SCIENTIST EXCHANGES

BASIC SCIENCE

(1) Dr. Misako HARAGUCHI, Institute for Cancer Research, Faculty of Medicine, Kagoshima University
Subject of Research: Formation pf Acetaldehyde from the 2-deoxy-d-ribose and it's Physiological Function
Dates: April 5-12, 2002
Host: Dr. Takayuki SHIBAMOTO, Department of Environmental Toxicology, University of California, Davis

(2) Dr. Akio SUGINO, Graduate School of Frontier Biosciences, Osaka University
Subject of Research: Isolation of Mutators of DNA Polymerase e and their Effect on Cancer Susceptibility in Mice and Human
Dates: July 22- September 20, 2002
Host: Dr. Vladimir V. LARIONOV, Laboratory of Biosystems and Cancer, NCI
Dr. Michael A. Resnick, Laboratory of Molecular Genetics, NIH

(3) Dr. Kazuo SHIN-YA, Institute of Molecular and Cellular Biosciences, The University of Tokyo
Subject of Research: Studies on the Role of Telomerase in the Development of Neural Progenitor Cells Using a Telomerase Inhibitor, and Par-4 in Apoptosis Observed in Cancer and Neurodegenerative Disorders
Dates: May 10- August 9, 2002
Host: Dr. Mark P. MATTSON, Gerontology Research Center, NIH

(4) Dr. Sayoko IHARA, Graduate School of Agricultural and Life Sciences, The University of Tokyo
Subject of Research: Study of Swap-70, a Novel Rac-GEF, in Formation of Tumors
Dates: June 2- August 30, 2002
Host: Dr. Rolf JESSBERGER, Mount Sinai School of Medicine

(5) Dr. Masato HIRATA, Faculty of Dental Science, Kyushu University
Subject of Research: Prevention of Oncogenesis by Inositol Phosphates
Dates: June 10- August 10, 2002
Host: Dr. Stephen B. SHEARS, National Institute of Environmental Health Science

(6) Dr. Ken-ichi SATO, Research Center for Environmental Genomics, Kobe University
Subject of Research: Studies on the Molecular Mechanism of Fertilization and Activation of Development Regulated by Proto-oncogene Src
Dates: June 13- August 31, 2002
Host: Dr. Rafael A. FISSORE, University of Massachusetts

(7) Dr. Yoshiko AKAMATSU, Institute for Genome Rsearch, The University of Tokushima
Subject of Research: Molecular Mechanism and Regulation of V(D)J Rearrangement
Dates: June 27- July 27, 2002
Host: Dr. Marjorie A. OETTINGER, Massachusetts General Hospital, Harvard Medical School

(8) Dr. Katsunori TANAKA, Faculty of Life and Environmental Science, Shimane University
Subject of Research: Cell-cycle Checkpoint Functions of ATM-related Kinase in Cancer Cells
Dates: July 1- September 30, 2002
Host: Dr. Paul RUSSELL, Department of Molecular and Cell Biology, The Scripps Research Institute

(9) Dr. Naofumi MUKAIDA, Cancer Research Institute, Kanazawa University
Subject of Research: Clarification on the Pathophysiological Roles of Chemokines in Tumor Progression
Dates: July 4- 20, 2002
Host: Dr. Joost J. OPPENHEIM, NCI-FCRDC
Dr. Isiah J. FIDLER, M.D.Anderson Cancer Center

(10) Dr. Katsuhide IGARASHI, Cellular & Molecular Toxicology Division, National Institute of Health Sciences
Subject of Research: Identification of Tumor Related Gene Expression Profile Induced by Carcinogenic Chemicals Using p53 Knock Out Mouse
Dates: July 29- September 29, 2002
Host: Dr. Tennant W. RAYMOND, National Institutes of Environmental Health Sciences

(11) Dr. Tohru KAMATA, Department of Molecular Biology and Biochemistry, Shinshu University
Subject of Research: The Transcriptional Regulation of Superoxide-producing Oxidase Nox1 Mediating the Oncogenic Cell Transformation
Dates: August 4-25, 2002
Host: Dr. Nancy H. COLBURN, NCI-FCRDC

(12) Dr. Shigeru CHIBA, Graduate School of Medicine, The University of Tokyo
Subject of Research: Cross-talk Between Notch Signaling and DLX Homeobox Genes and its Significance in Leukemogenesis
Dates: August 4-25, 2002
Host: Dr. Kenichi TAKESHITA, Hematology Division, Department of Medicine, New York University

(13) Dr. Tadashi MATSUDA, Dept. of Innunology, Grad. Sch. Of Pharm. Sci., Hokkaido University
Subject of Research: IL-12 Signaling in Tyk2 Knockout Mice
Dates: August 15- 23, 2002
Host: Dr. Shigemi KINOSHITA, UT southwestern Med. Ctr. At Dallas, USA

(14) Dr. Hideki KAMITANI, Institute of Neurological Sciences, Tottori University
Subject of Research: Gene Expression Profile in Tumor Cells after Treatment with Eicosanoids by Microarray Experiment
Dates: August 26- September 14, 2002
Host: Dr. Thomas Eling, National Institute of Environmental Health Sciences

(15) Dr. Kazuo OHMORI, Toyama Medical and Pharmaceutical University
Subject of Research: Gain and Loss of DNA Sequence in the Primary Malignant Bone Tumor by Comparative Genomic Hybridization
Dates: October 15, 2002 - January 13, 2003
Host: Dr. Julia A. BRIDGE, Department of Pathology and Microbiology, University of Nebraska Medical Center

(16) Dr. Kenzo TAKADA, Institute for Genetic Medicine, Hokkaido University
Subject of Research: Development of BAC-cloning and Mutagenesis System for Studying Epstein-Barr Virus Oncognenesis
Dates: October 29, 2002 - November 4, 2002
Host: Dr. Bill SUGDEN, University of Wisconsin-Madison


EPIDEMIOLOGY/BEHAVIORAL SCIENCE
(17) Dr. Toshiro TAKEZAKI, Aichi Cancer Center
Subject of Research: Investigation of the Design and Analyses for International Joint Study: with Special Reference to Epidemiological Studies on Lung Cancer with the US and on GI-tract Cancer with China
Dates: June 10 - 23, 2002
Host: Dr. Walter WILLET, Harvard School of Public Health, Harvard University

(18) Dr. Shinji YOSHINAGA, National Institute of Radiological Sciences
Subject of Research: Combined Analysis of Cancer Mortality Among Radiological Technologists in the U.S. and Japan
Dates: July 25 - August 31, 2002
Host: Dr. Alice SIGURDSON, National Cancer Institute, National Institutes of Health


(1) Haraguchi Misako
Institute for Cancer Research, Faculty of Medicine, Kagoshima University.

