Scientist Exchanges

JAPAN (NCI-JSPS) COOPERATIVE CANCER RESEARCH PROGRAM SCIENTIST EXCHANGE SUMMARY REPORT

(1) Michiaki Matsushita
Hokkaido University Graduate School of Medicine

SPONSOR AND HOST INSTITUTION:
Professor MM. Ziegler at Harvard Medical School
DATES OF VISIT:
From May 15, 2000 to August 11, 2000

SUMMARY OF ACTIVITIES:
Objectives:
To participate in the project in which angiogenetic effects of AGM-1470, BB-94, angiostatin, endostatin were evaluated
Evaluation and results
AS animals, RIP1-Td-g2 transgenic mice were used. Four different anti-angiogenetic agents, such as AGM-1470, BB-94, angiostatin, endostatin were evaluated in the effects of anti-tumor growth and Angiogenesis. In the prevention trial, the effects of tumor reduction were 50% in BB-94, 61% in endostatin and 63% in a combination of angiostatin and endostatin. In the interventional trial, the effects of tumor reduction were 60% in angiostatin, 82% in AGM-1470, 83% in BB-94, and 84% in endostatin. In the regression trial, angiostatin and a combination of angiostatin and endostatin showed significant prolongation in terms of life saving of tumor bearing animals.


(2)Noriko Yamaguchi
Tokyo Medical and Dental University, Medical Research Institute

SPONSOR AND HOST INSTITUTION:
Prof. Bjorn R. Olsen, Harvard Medical School
DATES OF VISIT:
From June 14, 2000 to July 1 5, 2000

SUMMARY OF ACTIVITIES:
Endostatin was isolated as an angiogenesis inhibitor and considered to be one of the most promising anti-cancer reagents. The final goal of our study is to understand the mechanism of endostatin. One approach is to define the active center of endostatin by comparing the activity among the various mutant endostatins. The other approach is to identify the receptor molecule of endostatin and characterize the reactions induced by the binding of endostatin. I have already discovered that one mutant showed reduced activity estimated by endothelial cell migration assay. Using this mutant we planned to confirm its reduced activity in endothelial cell outgrowth assay and mouse tumor model in cooperative way. Bjorn suggested that binding assay should be done to estimate the probability of the receptor. He also recommended me to use labeled kemptide-endostatin fusion protein for the binding assay, which peptide has the substrate sequence of cAMP-dependent protein kinase. We can expect the high sensitivity to detect the binding ability of endostatin to endothelial cells using isotope labeled tracer. Therefore we discussed to proceed the binding assay and if it will be done successfully, we could get the information of binding affinity and the number of the receptors.

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

SPONSOR AND HOST INSTITUTION:
Dr.Ira Daar National Cancer Institute-FCRDC, Regulation of Cell Growth Laboratory
DATES OF VISIT:
7/22 - 8/22, 2000

SUMMARY OF ACTIVITIES:
The Objective is to investigate the role of Rac1 in cell adhesion and migration by using Xenopus embryo systems.
Achievements: We constructed a dominant negative (DN)-Rac1 mutant and subcloned it into pcS2+ vector to prepare mRNAs of DN-Rac1 by in vitro transcription. The synthesized mRNAS were microinjected into the fertilized Xenopus eggs together with mRNAs of a Eph receptor tyrosin kinase (EphA4) mutant. Then, we examined whether the induction of abnormal cell migration by the Eph 4 mutant is blocked by DN-Rac1. If DN-Rac1 has a blocking effect, the data suggest that Rac1 is a regulator for Eph receptor signaling involved in cell adhesion and migration. We have not got the conclusive data yet, possibly due to failure in introduction of a correct mutation into Rac1. The repeated experiments are under way.



(4)TANI, Fumito
Research Institute for Food Science, Kyoto University

SPONSOR AND HOST INSTITUTION:
Chief, David H. Margulies, MD, PhD
National Institute of Allergy and Infectious Diseases, NIH
DATES OF VISIT:
13 July, 2000-10 September, 2000

