucleotide sugar transporters in the Golgi apparatus were recently cloned, including the transporters for UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine (UDP-GlcNAc) and CMP-sialic acid (CMP-SA). We have examined the mRNA expression of these three transporters in human colon cancer tissues by reverse transcrip-tion-PCR analysis in comparison with that in non-malignant colonic mucosa prepared from the same patients. The amount of mRNA for UDP-Gal trans-porter was significantly increased in colon cancer tissues compared with nonmalignant mucosa (P = 0.035; n = 20) (Fig. 2). The increase was more prominent in patients with advanced colorectal can-cer of Dukes’ stages C and D, in which the amount of UDP-Gal transporter mRNA showed on average about a 3.6-fold increase over paired nonmalignant samples (statistically significant at P = 0.004; n = 14). The mRNA content of the other two transporters showed no significant difference be-tween the paired cancer and normal tissues. When UDP-Gal transporter cDNA was stably transfected into cultured human colon cancer cells, expression of Thomsen-Friedenreich (TF) antigen and of sialyl Lewis A (NeuAcα2→3Galβ1→3[Fucα1→4]Glc NAcβ1→ R) and sialyl Lewis X (NeuAcα2→3Gal β1→4[Fucα1→3]GlcNAcβ1→R) determinants was significantly induced on transfectant cells, resulting in markedly enhanced cell adhesion to vascular E-selectin. These findings suggest that increase of UDP-Gal transporter mRNA is involved in en-hanced expression of cancer-associated carbohy-drate determinants such as TF and sialyl Lewis A/X

Fig. 2. Typical examples of RT-PCR analyses of nucleotide sugar transporter gene expression in human colon cancer tissues and non-malignant mucosa. After PCR reaction using the specific primers, the aliquots of products were electrophoresed in 2% agarose gel and were stained with ethidium bromide. Sizes of each band are indicated in basepairs (bp). Ca, cancer tissues; N, non-malignant mucosa prepared from the same patient. UDP-Gal T, UDP-Gal transporter; G3PDH, glyceraldehyde 3-phosphate dehydrogenase.

46

antigens in colon cancers. *1 Department of Physiological Chemistry, The Tokyo

Metropolitan Institute of Medical Science, Tokyo. 3. Regulatory mechanisms for expression

of functional carbohydrate determi-nants on malignant and non-malignant cells:

3-2. A T-box transcriptional factor that syn-ergizes with HTLV-1 Tax in transacti-vating the selectin-ligand synthesizing enzyme, fucosyltransferase VII

Hiraiwa, N. and Kannagi, R.

Sialyl Lewis x is reportedly to be expressed on leukocytes as a ligand for selectins on vascular en-dothelium. Molecular-biological studies have re-vealed that its synthesis is critically regulated by the key enzyme, fucosyltransferase VII (Fuc-T VII) in leukocytes. We recently found that adult T-cell leukemia malignant cells express sialyl Lewis x and Fuc-T VII, increased expression of mRNA for the latter actually being generated by Tax protein de-rived from the human T-cell leukemic virus type 1 (HTLV-1) virus. We have demonstrated that Tax activates the promoter of Fuc-T VII at a CRE-like (cAMP-responsive element) site approx. 150-bp away from the initiator of the gene. Detailed studies revealed that the sequence of the CRE-like site where CREB-1, other CREB/ATF transcrip-tional factors can bind, resembles the 21-bp se-quence of the HTLV-1 long-terminal repeat (LTR) and is critical for Tax-activation. We therefore postulate the presence of unknown factor(s) with the ability to bind to the site and associate with other factors like members of CREB/ATF or Tax. To clarify this possibility, we conducted a one-hybrid experiment with a cDNA library from ATL-related cells, cloned several binding-factors, and demonstrated that a new T-box type transcrip-tional factor, F7CAF-1 (Fuc-T VII CRE-associated Factor), associates with CREB-1 and Tax at the CRE-like site to facilitate Tax-induced Fuc-T VII activation. The CRE-like site of the promoter of the Fuc-T VII is akin to the semi-palindromic se-quence of the reported T-box binding site. The exact sequence to which the F7CAF-1 binds re-mains to be elucidated. Using JPX-9 cells, a de-rivative of Jurkat T-cells carrying the transfected Tax gene under the control of metallothionein pro-moter, the induction of Tax was found to result in appearance of mRNA of F7CAF-1 along with the

Fuc-T VII. A reporter assay with expres-sion-plasmids for the F7CAF-1, CREB-1 or Tax showed F7CAF-1 to transactivate Tax-induced ex-pression of Fuc-T VII in cooperation with CREB-1. Immunoprecipitation studies revealed association of F7CAF-1 with Tax and CREB-1, indicating that the complex formed with these factors might carry out activation of Fuc-T VII in ATL cells. Northern blots showed that F7CAF-1 is expressed in lung, liver, and also in spleen, lymph nodes, and bone marrow, closely related to expression of Fuc-T VII. In ATL-related cell lines, F7CAF-1 was found to be remarkably transcribed in association with Fuc- T VII, but other kinds of T-cell leukemic/lymphoma cell lines and B-cell leukemic/lymphoma cell lines proved negative. These results indicate that ATL cells overex-press the Fuc-T VII gene that can be transactivated by HTLV-1 Tax in concert with a T-box factor, F7CAF-1. Cloning of F7CAF-1 should facilitate research on Tax-induced transactivation of various genes and elucidate underlying mechanisms 4. A murine model of tumor suppression

by vaccination with MUC1 DNA and dendritic cells

Kontani, K.*1 and Taguchi, O.

Among specific approaches to cancer immuno-therapy, DNA vaccines are thought to be more ap-plicable for clinical use than other methods, such as vaccines with peptides or autologous cancer cells, or adoptive transfer of cytotoxic T lymphocytes (CTLs), for the following reasons: 1) DNA vaccines are inexpensive and simple to use once the DNA vector is prepared; 2) adjuvants are usually unnec-essary for DNA vaccination; 3) high levels of anti-gen expression can be maintained; 4) autologous immune cells or cancer cells are not needed; and 5) facilities and techniques for cell culture are not nec-essary. Therefore, DNA vaccines targeting tumor antigens have great potential for anti-cancer immunotherapy. MUC1 antigen is abundantly expressed on breast, pancreas, lung, and colon cancer cells, elic-iting strong anti-tumor immunity in hosts. The tandem repeat domain on its core protein contains antigenic epitopes which are recognized by CTLs in both mice and humans. Therefore, MUC1 is suit-able target for cancer immunotherapy. In order to induce specific anti-tumor immunity in mice, we attempted to immunize C57BL/6 mice with a DNA vaccine encoding the MUC1 polypep-

47

tide. When mice immunized with MUC1 DNA were challenged with EL4-muc, MUC1-transfected syngeneic lymphoma cells, they completely pre-vented tumor growth. In contrast, when the vac-cine was given to EL4-muc tumor-bearing mice, suppression was not observed. However, activated, but non-primed dendritic cells (DCs) obtained from syngeneic mice, if applied simultaneously with the MUC1 DNA vaccine to the same sites of EL4-muc tumor-bearing mice, tumor growth was markedly suppressed with prolongation of survival. MUC1 antigen could be detected on the dendritic cells at the vaccination site and in regional nodes in the targeted mice, which showed strongly enhanced

cellular immune responses specific for MUC1 as compared to those in mice vaccinated with MUC1 DNA alone. No significant difference in titers of antibodies to MUC1 between the two groups was observed, suggesting that non-primed DCs inocu-lated into DNA vaccine sites are essential for elic-iting strong anti-tumor cellular immunity to sup-press tumor growth efficiently. This animal model should prove useful for developing DNA vaccines for anti-cancer immunotherapy. *1 Second Department of Surgery, Shiga University of

Medical Science.

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From left to right First row: Dr. F. Hirose, Dr. K. Nagata, Mr. Y. Yasui, Dr. I. Izawa, Dr. M. Inagaki and Ms. M.

Nishizawa. Second row: Ms. T. Yuhara, Ms. N. Saitoh, Ms. A. Kawajiri, Ms. Y. Hayashi, Ms. N. Ohshima, Dr. N.

Hanai, Mr. T. Oguri, Dr. T. Yokoyama, Mr. T. Siromizu and Dr. K. Ohtakara. Insets: Dr. K. Ohno, Dr. M. Yamaguchi, Dr. H. Yoshida and Dr. M. Kato.

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Division of Biochemistry ________________________________________________________________________________ Masaki Inagaki, M.D. Chief Koh-ichi Nagata, M.D. Section Head Masamitsu Yamaguchi, Ph.D. Section Head (until June, 2001) Ichiro Izawa, M.D. Senior Researcher Hiroyasu Inada, M.D. Senior Researcher (until June,2001) Fumiko Hirose, Ph.D. Senior Researcher Miwako Nishizawa, B.P. Senior Research Assistant Yoshihiro Yasui, M.P. Research Assistant Noriko Saito, B.M.T. Research Assistant Yoshio Nishimoto, B.S. Senior Research Assistant (until March 2001) Yuko Hayashi, B.S. Research Assistant Visiting Scientists Hidemasa Goto, Domestic Research Fellow, Japan Science and Technology Corporation (until Octo-ber,2001) Visiting Trainees Hideaki Togashi, M.S. Faculty of Science,Nagoya University,Graduate School of Science (until March,2001) Mihoko Takagishi, B.P. Faculty of Pharmaceutical Science, Nagoya City University (until March,2001) Seiaya Matsui, B.P. Faculty of Pharmaceutical Science, Nagoya City University (until March,2002) Kazuhiro Ohtakara, M.D. Department of Pediatrics, Mie University School of Medicine Aie Kawajiri, M.S. Department of Pathology, Nagoya University School of Medicine (as of April,2000) Katsuhiko Ohno, Ph.D. Graduate School of Science, Nagoya University (until September, 2001) Eun-Jeong Kwon, M.S. Graduate School of Science, Nagoya University Hideki Yoshida, M. S. Faculty of Science and Technology, Tokyo Science University (until September, 2001) Masaki Kato, M.S. Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology (until September, 2001) General Summary Research in Division of Biochemistry is concerned with the regulation of tumor development, invasion and metastasis, and our attention is focused on four specific areas: (1) Phosphorylation-dependent regulation of elements of the cytoskeleton such as intermediate filaments, intermediate filament associated proteins and septin proteins and cell adhesion molecules; (2) Identification and functional analysis of protein kinases in-volved in cell division; (3) A search for intermediate filament binding proteins to elucidate mechanism of regulation of the cell adhesion machinery, including hemidesmosome and desmosome functions; and (4) Transcriptional regulation of genes related to cell proliferation. 1. Aurora B and Rho-kinase regulate

cleavage furrow-specific vimentin phosphorylation in the cytokinetic pro-cess

Yasui, Y., Goto, H., Kawajiri, A.*1, Nigg, E.A.*2, Terada, Y.*3, Tatsuka, M.*4, Matsui, S.*5, Manser, E.*6, Lim, L.*6,7, Nagata, K. and Inagaki, M.

Vimentin, one of the type III intermediate fila-ment (IF) proteins, is expressed not only in mesen-chymal cells but also in most types of tumor cells. We have introduced several types of vimentin mu-tated at putative phosphorylation sites in its

amino-terminal head domain into type III IF-negative T24 cells. Site-specific mutation in-duced the formation of an unusually long bridge-like IF structure between unseparated daughter cells, although these mutants formed a filament network similar to that in wild type inter-phase cells. Among the phosphorylation sites vimentin-Ser72 was one mutation site essential for this phenotype. We further demonstrated that the novel phosphorylation site, vimentin-Ser72, was phosphorylated specifically at the cleavage furrow during cytokinesis. Aurora B is a kinase involved in cytokinesis, but

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little is known about its target substrates. We pro-vided evidence that vimentin-Ser72 is phosphory-lated specifically at the border of the Aurora B-localized area from anaphase to telophase during mitosis. Expression of a dominant-negative mutant of Aurora B leads to a reduction in cleavage fur-row-specific phosphorylation. We have identified Ser6, Ser24, Ser38, Ser46, Ser64, Ser65, Ser72, and Ser86 on vimentin as Aurora B phosphorylation sites in vitro. Mutations in there were found to induce the formation of the unusually long bridge-like IF structure. Together with finding that the cyclic AMP-dependent protein kinase ac-tive form and p21-activated kinase, possible vimentin-Ser72 kinases, are not localized at cleav-age furrow during mitotic phase, the results indicate that Aurora B regulates cleavage furrow-specific vimentin phosphorylation and that functional al-teration ensues. We previously demonstrated vimentin-Ser71, which is phosphorylated by Rho-kinase, to be also phosphorylated specifically at the cleavage furrow. Thus, the cleavage furrow-specific vimentin phos-phorylation may be regulated by at least two dis-tinct kinases (Aurora B and Rho-kinase). We propose that continuous furrow ingression may in-crease vimentin accessibility not only to Aurora B in the spindle midzone but also to Rho-kinase in the plasma membrane of the cleavage furrow. *1 Department of Pathology, Nagoya University School

of Medicine, Nagoya, Aichi 466-8550, Japan. *2 Max-Planck Institute for Biochemistry, Department

of Cell Biology, D-82152 Martinsried, Germany. *3 Department of Genetics, Cell Biology and Develop-

ment, University of Minneapolis, MN55455, USA. *4 RIRBM, Hiroshima University, Hiroshima 734-8553,

Japan. *5 Department of Biological Chemistry, Faculty of

Pharmaceutical Science, Nagoya City University, Nagoya 467-8603, Japan.

*6 Glaxo-IMCB Group, Institute of Molecular & Cell Biology, Singapore 117609, Singapore.

*7 Institute of Neurology, University College London, London WC1N 1PJ, UK.

2. Aurora B phosphorylation of histone H3

at serine28 prior to the mitotic chro-mosome condensation

Goto, H.*1, Yasui, Y., Nigg, E.A.*2 and Inagaki, M.

Histone H3 (H3) phosphorylation plays impor-tant roles in mitotic chromosome condensation.

We have reported that H3 phosphorylation occurs not only at Ser10 but also at Ser28 during mitosis, at least in mammals. Aurora B was recently demonstrated to be responsible for Ser10 phos-phorylation in S. cerevisiae, C. elegans, Drosophila, and a Xenopus egg extract. To determine whether Aurora B might phosphorylate H3 not only at ser-ine10 but also at serine28 in mammals, we com-pared the distribution of the enzyme with that of H3 phosphorylation. Aurora B was found to be pri-marily localized in heterochromatin of late G2 phase cells where only Ser10 phosphorylation was observed. Treatment of such cells with calyculin A induced Ser28 phosphorylation in the Aurora B-localized area. During prophase to metaphase, Aurora B becomes distributed in condensing chro-mosomes demonstrating Ser10 and Ser28 phos-phorylation. Aurora B can phosphorylate H3-Ser10 and -Ser28 in nucleosomes in vitro, transfection of a dominant-negative mutant result-ing in reduction of H3 phosphorylation not only at Ser10 but also Ser28 during mitosis. This occurs during mitotic chromosome condensation, and the level of Ser28 phosphorylation is diminished to an undetectable level by PP1 phosphatase prior to en-try into mitosis. *1 Division of Signal Transduction, Nara Institute of

Science and Technology *2 Max-Planck Institute for Biochemistry, Department

of Cell Biology, D-82152 Martinsried, Germany. 3. Keratin attenuates tumor necrosis fac-

tor-induced cytotoxicity through asso-ciation with TRADD

Inada, H., Izawa, I., Nishizawa, M., Fujita, E.*1, Kiyono, T., Takahashi, T., Momoi, T.*1 and Inagaki, M.

Keratin 8 and 18 (K8/18) are the major compo-nents of intermediate filament (IF) proteins of sim-ple or single-layered epithelia. Recent data show that normal and malignant epithelial cells deficient in K8/18 are nearly 100 times more sensitive to tu-mor necrosis factor (TNF)-induced cell death. We have now identified the human TNF receptor 1 (TNFR1)-associated death domain protein (TRADD) to be a K18-interacting protein. Among IF proteins tested in two-hybrid systems, TRADD specifically bound to K18 and K14, type I (acidic) keratins, the COOH-terminal region of TRADD in-teracting with the coil Ia of the rod domain of K18. Endogenous TRADD coimmunoprecipitated with K18, and colocalized with K8/18 filaments in hu-

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man mammary epithelial cells. Overexpression of the NH2-terminus (aa 1-270) of K18 containing the TRADD-binding domain as well as overexpression of K8/18 in SW13 cells, which are devoid of kera-tins, render the cells more resistant to killing by TNF. We also showed that the overexpressed NH2-termini of K18 and K8/18 associate with en-dogenous TRADD in SW13 cells, resulting in inhi-bition of activation of caspase-8. These results in-dicate that K18 may sequester TRADD to attenuate its interactions with activated TNFR1 and moderate TNF-induced apoptosis in simple epithelial cells. *1 Division of Development and Differentiation, Na-

tional Institute of Neuroscience, NCNP, Kodaira, Tokyo 187-8502, Japan

4. ERBIN associates with p0071, an arma-

dillo protein, at cell-cell junctions of epi-thelial cells

Izawa, I., Nishizawa, M., Tomono, Y. *1, Ohtakara, K., Takahashi, T. and Inagaki, M.

ERBIN, an ErbB2 receptor-interacting protein, belongs to a recently described family termed LAP [leucine-rich repeats and PSD-95/dlg-A/ZO-1 (PDZ) domains], that plays essential roles in estab-lishment of cell polarity. To identify new ER-BIN-binding proteins, we screened a yeast two-hybrid library, using the carboxyl-terminal fragment of ERBIN containing a PDZ domain as bait, and isolated p0071 (also called plakophilin-4) as an ERBIN-interacting protein. p0071 is a member of the p120 catenin family, defined as pro-teins with 10 armadillo repeats, and is localized along cell-cell border. ERBIN PDZ domain binds the COOH-terminus of p0071 containing the PDZ domain-binding sequence, and endogenous ERBIN was found to be co-immunoprecipitated with p0071. In fully polarized Madin-Darby canine kidney (MDCK) cells, ERBIN co-localized largely with β-catenin and partly with desmoplakin along the lateral plasma membrane. At these cell-cell con-tact regions, ERBIN co-localizes with p0071. Overexpression of the dominant active forms of Cdc42, Rac1, or RhoA, Rho family small GTPases, resulted in the marked accumulation of ERBIN at cell-cell contacts of MDCK and HeLa cells. These results show that ERBIN interacts in vivo with p0071 and may be involved in the organization of adherens junctions and desmosomes of epithelia. In addition, we have demonstrated that the subcel-lular localization of ERBIN might be regulated by

Rho family small GTPases. These observations should pave the way toward further research on cell polarity and adhesion as well as generating under-standing of pathological mechanisms of cancer. *1 Division of Molecular and Cell Biology, Shigei

Medical Research Institute, Okayama 701-0202, Ja-pan

5. Characterization of a mammalian septin

MSF-A Nagata, K., Kawajiri, A.*1, Saito, N., Togashi,H., Takagishi, M., Matsui, S., Hotani, H.*2 and Inagaki, M.

Septins are a family of conserved proteins im-plicated in cell growth and cell cycle regulation, al-though their properties and modes of action are lar-gely unknown. A septin termed MSF (MLL septin-like fusion) has been identified as a fusion partner gene in mixed lineage leukemia (MLL) in a case of therapy-related acute myeloid leukemia with a t(11;17)(q23;q25). Two alternative splicing variants, MSF-A and MSF-B, have now been iden-tified while another report documented a compli-cated transcriptional pattern of MSF. MSF has been found to be mutated in some cases of breast and ovarian cancers and is considered to be a can-didate tumor suppressor gene. The mutations may be associated with allelic loss of the 17q25 region. Although these findings have provided insights into a possible role for MSF in leukemogenesis and on-cogenesis, and the molecular mechanism(s) linking MSF function and tumorigenesis and also bio-chemical and biological properties of MSF proteins remain to be elucidated. We therefore carried out an immunocytochemi-cal and biochemical characterization of MSF-A, using an antibody specific for MSF subfamily pro-teins. Expression was found to be predominantly in mammary HMEC cells, in associated with mi-crotubules in interphase but the mitotic spindle and bundle of microtubule in the midzone during mito-sis. Biochemical analysis revealed direct binding of MSF-A with polymerized tubulin through its central region containing guanine nu-cleotide-interactive motifs, although GTPase activ-ity was not required for the association. Condi-tions that disrupted the microtubule network also disrupted the MSF-A-containing filament structure, resulting in a punctate cytoplasmic pattern. Unlike Nedd5, a septin thought to be involved in cytokinesis, a MSF mutant deficient in GTPase ac-tivity was found to form filament indistinguishable

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from those of the wild type. These results indicate that the interaction of MSF-A with microtubule might be an important mechanism regulating a va-riety of septin-dependent cellular processes. *1 Department of Pathology, Nagoya University School

of Medicine *2 Division of Biological Sciences, Graduate School of

Science, Nagoya University 6. Functional analysis of DREF using

transgenic flies Hirose, F., Ohshima, N., Kwon, E-J.#1, Yoshida, H.#2, Inoue, Y.H.#3, Matsukage, A.#4 and Yamaguchi, M.#2

The promoters of Drosophila genes encoding DNA replication-related proteins contain transcrip-tion regulatory elements DRE (5'-TATCGATA) in addition to E2F recognition sites. A specific DRE-binding factor, DREF, positively regulates DRE-containing genes. In addition, it has been reported that DREF can bind to a sequence in the hsp70 scs' chromatin boundary element that is also recognized by boundary element-associated factor (BEAF) , and thus DREF may play a role in regu-lating insulator activity. To examine DREF func-tions in vivo, we have established transgenic flies in which ectopic expression of DREF is targeted to the eye imaginal discs. Adult flies expressing DREF exhibited a severe rough eye phenotype, featuring ectopic DNA and abolition of photoreceptor differ-entiation. Furthermore, DREF expression caused apoptosis in the imaginal disc cells. The DREF induced rough eye phenotype could be suppressed by a half dose reduction of the E2F gene, one of the genes under DREF regulation, indicating that the DREF overexpression phenotype is useful for screening for modifiers of DREF activity. Among Polycomb/trithorax-group genes, we found that half dose reduction of some of trithorax-group genes involved in determining chromatin structure or chromatin-remodeling (brahma, moira and osa) significantly suppressed while that of Distal-less enhanced the DREF-induced rough eye phenotype. The results suggest the possibility that DREF activ-ity might be regulated by protein complexes that play roles in modulating chromatin structure. To examine DREF functions in developing tis-sues, overexpression was accomplished in wing imaginal discs using a GAL4-UAS targeted expres-sion system in Drosophila. A notching wing phenotype was induced, associated with ectopic apoptosis. In addition, overexpression of the 32

kDa boundary element-associated factor (BEAF-32), suggested to compete against DREF for common binding sites in genomic regions, rescued the DREF-induced notching wing phenotype, while a half reduction of the genomic region, including the BEAF-32 gene, exerted enhancing effects. To our knowledge, this is the first evidence for any genetic interaction between DREF and BEAF-32. The DREF-induced notching wing phenotype is caused by induction of apoptosis in the Drosophila wing imaginal disc. Current addresses #1 Massachusetts General Hospital. #2 Division of Biotechnology, Kyoto Institute of Tech-

nology. #3 Drosophila Genetic Resource Center, Kyoto Institute

of Technology. #4 Chemical and Biological Sciences, Japan Women’s

University. 7. Functional analysis of BEAF32A using

transgenic flies Yamaguchi, M.#1, Yoshida, H.#1, Hirose, F., Inoue, Y.H.#2, Hayashi, Y., Yamagishi, M.#3, Nishi Y., Tamai, K.#4, Sakaguchi, K.#5 and Matsukage, A.#6

Transgenic flies were established in which ec-topic expression of boundary element-associated factor (BEAF) 32A was targeted to the Drosophila eye imaginal disc. The adult fly eyes were found to display a severe rough eye phenotype, most om-matidia being fused with generation of irregularly shaped rabdomeres. In the developing eye imagi-nal disc, expression of BEAF32A inhibited differ-entiation of photoreceptor cells and also induced extensive apoptosis of eye imaginal disc cells. Consistent with this, co-expression of baculovirus P35 in the eye imaginal disc suppressed the BEAF32A-induced rough eye phenotype. To in-vestigate the effects of BEAF32A on regulation of chromatin structure, genetic crosses of BEAF32A-overexpressing flies with loss-of-func-tion mutants for genes encoding other boundary element-binding factors or regulators of chromatin structure were conducted. Interestingly half-dose reduction of the su(Hw) gene strongly enhanced the rough eye phenotype induced by BEAF32A. Fur-thermore, genetic crosses of the transgenic flies with loss-of-function mutants for genes interacting with Polycomb revealed specific links between BEAF32A and genes such as Distal-less and kohtalo, suggesting a relation to the chromatin insulator

53

chromatin insulator function of BEAF. In addition, genetic crosses of transgenic flies expressing BEAF32A with a collection of Drosophila defi-ciency stocks allowed us to identify several ge-nomic regions, deletions of which caused enhance-ment or suppression of the BEAF32A-induced rough eye phenotype. The transgenic flies estab-lished in this study should be useful to identify tar-gets of BEAF32A and its positive or negative regu-lators in Drosophila. Present addresses #1 Division of Biotechnology, Kyoto Institute of Tech-

nology. #2 Drosophila Genetic Resource Center, Kyoto Institute

of Technology. #3 Neuroscience Research Institute, National Institute of

Advanced Industrial Science and Technology. #4 Medical and Biological Laboratories Corporation

Limited. #5 Department of Applied Biological Science, Science

University of Tokyo. #6 Chemical and Biological Sciences, Japan Women’s

University.