SPONSOR AND HOST INSTITUTION:
Dr. Takayuki Shibamoto, Department of Environmental Toxicology, University of California, Davis
DATES OF VISIT: April 5, 2002 -April 12, 2002

SUMMARY OF ACTIVITIES:
Objective of Study
Investigate the mechanism of physiological function and degradation pathway of 2-deoxy-d-ribose.

Achievements
1) Discussion about the mechanism of the formation of Acetaldehyde or Malonealdehyde from 2-deoxy-d-ribose.
2) See how to measure the concentration of Acetaldehyde and Malonealdehyde.
3) Analysis of the formation of Acetaldehyde or Malonealdehyde from 2-deoxy-d-ribose.

How study relates to future work
Measure the plasma concentration of 2-deoxy-d-ribose in TP kockout mice which are challenged by infection, inflammation and tumor implantation.


(2) AKIO SUGINO, Ph. D.
Graduate school of Frontier Biosciences, Osaka University

SPONSOR AND HOST INSTITUTION:
l) Dr. Vladimir V. Larionov, Laboratory of Biosystem and Cancer, National Cancer Institute, National Institutes of Health
2) Dr. Michael A. Resnick, Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health
DATES OF VISIT: From July 22, 2002 to September 20, 2002.

SUMMARY OF ACTIVITIES:
In order to introduce the same mutations that Sacharomyces cerevisiae DNA polymerase epsilon pol2-4, pol2C1089Y (rsa), and pol2-16 mutants have into the POLE gene encoding either mouse or human DNA polymerase epsilon catalytic subunit, we firstly isolated the entire region of mouse genomic DNA (including introns as well as 49 exons) by a TAR-cloning method which Dr. V. Larionov's group has previously developed. We also obtained BAC clones which contain the same region of mouse genomic DNA from Dr. Pieter J. de Jong. Then, using these genomic DNA clones, we constructed the vector DNA containing mouse POLE gene containing either pol2-4, pol2C1089Y, or pol2-16 mutations. Those mutations were confirmed by sequencing of the constructed plasmid DNA.
To determine the consequences of defective proofreading of DNA polymerase epsilon in mammals, we will create mice with pol2-4, pol2C1089Y (rsa), both pol2-4 pol2C1089Y (rsa), or pol2-16 mutations of DNA polymerase epsilon in collaboration with Dr. Okabe's group at Osaka University.
During my stay in both Institutes, I gave two seminars entitled "Molecular Dynamism at Eukaryotic chromosomal DNA Replication Forks" to explain why I have been in the Institute and doing above experiments. After the seminar, everybody congratulated our DNA polymerase epsilon's works and gave so many suggestions for our approaches to investigate the consequences of defective proofreading and/or accuracy of DNA polymerase epsilon in mammals.


(3) Kazuo Shin-ya
Institute of Molecular and Cellular Biosciences, The University of Tokyo

SPONSOR AND HOST INSTITUTION:
Dr. Marik P. Mattson, Gerontology Research Center, National Institute of Aging, National Institute of Health
DATES OF VISIT: May 10, 2002 - August 9, 2002

SUMMARY OF ACTIVITIES:
The objective of this study and achievements
The purposes of this study was to reveal the role of telomerase in neural stem and precursor cells, and examine the effects of versipelostatin, a novel inhibitor of ER stress-induced molecular chaperone expression.
Recent studies revealed that telomerase, which plays an important role to maintain telomere length, also acts as a survival factor in not only cancer cells but also in neurons. Telomerase was reported that it was down-regulated when cells differentiated. Thus, the inhibition of telomerase was thought to induce differentiation in neuronal stem and precursor cells. To reveal the role of telomerase in neural stem cells, the specific telomerase inhibitor telomestatin (TMS) was applied to mouse neural stem cells. Prior to apply for neural stem cells, we first observed the effects of telomestatin on rat primary hippocampal neurons. TMS is the most potent and specific G-quadruplex interacting agent. TMS induced cell-senescence in cancer cells, but showed limited effects on normal cells such as fibroblasts and bone marrow progenitor cells. Unexpectedly, TMS showed cytotoxic effect on rat primary hippocampal neurons when treated at one week after preparation. Telomerase activities were proved to down-regulated by one week in rat primary hippocampal cultures. These data supported that telomerase acted as a cap molecule to protect telomeres of immature neurons to facilitate their survival. TMS also induced differentiation in mice neural stem cells without showing cytotoxic effects at the same concentration.
GRP78, a major molecular chaperone induced by endoplasmic reticulum (ER) stress, increase survival of solid tumors to protect from anoxia. Thus, the inhibitors of GRP78 expression are expected to be promising antitumor drugs. Versipelostatin (VST) has been proved to be a specific inhibitor of GRP78 expression. Unlike the weak cytotoxic effect in vitro, it showed a lethal toxicity in mice. As the result of studies on Alzheimer's disease, neurons highly depend on GRP78 for their survival. VST itself did not induce neuronal cell death, but it showed potent toxicities when applied with ER stress inducing agents such as 2-deoxyglucose.
TMS is now expected to be a promising antitumor drug for clinical use. This study could be one of the useful results how DNA instability is induced by the inhibition of telomere function. VST is the first compound that specifically inhibits the expression GRP78 under ER stress not in normal condition. Molecular chaperone GRP78 is considered to be a new molecular target for antitumor drugs. But the inhibition of GRP78 expression in neuronal cells has not been fully examined. Thus, this study could be important to overcome their side effects on neuronal system. The preparation of these studies are now underway.