SUMMARY OF ACTIVITIES:
To test the hypothesis that natural killer (NK) cells recognize cell surface heat shock protein (hsp) molecules expressed on tumor cells, this proposal outlines a program to examine the cytotoxicity of NK cells to targets that have been transfected to express large amounts of a membrane-anchored form of hsp. Candidate molecules on the NK cell that might serve as the hsp receptor include glycolipids such as sulfatide and asialo-GM1 ganglioside. This possibility derives from the knowledge that the murine hsp70 homolog hsc70t which binds specifically to sulfogalactosylceramide during the fertilization process shows 90% amino acid sequence homology to the stress-induced hsp72. To examine the binding of hsp72 to a variety of cells in immune system, a biotinylated hsp72 molecule derived from a bacterially overexpressed hsp70A1 gene was prepared. Considering that the murine hsp72 is originally stress -induced in a cytosol, we constructed the expression vector with pET-20b(+), which lacks a signal peptide for extracellular secretion, and purified the cytosolic hsp72 using Ni-NTA and ATP-agarose affinity column. The ATP-binding ability was found to be a hallmark for the overexpressed hsp72 to be folded correctly. Using fluorescent staining experiments by FACS, we showed that the biotinylated hsp72 molecule bound to murine macrophage- monocyte lineage, J774A.1 and P388D1 cells, but not to T-cell hybridoma and rat NK cells. When P388D1 cells were treated with the protease K, by which extracellular polypeptide chains can be removed from the surface proteinaceous molecules, the staining of enzyme-treated cells was reduced. It thus follows that only the hsp72 with native conformation serves as a probe for binding to immunocytes and that, contrary to our expectation, a putative receptor for hsp72 is not glycolipids, but a proteinaceous molecule. Based on the experience which the NIH laboratory has in the use of SPR, in the future, a putative receptor will be characterized by studying the molecular interactions between the hsp72 immobilized onto a sensorchip and a membrane fraction solubilized from P388D1 cells which showed high capacity for binding hsp72.


(5)Yasuhiro Minami
Department of Biomedical Regulation and Parasitology Kobe University, School of Medicine

SPONSOR AND HOST INSTITUTION:
Dr. Richard D. Klausner, National Cancer Institute
DATES OF VISIT:
November 27, 2000 - December 3, 2000

SUMMARY OF ACTIVITIES:
The purpose of my visit in this scientific exchange program is to have stimulative mutual scientific interactions with outstanding scientists in the U.S. and to discuss about our future collaborative studies. First, I had an interview with Dr. Klausner (NCI), and we discussed about recent topics on "cell cycle checkpoint regulation & its relationship with tumorigenesis. I could also obtain several up-to-date information from Dr. Klausner about the advantage and disadvantage of a recently developed technique, the chemical genetics. At Dr. Samelson's laboratory (NCI), I gave a talk about our recent studies, and exchanged our recent findings on lymphocyte signaling with Dr. Samelson and his colleagues. At NIH, I obtained valuable information about Jak-Stat signaling from Dr. O'Shea. Drs. Samelson and O'Shea also gave me several important comments and suggestions on our on-going research. Finally, at Harvard University, I could also have fruitful scientific interactions with Drs. Hsu, Band, and Sugita. In particular, I have discussed with Dr. Hsu about the regulation of intracellular trafficking and of intercellular adhesion. We also planned about our future collaboration on this research subject.



(6)Nobuo Sakaguchi
Department of Immunology, Kumamoto University School of Medicine

SPONSOR AND HOST INSTITUTION:
Rockefeller University Dr. Michel Nussenzweig, Duke University Dr. Garnett Kelsoe, Oklahoma Medical Research Foundation Dr. Paul Kincade, Food and Drug Administration of USA Dr. Steven R. Bauer
DATE OF VISIT:
September 8th to 30th, 2000.

SUMMARY OF ACTIVITIES:
I visited four institutions in the USA from September 8th to 30th. I stayed in Oklahoma Medical Research Foundation in Oklahoma City and worked with Dr. Paul W. Kincade, Member and Head of Immunology and Cancer Center. We are both interested in the research of early B cell development and the activation and differentiation of B cells in the humoral immune response. We both proposed the projects with which we will collaborate. I also talked with Drs. Linda Thomson, Mark Coggeshall, Xiao-Hong Sun, Michel Centola, Bill Rogers, and Carol Webb. My presentation of our recent results as a seminal was successful in drawing their attention and their interests. The evaluation I heard was a favorite one. I visited Rockefeller University at Dr. Michel Nussenzweig, a prominent Immunologist concerning to Germinal center B cells. I could hear a lot of information not only about the Immunology but also concerning to the trend of the scientists in the international level, because he is an active editor of Journal of Experimental Medicine. I also visited Dr. Steven R. Bauer and Ed Max at the FDA in NIH campus. There, I could learn the rationale of the evaluation of the application of the gene technology, especially in the gene therapy by the retroviral introduction of the genes. I was also much impressed to know how Dr. Ed Max is familiar with our latest results.
Finally, I visited Dr. Garnett Kelsoe, who is also a prominent scientist in the closest field to my study about the activation of antigen-driven B cells in the peripheral lymphoid organs. He expressed much interest on the germinal center (GC) specific molecules that we are analyzing recently. He offered me a lot of important stuffs for the research, for which I am very much grateful. I made three informal seminars at the institutions I visited and could obtain a lot of important suggestion and the offer. The seminar has been accepted as a favorite one in every place. I consider that my visit was a successful one and I appreciate very much for this opportunity. I would like to make further progress of our study and would like to contribute for the medical research in Immunology.