54

From left to right First row : Ms. Y. Hayashi, Ms. H. Fukami, Ms. Y. Iwata, Ms. S. Matsumoto and Dr. Y. Yamane. Second row: Mr. H. Nakamura, Dr. K. Ishizaki, Ms. S. Abe, Mr. Y. Nishimoto and Dr. H. Kuminoto.

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Division of Central Laboratory & Radiation Biology ________________________________________________________________________________ Kannji Ishizaki, Ph.D. Chief Hiroshi Kumimoyo, Ph.D. Researcher Yoshio Nishimoto, B.S. Senior Research Assistant Hiroko Fukami, Research Assistant Nobuhiro Uchida, Ph.D. Researcher (until February 2001) Kennzou Ohtsuka, Ph.D. Section Head (until March 2001) Mami Hata, B.P. Senior Research Assistant (until March 2001) Visiting Trainees Hideaki Nakamura, M.S. School of Medicine, Nagoya University Yoshihiro Yamane, Ph.D. Faculty of Science , Nagoya University General summary Our main research project is analysis of the molecular genetics of human esophageal tumors. In resent years, our effort has been focused on identification of a candidate tumor suppressor gene on 13q12-13, which may be closely related to the prognosis of esophageal tumors. So far we have analyzed several candidate ge-nes located in or near this region, such as the Rb, BRCA2, AS3 and LATS2 genes. However, tumor specific mutations were not detected. Now, we are intensively searching for a new gene in this region by constructing a fine deletion map with esophageal tumors. We are also studying interaction provide between genetic polymorphism and life-style factors with refer-ence to esophageal tumors to provide clue to effective prevention. We have found that a specific allele of the L-myc gene is associated with induction of esophageal tumors in individual with smoking and drinking hab-its. We are also now analyzing polymorphisms in other genes. Another research project is to study genetic effects of low dose radiation on human cells. For this purpose we have established immortal cell lines derived from normal controls and patients with radiation sensitive genetic diseases by introducing the human telomerase gene. These cell lines are immortal but without any change in p53 and other genes that are involved in cellular signal transduction, exhibiting normal responses to low dose radiation. Using these cell lines we are now studying induced mutations. 1. Analysis of a candidate tumor sup-

pressor gene, LATS2, on 13q11-12 in esophageal squamous cell carcinoma

Ishizaki, K., Fujimoto, J.*1, Kumimoto, H., Nishimoto, Y., Shimada, Y.*2, Shinoda, M.*3 and Yamamoto, T. *1

We previously reported that loss of heterozy-gosity on 13q12-13 is related to a poor prognosis with esophageal cancer. We intensively screened esophageal tumor tissues and cell lines for genetic change in the Rb, BRCA2, and AS3 genes, located in or near this region. But none of these genes was found to exhibit a significant number of tumor spe-cific mutations, indicating the existence of another tumor-suppressor gene in this region. Recently, LATS2, a new human homologue of the Drosophila tumor suppressor gene (lats/warts) was found on 13q11-12. We therefore screened esophageal tumor cell lines and tumor tissues to detect tumor specific

mutations in this gene by PCR-SSCP and direct se-quencing with genomic DNA and cDNA. Although we found 5 different polymorphisms (4 single-base changes and a 6 bp insertion), a tumor specific mu-tation was identified in only one out of 60 tumor tissues. These results indicated that the LATS2 gene is inactivated only rarely in esophageal tumors, if at all, and that there is still another tumor suppressor gene in this region. We are now constructing a fine deletion map for its identification. *1 Department of Oncology, Institute of Medical Sci-

ence, University of Tokyo *2 Department of Surgery and Surgical Basic Science,

Graduate School of Medicine, Kyoto University *3 Department of Thoracic Surgery, Aichi Cancer Cen-

ter Hospital

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2. Different susceptibility of each L-myc genotype to risk factors for esophageal cancer or lung cancer

Kumimoto, H., Nishimoto , Y., Hamajima, N., Matsuo, K. and Ishizaki, K.

To understand the relationship between the L-myc genotypes and esophageal cancer risk, a po-lymerase chain reaction-based restriction fragment length polymorphism analysis was performed with 91 esophageal cancer patients diagnosed in the Ai-chi Cancer Center Hospital and 241 non-cancer outpatients as controls. No significant difference in the distribution of genotypes was observed be-tween the two groups; the figures were 18.7% for the LL genotype, 56.0% for LS and 25.3% for SS in patients, and 24.5%, 55.6% and 19.9%, respectively, in controls. The frequency of the s-allele in pa-tients (0.533) was slightly higher than in controls (0.477), but the difference was not statistically sig-nificant. However, the odds ratios (ORs) for smoking or heavy drinking were markedly higher for SS and LS genotypes than for the LL genotype; age-sex-adjusted ORs for smoking being 7.57 for the SS genotype, 6.40 for the LS genotype and 1.77 for the LL genotype. Age-sex-adjusted ORs for heavy drinking were 19.78, 18.20 and 7.40, respec-

tively (Fig.1). Values for both factors combined were 12.77, 18.45 and 1.44, suggesting that the L-myc polymorphism may strongly influence the induction of esophageal cancer in individuals with smoking and/or heavy drinking habits. We also analyzed the relationship between the L-myc genotypes and lung cancer risk from smok-ing with 191 lung-cancer patients and 241 non-cancer controls. Genotype distributions were not significantly different between patients and controls; 24.5% LL, 51.3% LS and 27.2 SS in pa-tients and 24.5%, 55.6% and 19.9%, respectively, in controls. The frequency of the s-allele in patients (0.529) was slightly higher than in controls (0.477). The ORs for smoking were markedly higher for the SS and LS genotypes than for LL; age-sex-adjusted values being 3.19, 2.30 and 0.92, respectively (Fig.2). This result suggests that the L-myc poly-morphism may also affect development of lung cancer in smokers. The patients were divided into two groups; one including patients less than 3 years after diagnosis and the other, 3 years or more after diagnosis. The OR for smoking in SS-genotype patients diagnosed less than three years was higher than that in other SS patients, suggesting that smoking-related lung cancer in SS genotype might exhibit a poorer prognosis.

20

10

0

OR for smoking

OR for drinking

5

0

Fig.1 ORs for smoking and drinking according to L-myc genotype in esophageal cancer

L L L S S STotal LL LS SS

Fig.2 ORs for smoking according to L-myc genotype in lung cancer : Age-sex-adjusted OR : Age-sex-drinking status-adjusted OR

0

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4OR

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3. Establishment of immortal normal and ataxia telangiectasia fibroblast cell lines by introduction of the hTERT gene

Nakamura, H., Fukami, H., Kiyono, T. *1 and Ishizaki, K.

For analyzing effects of low doses and low-dose-rate radiation on human cells, signal transduction system including p53 responses are important. However, they may not function nor-mally in cells by immortalized introduction of SV 40 or in cancer cells. Recently, however it has been reported that cellular response may be maintained in human cells immortalized by introduction of the human catalytic subunit of telomerase (hTERT) gene. Therefore we introduced the hTERT gene with a retrovirus vector into both normal (SuSa) and ataxia telangiectasia (AT10S) fibroblast cells to establish immortal cell lines (SuSa/T-n and AT10S/T-n). Af-ter hTERT introduction, these cells continued to grow beyond a population doubling number 300 with no indication of senescence, like flat shape and

reduced growth rate (Fig. 1). Induction of p53, phosphorylation of Ser15 in p53, and induction of p21 by X-ray irradiation in SuSa/T-n were not af-fected by the hTERT introduction. Both SuSa/T-n and AT10S/T-n cells exhibited an apparent inhibi-tion of growth, like the parental cells when they reached confluence. Karyotype analysis has re-vealed that they are in the diploid range. SuSa/T-n cells maintained their original radiosensitivity (Fig. 2), while the AT10S/T-n line appears to be slightly more resistant than the original cells. However, the difference is very small, and AT cells are still much more sensitive than normal cells. These results suggest that cells immortalized by hTERT introduction retain their original character-istics, except for immortalization, and that they may be useful for analyzing various effects of radiation. We are now analyzing the influence of low dose rate radiation on human cells using these immortal cells as today. *1 Division of Virology, Aichi Cancer Center Research

Institute

Fig. 1. Growth curves after hTERT-introduction. Both hTERT-introduced cells (SuSa/T-n and AT1OS/T-n) de-

monstrate growth far beyond the point at which control cells (SuSa) stopped growing. Fig. 2. X-ray sensitivity of original (SuSa and AT1OS) and immortalized cells (SuSa/T-n and AT1OS/T-n).

Both immortalized lines show radiosensitivity similar to their parental cells. Bars in figure indicate stan-dard deviations (n = 3).

0

50

100

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0 100 200 300 400 500 600 700 800

SuSa/neoSuSa/T-nAT1OS/T-n

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Fig. 1

0.001

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SuSa/T-nSuSaAT1OS/T-nAT1OS

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Fig. 2

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From left to right First row: Ms. Y. Kanie, Mr. M. Terashima and Ms. M. Yamamoto, second row: Dr. K. Tanabe, Dr. H.

Nakamura and Mr. Y. Minoura

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Central Service Unit ________________________________________________________________________________ Kazushi Tanabe, M.S., D.M.Sc. Section Head (until March 2002) Hiromu Nakamura, D.M.Sc. Section Head (as of April 2002) Morio Terashima, B.A. Senior Research Assistant (Technical photography specialist) Yasushi Minoura, B.P. Senior Research Assistant Masami Yamamoto, D.V.M. Research Assistant (as of April 2002) Mikio Hagino Research Assistant (Animal care specialist) General Summary

The Central Service Unit was formerly known as the Biophysics Unit, but the Unit name was changed in April 2000. In addition, the past section head of the Unit, Dr. K. Tanabe retired in March 2002, and Dr. H. Nakamura has succeeded him. Over the past several years, we concentrated on the planning and construction of our new Institute building, which was finally completed in January 2002. On the opening of new building, the maintenance and management of the fixtures and facilities of the new building, such as the air conditioning system, security system, water purifying system, waste water treatment system, carbon dioxide gas supply system and the experimental animal facilities became our duties. The Central Service Unit fulfills many functions in assisting the investigations performed by the Institute, and has responsibilities for the maintenance and operation of various instruments for biotechnology research. These are the protein sequencer (ABI 476A), DNA sequencer (ABI 3100), flow-cytometer (Becton-Dickinson FACSCalibur), high performance liquid chromatography equipment (Waters), imaging analyzers (Fujix BAS2500Mac, Amersham-Pharmacia Imagemaster and FluorImager 595), X-ray machine (Hitachi), electron microscopes (JEOL and Hitachi), confocal laser scanning microscope (Bio-Rad Radiance), real-time PCR analyzer (Roche Light Cycler), ultracentrifuges (Beckman and Hitachi), and computer system for image treatment. Furthermore, the Central Service Unit maintains and manages the radioisotope experiment facilities, SPF and conventional animal facilities, the laboratory of translational research, technical photographic work, hazardous chemical storage, ultra-low temperature freezers, cold rooms, liquid nitrogen storage room, LAN system, and contributes to many other aspects of the Institute's functions. Our activities provide background support to all of the investigations carried out by the Research Institute.

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Librarians ________________________________________________________________________________

From left to right Librarians, Ms. K. Adachi, Ms. S. Mori, Ms. M. Teratani and Ms. T. Yasuda, supporting

scientific and medical informations.

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A transmission electron micrograph of an adult compound eye from a wild-type fly elaborated by Dr. Fumiko Hirose, a senior researcher in the Division of Biochemistry, adopted as the cover photograph of Molecular and Cellular Biology, volume 22, issue 8. Ectopic expression of transcription factor DREF (for DNA replication-related element-binding factor) in Drosophila melanogaster imaginal discs induces DNA synthesis, apoptosis, and unusual morphogenesis and results in a severe rough eye phenotype (see Publication J043).

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Researches Supported by Special Project Programme ________________________________________________________________________________ 1. Identification of tumor-associated

antigens recognized by T cells infiltrating Epstein-Barr virus-positive gastric carcinomas

Kuzushima, K., Nakamura, S.*1, Nakamura, T.*2, Yamamura, Y.*3, Hayashi, N. and Tsurumi, T.

Gastric adenocarcinomas carrying the Epstein-Barr virus (EBV) are known to be accompanied by massive lymphocyte infiltration. To characterize the tumor infiltrating lymphocytes (TILs), we isolated and cultured such cells from surgically resected lesions. They were found to be positive for CD3 and CD8 and consists of HLA-class I-restricted CD8+ cytotoxic T lymphocytes (CTLs) which killed autologous EBV-transformed cells but not PHA blast cells and recognized HLA-A24 as a restriction molecule. However, the TILs did not recognize known EBV antigenic peptides presented by HLA-A24 nor HLA-A24 positive fibroblasts infected with vaccinia recombinant virus expressing each of the EBV latent proteins. EBV-positive gastric carcinomas do not express conventional target proteins of EBV-specific CTLs and the data suggest that some cellular proteins may be involved in the strong T cell response to EBV-associated gastric carcinomas. Frequencies of CD8+ antigen-specific T cells in TILs versus PBMCs were 1.9 % versus 0.013 % by limiting dilution assay, 7.0% versus undetected by intracellular interferon (IFN)-γ production assay and 22.8% versus 1.5 % by enzyme-linked immunospot (ELISpot) assay. These data demonstrate that class-I-restricted antigen-specific CD8+ CTLs are specifically expanded within EBV-positive gastric carcinoma tissue. To target EBV-positive gastric carcinomas, where EBV protein expression is limited, utilization of cytotoxic T lymphocytes recognizing latent membrane protein (LMP2) could be an option. We adopted an approach for determination of CTL epitopes through multiple screenings, consisting of a computer-assisted algorithm, a major histocompatibility complex (MHC) stabilization assay and ELISpot assay, an LMP2 epitope was identified. Further analysis using a CTL clone recognizing the epitope revealed that it is not presented on the surface of HLA A24-positive fibroblast cells infected with recombinant vaccinia

viruses expressing LMP2. ELISpot assays using the CTL clone and various antigen presenting cells demonstrated that the presentation was partially restored by pretreatment of the fibroblast cells with IFN-γ. The hydrophobic epitope, IYVLVMLVL, was presented on transporters associated with antigen processing-negative T2 cells transfected with plasmids encoding HLA A*2402 and the minimal epitope, indicating the presentation to be TAP-independent. The identified peptide could be a useful reagent to study and/or elicit CTL responses to EBV-positive gastric carcinomas expressing the LMP2 Departments of *1Pathology and Clinical Laboratories, *2Gastroenterology, *3Gastroenterological Surgery, Aichi Cancer Center Hospital

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Publications ________________________________________________________________________________ Journals

J001. Akatsuka, Y., Warren, E.H., Brickner, A.G., Engelhard, V.H. and Riddell, S.R.: Determination of Intronic Sequences Adjacent to an Exon Using Polymerase Chain Reaction and Genomic DNA Library Constructed by TA Cloning. Anal. Biochem. 289: 289-292, 2001.

J002. Ando, S., Gohara, R., Takasaki, Y., Masuda, A. and Nakanishi, H.: Measurement of protein tyrosine phosphatase activity using as substrates synthetic phosphotyrosine-containing peptides. Res. Commun. Biochem. Cell Mol. Biol. 5:75-84, 2001.

J003. Aoki, H., Mizuno, M., Natsume, A., Tsugawa, T., Tsujimura, K., Takahashi, To. and Yoshida, J.: Dendritic cells pulsed with tumor extract-cationic liposome complex increase the induction of cytotoxic T lymphocytes in mouse brain tumor. Cancer Immunol. Immunother. 50: 463-468, 2001.

J004. Arata, Y., Fujita, M., Ohtani, K., Kijima, S. and Kato, J.: Cdk2-dependent and -independent pathways in E2F-mediated S phase induction. J. Biol. Chem.275: 6337-6345, 2000.

J005. Arinaga, M., Shimizu, S., Gotoh, K., Haruki, N., Takahashi, To, Takahashi, Ta. and Mitsudomi, T.: Expression of human telomerase subunit genes in primary lung cancer and its clinical significance. Ann. Thor. Surg. 70: 401-405, 2000.

J006. Bora, R. S., Ichikawa, S., Kanamori, A. and Hirabayashi, Y.: Genomic structure and promoter analysis of putative mouse acetyl-CoA transporter gene. FEBS Lett. 473: 169-172, 2000.

J007. Bora, R. S., Ichikawa, S., Kanamori, A. and Hirabayashi, Y.: cDNA cloning of putative rat acetyl-CoA transporter and its expression pattern in brain. Cytogenet. Cell Genet. 89: 204-208, 2000.

J008. Brickner, A.G.*, Warren, E.H.*, Caldwell, J.A.*, Akatsuka, Y.*, Golovina, T.N., Zarling, A.L., Shabanowitz, J., Eisenlohr, L.C., Hunt, D.F., Engelhard, V.H. and Riddell, S.R.: The immunogenicity of a new human minor histocompatibility antigen results from differential antigen processing. J. Exp. Med. 193: 195-206, 2001. (* contributed equally)

J009. Chung, D.C., Brown, S.B., Graeme-Cook, F.,

Seto, M., Warshaw, A.L., Jensen, R.T. and Arnold, A.: Overexpression of cyclin D1 occurs frequently in human pancreatic endocrine tumors. Clin. Endocrinol. Metab. 85: 4373-4378, 2000.

J010. Crevel, G., Ivetic, A., Ohno, K., Yamaguchi, M. and Cotterill, S.: Nearest neighbour analysis of MCM protein complexes in Drosophila melanogaster. Nucleic Acids Res. 29: 4834-4842, 2001.

J011. Damiani, A. M., Matsumura, T., Yokoyama, N., Mikami, T., and Takahashi, E.: A deletion in the gI and gE genes of equine herpesvirus type 4 reduces viral virulence in the natural host and affects virus transmission during cell-to-cell spread. Virus Res. 67: 189-202, 2000.

J012. Dammann, R., Takahashi, Ta. and Pfeifer, G. P.: The CpG island of the novel tumor suppressor gene RASSF1A is intensely methylated in primary small cell lung carcinomas. Oncogene 20: 3563-3567, 2001.

J013. Delcommenne, M., Kannagi, R. and Johnson, P.: TNF-α increases the carbohydrate sulfation of CD44: induction of 6-sulfo N-acetyl lactosamine on N- and O-linked glycans. Glycobiology, in press.

J014. Dragani, T. A., Hirohashi, S., Juji,T., Kawajiri, K., Kihara, M., Ono-Kihara, M., Manenti, G., Nomoto, T., Sugimura, H., Genka, K., Yokota, J. and Takahashi, Ta., Mitsudomi, T. and Nagao, M.: Population-based mapping of pulmonary adenoma susceptibility 1 (PAS1) locus. Cancer Res. 60: 5017-5020, 2000.

J015. Eguchi, M., Eguchi, M., Seto, M., Morishita, K., Suzuki, K., Ueda, R., Ueda, K., Kamada, N. and Greaves, M.: GPHN, a novel partner gene fused to MLL in a leukemia with t(11;14)(q23;q24) Genes Chrom. Cancer, 32: 212-221, 2001.

J016. El-Fasakhany, F.M., Uchimura, K., Kannagi, R. and Muramatsu, T.: A novel human Gal-3-O-sulfotransferase: molecular cloning, char- acterization and its implications in biosynthesis of 3'-sulfo Lewisx. J. Biol. Chem. 276: 26988-26994, 2001.

J017. Fujii, K, Yokoyama, N., Kiyono, T., Kuzushima, K., Homma, M., Nishiyama, Y., Fujita, M., and Tsurumi, T.: The Epstein-Barr virus Pol

64

catalytic subunit physically interacts with the BBLF4/BSLF1/BBLF2/3 Complex. J. Virol. 74: 2550-2557, 2000.

J018. Fujita, M., Ishimi, Y., Nakamura, H., Kiyono, T. and Tsurumi, T.: Nuclear organization of DNA replication initiation proteins in mammalian cells. J. Biol. Chem. 277: 10354-10361, 2002.

J019. Fukumoto, M., Sugiyama, A., Ishida, K., Ikeno, T., Murakami, M., Kawasaki, S., Ota, H., Tatematsu, M. and Katsuyama, T.: Timing of N-methyl-N-nitrosourea administration affects gastric carcinogenesis in Mongolian gerbils infected with Helicobacter pylori. Cancer Lett.,160, 99-105, 2000.