(4)Sayoko Ihara
University of Tokyo

SPONSOR AND HOST INSTITUTION:
Mount Sinai School of Medicine
DATES OF VISIT: 2002.6.2-2002.8.30

SUMMARY OF ACTIVITIES:
Swap-70 is considered to be a guanine nucleotide exchange factor (GEF) for Rac GTPase. Since many of GEFs for Rho family GTPases have been suggested to exhibit oncogenic properties. it is possible that Swap-70 may play a role in signal transduction of cell transformation. This work is aimed to study this possibility.
Firstly, I prepared several clones of Swap-70 KO MEFs from heterozygous mouse. The growth of Swap-70 KO MEF was slightly slower than that of the WT MEF and the morphology of those cells was more flattened than that of WT cells, suggesting that the motility of the KO cells can be lower than that of WT cells. To confirm this point, I am currently trying to establish them as cell lines. After establishment of them, I am planning to examine the transforming properties of these cells by oncogenes such as v-Ras, v-Src, or v-ErbB2 to sec requirement of Swap-70 in cell transformation.
Secondly, I found that v-Src strongly phosphorylated Swap-70 on tyrosine. Endogenous c-Src immunoprecipitated with an anti-Src antibody also phosphorylated the recombinant Swap-70. In addition, Swap-70 coimmunoprecipitated with c-Src, suggesting strong interaction between c-Src and Swap-70. These results suggest that Swap-70 can be downstream of c-Src or v-Src in signaling of cell transformation. Further study is underway.


(5) Masato Hirata
Kyushu University

SPONSOR AND HOST INSTITUTION:
Dr. Steve Shears, National Institute of Environmental Health Sciences
DATES OF VISIT: 6/10/02-8/10/02

SUMMARY OF ACTIVITIES:
Aim: We have examined the effects of inositol hexakisphosphate (InsP6) on cell growth and cell survival, and found that it inhibited both events, indicating that it could be an effective molecule for preventing oncogenesis. We further explored that it inhibits Akt signaling to block the process of cell survival signals. However, the mechanisms by which extracellularly-applied InsP6 inhibits Akt signaling in cells are not known. Therefore, in the present study, we planned to examine whether extracellularly-applied InsP6 could be incorporated inside cells and metabolized to other inositol phosphates to inhibit the binding of Akt to PtdIns(3, 4, 5)P3 by the inositol phosphates.
Materials and Methods: HeLa cells at 1x106 cells were cultured in a 6-multiwell plate, and then starved for 12 hrs, followed by the incubation with 0.1 mM InsP6 containing 0.5 µCi [3H]InsP6 for another 6 hrs. After washing with phosphate-buffered saline twice, cells were collected in a 0.6 M percholic acid containing with 0.1 mg/ml InsP6 as a carrier. Cellular extract after the neutralization with 1 M K2CO3 (containing 5 mM EDTA) was applied to a SAX (strong anion exchange) column. Fractions were collected every 1 min, followed by a scintillation counting.
Results and Discussion: As shown in the figure below, several radioactive peaks were observed, but the peaks except for that of [3H] InsP6, did not coincide with those of the most popular InsP3, InsP4 and InsP5, [3H] Ins (1, 4, 5) P3, [3H] Ins (1, 3, 4, 5) P4, [3H] Ins (1, 3, 4, 5, 6) P5, which were applied to the same column in advance. According to the suggestion by Dr. Shears, I tentatively concluded the radioactive peaks found were Ins (1, 2, 4/6) P3, Ins (1, 2, 3, 4/6) P4, Ins (1. 2. 3, 4, 6) P5, Ins (1, 2, 3, 4, 5) P5, Ins (1, 3, 4, 5, 6) P5 and InsP6, respectively. Great majority of [3H] InsP6 would be derived from the non-specific binding to the extracellular plasma membranes. Therefore, it would be assumed that these inositol phosphates metabolized from intracellularly incorporated InsP6 is involved in the inhibition of Akt cell survival pathway.


(6) KEN-ICHI SATO
KOBE UNIVERSITY

SPONSOR AND HOST INSTITUTION:
RAFAEL A. FISSORE, Ph.D.
UNIVERSITY of MASSACHUSETTS at AMHERST
DATES OF VISIT: JUNE 13, 2002 -AUGUST 31, 2002 (80 DAYS)

SUMMARY OF ACTIVITIES:
The present study was aimed to understand the role played by Src family protein-tyrosine kinases/ proto-oncogene products in egg fertilization of mammalian species. Research conducted involves three experimental projects and the results and achievements are noted as follows.
1) Effect of Src-specific inhibitors on sperm-induced activation of mouse eggs:
Mouse unfertilized eggs were microinjected with several Src-specifc or tyrosine kinase-specific inhibitors and examined for the ability to activate in response to insemination, intracytoplasmic sperm injection, or injection of sperm cytoplasmic extracts. Egg activation was assayed by monitoring calcium oscillating signals and formation of pronucleus in eggs. ATP-competitive inhibitors such as PP2 and lavendustin A did not block egg activation by any treatments. However, ATP non-competitive inhibitors peptide A and peptide A7, both of which are capable of binding to the catalytic domain of Src (ref. 1,2), showed an inhibitory effect. The peptide inhibition of egg activation was most notably when sperm cytoplasmic extracts were used to activate eggs.
2) Molecular identification of peptide A/A7-binding proteins in sperm cytoplasmic extracts: Results described above suggest that sperm cytoplasmic extracts contain peptide A/A7-binding protein(s) that are crucial for its egg-activating property. Therefore we prepared biotinylated version of peptide A7 and used it for affinity purification of the binding proteins. Specific binding proteins were identified by SDS-polyacrylamide gel electrophoresis and silver staining of proteins. Molecular identity of these proteins was determined by mass spectrometry and database search. Three sperm proteins were identified by this method to date. They include b-galactosidase, chaperonin subunit, and zona pellucida-binding protein. Immunochemical approach has demonstrated that Hck, a member of Src family kinases, is also a constituent in a pool of the peptide-binding proteins. Functional importance of these proteins is now under investigation.
3) Expression, subcellular localization, and activation of Src tyrosine kinase in mouse eggs: This is a fundamental question regarding the role played by Src family kinases in egg fertilization of mice. To access this issue, we conducted cDNA cloning of Xenopus Src gene, whose gene product has been suggested to be important in egg fertilization of Xenopus, and constructed mammalian expression vectors that will express xSrc gene of either wild type, active form (via substitution of Tyr-527 to Phe), or inactive form (via substitution of Lys-295 to Met). These constructs were examined for their functional expression in COS7 cells (transfection) and in Xenopus oocytes (injection of mRNA). We think it will be valuable to employ Xenopus Src gene to test the physiological importance of Src in mouse egg fertilization. Further study will be continued to express green fluorescent protein-tagged xSrc gene product in mouse eggs and examine its expression, localization, and active-inactive transition in the course of fertilization.