(7)Masami Suganuma
Saitama Cancer Center Research Institute.

SPONSOR AND HOST INSTITUTION:
Dr. Mike Marino Memorial Sloan-Kettering cancer center
DATES OF VISIT:
Feb. 15, 2001- March 21, 2001

SUMMARY OF ACTIVITIES:
The highlights of this science exchange program were as follows:
1. Discussed a new project to study mechanisms of action of TNF-!!!in tumor promotion with Dr. M. Rosner, University of Chicago.
2. Learned new techniques for studying TGF-!!!resistance from Dr. S-J. Kim, National Cancer Institute.
3. Presented results of our research on cancer prevention with green tea at Keystone Symposium.
4. Studied differences in gene expression between TNF-!!!deficient and wild mice using DNA microarray, in collaboration with Dr. M. Marino, Memorial Sloan-Kettering Cancer Center.
5. Studied role of TNF-!!!in carcinogenesis in IGF-1 transgenic mice, in collaboration with Dr. J. DiGiovannni, University of Texas, M. D. Anderson Cancer Center.
The initial plans for these studies were mostly mad during this trip, I gave lectures on "Essential role of TNF-!!!in tumor promotion and cancer prevention with green tea" and had fruitful discussions with many scientists. New projects for the study of mechanisms of TNF-!!!as a tumor promoter have commenced with Dr. Marino, Memorial Sloan-Kettering Cancer Center, and Dr. DiGiovanni, M. D. Anderson Cancer Center. I am sure that new knowledge and techniques gained during my stay in the United States will bring new insights for our future projects. I am grateful to the Japan Society for the Promotion of Science for their kind support for Scientific traveling in Scientist Exchange under the U.S.-Japan Cooperative Cancer Research Program.



(8)Fumiaki Uchiumi
Dept Of Pharmaceutical Sciences, Science Univ. of Tokyo

SPONSOR AND HOST INSTITUTION:
Professor Ellen Fanning, Ph. D.
DATES OF VISIT:
From 05/11/00 to 08/10/00

SUMMARY OF ACTIVITIES:
DNA replication reaction is accomplished by a lot of protein factors. DNA helicase is one of those DNA replication associated factors. Few years ago, a novel DNA helicase was identified as mouse DNA helicase B (MDHB) from a temperature sensitive mouse cell line in Dr. Enomoto's Lab (Tohoku Univ. Japan). Since the human homologue CDNA has been cloned at Dr, Fanning's Lab very recently, my objectives of study was settled to analyze the gene expression of the human DNA helicase B (HDHB) and to investigate HDHB binding proteins.
Because the HDHB transcripts were too little to be detected by Northern Blotting, quantitative analysis was developed using RT-PCR. At first, I tried to perform the experiments treating HeLa cells with various reagents that regulate the cell cycle. The results suggested that the HDHB transcripts were accumulated at G1 phase. Similar experiments using DNA damaging reagent MMS was carried out to show that the HDHB gene expression could be elevated after the removal of the reagent. These results suggest that the HDHB is involved in the replication as well as DNA repair synthesis.
HDHB protein has a certain homology with bacterial Rec D protein which is thought to be involved in homologous recombination reaction, Although, it is a hypothesis that HDHB plays a part in the DNA recombination in mammalian cells, the protein might associate with other recombination associating factors in mammalian cells. Now I am trying to establish a system to indicate HDHB association with specific proteins by performing Immuno Precipitation-Western Blotting analysis.
At present, the precise biological function of the HDHB has not been clarified yet, However, I believe that DNA replication, repair, or recombination may have some association with the HDHB protein function. In the future, the possibility would be discussed more in detail by the cooperation with the two Labs. (Vanderbilt and Tohoku Univ).