J020. Futamura, N., Nakamura, S., Tatematsu, M., Yamamura, Y., Kannagi, R. and Hirose, H.: Clinicopathologic significance of sialyl Lex expression in advanced gastric carcinoma. Br. J. Cancer 83: 1681-1687, 2000.

J021. Gao, C., Li, Z., Ding, J., Wang, J., Hu, X., Liu, T., Xue, T., Li, H., Fujiyoshi, T., Takezaki, T. and Tajima, K.: Study on the relation between HLA-DRB1 alleles and Helicobacter pylori infection. Chin. J. Epidemiol. 21: 417-419, 2000.

J022. Gohara, R., Tang, D., Inada, H., Inagaki, M., Takasaki, Y. and Ando, S.: Phosphorylation of vimentin head domain inhibits interaction with the carboxyl-terminal end of alpha-helical rod domain studied by surface plasmon resonance measurements. FEBS Lett. 489:182-186, 2001.

J023. Hamada, T., Hirota, H., Yokoyama, S., Otsubo, N., Ishida, H., Kiso, M., Kanamori, A. and Kannagi, R.: NMR analysis of novel ganglioside GM4 analogues containing de-N-acetyl and lactamized sialic acid: probes for searching new ligand structures for human L-selectin. Magn. Reson. Chem. 40: 517-523, 2002.

J024. Hamajima, N. and Matsuo, K.: Subtle instruction to quit smoking may be efficacious for certain smokers. Asian Pacific J. Cancer Prev. 1: 257-258, 2000.

J025. Hamajima, N., Fukumitsu, T., Odauchi, S., Akashi, T., Usui, T. and Ido, M.: A large-scale follow-up study of smokers visiting medical facilities in Japan. Asian Pacific J. Cancer Prev. 2: 185-191, 2001.

J026. Hamajima, N., Iwata, H., Obata, Y., Matsuo, K., Mizutani, M., Iwase, T., Miura, S., Okuma, K.,

Ohashi, K. and Tajima, K.: No association of the 5’ promoter region polymorphism of CYP17 with breast cancer risk in Japan. Jpn. J. Cancer Res. 91: 880-885, 2000.

J027. Hamajima, N., Katsuda, N., Matsuo, K., Saito, T., Ito, L.S., Ando, M., Inoue, M., Takezaki, T. and Tajima, K.: Smoking habit and Interleukin 1B C-31T polymorphism. J. Epidemiol. 11: 120-125, 2001.

J028. Hamajima, N., Matsuo, K. and Yuasa, H.: Adjustment of prognostic effects in prevalent case-control studies on genotype. J. Epidemiol. 11: 204-210, 2001.

J029. Hamajima, N., Matsuo, K., Saito, T., Tajima, K., Okuma, K., Yamao, K. and Tominaga, S.: Interleukin 1 polymorphisms, lifestyle factors, and Helicobacter pylori infection. Jpn. J. Cancer Res. 92: 383-389, 2001.

J030. Hamajima, N., Matsuo, K., Suzuki, T., Nakamura, T., Matsuura, A., Tajima, K. and Tominaga, S.: Low expression myeloperoxidase genotype negatively associated with Helicobacter pylori infection. Jpn. J. Cancer Res. 92: 488-493, 2001.

J031. Hamajima, N., Matsuo, K., Tajima, K., Mizutani, M., Iwata, H., Iwase, T., Miura, S., Oya, H. and Obata, Y.: Limited association between a catechol-O-methyltransferase (COMT) poly- morphism and breast cancer risk in Japan. Int. J. Clin. Oncol. 6: 13-18, 2001.

J032. Hamajima, N., Saito, T., Matsuo, K., Kozaki, K. Takahashi, Ta. and Tajima, K.: Polymerase chain reaction with confronting two-pair primers for polymorphism genotyping. Jpn. J. Cancer Res. 91: 865-868, 2000.

J033. Hamajima, N., Takezaki, T., Matsuo, K., Saito, T., Inoue, M., Hirai, T., Kato, T., Ozeki, J. and Tajima, K.: Genotype frequencies of cyclooxygenase 2 (COX2) rare polymorphisms for Japanese with and without colorectal cancer. Asian Pacific J. Cancer Prev. 2: 57-62, 2001.

J034. Hara, S., Kami, M., Miyakoshi, S., Suzuki, R., Takeuchi, K., Seki, T., Oki, Y., Kishi, Y., Ueyama, J., Morinaga, S. and Muto, Y.: Central nervous system involvement in pyothorax- associated lymphoma: ring enhancement on CT scan. Ann. Hematol. 80: 174-177, 2001

J035. Harada, H., Uchida, N., Shimada, Y., Kumimoto, H., Shinoda, M., Imamura, M. and

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Ishizaki, K.: Polymorphism and allelic loss at the AS3 locus on 13q12-13 in esophageal squamous cell carcinoma. Int. J. Oncol. 18: 1003-1007, 2001.

J036. Haruki, N., Harano, T., Masuda, A., Kiyono, T., Takahashi, T., Tatematsu, Y., Shimizu, S., Mitsudomi, T., Konishi, H., Osada, H., Fujii, Y. and Takahashi, Ta.: Persistent increase in chromosome instability in lung cancer : Possible indirect involvement of p53. Am. J. Pathol. 159: 1345-1352, 2001.

J037. Haruki, N., Saito, H., Harano, T., Nomoto, S., Takahashi, Takao, Osada, H., Fujii, Y. and Takahashi, Ta.: Molecular analysis of the mitotic checkpoint genes BUB1, BUBR1 and BUB3 in human lung cancers. Cancer Lett. 162: 201-205, 2001.

J038. Haruki, N., Saito, H., Tatematsu, Y., Konishi, H., Harano,T., Masuda, A., Fujii, Y. and Takahashi, Ta.: Histological type-selective, tumor-predominant expression of a novel CHK1 isoform and infrequent in vivo somatic CHK2 mutation in small cell lung cancer. Cancer Res. 60: 4689-92, 2000.

J039. Haruki, N., Yatabe, Y., Travis, W. D., Nomoto, S., Osada, H., Nakamura, S., Nakao, A., Fujii, Y. and Takahashi, Ta.: Characterization of high-grade neuroendocrine tumors of the lung in relation to menin mutations. Jpn. J. Cancer Res. 91: 317-323, 2000.

J040. Hata, M. and Ohtsuka, K.: Cloning and expression of murine Hsp40 gene: Differences in initiation sites between heat-induced and constitutive transcript. DNA Seq. 11: 213-223, 2000.

J041. Hata, M. and Ohtsuka, K.: Murine cDNA encoding a novel type I HSP40/DNAJ homolog, mmDjA4. Biochim. Biophys. Acta 1493(1-2): 208-210, 2000.

J042. Hida, T., Kozaki, K., Muramatsu, H., Masuda, A., Shimizu, S., Mitsudomi, T., Sugiura, T., Ogawa, M. and Takahashi, Ta.: Cyclooxygenase (COX)-2 inhibitor induces apoptosis and enhances cytotoxicity of various anticancer agents in non-small cell lung cancer cell lines. Clin. Cancer Res. 6: 2006-2011, 2000.

J043. Hirose, F., Ohshima, N., Shiraki, M., Inoue, Y. H., Taguchi, O., Nishi, Y., Matsukage, A. and Yamaguchi, M.: Ectopic expression of DREF

induces DNA synthesis, apoptosis and unusual morphogenesis in the Drosophila eye imaginal disc: possible interaction with Plycomb and trithorax group proteins. Mol. Cell. Biol. 21: 7231-7242, 2001.

J044. Hirose, K., Tajima, K., Hamajima, N., Takezaki T., Inoue, M., Kuroishi T., Miura, S., and Tokudome, S.: Association of family history and other risk factors with breast cancer risk among Japanese premenopausal and postmenopausal women. Cancer Causes Cont. 12: 349-358, 2001.

J045. Hirota, T., Morisaki, T., Nishiyama, Y., Marumoto, T., Tada, K., Hara, T., Masuko, N., Inagaki, M., Hatakeyama, K. and Saya, H.: Zyxin, a regulator of actin filament assembly, plays a role in cell division by interacting with LATS1/h-warts tumor suppressor on mitotic apparatus. J. Cell Biol. 149: 1073-1086, 2000.

J046. Hoshino, Y., Kimura, H., Kuzushima, K., Tsurumi, T., Nemoto, K., Kikuta, A., Nishiyama, Y., Kojima, S., Matsuyama, T. and Morishima, T.: Early intervention in post-transplant lympho- proliferative disorders based on Epstein-Barr viral load. Bone Marrow Transplant. 26:199-201, 2000.

J047. Hosokawa, Y., Maeda, Y. and Seto, M.: Low frequency of expression of dominant-negative Ikaros isoforms in human leukemia and lymphoma cell lines. Leukemia Res. 24: 263-264, 2000.

J048. Hosokawa, Y., Maeda, Y. and Seto, M.: Target genes downregulated by the BCL-6/LAZ3 oncoprotein in mouse Ba/F3 cells. Biochem. Biophys. Res. Commun. 283: 563-568, 2001.

J049. Hosokawa, Y., Maeda, Y., Ishinohasama, R., Miura, I., Taniwaki, M. and Seto, M.: The Ikaros gene, a central regulator of lymphoid differentiation, fuses to the BCL6 gene as a result of t(3;7)(q27;p12) translocation in a patient with diffuse large B-cell lymphoma. Blood 95: 2719-2721, 2000.

J050. Hosokawa, Y., Nagai, E. and Seto, M.: Truncated TSG101 transcripts in human leukemia and lymphoma cell lines. J. Cancer Res. Clin. Oncol. 126: 79-84, 2000.

J051. Hosokawa, Y., Papanikolaou, A., Cardiff, R.D., Yoshimoto, K., Bernstein, M., Wang, T.C., Schmidt, E.V. and Arnold, A.: In vivo analysis of mammary and non-mammary tumorigenesis in MMTV-cyclin D1 transgenic mice deficient in p53. Transgenic Research, 5: 471-478, 2001.

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J052. Huang X.-E., Tajima, K., Hamajima, N., Kodera, Y., Yamamura, Y., Xiang, J., Tominaga, S. and Tokudome, S.: Effects of dietary, drinking, and smoking habits on the prognosis of gastric cancer. Nutr. Cancer 38: 30-36, 2001.

J053. Huang, X.-E., Hamajima, N., Saito, T., Matsuo, K., Mizutani, M., Iwata, H., Iwase, T., Miura, S., Mizuno, T., Tokudome, S. and Tajima, K.: Possible association of β 2- and β3-adrenergic receptor gene polymorphisms with susceptibility to breast cancer. Breast Cancer Res. 3: 264-269, 2001.

J054. Huang, X.-E., Tajima, K., Hamajima, N., Xiang, J., Inoue, M., Hirose, K., Tominaga, S., Takezaki, T., Kuroishi, T. and Tokudome, S.: Comparison of lifestyle and risk factors among Japanese with and without gastric cancer family history. Int. J. Cancer 86: 421-424, 2000.

J055. Ikeda, A., Sun, X., Li, Y., Zhang, Y-K., Eckner, R., Doi, T.S., Takahashi, To., Obata, Y., Yoshioka, K. and Yamamoto, K.: p300/CBP-dependent and -independent trans- criptional interference between NF-κB RelA and p53. Biochem. Biophys. Res. Commun. 272: 375-379, 2000.

J056. Ikeda, N., Eguchi, H., Nishihara, S., Narimatsu, H., Kannagi, R., Irimura, T., Ohta, M., Matsuda, H., Taniguchi, N. and Honke, K.: A remodeling system of the 3'-sulfo Lewis a and 3'-sulfo Lewis x epitopes. J. Biol. Chem. 276: 38588-38594, 2001.

J057. Ikehara, Y., Nishihara, S., Yasutomi, H., Kitamura, T., Matsuo, K., Shimizu, N., Inada, K., Kodera, Y., Yamamura, Y., Narimatsu, H., Hamajima, N. and Tatematsu, M.: Polymorphisms of two fucosyltransferase genes (Lewis and Secretor genes) involving type I Lewis antigens are associated with the presence of anti-Helicobacter pylori IgG antibody. Cancer Epidemiol. Biomarkers Prev. 10: 971-977, 2001.

J058. Imanishi, Y.*, Hosokawa, Y.*, Yoshimoto, K*., Schipani, E., Mallya, S., Papanikolaou, A., Kifor, O., Takahiko, T., Sablosky, M., Ledgard, F., Gronowicz, G., Wang, T.C., Schmidt, E.V., Hall, C., Brown, E.M., Bronson, R. and Arnold, A.: (* equal contributor) Primary hyperparathyroidism caused by parathyroid-targeted overexpression of cyclin D1 in transgenic mice. J. Clin. Invest. 9, 1093-1102, 2001.

J059. Inada, H., Izawa, I., Nishizawa, M., Fujita,

E., Kiyono, T., Takahashi, To., Nomoi, T. and Inagaki, M.: Keratin attenuates tumor necrosis factor-induced cytotoxicity through association with TRADD. J. Cell Biol. 155: 415-425, 2001.

J060. Inada, K., Tanaka, H., Nakanishi, H., Tsukamoto, T., Ikehara, Y., Tatematsu, K., Nakamura, S., Porter, E.M. and Tatematsu, M.: Identification of Paneth cells in pyloric glands associated with gastric and intestinal mixed-type intestinal metaplasia of the human stomach. Virchows Arch, 439, 14-20, 2001.

J061. Inagaki, H., Okabe, M., Seto, M., Nakamura, S., Ueda, R. and Eimoto, T.: API2-MALT1 fusion transcripts involved in mucosa-associated lymphoid tissue lymphoma: multiplex RT-PCR detection using formalin-fixed paraffin-embedded specimens. Am. J. Pathol. 158: 699-706, 2001.

J062. Inagaki, N., Nishizawa, M., Minakata, Y., Yamamoto, H., Takeuchi, Y., Miyamoto, E., Kaibuchi, K. and Inagaki, M.: Activation of Ca2+/calmodulin-dependent protein kinase II within post-synaptic dendritic spines of cultured hippocampal neurons. J. Biol. Chem. 275: 27165-27171, 2000.

J063. Inoue, Y. H., do Carmo Avides., M., Shiraki, M., Deak, P. Avides, M. C., Yamaguchi, M., Nishimoto, Y., Matsukage, A. and Glover, D. M.: Orbit, a novel microtubule-associated protein essential for mitosis in Drosophila. J. Cell Biol. 149: 153-165, 2000.

J064. Inoue, M., Tajima, K. and Tominaga, S.: Probabilities of developing cancer over the whole life span of a Japanese. Asian Pacific J. Cancer Prev, 1: 333-336, 2000.

J065. Inoue, M., Tajima, K., Matsuura, A., Suzuki, T., Nakamura, T., Ohashi, K., Nakamura, S. and Tominaga, S.: Severity of chronic atrophic gastritis and subsequent gastric cancer occurrence: a 10-year prospective cohort study in Japan., Cancer Lett. 161: 105-112, 2000.

J066. Inoue, M., Tajima, K., Mizutani, M., Iwata, H., Iwase, T., Miura, S., Hirose, K., Hamajima, N. and Tominaga, S.: Regular consumption of green tea and the risk of breast cancer recurrence: follow-up study from the hospital-based epidemiologic research program at Aichi Cancer Center (HERPACC), Japan. Cancer Lett. 167: 175-182, 2001.

J067. Inoue, M., Tajima, K., Tominaga, S., Aoki,

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K. and Kato, M.: A new model population-based cancer registration system in Aichi Prefecture, Japan. Asian Pacific J. Cancer Prev. 1: 67-71, 2000.

J068. Inoue, M.: Information feedback by means of electronic media. JACR Monogr. 5: 44-47, 2000 (in Japanese).

J069. Inoue, T., Nakanishi, H., Inada, K., Hioki, T., Tatematsu, M. and Sugimura, Y.: Real time revwrse transcriptase polymerase chain reaction of urinary cytokeratin 20 detects transitonal cell carcinoma cells. J. Urol., 166, 2134-2141, 2001.

J070. Ishiguro, K., Kadomatsu, K., Kojima, T., Muramatsu, H., Tsuzuki, S., Nakamura, E., Kusugami, K., Saito, H. and Muramatsu, T.: Syndecan-4 deficiency impairs focal adhesion formation only under restricted conditions. J. Biol. Chem. 25: 5249-5252, 2000.

J071. Ishii, K., Usui, S., Sugimura, Y., Yoshida, S., Hioki, T., Tatematsu, M., Yamamoto, H. and Hirano, K.: Aminopeptidase N regulated by zinc in human prostate participates in tumor cell invasion. Int. J. Cancer, 92, 49-54, 2001.

J072. Ishii, K., Usui, S., Yamamoto, H., Sugimura, Y., Tatematsu, M. and Hirao, K.: Decreases of Metallothionein and Aminopeptidase N in Renal Cancer Tissues. J. Biochem, 129, 253-258, 2001.

J073. Ishizaki, K., Nishizawa, K., Kato, T., Kitao, H., Han, Z-B., Hirayama, J., Suzuki, F., Cannon T. F., Kamigaichi, S., Tawarayama, Y., Masukawa, M., Shimazu, T. and Ikenaga, M.: Genetic changes induced in human cells in space shuttle experiment (STS-95). Aviat. Space Environ. Med. 72: 794-798, 2001.

J074. Ito, H., Matsuo, K., Saito, T., Hirose, K., Inoue, M., Takezaki, T., Hamajima, N., Kuroishi, T., and Tajima, K.: Valid responses to ABO blood type question in self-reporting questionnaire. Asian Pacific J. Cancer Prev. 2: 315-317, 2001.

J075. Ito, K., Ye, C.L., Hibi, K., Mitsuoka, C., Kannagi, R., Hidemura, K. ando, H., Kasai, Y., Akiyama, S. and Nakao, A.: Paired tumor marker of soluble E-selectin and its ligand sialyl Lewis A in colorectal cancer. J. Gastroenterol. 36: 823-829, 2001.

J076. Ito, L.S., Oba, S.M., Hamajima, N., Marie, S.K.N., Uno, M., Shinjo, S.K., Kino, A., Lavilla, F., Inoue, M., Tajima, K. and Tominaga, S.: Helicobacter pylori seropositivity among 963 Japanese Brazilians according to sex, age,

generation, and lifestyle factors. Jpn. J. Cancer. Res. 92: 1150-1156, 2001.

J077. Ito, S., Nakanishi, H., Ikehara, Y., Kato, T., Kasai, Y., Ito, K., Akiyama, S., Nakao, A. and Tatematsu, M.: Real-time observation of micrometastasis formation in the living mouse liver using a green fluorescent protein gene-tagged rat tongue carcinoma cell line. Int. J. Cancer, 93, 212-217, 2001.

J078. Iwase, S., Tsujimura, K., Matsudaira, Y., Ozeki, S., Onozaki, K., Obata, Y. and Takahashi, To.: Comparison of anti-tumor responses against TL positive lymphoma induced by skin grafting and dendritic cell immunization. Microbiol. Immunol. 44: 609-618, 2000.

J079. Iwata, H., Yamamoto, M., Nemori, R., Mizutani, M., Iwase, T., Miura, S., Obata, Y., Hara, Y., Omoto, Y., Toyama, T., Yamashita, H., Iwase, H. and Kobayashi, S.: Localization of gelatinolytic activity can be detected in breast cancer tissues by film in situ zymography. Breast Cancer 8: 111-115, 2001.

J080. Izawa, I., Nishizawa, M. Ohtakara, K., Ohtsuka, K., Inada, H. and Inagaki, M.: Identification of Mrj, a DnaJ/Heat shock protein 40 family protein, as a keratin 8/18 filament-regulatory protein. J. Biol. Chem. 275: 34521-34527, 2000.

J081. Izawa, M., Kumamoto, K., Mitsuoka, C., Kanamori, A., Ohmori, K., Ishida, H., Nakamura, S., Kurata-Miura, K., Sasaki, K., Nishi, T. and Kannagi, R.: Expression of sialyl 6-sulfo Lewis x is inversely correlated with conventional sialyl Lewis x expression in human colorectal cancer. Cancer Res. 60: 1410-1416, 2000.

J082. Izumi, M., Yokoi, M., Nishikawa, N. S., Miyazawa, H., Sugino, A., Yamagishi, M., Yamaguchi, M., Matsukage, A., Yatagai, F. and Hanaoka, F.: Transcription of the catalytic 180-kDa subunit gene of mouse DNA polymerase αis controlled by E2F, an Ets-related transcription factor, and Sp1. Biochem. Biophys. Acta. 1492 : 341-352, 2000.

J083. Kagami, Y., Jung, J., Choi, YS., Osumi, K., Nakamura, S., Morishima, Y. and Seto, M.: Establishment of a follicular lymphoma cell line (FLK-1) dependent on follicular dendritic cell-like cell line HK. Leukemia 15: 148-156, 2001.

J084. Kanamori, A., Kojima, N., Uchimura, K., Muramatsu, T., Tamatani, T., Berndt, M.C.,

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Kansas, G.S. and Kannagi, R.: Distinct sulfation requirements of selectins disclosed using cells which support rolling mediated by all three selectins under shear flow: L-SELECTIN PREFERS CARBOHYDRATE 6-SULFATION TO TYROSINE SULFATION WHEREAS P-SELECTIN DOES NOT. J. Biol. Chem. in press.

J085. Kanda, Y., Mineishi, S., Saito, T., Seo, S., Saito, A., Ohnishi, M., Suenaga, K., Niiya, H., Nakai, K., Takeuchi, T., Kawahigashi, Y., Shoji, N., Ogasawara, T., Tanosaki, R., Kobayashi, Y., Tobinai, K., Kami, M., Mori, S., Suzuki, R., Kunitoh, H. and Takaue, Y.: Pre-emptive therapy against cytomegalovirus (CMV) diseases guided by CMV antigenemia assay after allogeneic hematopoietic stem cell transplantation: a single-center experience in Japan. Bone Marrow Transplant. 27: 437-444, 2001.

J086. Kannagi, R.: Monoclonal anti-glycosphingo- lipid antibodies in sphingolipid metabolism and cell signaling. Method. Enzymol. 312: 160-179, 2000.

J087. Kannagi, R.: Use of liposomes containing carbohydrates for production of monoclonal antibodies. Method. Mol. Biol. 199: 203-218, 2002.

J088. Katsuda, N., Hamajima, N., Matsuo, K., Saito, T., Ito, S.L., Inoue, M., Takezaki, T., Tajima, K. and Tominaga, S.: Association between the interleukin 1B (C-31T) polymorphism and Helicobacter pylori infection in health checkup examinees. Jpn. J. Public Health 48: 604-611, 2001 (in Japanese).

J089. Kikuchi, Y., Hirano, M., Seto, M. and Takatsu, K.: Identification and characterization of a molecule, BAM11, that associates with the pleckstrin homology domain of mouse Btk. Int. Immunol. 12: 1397-1408, 2000.

J090. Kimura, H., Nagasaka, T., Hoshino, Y., Hayashi, N., Tanaka, N., Xu, J.L., Kuzushima, K. and Morishima T.: Severe Hepatitis caused by Epstein-Barr virus without infection of hepatocytes. Hum. Pathol. 32:757-762,2001.