(7) Yoshiko Akamatsu, Ph.D.
Institute for Genome Research, The University of Tokushima

SPONSOR AND HOST INSTITUTION:
Marjorle A. Oettinger, Ph.D., Massachusetts General Hospital, Harvard Medical School
DATES OF VISIT: June 28, 2002 – July 27, 2002

SUMMARY OF ACTIVITIES:
My first objective of this visit was to complete the study of Dr. Oettingr and I have been working for last few years. We submitted our paper about the function of the RAG2 C-terminal region a month before visiting her laboratory. As expected, the reviewer's comments came back at the right timing of my visit. The study was also contributed by the members of Dr. Frederick W. Alt's laboratory. So, we all first discussed about the comments and what to do. As a results, we figured out we'd better show the efficiency of V(D)J rearrangement more accurately. To analyze the developmental stage-specific recombination efficiency, I prepared genomic DNA from the fetal livers of various developmental stages that contains lymphoid progenitors after the mating of knock-in mice, I could not finish testing the efficiency since it took unexpectedly more time to get approval to use their animal facility. The actual PCR analysis is to be completed after the visit.
We also found that C-terminal deletion of RAG2 causes certain defect on DNA the end-processing and resolution. I was able to obtain some genomic DNA derived from lymphoid organ of mutant mice of which N-terminal RAG1 was deleted for our future study. We are going to compare the joints processed by those mutant RAGs to analyze the function of N-terminal RAG1 and C-terminal RAG2. I purified CD4+CD8+T cell by cell sorter and spleen T cells by MACS, as well as total thymus DNA of mutant mice and a WT mouse to analyze the junctions caused by TCR!!!rearrangement.
In addition, I have finished writing the manuscript of our recent report about the effects of DNA methylation on the activities of RAG1 and RAG2, and had Dr. Oettinger and two more researchers in her group to read. I got very helpful comments from all of them and I am currently working for the additional experiments they suggested to improve the study. We exchanged the useful protocols, shared our new data each other and got the idea what to be studied next. I believe my visit was very fruitful for the both groups, Dr. Oettinger's and mine.



(8)Katsunori Tanaka
Shimane University

SPONSOR AND HOST INSTITUTION:
Sponsor: JSPS
Host Institution: The Scripps Research Institute
DATES OF VISIT: 2002. 7. 1 – 2002. 9. 30

SUMMARY OF ACTIVITIES:
Research Project: Cell-cycle checkpoint functions of ATM-related kinase in cancer cells
(Research plan)
Cell cycle checkpoints ensure that chromosomal DNA is replicated and repaired before nuclear division. In mammalian cells, loss of checkpoint control results in rearrangements, amplification, and loss of chromosomes, events that are causally associated with cancer.
Checkpoint responses change as cells proceed through the cell cycle. I isolated a novel checkpoint gene in fission yeast, mrc1 (mediator of replication checkpoint), that confers activation of the checkpoint kinase Cds1 to DNA synthesis (S) phase. Mrc1 associates with Cds1 and is required for regulation of Cds1 by the checkpoint kinase Rad3. Mrc1 is cell cycle regulated, with the appearance of Mrc1 mRNA and protein coinciding with S phase. I propose that coordinated expression of Mrc1 with replication control proteins helps to insure activation of the appropriate checkpoint response during DNA replication.
To elucidate this model, I will try to purify and characterize the protein complex that contains Mrc1 protein. This purification will make how Mrc1 channels DNA replication arrest signal to checkpoint kinase Cds1 clear.
(Results)
To purify and characterize the protein complex that contains Mrc1 or Cds1 protein in the replication checkpoint-active or checkpoint-nonactive condition, TAP-tag purifications have been done for Mrc1 or Cds1 from 12L fission yeast cultures in the presence or the absence of HU, DNA replication inhibitor. As shown in Figure, Mrc1 and Cds1 have been successfully purified and several associated proteins could be recognized on SDS-PAGE gel detected by silver staining. I am currently trying to characterize the proteins involved in these compleses by multidimensional protein identification technology (MudPIT).