(9)Kaoru Nemoto
Department of Urology, Nippon Medical School

SPONSOR AND HOST INSTITUTION:
John S. Lazo Department of Pharmacology, University of Pittsburgh, School of Medicine
DATES OF VISIT:
5/22/2000 - 8/22/2000

SUMMARY OF ACTIVITIES:
This is a brief report of my research project entitled "Investigation of telomerase activation mechanism after administration of essential trace elements in cancer cells "carried out under the U.S.-Japan cooperative cancer program. The experiments were performed in University of Pittsburgh in collaboration with Prof. J. S. Lazo, Department of Pharmacology. The experimental design was slightly modified after the discussion with Prof. Lazo. Human prostate cancer DU145 cells and human renal cancer ACHN ceils were used in all experiments.
1. Subcellular localization of metallothionein in cancer cells:
I have already reported that Zn induced enhancement of telomerase activity in human prostate and renal cancer cells. To explore the possibility that metallothionein (MT) is involved in Zn-induced telomerase activation by mediating the translocation of Zn into the nucleus changes in subcellular localization of MT by the addition of Zn was investigated. However, Zn did not induce translocation of MT after the addition of Zn in either ceil lines.
2. Modulation of c-myc expression by Zn in cancer cells:
Telomerase reverse transcriptase (TERT) is a catalytic subunit in telomerase, and is known to be activated by c-myc. Modulation of c-myc expression by the addition of Zn into the medium of cancer cells was examined using Western blotting. Expression of c-myc was enhanced dose-dependently 3 h after the addition of Zn in both cell lines.
3 Modulation of TERT expression by Zn in cancer cells:
Modulation of TERT expression by Zn addition in cancer cells was examined using RT-PCR. TERT was detected by RT-PCR in both cells. However, the quantification of TERT by PCR has not yet been achieved because suitable PCR conditions could not be established.
Future studies
1. Establishment of RT-PCR conditions for quantification of TERT.
2. Examination of effects of other metals on c-myc and TERT expression.
3. Examination of Zn-induced telomerase activation in bladder tumor cells derived from MT+/+ and MT-/- mice.




(10)NORIAKI OHUCHI
DIVISION OF SURGICAL ONCOLOGY, TOHOKU UNIVERSITY GRADUATE SCHOOL OF MEDICINE

SPONSOR AND HOST INSTITUTION:
DIVISION OF CANCER CONTROL AND PREVENTION, NCI, NIH
DATES OF VISIT:
MARCH 5 TO MARCH 16

SUMMARY OF ACTIVITIES:
We have outlined to enhance understanding of intermediate measures of screening mammography performance and variations across countries. The initial focus of the research assessed recall rates and compared these with cancer incidence. We discussed how to move this process forward. The following issues were proceeded. 1) To design a questionnaire, or data form, in order to request specific data from each country. 2) We revised and finalized the questionnaire. At this point, we met in person to work out any details. 3) It is clear that what we have in common across our different programs are: a) the ability to categorize screening visits into those recalled/ hose not recalled for further investigation of any type, b) this can be done for the age group 40-69. 4) We discussed the following: a) all women appearing for screening would be included in the denominator, b) the major outcome of interest would be cancer incidence in the recall group, c) we would also collect data to enable us to calculate sensitivity, and PPV and other data for subset analyze, d) it would be important to have information to classify the screening mammograms by round of screening. We had meetings with individual investigators on the topics listed in the attached sheet (Additional Information related to the exchanged program). In addition,
We had discussion on Mammography Quality Standard Act (MQSA) Program, at the Food and Drug Administration (FDA) with Dr. Charles Finder, to create quality standard for mammography screening in Japan.

I submit additional information related to the exchanged program.
Additional Information related to the exchanged program.
Agenda and outcome for visit to NIH/NCI by Dr. Noriaki Ohuchi, Professor, Division of Surgical Oncology, Tohoku University School of Medicine, Country Representative (Japan), International Breast Cancer Screening Network (IBSN); March 8-16, 2001

Date Activity

(11)Hironobu Yanagie, M.D., Ph.D.
Department of Surgery Teikyo University School of Medicine Ichihara Hospital
3426-3 Anesaki, Ichihara, Chiba 299-0111. JAPAN

SPONSOR AND HOST INSTITUTION:
Professor Leaf Huang. Ph.D., University of Pittsburgh
Department of Molecular Genetics and Biochemistry and Department of Pharmaceutical Sciences. University of Pittsburgh
DATES OF VISIT:
March 11th, 2001 ~ March 19th, 2001