J091. Kishi, Y., Kami, M., Oki, Y., Kazuyama, Y., Kawabata, M., Miyakoshi, S., Morinaga, S., Suzuki, R., Mori, S. and Muto, Y.: Donor lymphocyte infusion for treatment of life-threatening respiratory syncytial virus infection following bone marrow transplantation. Bone Marrow Transplant. 26: 573-576, 2000.

J092. Kobayashi, Y., Kume, A., Li, M., Doyu, M., Hata, M., Ohtsuka, K. and Sobue, G.: Chaperones Hsp70 and Hsp40 suppress aggregate formation and apoptosis in cultured neuronal cells expressing truncated androgen receptor protein with expanded polyglutamine tract. J. Biol. Chem. 275: 8772-8778, 2000.

J093. Kohno, A., Tsuzuki, S., Kasai, M., Miyamura, K., Emi, N., Tanimoto, M. and Saito, H.: Acute promyelocytic leukemia with apparently normal karyotype: molecular findings and response to all-trans retinoic acid. Leuk. Lymphoma, 42: 151-61, 2001.

J094. Koike, C., Luppi, P., Sharma, S.B., Kannagi, R., Nakashima, I., Starzl, T.E. and Trucco, M.: Molecular basis of evolutionary loss of α1,3-galactosyltransferase gene in higher primates. J. Biol. Chem. 277: 10114-10120, 2002.

J095. Kondo, E., Ogura, M., Kagami, Y., Taji, H., Miura, K., Takeuchi, T., Maeda, S., Asakura, S., Suzuki, R., Nakamura, S. and Morishima, Y.: Assessment of prognostic factors in follicular lymphoma patients. Int. J. Hematol. 73: 363-368, 2001.

J096. Kontani, K., Taguchi, O., Narita, T., Hiraiwa, N., Sawai, S., Hanaoka, J., Ichinose, M., Tezuka, N., Inoue, S., Fujino, S. and Kannagi, R.: Autologous dendritic cells or cells expressing both B7-1 and MUC1 can rescue tumor-specific cytotoxic T lymphocytes from MUC1-mediated apoptotic cell death. J. Leukocyte Biol. 68: 225-232, 2000.

J097. Kontani, K., Taguchi, O., Narita, T., Izawa, M., Hiraiwa, N., Zenita, K., Takeuchi, T., Murai, H., Miura, S. and Kannagi, R.: Modulation of MUC1 mucin as an escape mechanism of breast cancer cells from autologous cytotoxic T-lymphocytes. Br. J. Cancer 84: 1258-1264, 2001.

J098. Kosako, H., Yoshida, T., Matsumura, F., Ishizaki, T., Narumiya, S. and Inagaki, M.: Rho-kinase/ROCK is involved in cytokinesis through the phosphorylation of myosin light chain and not ezrin/radixin/moesin proteins at the cleavage furrow. Oncogene 19: 6059-6064, 2000.

J099. Kozaki, K., Koshikawa, K., Tatematsu, Y., Miyaishi, O., Saito, H., Hida, T., Osada, H. and Takahashi, Ta.: Multi-faceted analyses of a highly metastatic human lung cancer cell line NCI-H460-LNM35 suggest mimicry of

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inflammatory cells in metastasis. Oncogene 20: 4228-4234, 2001.

J100. Kozaki, K., Miyaishi, O., Tsukamoto, T., Tatematsu, Y., Hida, T., Takahashi, To. and Takahashi, Ta.: In vivo selected human lung cancer cell line H460-LNM35 consistently exhibits lymphogenous metastasis via both subcutaneous and orthotopic propagation. Cancer Res. 69: 2535-2540, 2000.

J101. Kumamoto, K., Goto, Y., Sekikawa, K., Takenoshita, S., Ishida, N., Kawakita, M. and Kannagi, R.: Increased expression of UDP-galactose transporter mRNA in human colon cancer tissues and its implication in synthesis of Thomsen-Friedenreich antigen and sialyl Lewis A/X determinants. Cancer Res. 61: 4620-4627, 2001.

J102. Kumimoto, H., Hamajima, N., Nishizawa, K., Nishimoto, Y., Matsuo, K., Harada, H., Shinoda, M., Hatooka, S. and Ishizaki, K.: Different susceptibility of each L-myc genotype to esophageal cancer risk factors. Jpn. J. Cancer Res. 92: 735-739, 2001.

J103. Kumimoto, H., Hamajima, N., Nishizawa, K., Nishimoto, Y., Matsuo, K., Harada, H., Shimada, Y., Imamura, M., Shinoda, M., Hatooka, S. and Ishizaki, K.: Different susceptibility of each L-myc genotype to esophageal cancer risk factors. Jap. J. Cancer Res., 92:735-739, 2001.

J104. Kuroishi, T., Hirose, K., Suzuki, T. and Tominaga, S.: Effectiveness of mass screening for breast cancer in Japan. Breast Cancer 7: 1-8, 2000.

J105. Kuzushima, K., Hayashi, N., Kimura, H. and Tsurumi, T.: Efficient identification of HLA A*2402-restricted cytomegalovirus-specific CD8+ T cell epitopes by a computer algorithm and an enzyme-linked immunospot assay. Blood 98: 1872-1881, 2001.

J106. Kuzushima, K., Kimura, H., Hoshino, Y., Yoshimi , A., Tsuge, I., Horibe, K., Morishima, T., Tsurumi, T. and Kojima, S.: Longitudinal dynamics of Epstein-Barr virus-specific cytotoxic T lymphocytes during the posttransplant lympho- proliferative disorder. J. Infect. Dis. 182: 937- 940, 2000.

J107. Kwon, E.-J., Oh, E.-J., Kim, Y.-S., Hirose, F., Ohno, K., Nishida, Y., Matsukage, A., Yamaguchi, M. and Yoo. M.-A.: Transcriptional regulation of the Drosophila raf proto-oncogene

by the transcription factor dE2F. Nucleic Acids Res. 29: 1808-1814, 2001.

J108. Kwon, E.-J., Park, H.-S., Kim, Y.-S., Oh, E.-J., Nishida, Y., Matsukage A., Yoo, M.-A. and Yamaguchi, M.: Transcriptional regulation of the Drosophila raf proto-oncogene by D-STAT during development and immune response. J. Biol. Chem. 275:19824-19830, 2000.

J109. Lu, J., Landerholm, T.E., Wei, J.S., Dong, X.-R., Wu, S.-P., Liu, X., Nagata, K., Inagaki, M. and Majesky, M.W.: Coronary smooth muscle differentiation from proepicardial cells requires Rho-a-mediated actin reorganization and p160 Rho-kinase activity. Dev. Biol. 240: 404-418, 2001.

J110. Lucas, PC., Yonezumi, M., Inohara, N., McAllister-Lucas, L.M., Abazeed, M.E., Chen, F.F., Yamaoka, S., Seto, M. and Nunez, G.: Bcl10 and MALT1, independent targets of chromosomal translocation in malt lymphoma, cooperate in a novel NF-kappa B signaling pathway. J. Biol. Chem. 276: 19012-19019, 2001.

J111. Lunevicius, R., Nakanishi, H., Ito, S., Kozaki, K., Kato, T., Tatematsu, M. and Yasui, K.: Clinicopathological significance of fibrotic capsule formation around liver metastasis from colorectal cancer. J. Cancer Res. Clin. Oncol., 127,193-199, 2001.

J112. Lymphoma Study Group of Japanese Pathology: The World Health Organization classification of malignant lymphomas in Japan: Incidence of recently recognized entities. Pathol. Int. 50: 696-702, 2000.

J113. Maeda, S., Kagami, Y., Ogura, M., Taji, H., Suzuki, R., Kondo, E., Asakura, S., Takeuchi, T, Miura K., Ando, M., Nakamura, S., Ito, T., Kinoshita, T., Ueda, R. and Morishima, Y.: CD34+-selected autologous peripheral blood stem cell transplantation conditioned with total body irradiation for malignant lymphoma: increased risk of infectious complications. Int. J. Hematol. 74: 214-221, 2001.

J114. Maruta, F., Ota, H., Genta, M., Sugiyama, A., Tatematsu, M., Katsuyama, T. and Kawasaki, S.: Role of N-methyl-N-nitrosourea in the induction of intestinal metaplasia and gastric adenocarcinoma in Mongolian Gerbils infected with Helicobacter Pylori. Scand. J. Gastroenterol. , 3, 283-290, 2001.

J115. Masuda, A., Osada, H., Yatabe, Y., Kozaki, K., Tatematsu, Y., Takahashi , T., Hida, T.,

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Takahashi, To. and Takahashi, Ta.: Protective function of p27KIP1 against apoptosis in small cell lung cancer cells in unfavorable microenvironments. Am. J. Pathol. 158: 87-96, 2001.

J116. Matsuo, K., Hamajima, N., Hirose, K., Inoue, M., Takezaki, T., Kuroishi, T. and Tajima, K.: Alcohol, smoking, and dietary status and susceptibility to malignant lymphoma in Japan: Results of a hospital-based case-control study at Aichi Cancer Center. Jpn. J. Cancer Res. 92: 1011-1017, 2001.

J117. Matsuo, K., Hamajima, N., Morishima, Y. and Harada, M.: Hospital capacity and post-transplant survival after allogeneic marrow transplantation: analysis of data from Japana Society for Hematopoietic Cell Transplantation. Bone Marrow Transplant. 26: 1061-1067, 2000.

J118. Matsuo, K., Hamajima, N., Shinoda, M., Hatooka, S., Inoue, M., Takezaki, T. and Tajima, K.: Gene-environment interaction between an aldehyde dehydrogenase-2 (ALDH2) polymorph- ism and alcohol consumption for the risk of esophageal cancer. Carcinogenesis 22: 913-916, 2001.

J119. Matsuo, K., Hamajima, N., Shinoda, M., Hatooka, S., Inoue, M., Takezaki, T. and Tajima, K.: Possible risk reduction in esophageal cancer associated with MPO -463 A allele. J. Epidemiol. 11: 109-114, 2001.

J120. Matsuo, K., Hamajima, N., Suzuki, R., Nakamura, S., Seto, M., Morishima, Y. and Tajima, K.: No substantial difference in genotype frequencies of interleukin and myeloperoxidase polymorphsisms between malignant lymphoma patients and non-cancer controls. Haematologica 86: 602-608, 2001.

J121. Matsuo, K., Hamajima, N., Tominaga, S., Suzuki, T., Nakamura, T., Matsuura, A. and Kitayama, K.: Helicobacter pylori IgG antibody test established in the United States showed a substantially lower sensitivity for Japanese population. Am. J. Gastroenterol. 95: 1597-1598, 2000.

J122. Matsuo, K., Suzuki, R., Hamajima, N., Ogura, M., Kagami, Y., Taji, H., Kondoh, E., Maeda, S., Asakura, S., Kaba, S., Nakamura, S., Seto, M., Morishima, Y. and Tajima, K.: Association between polymorphisms of folate- and methionine-metabolizing enzymes and susceptibility to malignant lymphoma. Blood 97:

3205-3209, 2001.

J123. Matsuoka, S., Nakagawa, T., Masuda, A., Haruki, N., Elledge, S. J. and Takahashi, Ta.: Reduced expression and impaired kinase activity of a Chk2 mutant identified in human lung cancer. Cancer Res. 61: 5362-5365, 2001.

J124. Meloni, G., Capria, S., Vignetti, M., Alimena, G., de Fabritiis, P., Montefusco, E., Mandelli, F., Matsuo, K, Hamajima, N., Suzuki, R., Nakamura, S., Seto, M., Morishima, Y. and Tajima, K.: Ten-year follow-up of a single center prospective trial of unmanipulated peripheral blood stem cell autograft and interferon-a in early phase chronic myeloyd leukemia. Haematologica 86: 596-601, 2001.

J125. Mitsudomi, T., Hamajima, N., Ogawa, M. and Takahashi, Ta.: Prognostic significance of p53 alterations in patients with non-small cell lung cancer: a meta-analysis. Clin. Cancer Res. 6: 4055-4063, 2000.

J126. Mizoshita, T., Inada, K., Tsukamoto, T., Kodera, Y., Yamamura, Y., Hirai, T., Kato, T., Jou, T., Itoh, M. and Tatematsu, M.: Expression of Cdx1 and Cdx2 mRNAs and relevance of this expression to differentiation in human gastrointestinal mucosa-with special emphasis on participation in intestinal metaplasia of the human stomach. Gastric Cancer, 4, 185-191, 2001.

J127. Mizutani, K., Matsubayashi, T., Iwase, S., Doi, T.S., Kasai, K., Yazaki, M., Wada, Y., Takahashi, To. and Obata, Y.: Murine delta homologue, mDelta1, expressed on feeder cells controls cellular differentiation. Cell. Struct. Funct. 25: 21-31, 2000.

J128. Moore, M.A. and Tatematsu, M.: Are the phenotypes of preneoplastic lesions of significance for cancer prevention? 1. Liver. Asian Pacific J. Cancer. Prev., 2, 27-42, 2001.

J129. Motegi, M., Yonezumi, M., Suzuki, H., Suzuki, R., Hosokawa, Y., Hosaka, S., Kodera, Y., Morishima, Y., Nakamura, S. and Seto, M.: API2-MALT1 chimeric transcripts involved in mucosa-associated lymphoid tissue type lymphoma predict heterogenous products. Am. J. Pathol. 156: 807-812, 2000.

J130. Nakagawa, A., Hara, K., Takeuchi, K., Seto, M. and Nakamura, S.: Sarcomatoid variant of anaplastic large cell lymphoma with cytoplasmic ALK and a-smooth muscle actin expression: a

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mimic of inflammatory myofibroblastic tumor. Am. J. Pathol. in press.

J131. Nakamura, S., Kato, M., Ichimura, K., Yatabe, Y., Kagami, Y., Suzuki, R., Taji, H., Kondo, E., Asakura, S., Kojima, M., Murakami, S., Yamao, K., Tsuzuki, T., Adachi, K., Miwa, A. and Yoshida, T.: Peripheral T/natural killer-cell lymphoma involving female genital tract: a clinicopathologic study of 5 cases. Int. J. Hematol. 73: 108-114, 2001.

J132. Nakamura, T., Nakamura, S., Yonezumi, M., Seto, M. and Yokoi, T.: The t(11; 18)(q21; q21) translocation in H. pylori-negative low-grade gastric MALT lymphoma. Am. J. Gastroenterol. 95: 3314-3315, 2000.

J133. Nakamura, T., Nakamura, S., Yonezumi, M., Suzuki, T., Matsuura, A., Yatabe, Y., Yokoi, T., Ohashi, K. and Seto, M.: Helicobacter pylori and the t(11;18)(q21;q21) translocation in gastric low-grade B-cell lymphoma of mucosa-associated lymphoid tissue type. Jpn. J. Cancer Res. 91: 301-309, 2000.

J134. Nakamura, Y., Hashimoto, R., Amano, M., Nagata, K., Matsumoto, N., Goto, H., Fukusho, E., Mori, H., Kashiwagi, Y., Kudo, T., Inagaki, M. and Takeda, M.: Localized phosphorylation of vimentin by Rho-kinase in neuroblastoma N2a cells. Genes Cells 5:823-837, 2000.

J135. Nakanishi, H., Kodera, Y., Yamamura, Y., Ito, S., Kato, T., Ezaki, T. and Tatematsu, M.: Rapid quantitative detection of carcinoembryonic antigen-expressing free tumor cells in the peritoneal cavity of gastric-cancer patients with real-time RT-PCR on the lightcycler. Int. J. Cancer, 89: 411-417, 2000.

J136. Nakayashiki, N., Yoshikawa, K., Nakamura, K., Hanai, N., Okamoto, K., Okamoto, S., Mizuno, M., Wakabayashi, T., Sega, S., Yoshida, J. and Takahashi, To.: Prodauction of a single chain variable fragmanet recognizing type III mutant epidermal growth factor receptor. Jpn. J. Cancer Res. 91: 1035-1043, 2000.

J137. Naoe, T., Takeyama, K., Yokozawa, T., Kiyoi, H., Seto, M., Uike, N., Ino, T., Utsunomiya, A., Maruta, A., Jin-nai, I., Kamada, N., Kubota, Y., Nakamura, H., Shimazaki, C., Horiike, S., Kodera, Y., Saito, H., Ueda, R., Wiemels, J. and Ohno, R.: Analysis of genetic polymorphism in NQO1, GST-M1, GST-T1, and CYP3A4 in 469 Japanese patients with therapy-related leukemia/

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J138. Naruse, T., Yuzawa, Y., Akahori, T., Mizuno, M., Maruyama, S., Kannagi, R., Hotta, N., Matsuo, S.: P-selectin-dependent macrophage migration into the tubulointerstitium in unilateral ureteral obstruction. Kidney Int. 62: 94-105, 2002.

J139. Natume, A., Tsujimura, K., Mizuno, M., Takahashi, To. and Yoshida, J.: IFN-β gene therapy induces systemic antitumor immunity against malignant glioma. J. Neurooncol. 47: 117-124, 2000.

J140. Nishida, K., Seto, M. and Ishida, R.: Different susceptibilities of post-mitotic checkpoint-proficient and -deficient BALB/ 3T3 cells to ICRF-193, a catalytic inhibitor of DNA topoisomerase II. Jpn. J. Cancer Res. 92: 193-202, 2001.

J141. Nomoto, S., Haruki, N., Tatematsu, Y., Konishi, H., Mitsudomi, T., Takahashi, To. and Takahashi, Ta.: Frequent allelic imbalance suggests the involvement of a tumor suppressor gene at 1p36 in the pathogenesis of human lung cancers. Genes Chrom. Cancer 28: 342-346, 2000.

J142. Obata, Y., Takahashi, To., Sakamoto, J., Tamaki, H., Tominaga, S., Hamajima, N., Chen, Y.-T. and Old, L.J.: SEREX analysis of gastric cancer antigens. Cancer Chemother. Pharmacol. 46 (suppl): S37 - S42, 2000.

J143. Ogawa, K., Nakanishi, H., Takeshita, F., Futkuchi, M., Asamoto, M., Imaida, K., Tatematsu, M. and Shirai, T.: Establishment of rat hepatocellular carcinoma cell lines with differing metastatic potential in nude mice. Int. J. Cancer, 91: 797-802, 2001.

J144. Ohgaki, H., Fukuda, M., Tohma, Y., Huang, H., Stoica, G., Tatematsu, M. and Donehowr, L.A.: Effect of intragastric application of N-methylnitrosourea in p53 knockout mice. Molecular Carcinogenesis. 28: 97-101, 2000.

J145. Ohmori, K., Kanda, K., Mitsuoka, C., Kanamori, A., Kurata-Miura, K., Sasaki, K., Nishi, T., Tamatani, T. and Kannagi, R.: P- and E-Selectins recognize sialyl 6-sulfo Lewis X, the recently-identified L-selectin ligand. Biochem. Biophys. Res. Commun. 278: 90-96, 2000.

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J147. Ohshige, K., Morio, S., Mizushima, S., Kitamura, K., Tajima, K., Suyama, A., Usuku, S., Tia, P., Hor, L.B., Heng, S., Sapjonn, V., Tochikubo, O. and Soda, K.: Behavioral and serological human immunodeficiency virus risk factors among female commercial sex workers in Cambodia. Int. J. Epidemiol. 29: 344-354, 2000.

J148. Ohshige, K., Morio, S., Mizushima, S., Kitamura, K., Tajima, K., Ito, A., Usuku, S., Suyama, A., Saphonn, V., Heng, S., Hor, L.B., Tia, P. and Soda, K.: Cross-sectional analysis on risk factors of HIV among female commercial sex workers in Cambodia. Epidemiol. Infect. 124: 143-152, 2000.

J149. Ohta, T., Haga, H., Osada, H., Tanaka, K., Maeda, K., Takazaki, T., Seki, N., Ohyama, Y, Nakanishi, Y. and Ishikawa, K.: Development and evaluation of a QOL questionnaire for elderly subject living in a community. Jpn. J. Public Health 48: 258-267, 2001 (in Japanese).

J150. Ohtakara, K., Inada, H., Goto, H., Taki, W., Manser., E., Lim, L., Izawa, I. and Inagaki, M.: p21-activated kinase PAK phosphorylates desmin at the different sites from those for Rho-associated kinase. Biochem. Biophys. Res. Commun. 272: 712-716, 2000.

J151. Ohtsuka, K. and Hata, M.: Mammalian HSP40/DNAJ homologs: Cloning of novel cDNAs and a proposal for their classification and nomenclature. Cell Stress & Chaperones 5: 98-112, 2000.

J152. Okino, T., Onda, M., Matsukura, N., Inada, K., Tatematsu, M., Suzuki, S. and Shimada, T.: Sequential Histopathological Changes in vivo after suicide gene therapy of gastric cancer induced by N-methyl-N’-nitro-N-nitrosoguanidine in Rats. Jpn. J. Cancer Res, 92, 673-679, 2001.

J153. Osada, H., Tatematsu Y., Masuda, A., Saito, T., Sugiyama, M., Yanagisawa, K. and Takahashi, Ta.: Heterogeneous TGF-β unresponsiveness and loss of TGFβRII expression caused by histone deacetylation in lung cancer cell lines. Cancer Res. 61: 8331-8339, 2001.

J154. Oshige, M., Yoshida, H., Hirose, F., Takata, K., Inoue, Y. H., Aoyagi, N., Yamaguchi, M., Koiwai, O., Matsukage, A. and Sakaguchi, K.:

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J155. Oyama, T., Kagami, Y., Seto, M. and Morishima, Y.: Mechanism of action on B cell lymphoma by chimeric anti-CD20 monoclonal antibody. Leukemia 15: 1667, 2001.

J156. Ozawa, Y., Towatari, M., Tsuzuki, S., Hayakawa, F., Maeda, T., Miyata, Y., Tanimoto, M. and Saito, H.: Histone deacetylase 3 associates with and represses the transcription factor GATA-2. Blood 98: 2116-23, 2001.

J157. Pei, J., Akatsuka, Y., Anasetti, C., Lin, M.-T., Petersdorf, E.W., Hansen, J.A. and Martin, P.J.: Marrow graft rejection through recognition of an HLA-C alloantigen: analysis of alloimmunity by T cell cloning and testing of T cell repertoire rearrangements. Biol. Blood Marrow Transplant. 7: 378-383, 2001.

J158. Sakai, H., Tsukamoto, T., Yamamoto, M., Shirai, N., Iidaka, T., Yanai, T., Masegi, T., and Tatematsu, M.: Differential effects of partial hepatectomy and carbon tetrachloride administra- tion on induction of liver cell foci in a model for detection of initiation activity. Jpn. J. Cancer Res, 92, 1018-1025, 2001.

J159. Sakai, H., Tsukamoto, T., Yamamoto, M., Yanai, T., Masegi, T., Inada, K., Nakanishi, H. and Tatematsu, M.: Summation of initiation activities of low doses of the non-hepatocarcinogen 1,2-dimethylhydrazine in the liver after carbon tetrachloride administration. Cancer Lett.,148, 59-63, 2000.