(9) Naofumi MUKAIDA
Cancer Research Institute, Kanazawa University

SPONSOR AND HOST INSTITUTION:
Joost J. Oppenheim, National Cancer Institute-FCRDC
Isiah J. Fidler, M. D. Anderson Cancer Center
DATES OF VISIT:
Date Departure Arrival
July 4 Narita Washington D.C.
July 6 Washington D.C. Boston
July 13 Boston Houston
July 17 Houston San Diego
July 19 San Diego
July 20 Kansai Airport

SUMMARY OF ACTIVITIES:
a. National Cancer Institute-FCRDC (Frederick, MD)
I discussed our collaborative works to elucidate the pathophysiological roles of chemokines with Dr. Oppenheim and his colleagues. I gave a seminar on the roles of proinflammatory cytokines in the tumor metastasis processes.
b. Dana Farber Cancer Institute
I discussed with Dr. Barret Rollins, our collaborative works to clarify the roles of chemokines, particularly monocyte chemoattractant protein (MCP)-1 in the tumor progression and the host defense against tumors. I exchanged the recent information on the above-mentioned topics with him.
c. M.D. Anderson Cancer Center
1) I discussed with Dr. Fidler, our collaborative works to clarify the roles of proinflammatory cytokines, including chemokines, in tumor metastasis processes. I gave a seminar on our recent progress on the roles of proinflammatory cytokines in liver metastasis.
2) I discussed with Dr. Bingliang Fang, our collaborative works on cancer immuno-gene therapy using MCP- 1 gene.
d. Scripps Research Institute
1) I discussed with Dr. Francis Chisari, the pathophysiological roles of chemokines in hepatoma progression.
2) I discussed with Dr. Zhixing K. Pan, our collaborative works to delineate the roles of chemokines in tumor metastasis processes.


(10) KATSUHIDE IGARASHI
NATIONAL INSTITUTE OF HEALTH SCIENCES

SPONSOR AND HOST INSTITUTION:
NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCE
DATE OF VISIT: JULY 29, 2002 - SEPTEMBER 29, 2002

SUMMARY OF ACTIVITIES:
The main purpose of this research is to compare the platforms of gene expression profiling between National Center for Toxicogenomics / National Institute of Environmental Health Sciences / NIH of the United States and our institute, including the procedure of (1) purification of RNA from tissues, (2) labeling of cDNAs, (3) analysis of cDNA microanay, (4) data analysis and construction of database. In order to conduct this research, samples from a model study described below were used. In practice, two chemicals, phenobarbital (PB) and TCDD, nongenotoxic carcinogens were administered to p53 hetero knock out mice and wild type mice for 24 hrs and gene expression profiles from the liver were generated. The following table is the comparison between NIEHS/NCT system and NIHS (Japan) system.

Step
NIEHS/NCT
NIHS (Japan)
RNA purification Silica gel method Silica gel method
Microarray chip cDNA microarray, Long oligo array Genechip (25mer oligo DNA)
cDNA label Direct cDNA labeling Cy3, Cy5 Two color cRNA linear amplification PE single color
Hybridization Hybrichamber Genechip
Wash Manual Auto
Scan Slide glass scanner Genechip scanner
Data analysis MAPS (NCT original) Microarray suite (Affymetrix Inc.)
Database MAPS Under construction

I analyzed two pairs of samples (Phenobarbital treated one and TCDD treated one) with NIEHS microarray system and compared the results with our Affymetrix system results. In summary, the results between them were similar, especially for TCDD treated sample. I listed up those genes as follows.
<Phenobarbital>
More than 2 fold increase:
Aminolevulinicacidsynthasel, ApolipoproteinA-IV, Cytochrome P450 oxidoreductase, Cytochrome P450, 2a4, Cytochrome P450, 2b10, Epoxide hydrolase 1, microsomal, Glutathione S-transferase, alpha2[Yc2], Glutathione S-transferase mu2, GrowtharrestandDNA-damage-inducible45alpha
Less than 0.5 fold decrease:
Betaine-homocysteinemethyltransferase, Complementcomponent9, Glucose-6-phosphatase, catalytic, Majorurinaryprotein5, Presenilin2, SerumamyloidA2, SerumamyloidA3, Stearoyl-CoenzymeAdesaturasel, Urateoxidase
<TCDD 0.1ug>
More than 2 fold increase:
Aminolevulinicacidsynthasel, CaseinolyticproteaseX[E. coli], Cytochrome P450, lal, Cytochrome P450, la2, Cytochrome P450, 2a4, Cytochrome P450, 4al4
Less than 0.5 fold decrease:
SerumamyloidA2, SerumamyloidA3, Tubulin, beta 2
From these results, I think I could get the basic data which is required for collaboration between Japan and United States using DNA microarray.



(11) Tohru Kamata
Department of Molecular Biology and Biochemistry, Shinshu University School of Medicine

SPONSOR AND HOST INSTITUTION:
Dr. Nancy Colburn, National Cancer Institute-FCRDC, Gene Regulation Section
DATES OF VISIT: 8/4-8/25, 2002

SUMMARY OF ACTIVITIES:
Objective: The objective of our study is to investigate the role of the mitogenic oxidase, Nox1 in tumor promotion.
Achievements:
1. We examined the expression level of Nox1 in TPA-treated mouse JB6P+ cells by using RT-PCR. The data show that TPA induces the Nox1 gene expression at least at 6hr posttreatment.
2. Both a Nox1 inhibitor, DPI and a chemical antioxidant, N-acetyl cystine blocked the TPA-induced activation of the transcription factor, AP-1 activity. These results imply that the generation of reactive oxigen species by Nox1 is involved in the TPA-AP-1-mediated signaling pathway which has a pivotal role in tumor promotion. Based on the findings, the further study to clearify the mediating role of Nox1 in tumor promotion is under way.