SUMMARY OF ACTIVITIES:
The complex of cationic liposome and plasmid DNA, so called, "Lipoplex" are applied to gene therapy as non-viral vector.
Recently cationic liposome is used to clinical trials of gene therapy, so it is the most important issue that how the transfection efficiency using lipoplex is increased to the level of that of viral vector in non-viral vector gene therapy. In order to increase the transfection efficiency, the LPD is composed with cationic liposome and plasmid DNA which is condensed with protanline salfate in the Leaf Huang's Lab, A novel fusogenic peptide, JTS-1, in Influenza virus are also combined to plasmid DNA to increase the transfection efficiency in his Lab.
In this visiting period, I prepared the LPD conjugated with JTS-1 and evaluate the expression of NG-1 gene, a novel tumor suppressor, to hepatic cancer cell line, HepG-2. NG-1 is a newly identified tumor suppressor that may induce apoptosis. We have reported that NG gene have cloned in the cytotoxicity inducing region of hepatitis C virus, and have homology from 1 to 5. The effect of apoptosis is strongest by NG-1 transfection. Every cancer cell line that was transfected with lipofection of NG genes showed the apoptosis in 72 hours in vitro.
The concentrations of cationic lipid and plasmid DNA in LPD were as below;
DCchol: plasmid DNA: protamine: JTS-1 = 3nmol: 1µg: 2 µg: 0.1 µg
The mean diameter of LPD is 50 nm and it is so compact because the diameter of lipoplex is between 300nm and 1000nm.
I evaluate the expression of NG-1/GFP protein and apoptosis in HepG-2 cells by flow cytometer(FACS) and fluorescent microscopy. The induction of apoptosis by NG-1 was strongest according to the expression of NG-1/GFP protein by the double staining of FACS with Annexin V and PI.
The expression of NG/GFP protein was strongest in NG-1 gene transfectant cells by fluorescent microscopy. Almost cells were dead by induction of apoptosis that was exprained relative low numbers of PI staining of nucleus after 48 hour transfection of NG-1 gene.
These results suggest that the "LPD/JST-1" which prepared with cationic lipid and DNA plasmid which was condensed with protamine and fusogenic peptide has become an strong candidate for non viral vector for gene therapy. The tumor suppressor gene delivery using "LPD " system has applied to nonviral vector targeting for cancer gene therapy.




(12)Masahiko Kanamori
Department of Orthopaedics Toyama Medical and Pharmaceutical University

SPONSOR AND HOST INSTITUTION:
Department of Pathology and Microbiology
University of Nebraska Medical Center
DATES OF VISIT:
July 6 2000 - August 29 2000

SUMMARY OF ACTIVITIES:
A global overview of genetic changes can be obtained from the fresh or frozen samples of soft put sarcoma (rhabdomyosarcoma: 12 cases, malignant fibrous histiocytoma: 8 cases, dedifferentiated chondrosarcoma: 4 cases) using comparative genomic hybridization. Genomic DNA was isolated by routine methods with phenol/chloroform after proteinase K digestion. Tumor DNA was direct labeled with fluorescein-dUTP. Labeled tumor DNA and labeled reference DNA were coprecipitated with Cot-1 DNA, and hybridized. Images were aquired with CCD camera and analyzed. As a result, the gain of gene was observed in chromosome 2 (50%), chromosome 7 (42%), chromosome 8 (67%), chromosome 11 (42%), chromosome 12 (58%), chromosome 13 (33%) and chromosome 20 (33%). On the other hand, the loss of gene was detected in chromosome 1 (42%), chromosome 6 (33%), chromosome 9 (33%), chromosome 14 (25%) and chromosome 17 (25%). In chromosome 8 aberration, which was most common region, there was involved 8q24 located c-myc gene. In the chromosome 1 aberretion, there was a PAX7 gene aberration mapped on 1p35-36. In summary, this study reveales recurrent genetic gains and losses in soft part sarcoma by comparative genomic hybrdization analysis. The gain of chromosome 2, 7, 8, 11, 12, 13, 20 and the loss of chromosome 1, 6, 9, 14, 17 may harbor gene important for pathogenesis or progression of soft part sarcoma.
Publication
M. Kanamori, et al. Comparative genomic hybridization analysis of soft part sarcoma. Cancer Genet Cytogenet 2000 (submitted).



(13)Hideo Tsurushima
The University of Ryukyu, School of Medicine

SPONSOR AND HOST INSTITUTION:
Johns Hopkins University, School of Medicine
DATES OF VISIT:
49 days from Feb. 11, 2001 to March 31, 2001

SUMMARY OF ACTIVITIES:
Objectives: Vaccination for Brain Tumors using the slow-releasing materials of Cytokines.
Achievement: Making the basic plasmids, which are going to be used in this project.
Future work: Checking the functions of the slow-releasing materials.