J160. Sato, E., Yokoyama, N., Miyazawa, T, Maeda, K, Ikeda, Y., Nishimura, Y), Fujita, K, Kohmoto, M ., Takahashi, E., and Mikami, T.: Efficient expression of the envelope protein of feline immunodeficiency virus in a recombinant feline herpesvirus type 1 (FHV-1) using the gC promoter of FHV-1. Virus Res. 70: 13-23. 2000.

J161. Sawada, M., Moriya, S., Saito, S., Shineha, R., Satomi, S., Yamori, T., Tsuruo, T., Kannagi, R. and Miyagi, T.: Reduced sialidase expression in highly metastatic variants of mouse colon adenocarcinoma 26 and retardation of their metastatic ability by sialidase overexpression. Int. J. Cancer 97: 180-185, 2002.

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J163. Sawada, M., Nakashima, S., Kiyono, T., Yamada, J., Hara, S., Nakagawa, M., Shinoda, J., Sakai, N.: Acid sphingomyelinase activation requires caspase-8 but not p53 nor reactive oxygen species during Fas-induced apoptosis in human gioma cels. Exp. Cell Res. 273:157-168, 2002.

J164. Sekine, M., Taya, C., Kikkawa, Y., Yonekawa, H., Takenaka, M., Matsuoka, Y., Imai, E., Izawa, M., Kannagi, R. and Suzuki, A.: Regulation of mouse kidney tubular epithelial cell-specific expression of core 2 GlcNAc transferase. Eur. J. Biochem. 268: 1129-1135, 2001.

J165. Shimizu, K., Hashimoto, T., Harihara, S., Tajima, K., Sonoda, S. and Zaninovic, V.: B-globin gene haplotype characteristics of Colombian Amerinds in South America. Human Heredity 51: 54-63, 2001.

J166. Shimizu, N., Ikehara, Y., Inada, K., Nakanishi, H., Tsukamoto, T., Nozaki, K., Kaminishi, M., Kuramoto, S., Sugiyama, T., Katsuyama, T. and Tatematsu, M.: Eradication diminishes enhancing effects Helicobacter pylori infection on glandular stomach carcinogenesis in mongolian gerbils. Cancer Res., 60, 1512-1514, 2000.

J167. Shimizu, N., Ikehara, Y., Nozaki, K., Kaminishi, M., Tsuda, H., Sugiyama, T., Katsuyama, T. and Tatematsu, M.: Effects of lactoferrin administration in Helicobacter pylori infection animal model. In: K. Shimazaki, H. Tsuda, M. Tomita, T. Kuwata and Perraudin, J. P. (eds.), Lactoferrin: Structure, Function and Applications, pp. 209-215, Oxford, UK: Elsevier Science B.V., 2000.

J168. Shimizu, S., Yatabe, Y., Koshikawa, T., Haruki, N., Hatooka, S., Shinoda, M., Suyama, M., Ogawa, M., Hamajima, N., Ueda, R., Takahashi, T. and Mitsudomi, T.: High frequency of clonally related tumors in cases of multiple synchronous lung cancers as revealed by molecular diagnosis. Clin. Cancer Res. 6: 3994-3999, 2000.

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J170. Song, J., Ugai, H., Ogawa, K., Wang, Y., Sarai, A., Obata, Y., Kanazawa, I., Sun, K., Itakura, K. and Yokoyama, K. K.: Two consecutive zinc fingers in Sp1 and in MAZ are essential for interactions with cis-elements. J. Biol. Chem. 276: 30429-30434, 2001.

J171. Sonoda, S., Li, H.-C., Cartier, L., Nunez, L. and Tajima, K.: Ancient HTLV type I provirus DNA of Andean mummy. AIDS Res. Human Retrovirus 16: 1753-1756, 2000.

J172. Stellman, S.D., Takezaki, T., Wang, L., Chen, Y., Citron, M.L., Djordjevic, M.V., Harlap, S., Muscat, J.E., Neugut, A.I., Wynder, A.L., Ogawa, H., Tajima, K. and Aoki, K.: Smoking and Lung Cancer Risk in American and Japanese Men: An International Case-control Study. Cancer Epidemiol. Biomarkers Prev, 10: 1193-1199, 2001.

J173. Sugaya, Y., Ihara, K., Masuda, Y., Ohtsubo, E. and Maki, H.: Hyper-processive and slower DNA chain elongation catalysed by DNA polymerase III holoenzyme purified from the dnaE173 mutator mutant of Escherichia coli. Genes Cells 7: 385-99, 2002

J174. Sugiyama,T., Saka,K., Nakamura, S., Yonezumi, S. and Seto, M.: API2-MALT1 chimeric transcript is a predictive marker for the responsiveness of H. pylori eradication treatment in low-grade gastric MALT lymphoma. Gastroenterology 120: 1884-1889, 2001.

J175. Suzuki, R., Kagami, Y., Takeuchi, K., Kami, M., Okamoto, M., Ichinohasama, R., Mori, N., Kojima, M., Yoshino, T., Yamabe, H., Shiota, M., Mori, S., Ogura, M., Hamajima, N., Seto, M., Suchi, T., Morishima, Y. and Nakamura, S.: Prognostic significance of CD56 expression for ALK-positive and ALK-negative anaplastic large-cell lymphoma of T/null cell phenotype. Blood 96: 2993-3000, 2000.

J176. Suzuki, R., Seto, M. and Nakamura, S.: Idiopathic eosinophilia. N. Engl. J. Med. 342: 660-661, 2000. (discussion)

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J178. Suzuki, R., Takemura, K., Tsutsumi, M., Nakamura, S., Hamajima, N. and Seto, M.: Detection of cyclin D1 overexpression by real-time reverse-transcriptase-mediated quantitative poly- merase chain reaction for the diagnosis of mantle cell lymphoma. Am. J. Pathol. 159: 425-429, 2001.

J179. Tagawa, H., Miura, I., Suzuki, R., Suzuki, H., Hosokawa, Y. and Seto, M.: Molecular cytogenetic analysis of the breakpoint region at 6q21-22 in T-cell lymphoma/leukemia cell lines. Genes Chrom. Cancer, 34: 175-185, 2002.

J180. Tajima, K. and Sonoda, S.: Origins of HTLV-I with South America. Nat. Med. 6: 232-233, 2000.

J181. Takahashi, H., Maeda, Y., Seto, M. and Hosokawa, Y.: Nucleotide insertions and deletions within the homopolymeric runs of adenines and thymidines of BCL10 cDNAs in normal peripheral blood leukocytes. Blood 95: 2728-2729, 2000.

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J183. Takezaki, T., Gao, C.M., Wu, J.Z., Ding, J.H., Liu, Y.T., Zhang, Y., Li, S.P., Su, P., Liu, T.K. and Tajima, K.: Dietary protective and risk factors for esophageal and stomach cancers in a low-epidemic area for stomach cancer in Jiangsu Province, China; comparison with those in a high-epidemic area. Jpn. J. Cancer Res. 92: 1157-1165, 2001.

J184. Takezaki, T., Hamajima, N., Matsuo, K., Tanaka, R., Hirai, T., Kato, T., Ohashi, K. and Tajima, K.: Association of polymorphisms in the beta-2 and beta-3 adrenoceptor genes with risk of colorectal cancer in Japanese. Int. J. Clin. Oncol. 6: 117-122, 2001.

J185. Takezaki, T., Hirose, K., Inoue, M., Hamajima, N. Yatabe, Y., Mitsudomi, T., Sugiura, T., Kuroishi, T., and Tajima, K.: Dietary factors and lung cancer risk in Japanese: with special

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J189. Tamakoshi, A., Ohno, Y., Yamada, T., Aoki, K., Hamajima, N., Wada, M., Kawamura, T., Wakai, K. and Lin, Y.S.: Depressive mood and suicide among middle-aged workers: findings from a prospective cohort study in Nagoya, Japan. J. Epidemiol. 10: 173-178, 2000.

J190. Tamura, A., Miura, I., Iida, S., Yokota, S., Horiike, S., Nishida, K., Fuji, H, Nakamura, S., Seto, M., Ueda, R. and Taniwaki, M.: Interphase detection of immunoglobulin heavy chain gene translocations with specific oncogene loci in 173 patients with B-cell lymphoma. Cancer Genet. Cytogenet. 129: 1-9, 2001.

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J192. Tauchi, H., Komatsu, K., Ishizaki, K., Yatagai, F. and Kato, T.: Mutation spectrum of MSH3-deficient HHUA/chr.2 cells reflects in vivo activity of the MSH3 gene product in mismatch repair. Mutation Res., 447: 155-164, 2000.

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J195. Tomita, A., Towatari, M., Tsuzuki, S., Hayakawa, F., Kosugi, H., Tamai, K., Miyazaki, T., Kinoshita, T. and Saito, H.: c-Myb acetylation at the carboxyl-terminal conserved domain by transcriptional co-activator p300. Oncogene 20: 444-451. 2000.

J196. Toyoda, H., Nakamura, T., Shinoda, M., Suzuki, T., Hatooka, S., Kobayashi, S., Ohashi, K., Seto, M., Shiku, H. and Nakamura, S.: Cyclin D1 expression is useful as a prognostic indicator for advanced esophageal carcinomas, but not for superficial tumors. Dig. Dis. Sci. 45: 864-869, 2000.

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J213. Yanagisawa, K., Uchida, K., Nagatake, M., Masuda, A., Sugiyama, M., Saito, T., Yamaki, K., Takahashi, Ta. and Osada, H.: Heterogeneities in the biological and biochemical functions of Smad2 and Smad4 mutants naturally occurring in human lung cancers. Oncogene 19: 2305-2311, 2000.

J214. Yashiki, S., Fujiyoshi, T., Arima, N., Osame, M., Yoshinaga, M., Nagata, Y., Tara, M., Nomura, K., Utsunomiya, A., Hanada, S., Tajima, K. and Sonoda, S.: HLA-A26, HLA-B4002, HLA-B4006, and HLA-B4801 alleles predispose to atult T-cell

leukemia: The limited recognition of HTLV type 1 tax peptide anchor motifs and epitopes to generate anti-HTLV type 1 tax CD8+ cytotoxic T lymphocytes. AIDS Res. Human Retrovirol. 17: 1047-1061, 2001.

J215. Yasui, Y., Goto, H., Matsui, S., Manser, E., Lim, L., Nagata, K. and Inagaki, M.: Protein kinases required for segregation of vimentin filaments in mitotic process. Oncogene 20: 2868-2876, 2001.

J216. Yoshida, H., Inoue, Y. H., Hirose, F., Sakaguchi, K., Matsukage, A. and Yamaguchi, M.: Overexpression of DREF in the Drosophila wing imaginal disc induces apoptosis and a notching wing phenotype. Genes to Cells 6: 1-11, 2001.

J217. Yatabe, Y., Konishi, H., Mitsudomi, T., Nakamura, S. and Takahashi, Ta.: Topographical distributions of allelic loss in individual non-small cell lung cancers. Am. J. Pathol. 157: 985-93, 2000.

J218. Yatabe, Y., Suzuki, R., Matsuno, Y., Tobinai, K., lchinohazama, R., Tamaru, J., Mizoguchi, Y., Hashimoto, Y., Yamaguchi, M., Kojima, M., Uike, N., Okamoto, M., Isoda, K., lchimura, K., Morishima, Y., Seto, M., Suchi, T. and Nakamura, S.: Original Article. Morphological spectrum of cyclin D1 -positrve mantle cell lymphoma: Study of 1 68 cases. Pathol. Int., 51: 747-761, 2001.

J219. Yatabe, Y., Suzuki, R., Tobinai, K., Matsuno, Y., Ichinohasama, R., Okamoto, M., Yamaguchi, M., Tamaru, J., Uike, N., Hashimoto, Y., Morishima, Y., Suchi, T., Seto, M. and Nakamura, S.: Significance of cyclin D1 overexpression for the diagnosis of mantle cell lymphoma (MCL): a clinicopathologic comparison of cyclin D1-positive MCL and cyclin D1-negative MCL-like B-cell lymphoma. Blood 95: 2253-2261, 2000.

J220. Yazaki, M., Takahashi, To., Ito, Y., Mori, C. and Wada, Y.: Generation of HLA-A2 subtype specific T lymphocytes from cord blood used for cord blood stem cell transplantation. Bone Marrow Transplant. 26: 451-454, 2000.

J221. Yokoyama, N., Hirata, M., Ohtsuka, K., Nishiyama, Y., Fujii, K, Fujita, M., Kuzushima, K., Kiyono, T., and Tsurumi, T.: Co-expression of human chaperone Hsp70 and Hsdj or Hsp40 co-factor increases solubility of overexpressed target proteins in insect cells. Biochim. Biophys. Acta. 1493: 119-124, 2000.

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J222. Yonezumi, M., Suzuki, R., Suzuki, H., Yoshino, T., Oshima, K., Hosokawa, Y., Asaka, M., Morishima, Y., Nakamura, S. and Seto, M.: Detection of AP12-MALT1 chimaeric gene in extranodal and nodal marginal zone B-cell lymphoma by reverse transcription polymerase chain reaction (PCR) and genomic long and accurate PCR analyses. Br. J. Haematol. 115: 588-594, 2001.

J223. Yoo, K.-Y., Tajima, K., Park, S.-K., Kang, D., Kim, S.-U., Hirose, K., Takeuchi, T. and Miura, S.: Postmenopausal obesity as a breast cancer risk factor according to estrogen and progesteron receptor status (Japan). Cancer Letters, 167: 57-63, 2001.

J224. Yoshida, K., Hamajima, N., Kozaki, K., Saito, H., Maeno, K., Sugiura, T., Ookuma, K. and Takahashi, Ta.: Association between the dopamine D2 receptor A2/A2 genotype and smoking behavior in the Japanese. Cancer Epidemiol. Biomarkers Prev. 10: 403-405, 2001.

J225. Yuasa, H., Hamajima, N. and Matsuo, K.: Investigation for smoking cessation support on internet survey of health and smoking awareness of users. Jpn. J. Public Health 47: 820-827, 2000 (in Japanese).

J226. Zhao, S.-M., Li, H.-C., Lou, H., Lu, X.-X., Yu, X.-F., Gao, D.-H., Hu, J., Chiba, H., Takezaki, T., Yashiki, S., Fujiyoshi, T., Sonoda, S. and Tajima, K.: High prevalence of HBV in Tibet, China. Asian Pacific J. Cancer Prev. 2: 299-304, 2001.

J227. Zhong, S., Zhange, Y., Jansen, C., Goto, H., Inagaki, M. and Dong, Z.: MAP kinases mediate UVB-induced phosphorylation of histone H3 at Serine 28. J. Biol. Chem. 276: 12932-12937, 2001.

J228. Zhong, S., Jansen, C., She, Q.B., Goto, H., Inagaki, M., Bode, A.M., Ma, W.Y. and Dong, Z.: Ultraviolet B-induced phosphorylation of histone H3 at serine 28 is mediated by MSK1. J. Biol. Chem. 276: 33213-33219, 2001.

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Reviews and Books

R001. Akatsuka, Y.: Minor histocompatibility antigens and their clinical significance. Saishin Igaku 56: 186-191, 2001. (in Japanese)

R002. Ando, S. and Inagaki, M.: Protein kinase domain structure and phosphorylation specificity. Experimental Medicine 18(1): 7-12, 2000. (in Japanese)

R003. Chen Y.-T., Scanlan, M.J., Obata, Y. and Old, L.J.: Identification of human tumor antigens by serological expression cloning. In Principles and Practice of the Biologic Therapy of Cancer. Philadelphia; Lippincott Williams & Wilkins, Rosenberg, S.A., ed., pp. 557-570, 2000.

R004. Fujita, M.: Cell cycle regulation of DNA replication initiation proteins bycyclin/CDK. Experimental Medicine 20: 540-545, 2002. (in Japanese)

R005. Fujita, M.: Function and cell cycle regulation of DNA replication initiation proteins in mammalian cells. Experimental Medicine 18: 933-939, 2000. (in Japanese)

R006. Goto, H., Kosako, H. and Inagaki, M.: Regulation of intermediate filament organization during cytokinesis: possible roles of Rho-associated kinase. Microsc. Res. Tech. 49:173-182, 2000.

R007. Hamajima, N., Matsuo, K., Saito, T., Hirose, K., Inoue, M., Takezaki, T., Kuroishi, T. and Tajima, K.: Gene-environment interactions and polymorphism studies of cancer risk in the Hospital-based Epidemiologic Research Program at Aichi Cancer Center II (HERPACC-II). Asian Pacific J. Cancer Prev. 2: 99-107, 2001.

R008. Hamajima, N.: PCR-CTPP: a new genotyping technique in the era of genetic epidemiology. Expert Rev. Mol. Diagn. 1: 119-123, 2001.

R009. Hida, T. and Takahashi, Ta.: Development of novel treatments of lung cancer 2: application of COX-2 inhibitors. Chiryogaku, 35: 70-73, 2001. (in Japanese)

R010. Ikenaga, M., Ishizaki, K., Nishizawa, K., Han, Z.-B., Kitao, H., Hirayama, J. and Kato, T.: Genetic effects of space radiation and microgravity on cultured human tumor cells. In: Exploring Future Research Strategies in Space Radiation

Sciences, Eds. H. J. Majima and K. Fujitaka, pp. 86-91, Iryokagakusha, Co. Ltd., Tokyo, 2000.

R011. Inada, H., Nagata, K., Goto, H. and Inagaki, M.: Regulation of intermediate filament dynamics: A novel approach using site-and phosphorylation state-specific antibodies. Cytoskeleton: Signalling and Cell Regulation: A Practical Approach. (The practial Approach Series), eds. Carraway, K.L. and Carraway, C.A.C. Oxford University Press 183-207, 2000.

R012. Inoue, M.: Recent observations in the epidemiology of gastric cancer in Japan. 4th International Gastric Cancer Congress, eds., M.F. Brennan, M.S. Karpeh, p. 63-67, Monduzzi Editore, Italy, 2001.

R013. Kannagi, R. and Hakomori, S.: A guide to monoclonal antibodies directed to glycotopes. Adv. Exp. Med. Biol. 491: 587-630, 2001.

R014. Kannagi, R., Kumamoto, K., Tei, K., Saitou, S., Izawa, M., Goto, Y. and Fukui, F.: Carbohydrate determinants in cancer. Molecular Medicine, 38: 1190-1199, 2001. (in Japanese)

R015. Kannagi, R., Mitsuoka, C., Kanamori, A., Uchimura, K., Muramatsu, T., Imai, T., Yoshie, O., Matsushima, K. and Ohmori, K.: Expression and selectin-binding activity of sialyl 6-sulfo Lewis X determinant on human helper memory T lymphocytes. In: D.Y. Mason (ed.), Leukocyte Typing VII, pp. 39-41, Oxford: Oxford University Press, 2002.

R016. Kannagi, R.: Regulatory roles of carbohydrate ligands for selectins in homing of lymphocytes. Curr. Opin. Struct. Biol., in press.

R017. Kannagi, R.: Transcriptional regulation of expression of carbohydrate ligands for cell adhesion molecules in the selectin family. Adv. Exp. Med. Biol. 491: 267-278, 2001.

R018. Kannagi, R.: Fucosyltransferase V. In: N. Taniguchi, K. Honke and M. Fukuda (eds.), Handbook of Glycosyltransferases and Their Related Genes, pp. 232-236, Tokyo: Springer-Verlag, 2001.

R019. Kannagi, R.: Fucosyltransferase VI. In: N. Taniguchi, K. Honke and M. Fukuda (eds.), Handbook of Glycosyltransferases and Their Related Genes, pp. 237-245, Tokyo: Springer-Verlag, 2001.

R020. Kannagi, R.: Selectins and their

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carbohydrate ligands in cellular recognition. Seikagaku, 72: 1399-1415, 2000. (in Japanese)

R021. Kiyono, T.: Cell cycle perturbation by proteins of tumor viruses. In Mechanisms of Cell Cycle and Cancer ed. by Taya, Y. pp. 93-100, Yodosha, Tokyo, 2000. (in Japanese)

R022. Kiyono, T.: DNA virus and ageing. In Frontier in Aging Research ed. by Ishikawa, F. Springer-Verlag, Tokyo, in press. (in Japanese)

R023. Kiyono, T.: Functions of human papillomavirus early genes and carcinogenesis: Gendai Igaku, 32: 39-45, 2000. (in Japanese)

R024. Kiyono, T.: Mechanisms of carcinogenesis by human Papillomaviruses. J. Jpn. Soc. Gynecol. Oncol., 18: 131-138, 2000. (in Japanese)

R025. Kudo, T., Egawa, H., Kuzushima, K., Kimura, H., Morishima, T. and Ichiyama, S.: Diagnostic testing in Epstein-Barr virus infection. Clin. Chem. Lab. Med. 39: 789-794, 2001.

R026. Kuzushima, K.: Behavior of antigen-specific CD8+ T cells in Epstein-Barr virus-related disease. Uirusu. 2001 51:43-49. (in Japanese)

R027. Kuzushima, K.: Characterization of Epstein-Barr virus-specific CD8+ T cells in infectious mononucleosis. Rinsho Ketsueki. 42:458-463, 2001. (in Japanese)

R028. Kuzushima, K.: Quantitative analysis of Epstein-Barr virus (EBV)-specific CD8+ T lymphocytes in EBV-related diseases. Rinsho Ketsueki. 41: 487-90, 2000. (in Japanese)

R029. Kuzushima, K. and Tsurumi, T.: The CD8+ response to Epstein-Barr Virus (EBV): Development of an efficient methods to detect EBV-specific CD8+ T cells by flow cytometry and its clinical application. Current Topics in Virology, in press.

R030. Kuzushima, K.: Dynamics of Epstein-Barr virus-specific CD8+ T lymphocytes. Rinsho Men-eki. 34: 231-238, 2000. (in Japanese)

R031. Kuzushima, K.: Tetrameric MHC-peptide complex identifying CD8+ T cells specific to Epstein-Barr virus and human cytomegalovirus. Nihon Rinsho Men-eki Gakkai-shi 25: 70-78, 2001. (in Japanese)

R032. Matsuo, K., Hamajima, N., Suzuki, T., Nakamura, T., Matsuura, A. and Tominaga, S.: Better ROC Curves for a regionally developed

Helicobacter pylori antibody test. Asian Pacific J. Cancer Prev. 2: 155-166, 2001.

R033. Matsuo, K., Suzuki, R., Morishima, Y. and Hamajima, N.: Attribution of posttransplantation toxicity to methotrexate regarding genotype of methylenetetrahydrofolate reductase gene (MTHFR) polymorphism needs further clarification. Blood 98: 2283-2284, 2001.