(12) Shigeru Chiba
University of Tokyo

SPONSOR AND HOST INSTITUTION:
Ken-ichi Takeshita, New York University
DATES OF VISIT: Aug. 4 – Aug. 25, 2002

SUMMARY OF ACTIVITIES:
[Background] I have been studying the role of Notch signaling and its deregulation in normal and leukemic hematopoiesis. In the University of Tokyo, we recently found that activation of Notch results in block of TGF-!!!-induced growth suppression of TGF-!!!-responsive cells. This observation explains, at least in part, the mechanism how an excess of Notch activation contributes development of tumors (paper submitted). Meanwhile, Dr. Takeshita and I recently found that the product of a homeobox gene DLX1 blocks multiple signaling pathways from TGF-!!!superfamily members by interfering with Smad4, the common Smad (paper in preparation). These two findings together encouraged us to study the signal cross-talk among TGF-!!!superfamily members, activated Notch proteins and DLX proteins.
[Purposes, plans and achievements of collaboration] To further extend our findings of the cross-talk between TGF-!!! family of cytokines and DLX proteins, Dr. Takeshita and I planned a short period of collaboration in the New York University. Our research plans during my 3 wks stay in New York were (1) retroviral transdcution of mouse embryonic stem cells (ES cells) and cells from paraaorta-splanchnopleura (P-Sp) of E9.5 mouse embryo with DLX1 and DLX7. The transduced cells would be used to see if the hematogenic effects of TGF-b and one of its members BMP4 on in vitro hematopoiesis from ES and P-Sp cells co-cultured with a stromal cell line, OP9, is interfered by upregulation of DLX1 and DLX7; and (2) use of anti-sense oligos of DLX1 and DLX7 to prove that downregulation of DLX proteins would enhance the sensitivity of the cells to TGF-b and its family cytokines. To carry this out, we planed to see the TGF-!!!-induced growth suppression of several leukemia cell lines that express DLX1 or DLX7 and are insensitive to TGF-!!!. We could successfully initiate these experiment and now are continuing them.
In these processes, it was important that I could successfully transfer a technique for P-Sp preparation to Dr. Takeshita and his postdoc.
[Other activities] In the New York University, I made a seminar in the cross-talk between TGF-b and Notch, as we previously planned.
In addition, one day I visited Boston (August 10) to have a discussion with Dr. Spyrus Artavanis-Tsakonas about Notch studies, although this was not previously planned.



(13) Tadashi Matsuda, Ph.D.
Dept. of Immunology, Grad. Sch. of Pharm. Sci., Hokkaido University

SPONSOR AND HOST INSTITUTION:
Shigemi Kinoshita Ph.D.
UT southwestern Med. Ctr. At Dallas, USA
DATES OF VISIT: August 15, 2002 - Aug 23, 2002

SUMMARY OF ACTIVITIES:
We had established Tyk2 knockout mice (Immunity 13: 561-571, 2000). IL-12-induced NK cell activity in cells from Tyk2 knockout mice was drastically reduced compared to that in cells from wild-type mice (Blood 99: 2094-2099, 2002). Using these mice, we are now working on experiments to identify novel genes involved in IL-12 signaling. To examine whether newly identified genes can rescue the defect of IL-12 signaling, we are planning to introduce those genes into cells in Tyk2 knockout. For this purpose, we will apply the novel and powerful retrovirus systems under collaboration with Dr. Kinoshita. We will also examine whether those gene products can rescue the defect of IL-12 signaling in Tyk2 knockout mice Retroviruses are an efficient means to deliver single DNA expression constructs to a wide range of mammalian cell types. They are by far the easiest and fastest means to deliver genes stably to mammalian cells. To prepare retrovirus system, we have started a collaboration with Dr. Kinoshita (UT Southwestern Med. Ctr, Dallas, USA). She had developed and reported about improved retrovirus systems with Dr. Nolan (Stanford Univ. Med. Sch.) (Kinoshita, S. et al. Cell 95:595-604, 1998, Kinoshita, S. et al. Immunity 6:235-244, 1997). One of them is Phoenix retrovirus system. Phoenix-MMULV vectors is currently employed in over 2500 laboratories worldwide for delivery of genes and libraries to cells for biomedical research. It is based on Moloney Murine Leukemia Virus (MMULV) and allows for delivery of genes to most mammalian cell types. The system comes as either an Ecotropic packaging system (capable of delivering genes to dividing murine or rat cells) or an Amphotropic system (capable of delivering genes to dividing cells of most mammalian species, including human). This time, I visited Dr. Kinoshita at UT Southwestern Med. Ctr. At Dallas and made a retrovirus construct bearing Tyk2 cDNA. Then we have now started making retrovirus to introduce Tyk2 into Tyk2-deficiernt embryonic fibroblasts. This system would be a great help to identify novel functions in the downstream of Tyk2.


(14) KAMITANI HIDEKI
Dept. of Neurosurgery, Institute of Neurological Science, Tottori University

SPONSOR AND INSTITUTION:
Dr. Thomas Eling, National Institute of Environmental Health Sciences
DATES OF VISIT: 26/08/2002 TO 14/09/2002

SUMMERY OF ACTIVITIES:
I visited NIEHS to summarize and discuss the ongoing project entitled "Gene expression profile in tumor cells after treatment with eicosanoids by microarray experiments" During my stay in NIEHS, I checked the whole dates regarding the profiles in which gene expressions were altered. I again summarize the dates in details, and then I discussed the crucial genes with Dr. Thomas Eling, Dr. Jennifer Nixon and Dr. Frank. As the result of discussion, we selected a few critical genes, which affect the tumor cells after treatment with 13-HODE, which is the major metabolite of linoleic acid. On the other hand, we need to show the additional dates in which tumor cells produced in acute phase after treatment with eicosanoids. Additionally, I provided the cells except for colorectal carcinoma cells such as brain tumor cells by culturing in this laboratory.
We agree to continue to this project between the Laboratory of Molecular Carcinogenesis in NIEHS in USA and Tottori University in Japan. The manuscript of the project is partially under preparation.



(15) Kazuo Ohmori
Department of Orthopaedic Surgery, Toyama Medical and Pharmaceutical University

SPONSOR AND HOST INSTITUTION:
Department of Pathology and Microbiology, University of Nebraska Medical Center
DATES OF VISIT: October 15, 2002 - January 13, 2003

SUMMARY OF ACTIVITIES:
Chromosomal abnormalities of 41 specimens of the primary malignant bone tumor were cytogenetically investigated. Clonal chromosomal abnormalities were detected in 11 of these 41 specimens including involvement of 17p in seven. A 16;17 translocation was observed in 4 cases and included one case in which a three-break translocation was observed [t(7;17;16)(q21;p13;q22)]. Moreover, three additional cases exhibited 17p13 rearrangements, but with chromosomal partners other than chromosome 16. Notably, a 1;7 translocation involving the same breakpoint on the long arm of chromosome 7q22 was detected in two cases. These results suggest that chromosome translocation is related to the etiology of the primary malignant bone tumor.