R034. Mitsudomi, T., Takahashi, Ta.: Cancer treatment and novel molecular targets. Cancer up to date. 3: 10-13, 2001. (in Japansese)

R035. Nagata, K., Izawa, I. and Inagaki, M.: A decade of site-and phosphorylation state-specific antibodies: recent advances in studies of spatiotemporal protein phosphorylation. Genes Cells 6: 653-664, 2001

R036. Obata, Y., Takahashi, To., Sakamoto, J., Tamaki, H., Tominaga, S., Hamajima, N., Chen, Y.-T. and Old, L.J.: SEREX analysis of gastric cancer antigens. Cancer Chemother. Pharmacol. 46 (suppl.): S37-S42, 2000.

R037. Ohmori, K., Mitsuoka, C., Kanamori, A., Adachi, K., Kameyama, A., Nonoyama, S. and Kannagi, R.: Novel anti-CD15 antibodies with distinct specificity towards Lewis X determinants carried by mucin core carbohydrates. In: D.Y. Mason (ed.), Leukocyte Typing VII, pp. 179-182, Oxford: Oxford University Press, 2002.

R038. Ohtsuka, K. and Hata, M.: Induction of heat shock proteins and their biological functions. Kosaka, M., Sugahara, T., Schmidt, K.L. and Simon, E. (Eds), Thermotherapy for Neoplasia, Inflammation, and Pain, Springer-Verlag, Tokyo, pp. 328-334, 2000.

R039. Ohtsuka, K. and Hata, M.: Molecular chaperone function of mammalian Hsp70 and Hsp40 - A review. Int. J. Hyperthermia 16: 231-245, 2000.

R040. Ohtsuka, K. and Suzuki, T.: Roles of molecular chaperones in the nervous system. Brain Res. Bull. 53: 141-146, 2000.

R041. Osada, H. and Takahashi, Ta.: Molecular biology of lung cancer. Gendai igaku, 47: 515-518, 2000. (in Japanese)

R042. Osada, H. and Takahashi, Ta.: Molecular pathogenesis of lung cancer. Nippon Rinsyo 58: 1012-1016, 2000. (in Japanese)

R043. Riddell, S.R., Gavin, M., Akatsuka, Y.,

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Murata, M., Greenberg, D.G. and Warren, E.H.: Minor histocompatibility antigens in graft-vs.-leukemia reactions. In: K.A. Dicke and A. Keating (eds.), Autologous Blood and Marrow Transplantation X: Proceedings of the Tenth International Symposium, pp.333-339, Charlottesville: Carden Jennings Publishing, 2001.

R044. Riddell, S.R., Warren, E.H., Gavin, M.A., Akatsuka, Y., Lewinsohn, D., Mutimer, H., Cooper, L., Topp, M.S., Bonini, C. and, Greenberg, P.D.: Immunotherapy of human viral and malignant diseases with genetically modified T-cell clones. Cancer. J. Sci. Am. Suppl. 3: S250-258, 2000.

R045. Tajima, K. and Moore, M.: Screening efficacy in Asia – What of problems of non-presentation at hospital for treatment? Asian Pacific J. Cancer Prev. 2: 237-239, 2001.

R046. Tajima, K., Hirose, K., Hamajima, N., Inoue, M., Takezaki, T. and Kuroishi, T.: Asian Pacific cancer prevention in the 21st ccentury: five rule points form the Hospital-based Epidemiologic Research Program at Aichi Cancer Center (HERPACC). Asian Pacific J. Cancer Prev, 2: 81, 2001.

R047. Tajima, K., Hirose, K., Inoue, M., Takezaki, T., Hamajima, N. and Kuroishi, T.: A model of practical prevention for out-patients visiting hospital: the hospital-based epidemiologic research program at Aichi Cancer Center (HERPACC). Asian Pacific J. Cancer Prev, 1: 35-47, 2000.

R048. Tajima, K., Li, H.-C., Yashiki, S. and Sonoda, S.: HTLV-I provirus DNA in Andean mummies. Asian Pacific J. Cancer Prev. 1 (Supplement): 7-9, 2000.

R049. Tajima, K., Takezaki, T., Fujiyoshi, T. and Sonoda, S.: Geographical distribution of HTLV-I/II in south America. Asian Pacific J. Cancer Prev., 1(Supplement): 7-9, 2000.

R050. Tajima, K.: 16th Asian Pacific cancer conference, Manila. Asian Pacific J. Cancer Prev. 2: 259-260, 2001.

R051. Tajima, K.: Public symposium on forefront of cancer epidemiology and prevention by Study Areas of Cancer Epidemiology (SACE) in the Special Priority Area (C) sponsored by the Japanese Ministry of Education, Science, Culture, Sports and Technology. Asian Pacific J. Cancer Prev. 2: 9-14, 2001.

R052. Takahashi, Ta. and Mitsudomi, T.:

Molecular diagnosis of lung cancer. Igaku-no-ayumi, 197: 1189-1192, 2001. (in Japanese)

R053. Takahashi, Ta.: Oncogenes and tumor suppressor genes. In Clinical Oncology 2nd edition, Japanese Society of Medical Oncology (ed.), pp73-85, 1999. (in Japanese)

R054. Takezaki, T.: The JICA training course, community-based cancer prevention (epidemiol- ogical approach). Asian Pacific J. Cancer Prev. 2: 93-97, 2001.

R055. Tokudome, S., Nagaya, T., Okuyama, H., Tokudome, Y., Goto, C., Imaeda, N., Kitagawa, I., Fujiwara, N., Ikeda, M., Ichikawa, H., Kuriki, K., Takekuma, K., Shimoda, A., Hirose, K. and Usui, T.: Japanese versus mediteranean diets and cancer. Asian Pacific J. Cancer Prev. 1: 61-66, 2000.

R056. Tsurumi, T. and Fujita, M.: Molecular basis for Epstein-Barr Virus DNA replication. Current Topics in Virology, in press.

R057. Tsurumi, T.: Epstein-Barr virus and human cancer: EBV replication enzymes. Curr. Top. Microbiol. Immunol., 258: 65-87, 2001.

R058. Tsurumi, T.: Microbiology (Edited by Hatanaka, M. and Shimada. J., Bunkodo): Herpesviridae, pp. 491- 500, 2000. (in Japanese)

R059. Tsurumi, T.: Molecular mechanism of Epstein-Barr Virus DNA replication. Uirusu. in press. (in Japanese)

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Abstracts for International Conferences

A001. Akatsuka, Y., Warren, E.H., Brickner, A.G., Lin, M-T., Gooly, T., Martin, P.J., Hansen, J.A., Engelhard, V.H. and Riddell, S.R.: Effect of disparity in the newly identified minor histocompatibility antigen SKH13 on the development of graft-versus-host disease after marrow transplantation from an HLA-identical sibling. The 42nd Annual Meeting of the American Society of Hematology. abstract p.202, San Francisco, California, 2000.

A002. Akatsuka, Y.: Identification of human new histocompatibility antigens and their clinical application. The fourth Nagoya International Blood and Marrow Transplantation Symposium. abstract pp.36-37, Nagoya, 2001.

A003. Das, K., Basu, M., Henshaw, J., Li, S-C., Kannagi, R. and Basu, S.: GalNAcT-1 activity isolated from guinea pig bone marrow. XVIth International Symposium on Glycoconjugates, The Hague, Abstracts, pp. 71, Glycoconjugate J., 18: 92, 2001.

A004. Fujita, M. and Tsurumi, T.: Intranuclear organization of DNA replication initiation proteins in mammalian cells. Abstract of the Meeting on Eukaryotic DNA Replication at The Salk Institute. pp. 37, 2000.

A005. Fujita, M.: Cell Cycle regulation of DNA replication initiation proteins in mammalian cells. Abstract of Symposium on Molecular Network of G1-S Regulation in Eukaryotic Cells: From G1 Regulaters to DNA Replication Machinery. pp. 13. 2001.

A006. Hamajima, N. and Tajima, K.: Estimation of cancer susceptibility based on gene-environment interaction. Aichi Cancer Center International Symposium VII, p. 36-37, 2001.

A007. Hamajima, N., Katsuda, N., Matsuo, K., Saito, T., Ito, L.S., Ando, M., Inoue, M., Takezaki, T. and Tajima, K.: Smoking habit and interleukin 1B C-31T polymorphism, Program & Abstract of the 3rd Asian-Pacific Congress of Epidemiology, 2001, p. 48, 2001.

A008. Hamajima, N., Matsuo, K., Mizutani, M., Iwata, H., Iwase, T., Miura, S., Obata, Y. and Tajima, K.: Catechol-O-methyltransferase polymorphism and breast cancer risk in Japan. Abstract of the Twentieth International Symposium of the Sapporo Cancer Seminar Foundation: Gene

Environment Interaction and Cancer Prevention, p. 25, 2000.

A009. Hamajima, N., Tajima, K., Fukumitsu, T., Odauchi, S., Usui, T. and Akashi, T.: A large-scale follow-up study of smokers who visited medical facilities in Japan. The 22nd Annual Meeting of the International Association of Cancer Registries, Book of Abstract, p. 81, 2000.

A010. Haruki, N., Harano, T., Masuda, A., Kiyono, T., Tatematsu, Y., Shimizu, S., Mitsudomi, T., Konishi, H., Osada, H., Fujii, Y. and Takahashi, Ta.: Persistent increase of chromosome instability in lung cancer: possible indirect involvement of p53 inactivation. The 5th Joint Conference of the American Association for Cancer Research and the Japanese Cancer Association. pp. C-28, Maui, USA, 2001.

A011. Hirose, F., Yamaguchi, M., Taguchi, O. and Matsukage, A.: Transcriptional regulatory factor, DREF is involved in regulation of human CMV US3 gene expression. A Keystone Symposium Chromatin Structure and Function, Abstract pp. 80, 2000.

A012. Ichinohe, T., Matsuo, K., Tamaki, S., Hamajima, N., Hirabahashi, N. and Dohi, H.: Superior outcome of blood and marrow stem-cell transplantations using maternal grafts over transplants using paternal grafts. Blood, Vol 96 Abstract of the Annual Meeting of the 42nd American Society of Hematology, p. 208a, 2000.

A013. Ikehara, Y., Kojima, N., Kurosawa, N., Kono, M., Nishihara, S., Itzkowitz, S.H., Tatematsu, M., Tsuji, S., and Narimatsu, H.: Cloning and Expression of the probable candidate for the cancer associated Sialyl-Tn antigen synthase (human ST6GalNAcI). XIII International Congress International Academy of Pathology, p. 181, 2000.

A014. Inada, K., Tanaka, H., Nakanishi, H., Tsukamoto, T., Ikehara, Y., Tatematsu, K., Nakamura, S., Porter, E.M., and Tatematsu, M.: Identification of Paneth cells in pyloric glands associated with gastric and intestinal mixed type intestinal metaplasia of the human stomach. XIII International Congress International Academy of Pathology, p85, 2000.

A015. Inoue, M., Tajima, K. and Tominaga, S.: Probabilities of developing cancer over the whole life span of a Japanese. The 22nd Annual Meeting of the International Association of Cancer Registries, Book of Abstract, p. 34, 2000.

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A016. Inoue, M., Tajima, K. and Tominaga, S.: The estimation of cancer incidence in Aichi Prefecture, Japan: use of model area with good quality registry data. The 23rd Annual Meeting of the International Association of Cancer Registries, Abstracts book, p. 54, 2001.

A017. Inoue, M., Tominaga, S., Tajima, K., Matsuura, A. and Nakamura, S.: Chronic atrophic gastritis and subsequent gastric cancer: prospective cohort study in Japan. The 22nd Annual Meeting of the International Association of Cancer Registries, Book of Abstract, p. 17, 2000.

A018. Inoue, M.: Recent observations in the epidemiology of gastric cancer in Japan. 4th International Gastric Cancer Congress, Abstract Book, p. 8, 2001.

A019. Kannagi, R., Mitsuoka, C., Kanamori, A., Uchimura, K., Muramatsu, T., Imai, T., Yoshie, O., Matsushima, K. and Ohmori, K.: Expression and selectin-binding activity of sialyl 6-sulfo Lewis X determinant on human helper memory T- lymphocytes. The 7th International Conference on Human Leukocyte Differentiation Antigens, Harrogate, Tissue Antigens 55(Suppl.): 8, 2000.

A020. Kannagi, R.: Glycoconjugate ligands and regulation of selectin-mediated cell adhesion. (Plenary Lecture). XVIth International Symposium on Glycoconjugates, The Hague, Abstracts p. 7, Glycoconjugate J., 18: 10, 2001.

A021. Kannagi, R.: Regulation of T-lymphocyte traffic by carbohydrate ligands for selectin. (Invited Speaker). International Symposium on Protein Traffic, Glycosylation, and Human Health, Interlaken, Abstracts p. 17, 2001.

A022. Kato, M., Hayashi, Y. and Yamaguchi, M.: Identification of dRFX2 that binds to a novel transcriptional regulatory element of the Drosophila PCNA gene. Abstracts of Eukaryotic DNA Replication pp.105, 2001.

A023. Kumamoto, K., Izawa, M., Mitsuoka, C., Takenoshita, S. and Kannagi, R.: Significant alteration of carbohydrate 6-sulfotransferases in colon cancer and modulation by histone deacetylase inhibitors. The 5th Joint Conference of the American Association for Research and the Japanese Cancer Association "Molecular Biology and New Therapeutic Strategies: Cancer Research in the 21st Century," Hawaii, Abstract, pp. B-2, 2001.

A024. Kwon, E.-J., Hirose, F., Ohshima, N. and Yamaguchi, M.: Transcriptional regulation of a gene for DREF, a key regulatory factor for DNA replication related genes in Drosophila. Abstracts of Eukaryotic DNA Replication pp.118, 2001.

A025. Matsuo, K., Hamajima, N. and Tajima, K.: Methylenetetrahydrofolate reductase (MTHFR) polymorphsims and life style in malignant lymphoma. Abstract of the Twentieth International Symposium of the Sapporo Cancer Seminar Foundation: Gene Environment Interaction and Cancer Prevention, p. 26, 2000.

A026. Nagata, K. and Inagaki, M.: Char- acterization of septin MSF: Possible involve- ment in cytoskeletal reorganization. 41th American Society of Cell Biology Annual Meeting. Molecular Biology of the Cell, 12: 435a, 2001.

A027. Nakanishi, H., Ito, S., and Tatematsu, M.: Sequential observation of peritoneal micro- metastasis formation by LacZ gene and GFP gene-tagged murine carcinoma cells. XIII International Congress International Academy of Pathology, 2000.

A028. Nakanishi, H.: Molecular Diagnostic detection of free cancer cells in the body fluids of gastrointestinal and bladder cancer patients with real-time PCR. Program and abstracts of Aichi Cancer Center International symposium VII, p. 26-27, 2001.

A029. Ohmori, K., Mitsuoka, C., Kanamori, A., Adachi, K., Kameyama, A., Takahashi, N., Nonoyama, S. and Kannagi, R.: Novel anti-CD15 antibodies with distinct specificity towards Lewis X determinants carried by mucin core carbohydrates. The 7th International Conference on Human Leukocyte Differentiation Antigens, Harrogate, Tissue Antigens 55(Suppl.):31, 2000.

A030. Ohmori, K., Tei, K., Kumamoto, K., Imai, T., Yoshie, O., Hasegawa, H., Matsushima, K. and Kannagi, R.: Site-specific recruitment of human helper T cells by synergistic action of sulfated ligand for selectins and chemokines. The 5th Joint Conference of the American Association for Research and the Japanese Cancer Association "Molecular Biology and New Therapeutic Strategies: Cancer Research in the 21st Century," Hawaii, Abstract, pp. A-62, 2001.

A031. Ohno, K., Takahashi, Y., Hirose, F., Inoue, Y. H., Taguchi, O., Nishida, Y., Matsukage, A. and Yamaguchi, M.: Functional analysis of the

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Drosophila MLF homologue isolated as a factor interacting with DREF. A Keystone Symposium Chromatin Structure and Function, Abstract pp. 69, 2000.

A032. Tajima, K. and Moore, M.: Forefront of cancer epidemiology and prevention in the Asian Pacific area, Program & Abstract of The 3rd Asian-Pacific Congress of Epidemiology, 2001, p. 27, 2001; Proceeding of the 16th Asia Pacific Cancer Conference, p. 27, 2001.

A033. Tajima, K., Hirose, K., Hamajima, N., Inoue, M., Takezaki, T. and Kuroishi, T.: Lifestyles and cancer in Japan: with special reference to results from the Hospital-based Epidemiologic Research Program at Aichi Cancer Center (HERPACC). Proceedings of the 3rd Korea-Japan Joint Epidemiology Seminar, Diet and Health, p. 5, 2001.

A034. Tajima, K., Takezaki, T., Li, H.-C. and Sonoda, S.: Expansion of ethnoepidemiological studies on HTLV in Japan and the world. Abstract of the 8th Japanese-German Workshop on Molecular and Cellular Aspects of Carcinogenesis, p. 35-39, 2001.

A035. Tajima, K.: Adult T-cell leukemia/ lymphoma in Japan: Epidemiologic pattern and prevention strategy. Proceeding of Asian Cancer Conference in Shizuoka, p. 8, 2000.

A036. Takahashi, Ta., Haruki, N., Kozaki, K., Harano, T., Masuda, A., Konishi, H., Hida, T., Tatematsu, Y., Saito, H., Yatabe, Y., Yanagisawa, K., Yoshida, K., Mitsudomi, T. and Osada, H.: Multidirectional approach for studies on the carcinogenesis and progression of human lung cancers. Plenary session, The 9th World Conference of Lung Cancer. p. 35-36, Tokyo, Japan, 2000.

A037. Takahashi, Ta., Kozaki, K., Yatabe, Y., Achiwa, H. and Hida, T.: Increased expression of COX-2 in the development of human lung cancers. Cyclooxygenase-2, a Molecular Target for Cancer Chemoprevention. p.3, Seoul, Korea, 2001.

A038. Takahashi, Ta.: Altered cell cycle control in human lung cancer. Symposium on Cell Ceycle Control & Lung Cancer. p. 5, Hong Kong, China, 2000.

A039. Takahashi, Ta.: Smoking behavior and its consequences in the pathogenesis of lung cancer. Molecular Epidemiology of Nicotine Addiction and Lung Cancer. Maui, USA, 2001.

A040. Takahashi, Ta.: Updates on molecular pathogenesis of human lung cancer. Meet-the-Expert-Sunrise-Session. The 91st Annual Meeting of the American Association for Cancer Research. p. ?, San Francisco, USA, 2000.

A041. Takahashi, Ta.: Smoking behavior and it’s fingerprint in the precess of lung carcinogenesis. The 20th International Symposium of the Sapporo Cancer Seminar Foundation. pp16-17, Sapporo, Japan, 2001.

A042. Takezaki, T., Ikeda, S., Inoue, M., Tajima, K. and Tominaga, S.: Cooking methods and risk of stomach cancer incidence: a 14-year prospective study in a rural Area of Japan. Program & Abstract of the 3rd Asian-Pacific Congress of Epidemiology, 2001 -IEA Regional Scientific Meeting in Japan, p. 41, 2001.

A043. Tatematsu, M.: Analysis of the origin of gastric cancers using animal models. XIII International Congress International Academy of Pathology, p.56-63, 2000.

A044. Tatematsu, M.: Reversibility of gastric submucosal tumor-like lesions in Helicobacter pylori infected mongolian gerbils with eradication. U.S._Japan Cooperative Medical Science Program. Environmental Mutagenesis and Carcinogenesis Panel, p.10, 2001.

A045. Tsukamoto, T., Fukami, H., Yasutomi, H., Yamanaka, S., Nakanishi, H., Aoki, I., and Tatematsu, M.: Transcriptional down regulation of hexosaminidase alpha and beta subunits in abrrant crypts in the 1, 2-dimethylhydrazine treated rat colon. XIII International Congress International Academy of Pathology, p103, 2000.

A046. Tsukamoto, T., Inada, K., Fukami, H., Yamamoto, M., Tanaka, H., Kusakabe, M, Bishop, C.E., and Tatematsu, M.: Mouse stain susceptibility to diethylnitrosamine induced hepatocarcinogenesis is cell autonomous whereas sex-susceptibility is due to the micro-environment. The 5th Joint Conference of the American Association for Research and the Japanese Cancer Association "Molecular Biology and New Therapeutic Strategies: Cancer Research in the 21st Century," Hawaii, Abstract, pp. A-90, 2001.

A047. Tsurumi, T.: Epstein-Barr virus and human cancer: Epstein-Barr virus replication proteins. The 21st International Cancer Symposium in Sapporo. pp. 52-53. 2001 (Sapporo, Japan)

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A048. Uchimura, K., El-Fasakhany, F.M., Kannagi, R. and Muramatsu, T.: Molecular cloning, characterization and its implications in biosynthesis of 3'sulfo-Lewisa. XVIth International Symposium on Glycoconjugates, The Hague, Abstracts, pp. 941, Glycoconjugate J., 18: 120, 2001.

A049. Yamaguchi, M., Hayashi, Y. and Hirose, F.: The homeodomain protein, Distal-less negatively regulates the function of DREF, a master regulatory factor for DNA replication-related genes in Drosophila. Abstracts of Eukaryotic DNA Replication pp. 226, 2001.

A050. Yamaguchi, M., Hayashi, Y., Ohno, K., Kwon, E.-J., Yoshida, H., Hirose, F., Inoue, Y. H. and Matsukage, A.: Transcriptional regulatory network for Drosophila. DNA replication-related genes. Abstracts of Eukaryotic DNA Replication Meeting pp.122, 2000.

A051. Yoshida, H., Inoue, Y. H., Hirose, F., Sakaguchi, K., Matsukage, A. and Yamaguchi, M.: Ectopic DREF expression induces S phase and apoptosis in Drosophila wing imaginal discs. Abstracts of Eukaryotic DNA Replication Meeting. pp.125, 2000.

A052. Yoshioka, S., Sekine, M., Kannagi, R. and Suzuki, A.: Mouse kidney tubular epithelial cell-specific regulation of glycochains and megalin. XVIth International Symposium on Glyco- conjugates, The Hague, Abstracts, pp. 95, Glycoconjugate J., 18: 121, 2001.

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Record of Seminars ___________________________________________________________ Invited Speakers

2000

May 23 Qadota, H. (Nara Institute of Science and Technology) A protein complex regulating organ forma-

tion during the embryonic stage of C. elegans. Jul. 05 Dyson, N. (Laboratory of Molecular Oncology, Massachusetts General Hospital Cancer Center,

USA) The control of cell proliferation by E2F and pRB-family proteins. Aug. 16 Wei, M. (Laboratory of Dr. Stanley Korsmeyer, Dana Farber Cancer Institute and Harvard Medical

School, Boston, Massachusetts, USA ) Cell death induced by BID, a pro-apoptotic Bcl-2 family member.

Oct. 10 Carey, T.E. (Laboratory of Head and Neck Cancer Biology Department of Otolaryngology/Head and

Neck Surgery, University of Michigan, USA) Gene abnormalities of head and neck cancer. Oct. 18 Hess, J.L. (Department of Pathology and Laboratory Medicine, University of Pennsylvania School of

Medicine, USA) Transcriptional regulation by the mixed lineage leukemia protein (MLL). 2001 Jan. 26 Hoffman, E.W. (Office of Clinical Trial Management, Ludwig Institute for Cancer Research, New

York, New York, USA) Immunotherapy at the Ludwig Institute for Cancer Research. Feb. 28 Quinlan, R.A. (School of Life Sciences, MedicaI Sciences Institute, The University of Dundee, UK)

Crystallins and diseases of the heart and eye－Problems associated with protein misfolding and the formation of insoluble protein aggregates.