(16) Kenzo Takada
Hokkaido University

SPONSOR AND HOST INSTITUTION:
University of Wisconsin-Madison
DATES OF VISIT: Oct. 29, 2002 - Nov. 4, 2002

SUMMARY OF ACTIVITIES:
Since we had already succeeded in cloning of the Epstein-Barr virus genome into BAC plasmid, subsequent transfection of the resultant BAC plasmid into Akata cells and production of infectious virus in Akata cells. Based on these results, I discussed with Dr. Sugden about the method to increase the stability of BAC plasmid in E. coli. Furthermore, We discussed about the two methods of introducing mutations in BAC plasmid. One was developed in Germany, and another was developed in Australia. Our conclusion was that the method by Australian group was easy to perform and more suitable than that by German group. During this visit, I could get much important information, which will helpful for performing EBV research.



(17) Toshiro Takezaki
Aichi Cancer Center Research Institute

SPONSOR AND HOST INSTITUTION:
Harvard School of Public Health
American Health Foundation
DATE OF VISIT: June 10, 2002 - June 23, 2002

SUMMARY OF ACTIVITIES:
1) Investigation of dietary factors for lung cancer in Japanese
I'm involved in an international joint epidemiological study with the American Health Foundation (AHF) to compare the risk and protective factors for lung cancer between the US and Japan, because Japan shows paradoxically a lower mortality rate of lung cancer, in spite of high smoking rate in males. We have also reported frequent fish consumption was associated with decreased risk of lung adenocarcinomas in a Japanese population.
I introduced our findings to Drs. Walter Willet, Meir Stampfer and Ed Giovannucci at the Department of Nutrition and Epidemiology, Harvard School of Public Health (HSPH), and they had much interest in our hypothesis and results. They mentioned they would investigate this association, using their dataset. This further study will be beneficial to test our hypothesis.
I also visited AHF, and introduced our findings at the seminar. I met Dr. John H Weisburger, who encouraged our joint study and emphasized the importance of international comparison, such as geographical pathology. This concept is very acceptable for our joint studies with the US and with China. I also met Dr. Reddy and his group and discussed the anti-tumorgenic effects of NSAIDs and fish fat. I discussed further plan of analyses on our joint study with Dr. Steven D. Stellman, which was the most important objective of my visit. We listed the further interests on smoking habits and other lifestyles that were related to lung cancer risk in both countries, and decided to perform further analyses within several months each other. I also obtained the information of the methods how to estimate nutrient intake from AHF semi-quantitative food frequency questionnaire (SQFFQ), and decided the methods how to validate Japanese SQFFQ and link Japanese dataset of nutrient intake to American dataset. These procedures were the most fruitful outcome on this visit and I could clarify the difference of our SQFFQS between the US and Japan, and fix the plans for further analyses, that is, Japanese SQFFQ need to be validated by other data of food survey, although the US SQFFQ has already done for validity test by an other research group.
2) Investigation of food and nutrient intake in urban and rural areas of three different regions in China, and evaluation of semi-quantitative food frequency questionnaire in each region
We are developing area-specific SQFFQs in three areas of China, in order to investigate the association between nutrient intake and colorectal cancer risk. We have already collected the data of food intake in urban and rural residents of these areas, but have still questions on the method how to develop combined SQFFQ covering both residents. I discussed this matter with Dr. Willet, and concluded our method is acceptable for covering both populations, that is, food items that were independently obtained in the urban and rural SQFFQs can be combined in one SQFFQ. This information leads us to fix the appropriate method for development of the SQFFQs and encourage further case-control study.
I also discussed the methodological issues on international comparative studies with Dr. David Hunter at the Department of Epidemiology, Harvard Center for Cancer Prevention. He suggested the background information on nutrient intake in general population was useful to compare and combine the data in different study areas to adjust their geographical variations. Such standardization is also an important issue for our international comparative study in Korea, China and Japan.
3) Intervention trial for mother-to-child transmission of HTLV-I in Japan
Japan is an endemic area for Human T-lymphotropic Virus Type-I (HTLV-I) that is a causative virus for Adult T-cell Leukemia (ATL). As mother-to-child transmission via breast milk is a major route for HTLV-I transmission, we have conducted several epidemiological studies to clarify the risk of its transmission and establish the preventive method. We found short-term breast feeding revealed the lowest risk for HTLV-I infection among the children born to HTLV-I carrier mothers, being similar risk with bottle feeding. I introduced our findings to Drs. Nancy Mueller and Sherri Stuver at the Department of Epidemiology, HSPH, and made a presentation at the Seminar -Epidemiology library. My objective of the present visit is to discuss the appropriate methods for the prevention of HTLV-I infection and ATL development. We agreed the short-term breast feeding was a suitable choice for HTLV-I carrier mothers, due to beneficial effects of breast feeding for health with the lowest risk. We also discussed mother-to-child transmission of HIV via breast milk. The US researchers reported that short-term breast feeding of 11 months or less reduced the risk of HIV infection, but our case of HTLV-I was 6 months or less. This finding suggests protective effect of breast feeding may differ between HTLV-I and HIV.
Dr. Mueller also introduced the key person who was involved in the management of Japanese dataset. We agreed that such a person who could understand the counterpart language was needed to develop and advance the international joint study, being a similar situation with our joint study in China.
4) Molecular epidemiological study for GI-tract cancer
We are conducting an association study between gene polymorphisms and risk of esophageal and stomach cancer with environmental factors in China. I introduced several findings of our studies to Drs. David Hunter and Immaculata DeVivo at the Department of Epidemiology, Harvard Center for Cancer Prevention. They mentioned their major strategy for molecular epidemiological study was to focus on the gene polymorphism which biological function and activity were clarified and investigate its interaction with environmental factors, although they were trying a pilot study to screen the association between various gene polymorphisms and cancer risk. I have a same opinion on this matter and re-recognized the importance to have the working hypothesis with clarified biological function and activity for molecular epidemiological study, in addition to the information of gene polymorphisms.
I had sufficiently exchanged views and collected fruitful information on our epidemiological studies with counterpart and various other researchers of the counterpart institutes, with well-arranged schedule. I was impressed by the discussions with them, re-recognizing the importance of clear working hypothesis for research work. This visit was very valuable for advancement of my researches. I'd like to express my heartfelt gratitude for the persons concerned on the NCI-JSPA Cooperative Cancer Research Program to give me the present opportunity, and for the counterpart researchers to make fruitful meetings.