Mar. 13 Kotani, S. (Dept. of Intractable Diseases, International Medical Center of Japan) Molecular

mechanisms of regulation of APC/C. Apr. 18 Kitamura, T. (Division of Hematopoietic Factors, Institute of Medical Science, University of Tokyo) A

novel approach utilizing retroviruses in cytokine research / from cytokine to cytokinesis. Jul. 02 Goulmy, E. (Department of Immunohematology and Blood Transfusion, Leiden University Medical

Center, Leiden, The Netherlands) Minor histocompatibility antigens for hematopoietic tumor spe-cific cellular therapy.

Oct. 09 Inohara, N. (Department of Pathology and Comprehensive Cancer Center, University of Michigan

Medical School, USA ) Nod protein, a mediator of immune response and apoptosis: Crohn dis-ease and natural immunity.

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Institute Speakers 2000 Mar. 27 Haruki, N. (Molecular Oncology) Chromosomal instability in human lung cancers. Apr. 20 Ajiro, K. (Biochemistry) Regulation of chromatin structure and function by histone modification. Jun. 08 Ikehara, Y. (Oncological Pathology) The alteration of carbohydrate antigen expression in the cour-

se of carcinogenesis and cancer development in gastric mucosa. Jun. 30 Taguchi, O. (Molecular Pathology) The transplantation of the rat immune system to the scid

mouse. Aug. 05 Nagata, K. (Biochemistry) Identification and characterization of a novel Rho/Rho-Kinase activating

factor, KIAA0380, by a newly established detection system. Sep. 21 Kuzushima, K. (Virology) Quantification of Epstein-Barr virus and human cytomegalovirus-specific

CD8+T cells: Its clinical implication and prospects. Oct. 12 Suzuki, R. (Molecular Medicine) Identification of target genes for transcriptional regulator MLL. Nov. 09 Kumimoto, H. (Central Laboratory & Radiation Biology) Different susceptibility of each L-myc

genotype to esophageal cancer risk factors. Nov. 30 Hamajima, N. (Epidemiology and Prevention) Gene-environment interactions with IL-1B for Heli-

cobacter pylori infection. 2001 Jan. 31 Yanagisawa, K. (Molecular Oncology) Analysis of TGF-β induced apoptosis in hepatoma cell

lines. Apr. 26 Matsuo, K. and Hamajima, N. (Epidemiology and Prevention) Genetic polymorphisms for malig-

nant lymphoma risk. May 22 Nakanishi, H. (Oncological Pathology) Real-time analysis of micrometastasis formation in living

mice and its clinical application. Sep. 13 Akatsuka, Y.(I) and Kondo, E.(II) (Immunology) (I). Basic studies toward immunotherapy tar-

geting minor histocompatibility antigens. (II). Application of antigen-transduced CD40L-activated B cells as potent antigen presenting cells.

Oct. 30 Kiyono, T. and Yamashita, Y. (Virology) Immortalization and carcinogenesis of human cells. Nov. 22 Kanamori, A. (Molecular Pathology) Specificities of binding of selectins (CD62L, E, and P). Dec. 20 Izawa, I. (Biochemistry) Keratin attenuates tumor necrosis factor-induced cytotoxicity through as-

sociation with TRADD.

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Record of Symposia ___________________________________________________________ The 8th Aichi Cancer Center International Symposium “Prospects for Conquering Stomach Cancer in the 21st Century”

Organizing Committee: Masae Tatematsu, Tsuneya Nakamura, Takashi Suzuki, Hayao Nakanishi, Kenichi

Inada, Toshiro Takezaki, Akira Yamada, Yoko Nakashima February 16, 2002, International Conference Hall, Aichi Cancer Center.

Program of symposium Opening Remarks: Suketami Tominaga (Aichi Cancer Center) Molecular and Epigenetic Bases of Stomach Cancer 1. Epigenetic Alterations in Human Stomach Cancers Toshikazu Ushijima (National Cancer Center) 2. E-cadherin Germline Mutations in Familial Gastric Cancer Parry J. Guilford (University of Otago, New Zealand) 3. Molecular Classification of Stomach Cancer by Gene Expression Profiling Hiroyuki Aburatani (Tokyo University) The Battle against Helicobacter pylori for Stomach Cancer Prevention 4. Helicobacter pylori Seropositivity and Cardia Stomach Cancer: Positive Association in a Prospective,

Nested Case-cohort Study from Linxian, China Youlin Qiao (Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union

Medical College) 5. Polymorphisms of Fucosyltransferase Genes and Helicobacter pylori Infection Risk Nobuyuki Hamajima (Aichi Cancer Center) 6. Helicobacter pylori Is a Promoter of Stomach Cancer rather than an Initiator Masae Tatematsu (Aichi Cancer Center) 7. Effect of Helicobacter pylori Infection and Eradication on the Development of Gastric Cancer Naomi Uemura (Kure Kyosai Hospital) Strategies for Cure of Stomach Cancer 8. Endoscopic Mucosal Resection for Early Gastric Cancer － Indication and New Techniques, IT Knife

Method Hiroyuki Ono (National Cancer Center) 9. Current Status and Future Perspective of Laparoscopic Operation for Early Gastric Cancer Michitaka Fujiwara (Nagoya University) 10. Update of JCOG 9501 Study; a Randomized Controlled Trial to Evaluate Para-aortic Lymphadenec-

tomy for Gastric Carcinoma Yasuhiro Kodera (Nagoya University) 11. Surgery and Adjuvant Therapy for Gastric Carcinoma in the USA Roderich E. Schwarz (University of Medicine and Dentistry, New Jersey) 12. Ten Year Results of Prospective Randomized D1/D2 Gastric Cancer Trial Limited but Definitive Bene-

fits C. J. H. van de Velde (Leiden University Medical Center) Concluding Remarks: Ryuzo Ohno (Aichi Cancer Center)

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Abstracts

Epigenetic Alteration in Human Stomach

Cancers Toshikazu Ushijima

Carcinogenesis Division, National Cancer Center Re-search Institute, Tokyo, Japan CpG methylation plays important roles in car-cinogenesis. To search for tumor suppressor genes and genes with altered expressions using aberrant CpG methylation as a marker, we previously de-veloped a comprehensive genome-scanning method for differential methylations, methylation-sensitive- representational difference analysis (MS-RDA) [Ushijima et al., PNAS, 94; 2284, 1997]. A pair of a gastric cancer and its surrounding normal tissue was analyzed by MSRDA. Six DNA fragments were isolated as being flanked by a CpG island (CGI) and possibly hypermethylated. Three of the six flanking CGIs were confirmed to be hy-permethylated in the cancer, and two of them had known genes in their vicinities. DNA fragment 3A1 was derived from a CGI in the 5’ region of the Insulin-induced protein 1 (IN-SIG1/CL-6) gene. Hypermethylation of the CGI was present in 50% of the primary gastric cancers (11 of 22), and the hypermethylation was associated with reduced expression of INSIG1. When a cell line with hypermethylation and reduced expression of INSIG1 was treated with 5-aza-2’-deoxycytidine (aza-dC), a demethylating agent, demethylation of the CGI and re-expression of INSIG1 were ob-served. INSIG1 is known to be expressed when a fibroblast differentiates into an adipocyte, and it was suggested that the silencing of INSIG1 was re-lated to malignant phenotypes in gastric cancers. DNA fragment 3B4 was derived from intron 7, near exon 8, of the p41-Arc gene. A CGI spanning exon 8 was found to be hypermethylated in 10 of the 22 gastric cancers. p41-Arc expression was markedly reduced in seven cancers, five of which contained signet-ring cancer cells. A CGI in the p41-Arc 5’ upstream region was hypermethylated in one of these five cancers. p41-Arc is known to be essential in actin polymerization and cell-shape control. It was suggested that its decreased expres-sion was involved in gastric cancer cell morphology, especially in signet-ring cell cancers. The role of these new players in gastric car-cinogenesis is being studied by their introduction

into gastric cancer cell lines. E-cadherin Germline Mutations in Familial

Gastric Cancer Parry J. Guilford

Cancer Genetics Laboratory, Department of Biochemis-try, University of Otago, Dunedin, New Zealand

Hereditary diffuse gastric cancer (HDGC) is a cancer syndrome caused by inactivating germline mutations in the gene for the homophilic cell-to-cell adhesion protein E-cadherin (CDH-1). This syn-drome is typified by early-onset, histologically dif-fuse gastric cancer. HDGC families also have a six-fold increased risk of developing breast cancer, but do not present with the intestinal form of gastric cancer. The penetrance of HDGC is higher in fe-males than males (83% and 67% respectively) and the age of cancer onset ranges from 14 years up-wards. There is no evidence for any phenotype variation associated with mutations at different lo-cations in the CDH-1 gene. To date, about 25 families from a broad range of ethnic groups have been identified with inacti-vating CDH-1 germline mutations. However, in Asian populations, the only germline CDH-1 muta-tions found in gastric cancer families have been substitution mutations. It is probable that the high rate of sporadic gastric cancer in Asia is masking the “true” inherited gastric cancer families. For rea-sons which are not yet clear, HDGC is over-represented in the New Zealand Maori popula-tion. We speculate that the CDH-1 mutations may have led to a heterozygote advantage, perhaps re-lated to E-cadherin’s role as a receptor for the bac-terial pathogen Listeria monocytogenes. Unlike other familial cancers, LOH is not a common mechanism for inactivation of the second allele. In-stead, the dominant mechanism for the 2nd hit on CDH-1 appears to be promoter hypermethylation. The demonstration that the 2nd hit on CDH-1 need not be an irreversible event suggests that environ-mental or physiological factors which lead to sus-tained downregulation of E-cadherin can influence the genetics of tumor progression. Therefore, the maintenance of E-cadherin expression must be re-garded as a critical target for chemopreventative strategies. Recent detailed histological analyses of HDGC

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gastrectomies have shown that stomachs from HDGC patients develop multifocal clusters of sig-net ring cells at a relatively early age. One patient we analysed recently had >45 independent lesions. The existence of these multiple foci, which have lost all E-cadherin expression, suggests that the second CDH-1 hit has occurred in multiple cells at a similar moment in time. This broad 2nd hit argues strongly for the involvement of an environmental trigger such as a carcinogen, or a general physio-logical event such as inflammation, in the progres-sion of HDGC. The multifocal disease also suggests that few irreversible genetic hits are required for the development of signet ring cells. These lesions are, however, likely to be slow growing and may con-stitute precursor lesions that are not yet fully inva-sive. Molecular Classification of Stomach Cancer

by Gene Expression Profiling Hiroyuki Aburatani

Genome Science Division, Research Center for Ad-vanced Science and Technology, University of Tokyo, Tokyo, Japan Recent molecular analyses have clarified many genetic alterations in gastric carcinogenesis, such as p53, α-catenin, E-cadherin, TFF1 and c-met, but it is still hardly enough to understand common pathway of carcinogenesis and progres-sion of gastric cancer. Furthermore, gastric cancer shows diverse clinical properties such as histologi-cal type, metastatic status, invasiveness and respon-siveness to chemotherapy. Only a little is known about genes associated with these characteristics. To gain molecular understanding of carcino-genesis, progression and diversity of gastric cancer, 22 primary human advanced gastric cancer and 8 non-cancerous gastric tissues were analyzed by high-density oligonucleotide microarray in this study. Based on expression analysis of approxi-mately 6800 genes on HuFL array, a two-way clus-tering algorithm distinguished cancer tissues from non-cancerous tissues. Subsequently, differentially expressed genes between cancer and non-cancerous tissues were identified with Mann-Whitney's U-test; 162 and 129 genes highly expressed (P<0.05) more than 2.5 fold in cancer and non-cancerous tissues, respectively. In cancer tissues, genes related to cell cycle, growth factor, cell motility, cell adhesion and matrix remodeling were highly expressed, while genes related to gastrointestinal specific function and immune response were highly expressed in

non-cancerous tissues. Furthermore, we identified several genes associated with lymph node metasta-sis including Oct 2 or histological types including LI cadherin. These results provide not only a new molecular basis for understanding biological prop-erties of gastric cancer, but also useful resources for future development of therapeutic targets and diag-nostic markers for gastric cancer. Helicobacter Pylori Seropositivity and Car-

dia Stomach Cancer: Positive Associa-tion in a Prospective, Nested Caseco-hort Study from Linxian, China

Youlin Qiao

Department of Cancer Epidemiology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, PR China Background: H. pylori carriage is the well-known risk factor for non-cardia stomach adenocarcinoma. However, the association between H. pylori and cardia stomach cancer risk remains controversial. Objective: To explore the sub-site specific stomach cancer risks associated with H. pylori persistent infection by measuring seropositivity. Methods: A prospective, nested case-control study was designed. Subjects were identified from among the participants of a large (n=29, 584) nutrition in-tervention trial previously conducted in Linxian, China. Serum samples collected 9-12 months prior to the onset of intervention were used to determine H. pylori infectious status. Baseline characteristics were derived from a pre-trial evaluation. Incident stomach cancers were diagnosed over 5.25 years (duration of the intervention period). In total, 99 cardia stomach cancer cases, 82 non-cardia stomach cancer cases, and 192 cancer-free controls were in-cluded. H. pylori whole cell antibodies and H. py-lori cagA antibodies were measured using region-ally-validated enzyme-linked immunosorbent serum assays. Seropositivity was defined as one or both serum assays being positive. Odds ratios for stom-ach cancer were estimated using multivariate logis-tic regression analyses. All statistical comparisons were performed two-sided with alpha equal to 0.05. Results: Both types of H. pylori serum antibodies were more common among cases than controls. De-fined as one or both assay results positive, H. pylori seropositivity rates for subjects with gastric cardia cancer, non-cardia gastric cancer, and gastric cardia and non-cardia cancers combined were 70% (p=0.02), 72% (p=0.01), and 71% (p=0.003), versus 56% for controls. Odds ratio estimates based on H.

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pylori seropositivity were 1.87 (95% confidence in-terval [CI] = 1.10 to 3.17) for cardia stomach cancer, 2.29 (95% CI = 1.26 to 4.14) for non-cardia stom-ach cancer, and 2.04 (95% CI =1.31 to 3.18) for both stomach cancer sub-sites combined. Conclu-sions: H. pylori seropositivity was associated with similarly increased risks for both cardia and non-cardia stomach cancer in this well-character-ized cohort. Implications: Contrary to most earlier reports, the-se data suggest that the procarcinogenic potential of H. pylori carriage is whole stomach, rather than limited to the cardia stomach. Future studies should address whether or not H. pylori eradication repre-sents a logical stomach cancer prevention strategy in this high-risk population. Polymorphisms of Fucosyltransferase Ge-

nes and Helicobacter pylori Infection Risk

Nobuyuki Hamajima

Division of Epidemiology and Prevention, Aichi Cancer Center Research Institute, Nagoya, Japan Helicobacter pylori (HP) infection increases the risk of diseases including peptic ulcer and stomach cancer. Although the infection largely de-pends on the environmental factors, especially sanitary conditions in childhood, the genetic factors play a role in the infection, as a twin study shows. To date, as well as HLA types, polymorphisms of TNF-A, Lewis (Le, fucosyltransferase 3), secretor (Se, fucosyltransferase 2), IL-1B, and myeloper-oxidase have been reported to be possible genetic factors associated with the infection. The latter four genes were found out of fifty polymorphisms screened in Aichi Cancer Center. In this presenta-tion, I focus on the association of polymorphisms of Le and Se with the persistent HP infection. HP with babA2 gene encoding blood-group antigen-binding adhesin (BabA) has binding activ-ity to Leb and H type I antigens. Se and Le enzymes metabolize Type I precursor into H type I and Lea antigens, respectively. H type I is further metabo-lized into Leb by Le enzyme. Commonly observed in Japanese are Se1, Se2, Le, and le3 for functional alleles (Se and Le) and sej, se5, le1, and le2 for re-duced/no-functional allele (se and le), respectively. We examined the associations between anti-HP IgG antibody and the above genotypes for 241 non-cancer outpatients, and found that indi-viduals with se/se & Le/Le genotypes had the low-est seropositivity (33.3%, 9/27) and those with

Se/Se & le/le, Se/Se & Le/le, or Se/se & le/le geno-types the highest (83.8%, 31/37), and the rest in-termediate (62.3%, 109/175 excluding two not genotyped). Sex-age-adjusted OR of being infected relative to the lowest group was 3.34 for the inter-mediate and 10.21 for the highest. The expression of Leb and Lea antigens de-pending on the genotypes was confirmed in gastric foveolar epithelium. These findings suggest that Se and Le genotypes influence the risk of the contin-ued HP infection through the expression of ligands for BabA. Helicobacter pylori Is a Promoter of Stom-

ach Cancer rather than an Initiator Masae Tatematsu

Division of Oncological Pathology, Aichi Cancer Center Research Institute, Nagoya, Japan In 1994, the World Health Organiza-tion/International Agency for Research on Cancer concluded that Helicobacter pylori (Hp) is a defi-nite carcinogen based on the epidemiological evi-dence. For detailed analysis of the role of Hp in stomach carcinogenesis, it is essential to establish a small animal model. We have established experi-mental models of stomach carcinogenesis in Mon-golian gerbils (MGs) using the chemical carcino-gens, N-methyl-N'-nitro-N-nitrosoguanidine (MN- NG) and N-Methyl-N-nitrosourea (MNU). The le-sions were generally well differentiated, although poorly differentiated adenocarcinomas were also found. Hp infection enhances glandular stomach carcinogenesis in MGs treated with MNNG or MNU. Animals with high titers of anti-Hp antibod-ies are at greatest risk of developing neoplasms. Hp infection and high-salt diet administration are both considered being important factors for gastric car-cinogenesis in man. Hp infection exerts stronger promoting effects than a high-salt diet on gastric carcinogenesis, and that the two factors act as syn-ergistically to enhance development of stomach cancer. Eradication diminishes enhancing effects of Hp infection on glandular stomach carcinogenesis in MGs. Hp eradication may be useful as a preven-tion approach. On the other hand, submucosal pro-liferative tumor-like lesions are also induced in the glandular stomach with Hp infection alone, and of-ten they are similar to carcinomas. To explore if the role of Hp infection is promotion or initiation, we established an experimental model of long term Hp infection and eradication in MGs, without chemical

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carcinogens. Submucosal tumor-like lesions thereby develop with a phenotypic shift towards intestinali-zation. Eradication of the bacteria diminished the submucosal tumor-like lesions, so they were con-sidered to be reversible, rather than malignant in character. The role of Hp infection thus appears to be strong promotional influence, rather than initia-tion of gastric carcinogenesis. Effect of Helicobacter pylori Infection and

Eradication on the Development of Gas-tric Cancer

Naomi Uemura

Department of Gastroenterology, Kure Kyosai Hospital, Kure, Japan

Since the discovery of H. pylori, concept of diagnosis and treatment of upper gastrointestinal diseases has been changing greatly from conven-tional belief. Histological gastritis in human was found to have been attributable to infection with H. pylori, and subsequently various reports indicated that eradication of H. pylori markedly improved in-flammatory cell infiltration, characteristic of H. py-lori-related gastritis and inhibited recurrence of peptic ulcer. Recently, it has been reported that H. pylori infection is causally related to the incidence and growth of low grade of gastric MALT (mu-cosa-associated lymphoid tissue) lymphoma and that eradication leads to the regression of MALT lymphoma. With regard to association with gastric cancer, WHO/IARC stated in 1994 that there is suf-ficient evidence in humans for the carcinogenecity of infection with H. pylori based on epidemiologi-cal evidence and histopathological study. Also, as for impact of eradication on development of gastric cancer, based on a follow-up study on patients who underwent eradication therapy after endoscopic mucosal resection of early gastric cancer (EMR), a possibility of eradication to inhibit the development of metachronous cancer, which occurs in a site dif-ferent from treated lesion, was reported. Based on this report, remaining gastric mucosa after EMR is positioned as one of the indications of eradication therapy. In Japan, where the incidence of gastric cancer is the highest in the world, due to its different health care insurance system from Europe and the US, early-stage gastric cancer is more often discovered by endoscopy. In the symposium I will describe about dynamics of H. pylori infection in gastric cancer patients and effect of eradication in Japan.

Endoscopic Mucosal Resection for Early Gastric Cancer － Indication and New Techniques, IT Knife Method －

Hiroyuki Ono

Endoscopy and GI Oncology Division, National Cancer Center Hospital, Tokyo, Japan Endoscopic mucosal resection (EMR) has been extended for a treatment of early gastric cancer (EGC) in the world, especially in Japan from the beginning of the 1980s. It has an advantage over surgical gastrectomy in patient's quality of life. Because EMR is just a local treatment, we should choose appropriate candidates with low pos-sibility of lymph node metastasis. We analyzed about 1,700 patients with solitary, intramucosa, and surgical treated early gastric cancer. An early gas-tric cancer confined to the mucosa has to meet the following criteria in order to be resected endo-scopically: 1. Histologically differentiated adeno-carcinoma, 2. Size of less than 30 mm if the tumor has ulcerative changes. (no limitation of the size without ulcer), 3. Absence of lymphatic vascular involvement. Recently, dramatic developments have oc-curred in the operational mechanism and design of the accessory apparatus. To obtain the gcomplete resection histologically for large and difficult le-sions, we developed a special endoscopic knife in 1996 named Insulation-tipped electrosurgical knife (IT knife). This knife can cut submucosa safely and remove a lesion completely. We also developed an improved technique named PTA-EMR: Percutane-ous Traction-assisted EMR. We raise up a clip at-tached on the edge of a lesion by a thin retractor through the abdominal wall (like percutaneous en-doscopic gastrotomy), and then resect the lesion by IT knife. It can give counter traction to the lesion as well as surgical mucosectomy and can be carried out without systemic anesthesia. On the other hand, there is some possibility of complications, such as perforation of the gastric wall and active bleeding. Especially, up to now, a surgical operation has been required to treat them and it causes the deterioration of the patient's QOL. I introduce how to manage and rescue the patients endoscopically from perforation after EMR without surgical treatment. At all events, a picture is worth a thousand words. I will show video demonstration of EMR using IT knife and how to treat perforation endo-scopically.