(18) Shinji Yoshinaga
National Institute of Radiological Sciences, Chiba, Japan

SPONSOR AND HOST INSTITUTION:
Drs. Kiyohiko Mabuchi and Alice Sigurdson
Radiation Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda
DATES OF VISIT: from July 25, 2002 to August 31, 2002 (38 days)

SUMMARY OF ACTIVITIES:
[Research title]
Combined analysis of leukemia among the US and Japanese male radiological technologists
[Background]
Radiation-induced cancer risk has been extensively studied for atomic-bomb survivors, medically irradiated populations, etc who were exposed to acute single exposure to high dose and dose-rate of radiation. However, cancer risk due to protracted exposure to low- to moderate- dose of radiation exposure remained to be studied. Leukemia risk, which is considered as the most radiosensitive cancer, among radiological technologists in the US and Japan who represent one of the oldest occupational populations will be evaluated by using follow-up data from the two ongoing studies.
[Subjects for analyses]
1. US radiological technologists
Study population of the US radiological technologists study comprises 146,022 technologists who were certified by the American Registry of Radiologic Technologists for al least 2 years from 1926 through 1982. A total of 90,305 out of the entire cohort who responded to a questionnaire survey in 1983-89 were included in analyses.
2. Japanese radiological technologists
Study population of Japanese radiological technologists study comprises 12,174 male technologists who were licensed by Japanese government from 1968 to 1975, and were born in 1950 or earlier. Out of the entire cohort. a total of 5,087 technologists whose work history was available through several sources were included in analyses.
[Statistical analysis]
Standardized mortality ratio (SMR) and corresponding 95% confidence interval of leukemia were calculated by using sex-, race-, age-, calendar year-specific mortality rates from the Surveillance, Epidemiology, and End Results (SEER) data for the US technologists and from vital statistics for the Japanese technologists. SMRs were compared by several proxy measures of radiation exposure, including year first worked.

[Results and Discussion]
A total of 17 deaths from leukemia were observed among the 5,087 Japanese technologists in 1969-1998, while there were 57 leukemia deaths among the 90,305 US technologists in 1983-1997. As previously reported by Doody et al (1998), there seem to be some increases of leukemia risk for those who worked in earlier periods (Table 1). Among those who first worked after 1960, who were presumably exposed to lower radiation doses than those first worked before 1960, there were weak patterns that leukemia risks were decreased with increasing years since first work, and were increased with increasing age at first work or attained age (Tables 2-3). These patters are in contrast to findings from studies of atomic bomb survivors which show greater risks at younger age at exposure, and consistent with Richardson and Wing (1999) which suggested that sensitivity to the carcinogenic effects of ionizing radiation may increase with older ages at exposure. Detail evaluation of leukemia risk among the Japanese technologists was not performed because of low statistical power. Further analyses, such as Poisson regression analysis which incorporates several covariates simultaneously, on the basis of combined data oh medical radiation workers in the US, Japan, and other countries is promising for better description of cancer risk from the protracted exposure to ionizing radiation and for better understanding of carcinogenesis.

Table 1. Standardized mortality ratios (SMRs) of leukemia by year first worked among 90,305 US radilogic technologists, 1983-91
Year first worked
Year first worked
<1940
1940-49
1950-59
1960-69
1970+
Total
Number of cases
7
13
7
15
12
57
SMR
1.71
1.19
0.53
1.27
1.10
1.09
95% CI
0.69-3.52
0.63-2.03
0.22-1.10
0.71-2.10
0.57-1.92
0.82-1.41
Data on those who never worked or those with missing information on year first worked were not shown.

Table 2. Standardized mortality ratios (SMRs) of leukemia by year first worked and by age first worked, among 90,305 US radilogic technologists, 1983-97
Age first worked
Year first worked
<20
20-24
25-29
30+
Total
<1960
1.54 (0.77-2.76)
0.76 (0.33-1.50)
0.38 (0.06-1.39)
1.13 (0.42-2.46)
0.96 (0.63-1.40)
1960+
0.63 (0.21-1.46)
1.32 (0.70-2.26)
1.37 (0.30-4.02)
2.20 (0.81-4.80)
1.19 (0.78-1.73)
Total
1.06 (0.61-1.72)
1.03 (0.64-1.58)
0.68 (0.22-1.58)
1.49 (0.77-2.61)
1.09 (0.82- 1.41)
Data on those who never worked or those with missing information on year first worked were not shown.

Table 3. Standardized mortality ratios (SMRs) of leukemia by year first worked and by age first worked, among 90,305 US radilogic technologists, 1983-97
Attained age
Year first worked
<30
30-39
40-59
60+
Total
<1960
–
0 (0.00->1000)
0.45 (0.10-1.30)
1.12 (0.72-1.67)
0.96 (0.63-1.40)
1960+
0.00 (0.00-8.50)
0.82 (0.27-1.92)
1.31 (0.79-2.04)
1.79 (0.39-5.25)
1.19 (0.78-1.73)
Total
0.00 (0.00-8.50)
0.81 (0.27-1.89)
1.10 (0.71-1.64)
1.17 (0.78-1.69)
1.09 (0.82-1.41)
Data on those who never worked or those with missing information on year first worked were not shown.