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Current Status and Future Perspective of Laparoscopic Operation for Early Gas-tric Cancer

Michitaka Fujiwara

Department of Surgery II, School of Medicine, Nagoya University, Nagoya, Japan Background: Endoscopic mucosal resection (EMR) and laparoscopic wedge resection offer im-proved quality of life after treatment for early gas-tric cancer (EGC), but the degree of curability that can be obtained through these procedures in terms of systemic lymph node dissection is severely lim-ited. In 1993, laparoscopy-assisted distal gastrec-tomy (LADG) with lymphadenectomy emerged as a novel option for treating the EGCs of the middle to lower stomach with potential lymph node involve-ment. Due to recent refinements in the technique and instruments for laparoscopic surgery, some in-vestigators suggest that LADG can be applied to more advanced disease. Where we are today: Between 1995 and 2001, we have performed 120 laparoscopic operations, including 7 laparoscopic proximal gastrectomies, and one laparoscopic total gastrectomy, for gastric carcinoma that has been diagnosed as confined to the mucosa or the submucosa through endoscopic ultrasonography. The number of patients treated with laparoscopic wedge resection was relatively small at 20, because EMR is primarily indicated for a subset of EGC that is estimated not to have lymph node metastasis. LADG, the most frequently per-formed procedure under the laparoscopy, was per-formed in 92 patients. LADG was converted to open surgery in 4 of 92 cases because of uncontrol-lable bleeding, positive proximal margin, and mac-roscopic finding of lymph node metastasis (con-firmed by frozen section during surgery), but there was no mortality associated with this procedure. When the outcome of these patients was compared with that of the historical control consisting of 80 patients treated with conventional open surgery (1992~1997), no significant differences in the blood loss, morbidity, duration of postoperative fever elevation, and maximum value of CRP were ob-served. On the other hand, a smaller amount of an-algesics was required for the LADG patients who also had better postoperative recovery in terms of the duration before passing of the flatus and ambu-lation. LADG requires use of costly equipments, but has nevertheless proved less expensive in terms of the total cost required per a patient, due primarily to the shorter hospital stay. These patients have

been followed for a mean of 24.8 months (range: 2~58 months). One case from each group has so far died of the recurrent disease. There was no signifi-cant difference in the number of resected lymph nodes. We believe that D2 lymphadenectomy as de-fined by the Japanese Classification for Gastric Carcinoma can be performed adequately under the laparoscopy, although a longer follow-up time is needed for confirmation of the long-term conse-quences obtained through this approach. Future perspective: Advanced laparoscopic surgery requires intensive training as well as the use of costly surgical equipments, and can currently be performed only in specialized institutions. Besides constructing an adequate training program, further improvements in surgical devices and navigation systems may facilitate such operation. From this viewpoint, we have started the use of three-dimensional CT angiography for preoperative simulation as well as for navigation during surgery. Further progress in the field of optical technology is warranted. Update of JCOG 9501 Study; a Randomized

Controlled Trial to Evaluate Para-aortic Lymphadenectomy for Gastric Carci-noma

Yasuhiro Kodera

Department of Surgery II, School of Medicine, Nagoya University, Nagoya, Japan Background: Radical gastrectomy with D2 lymphadenectomy has been a standard procedure for treatment of gastric carcinoma in Japan and is considered responsible for the excellent stage-by-stage survival of these patients. Some pa-tients nevertheless have recurrences in the para-aortic lymph nodes. Long-term survivors have been reported among the population treated with systemic resection of these nodes in several pilot studies. Study design: A multi-institutional randomized controlled trial was performed to compare treatment results of para-aortic lymphadenectomy with those of standard Japanese-style D2 resection. Patients with histologically proven gastric adenocarcinoma who at laparotomy was found to have cancer inva-sion as far as or beyond the subserosa (T2), nega-tive cytology (CY0), and no distant or extensive node metastases (N0~2, M0) were randomized to receive either the standard D2 resection (Group A) or D2 plus extensive para-aortic lymph node dissec-tion (Group B). Primary endpoint of this trial is the

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overall survival that is to be evaluated after 5 years of follow-up. Secondary endpoints include recur-rence-free survival, morbidity, operative mortality, postoperative hospital stay, and QOL. Results: The patient accrual started in June 1995 and was completed in April 2001. A total of 523 patients were randomized into Groups A (n=263) and B (n=260). Cumulative 4-year survival rate was 69.7%. Difference in survival between the 2 groups has not been evaluated at this time. Two deaths due to postoperative complications and an-other two due to rapid disease progression were observed, hence the overall hospital mortality of 0.8%. Operative time was longer (300 mins versus 237 mins) and bleeding amount greater (660 mL versus 430 mL) for Group B, as has been expected. There was no difference between the groups in the incidence of major surgical complications such as leakage, pancreatic fistula, and intra-abdominal ab-scess, although complication as a whole was more frequent among Group B. Complications specific to Group B were paralytic ileus and prolonged lym-phorrhea. The mean number of lymph nodes re-trieved was 54 (range: 14~161) for Group A and 74 (range: 30~235) for Group B. The mean number of para-aortic lymph nodes resected by the su-per-extended lymphadenectomy was 25 (range: 4~75). Conclusion: Extended lymphadenectomy with and without para-aortic lymph node dissection is safe and feasible when performed at specialized centers in Japan. Super-extended lymphadenectomy was associated with longer operating time, greater blood loss, and higher incidence of surgical com-plications. Final survival analyses to assess whether these shortcomings can be compensated for by a significant survival benefit is eagerly awaited. Surgery and Adjuvant Therapy for Gastric

Carcinoma in the U.S.A. Roderich E. Schwarz

Cancer Institute of New Jersey and Department of Sur-gery, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, New Brunswick, U.S.A. Gastric cancer, still the predominant cause of cancer death in the U.S. 60 years ago, has signifi-cantly decreased in incidence and mortality. Physi-cians diagnosing or treating gastric cancer in the U.S. are facing specific characteristics and chal-lenges: significant social and ethnic patient hetero-geneity, geographic variations in incidence, ad-

vanced stages at diagnosis, significant comorbidity, a growing number of elderly patients, and a con-tinuing trend in the prevalence of cardia or proximal disease location. Treatment is increasingly influ-enced by managed care organizations, and access to specialized cancer- centers can be limited. Thus, the majority of patients continue to be treated in a low-volume setting. Gastrectomy remains the mainstay of therapy for potentially curable gastric cancer. Although radical regional resections, including extended lymph node dissection (ELND), had been utilized here since the middle of the 20th century, ELND is still not widely practiced throughout the country. In a nationwide survey, survival after gastrectomy in the U.S. remains inferior to that obtained in Japan or Western Europe. Specialized centers, however, in which ELND has been routinely applied, are generating stage-adjusted survival after gastrectomy which approaches that achieved in Japanese centers or series. Morbidity and length of hospital stay have continued to decrease during the past decade. Pat-terns of first recurrence after gastrectomy and ELND suggest that transserosal and hematogenous dissemination are operational in the vast majority of clinical relapses, but isolated regional (nodal) re-currences remain sparse. A recent U.S. Intergroup trial of postoperative adjuvant chemoradiation followed by chemotherapy (INT 116) has resulted in a significant overall sur-vival and relapse-free survival benefit. While the chemoradiation therapy (CRT) components were well quality-controlled, the operative treatment was not; only 10% of patients underwent formal ELND. CRT appeared to primarily reduce “local” and “re-gional” recurrences, and was associated with an in-crease in the relative frequency of distant relapses. It is unclear whether the radiation component could functionally substitute in part for the limited re-gional resection extent. In light of the specific characteristics of gastric cancer treatment in the U.S., one can conclude that: there is room for standardization of quality assur-ance of operative treatment; postoperative adjuvant chemoradiation therapy has shown a measurable benefit; CRT has not been validated for patients having undergone ELND; and routine use of CRT in different settings (outside the U.S., different re-currence patterns) appears not warranted at this time. Adjuvant therapy strategies should be tested for disease specific challenges as indicated by dominant relapse patterns.

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Ten Year Results of Prospective Random-ized D1/D2 Gastric Cancer Trial Limited but Definitive Benefits

C. J. H. van de Velde

Department of Surgery, Leiden University Medical Cen-ter, Leiden, the Netherlands

In the Netherlands 80 hospitals participated in a randomized trial to compare morbidity, hospital mortality survival and cumulative relapse risk after D1 versus D2 lymph node dissection for gastric cancer. Between 1989 and 1993, 996 patients were randomized by the Leiden University Medical Cen-ter in a study with extensive quality control in pa-tients with a mean age of 65 years and no age limit. A total of 711 patients underwent the allocated treatment with curative intend. Strict quality control

measures were undertaken to avoid interaction of both techniques. All operations were supervised and pathology revised. D2 operations were only per-formed under supervision of one out of 12 qualified D2-surgeons. D2-patients had higher postoperative mortality and significant more complications, sur-gery was performed according to the original rules of the JRSGC. Although 5-years survival rates were not different for D1 versus D2 patients(45 versus 47%), D2-patients with N1 disease generally with TNM-stages Ⅱ or ⅢA significantly benefited from a D2-dissection. Aspects of selection, sentinel node dissection will be discussed in view of the ten-year follow-up the Dutch Gastric Cancer Trial.

Drs. C.J.H. van de Velde, R. E. Schwarz and Youlin Qiao (from the left to right) at the 8th Aichi Cancer Center International Symposium held on February 16, 2002.

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Author index for research reports and publications ________________________________________________________________________________ Akatsuka, Y. 34, J001, J008, J157, R001, R043,

R044, A001, A002 Aoki, K. 7, J067, J189 Ariyoshi, Y. 7, J197 Doi, T. J055, J127 Fujii, K. 37, 37 Fujita, M. 37, 38, J004, J017, J018, J221,

R004, R005, R056, A004, A005 Fukami, H. 57, J201, J202, J203, A045, A046 Gao, C. J021, J183 Goto, H. 49, 50, J134, J150, J215, J227,

J228, R006, R011 Goto, Y. 43, 44, 45, J101, R014 Hagino, M. 59 Hamajima, N. 7, 10, 12, 20, 30, 56, J024, J025,

J026, J027, J028, J029, J030, J031, J032, J033, J044, J052, J053, J054, J057, J066, J074, J076, J088, J102, J103, J116, J117, J118, J119, J120, J121, J122, J124, J125, J142, J168, J169, J175, J178, J182, J184, J185, J186, J188, J189, J207, J224, J225, R007, R008, R032, R033, R036, R046, R047, A006, A007, A008, A009, A012, A025, A033

Harada, H. J035, J102, J103, J191, J205 Harano, T. 24, 25, J036, J037, A010, A036 Haruki, N. 25, J005, J036, J037, J038, J039,

J123, J141, J168, J169, A010, A036

Hasegawa, Y. J186 Hata, M. J040, J041, J092, J151, R038,

R039 Hatooka, S. J102, J103, J118, J119, J168, J169,

J186, J191, J196 Hayashi, N. 40, 61, J090, J105 Hayashi, Y. 52, 52, J211, A022, A049, A050 Hayashi, Y. J211, A022, A049, A050 Hida, T. 23, J042, J099, J100, J115, R009,

A036, A037 Hirai, T. J033, J126, J184 Hiraiwa, N. 46, J096, J097 Hirata, M. J221 Hirose, F. 52, J043, J107, J146, J154, J211,

J216, A011, A024, A031, A049, A050, A051

Hirose, K. 5, 10, 12, J044, J054, J066, J074, J104, J116, J185, J186, J223, R007, R046, R047, R055, A033

Hoshino, Y. 39, J046, J090, J106 Hosokawa, Y. 29, 30, 31, J047, J048, J049, J050,

J051, J058, J129, J179, J181, J222 Huang, X. J053, J054 Ichinose, M. J096 Iidaka, T. 19, J158 Ikehara, Y. 18, J057, J060, J077, J166, J167,

A013, A014 Imai, T. R015, A019, A030 Inada, H. 50, J022, J059, J080, J150, R011 Inada, K. 18, 19, 20, J057, J060, J069, J126,

J152, J159, J166, J201, A014, A046

Inagaki, M. 49, 50, 51, J022, J045, J059, J062, J080, J098, J109, J134, J150, J194, J208, J215, J227, J228, R002, R006, R011, R035, A026

Inagaki, N. J062 Inoue, M. 5, 7, 12, J027, J033, J044, J054,

J064, J065, J066, J067, J068, J074, J076, J088, J116, J118, J119, J185, J186, J202, J203, R007, R012, R046, R047, A007, A015, A016, A017, A018, A033, A042

Inoue, Y. 52, J043, J063, J146, J154, J211, J216, A031, A050, A051

Inoue, Y.H. 52 Ishida, H. 44, J023, J081 Ishida, R. J140 Ishizaki, K. 55, 56, 57, J035, J073, J102, J103,

J191, J192, J205, R010 Ito, H. J074 Ito, S. J077, J088, J111, J135, A027 Iwase, S. J078, J127, J199 Iwase, T. 34, J026, J031, J053, J066, J079,

A008 Iwata, H. J026, J031, J053, J066, J079,

A008 Izawa, I. 50, 51, J059, J080, J150, R035 Izawa, M. 44, J081, J097, J164, R014, A023 Kagami, Y. J083, J095, J113, J122, J131, J155,

J175, J210 Kanamori, A. 43, 44, J006, J007, J023, J081,

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J084, J145, J206, R015, R037, A019, A029

Kanda, K. 44, J145 Kannagi, R. 43, 44, 45, 46, J013, J016, J020,

J023, J056, J075, J081, J084, J086, J087, J094, J096, J097, J101, J138, J145, J161, J164, J187, J193, J206, R013, R014, R015, R016, R017, R018, R019, R020, R037, A003, A019, A020, A021, A023, A029, A030, A048, A052

Kato, M. J067, J131, A022 Kato, T. 18, J033, J073, J077, J111, J126,

J135, J184, J192, R010 Kawajiri, A. 49, 51 Kawakami-

Kimura, N. J193 Kitamura, T. 20, J057 Kiyono, T. 25, 37, 39, 50, 57, J017, J018,

J036, J059, J162, J163, J221, R021, R022, R023, R024, A010

Kobayashi, S. J079, J196 Kobayashi, T. J182 Kodera, Y. 20, J052, J057, J126, J129, J135,

J137, J212 Koiwai, O. J154 Kondo, E. 34, J095, J113, J131 Konishi, H. 23, 24, 25, 26, J036, J038, J141,

J217, A010, A036 Kosako, H. J098, R006 Koshikawa, K. 23, J099 Koshikawa, T. J168, J169 Kozaki, K. 23, J032, J042, J099, J100, J111,

J115, J224, A036, A037 Kumamoto, K. 44, 45, J081, J101, J187, J193,

J206, R014, A023, A030 Kumimoto, H. 55, 56, J035, J102, J103, J205 Kuroishi, T. 5, 7, 11, J054, J074, J104, J116,

J185, J186, R007, R046, R047, A033

Kuzushima, K. 37, 39, 40, 61, J017, J046, J090, J105, J106, J198, J221, R025, R026, R027, R028, R029, R030, R031

Kuzuya, K. 7 Kwon, E. -J. 52, J107, J108, A024, A050 Maeda, Y. J047, J048, J049, J181 Masuda, A. 24, 25, 26, J002, J036, J038, J042,

J115, J123, J153, J213, A010,

A036 Matsudaira, Y. 35, J078, J199, J200 Matsui, S. 49, 51, J215 Matsukage, A. 52, J043, J063, J082, J107, J146,

J154, J211, J216, A011, A031, A050, A051

Matsuo, K. 20, 56, J024, J026, J027, J028, J029, J030, J031, J032, J033, J053, J057, J074, J088, J102, J103, J116, J117, J118, J119, J120, J121, J122, J184, J188, J225, R007, R032, R033, A007, A008, A012, A025

Matsuura, A. J030, J065, J121, J133, R032, A017

Matsuura, H. J186 Minoura, Y. 59 Mitsudomi, T. 7, 23, 25, 34, J005, J014, J036,

J042, J125, J141, J168, J169, J185, J217, R034, R052, A010, A036

Mitsuoka, C. J075, J081, J145, R015, R037, A019, A023, A029

Miura, K. J081, J095, J145 Miura, S. 7, 34, J026, J031, J044, J053,

J066, J079, J097, J223, A008 Mizoshita, T. 19, J126 Mizuno, K. 25 Mizutani, K. J127 Mizutani, M. J026, J031, J053, J066, J079,

A008 Moore, M. J128, R045, A032 Morishima, Y. 34, J083, J095, J113, J117, J120,

J122, J124, J129, J155, J175, J210, J218, J219, J222, R033

Motegi, M. J129 Murai, H. J097 Nagata, K. 49, 51, J109, J134, J194, J208,

J215, R011, R035, A026 Nagatake, M. J213 Nakagawa, T. 24, 25, J123 Nakamura, H. 57, 59, J018, J137 Nakamura, S. 30, 44, 61, J020, J039, J060, J061,

J065, J081, J083, J095, J113, J120, J122, J124, J129, J130, J131, J132, J133, J174, J175, J176, J177, J178, J186, J190, J196, J210, J217, J218, J219, J222, A014, A017

Nakamura, T. 30, 61, J030, J065, J121, J132, J133, J196, R032

Nakanishi, H. 18, 19, J002, J060, J069, J077,

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J111, J135, J143, J159, J166, A014, A027, A028, A045

Nakasu, S. 38 Nakayashiki, N. 33, J136 Narita, T. J096, J097 Natsume, A. J003 Nishi, Y. J043, J211 Nishida, K. J140, J190 Nishida, T. 34 Nishimoto, Y. 55, J063, J102, J103 Nishizawa, K. J073, J102, J103, J205, R010 Nishizawa, M. 50, 51, J059, J062, J080 Nomoto, S. J037, J039, J141 Nozaki, K. 18, J166, J167 Obata, Y. 34, 35, J026, J031, J055, J078,

J079, J127, J142, J170, J199, J200, R003, R036, A008

Ogura, M. J095, J113, J122, J175 Ohashi, K. J026, J065, J133, J184, J196 Ohno, K. J010, J107, J146, A031, A050 Ohshima, N. 52, J043, A024 Ohtakara, K. 51, J080, J150 Ohtsuka, K. J040, J041, J080, J092, J151, J209,

J221, R038, R039, R040 Okuma, K. 7, J026, J029 Osada, H. 23, 24, 25, J036, J037, J039, J099,

J115, J149, J153, J213, R041, R042, A010, A036

Ozeki, S. 35, J078, J199, J200 Saito, H. 23, 25, J037, J038, J070, J093,

J099, J137, J156, J195, J204, J212, J224, A036

Saito, N. 51 Saito, S. J161 Saito, T. 7, J027, J029, J032, J033, J053,

J074, J088, R007, A007 Saito, T. 24, 26, J153, J213 Sakai, H. 19, J158, J159 Seto, M. 29, 30, 31, J009, J015, J047, J048,

J049, J050, J061, J083, J089, J110, J120, J122, J124, J129, J130, J132, J133, J137, J140, J155, J174, J175, J176, J177, J178, J179, J181, J182, J190, J196, J210, J218, J219, J222

Shimizu, N. 18, 20, J057, J166, J167 Shimizu, S. 25, J005, J036, J042, J168, J169,

A010 Shinoda, M. 55, J035, J102, J103, J118, J119,

J168, J186, J191, J196

Shirai, N. 19, J158 Shiraki, M. J043, J063 Suchi, T. J175, J218, J219 Sugaya, Y. J173 Sugiura, T. 7, J042, J185, J224 Sugiyama, M. 24, J153, J213 Suyama, M. J168, J169 Suzuki, H. 29, 30, J129, J179, J222 Suzuki, R. 30, 31, J034, J085, J091, J095,

J113, J120, J122, J124, J129, J131, J175, J176, J177, J178, J179, J210, J212, J218, J219, J222, R033

Suzuki, S. J152 Suzuki, T. 11, J030, J065, J104, J121, J133,

J196, R032, R040 Taguchi, O. 46, J043, J096, J097, J146, J193,

A011, A031 Taji, H. 34, J095, J113, J122, J131 Tajima, K. 5, 7, 10, 12, 34, J021, J026, J027,

J029, J030, J031, J032, J033, J044, J052, J053, J054, J064, J065, J066, J067, J074, J076, J088, J116, J118, J119, J120, J122, J124, J147, J148, J165, J171, J172, J180, J183, J184, J185, J186, J214, J223, J226, R007, R045, R046, R047, R048, R049, R050, R051, A006, A007, A008, A009, A015, A016, A017, A025, A032, A033, A034, A035, A042

Takagishi, M. 51, J194 Takahashi, H. J181 Takahashi, M. 34, J202, J203 Takahashi, Ta. 23, 24, 25, 26, J005, J012, J014,

J032, J036, J037, J038, J039, J042, J099, J100, J115, J123, J125, J141, J153, J169, J197, J213, J217, J224, R009, R034, R041, R042, R052, R053, A010, A036, A037, A038, A039, A040, A041

Takahashi, To. 33, 34, 35, J003, J055, J059, J078, J100, J115, J127, J136, J139, J141, J142, J199, J200, J220, R036

Takahashi, Y. J146, A031 Takeuchi, T. J085, J095, J097, J223 Takezaki, T. 7, 12, J021, J027, J033, J054,

J074, J088, J116, J118, J119, J172, J183, J184, J185, J186, J226, R007, R046, R047, R049, R054,

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A007, A033, A034, A042 Tamaki, H. 34, J142, R036 Tanabe, K. 59 Tanaka, H. J060, J191, J201, J202, J203,

A014, A046 Tatematsu, M. 18, 19, 20, J019, J020, J057, J060,

J069, J071, J072, J077, J111, J114, J126, J128, J135, J143, J144, J152, J158, J159, J166, J167, J201, J202, J203, A013, A014, A027, A043, A044, A045, A046

Tatematsu, Y. 23, 24, 25, J036, J038, J099, J100, J115, J141, A010, A036

Tei, K. J193, R014, A030 Terashima, M. 59 Togashi, H. J194 Tokumasu, S. J205 Tominaga, S. 5, 11, J029, J030, J052, J054,

J064, J065, J066, J067, J076, J088, J104, J121, J142, R032, R036, A015, A016, A017, A042

Tsujimura, K. 35, J003, J078, J139, J199, J200 Tsukamoto, T. 18, 19, J060, J100, J126, J158,

J159, J166, J201, J202, J203, A014, A045, A046

Tsurumi, T. 37, 38, 39, 40, 61, J017, J018, J046, J105, J106, J221, R029, R056, R057, R058, R059, A004, A047

Uchida, K. J213 Uchida, N. J035, J205 Ueda, R. J015, J061, J113, J137, J168,

J169, J182, J190, J210, J212 Yamagishi, M. 52, J082, J211 Yamaguchi, M. 52, J010, J043, J063, J082, J107,

J108, J146, J154, J210, J211, J216, J218, J219, A011, A022, A024, A031, A049, A050, A051

Yamamoto, M. 19, J079, J158, J159, J201, A046 Yamamura, Y. 20, 61, J020, J052, J057, J126,

J135 Yanagisawa, K. 24, J153, J213, A036 Yasui, K. J111 Yasui, Y. 49, 50, J215 Yasutomi, H. 20, J057, A045 Yatabe, Y. 24, 26, J039, J115, J131, J133,

J168, J169, J185, J217, J218, J219, A036, A037

Yokoyama, N. 37, J011, J017, J160, J221 Yonezumi, M. J110, J129, J132, J133, J222 Yoshida, H. 52, J154, J211, J216, A050, A051 Yoshida, M. 34 Yoshida, T. J098, J131 Yoshikawa, K. 33, 35, J136, J200 Yuasa, H. J028, J225 Zenita, K. J097