SUMMARY REPORTS OF EXCHANGE SCIENTISTS

(1) Minako Nagao
Carcinogenesis Division
National Cancer Center Research Institute, Tokyo

Sponsors and Host Institutions:
Prof. Herbert S. Rosenkranz
Department of Environmental Health Sciences
School of Medicine
Case Western Reserve University
Cleveland, Ohio
Prof. Marvin S. Legater
Department of Preventive Medicine and Community Health
Division of Environmental Toxicology
University of Texas Medical Branch
Galveston, Texas
Dates of Visit: July 8 - 22, 1989

Summary of Activities:
I have been studying environmental carcinogens, especially those present in food, to detect causes of human cancers. Using the Ames Salmonella test, we have surveyed various foods and identified several carcinogens. For evaluation of risks of these chemicals, we should know the exposure data for humans. It is also difficult to determine how to extrapolate animal carcinogenesis data to humans. For this purpose, information exchange between scientists who are working in this field were and will continue to be helpful.
Profs. Rosenkranz and Klopman set up CASE, the Computer-Automated Structure Evaluation System. During my visit to CASE Western Reserve University, I learned information obtained by CASE. CASE predicts correctly the carcinogenicity of nongenotoxic carcinogens, thereby suggesting a structural commonality in the action of this group of carcinogens. When the carcinogenic potency of chemicals studied by the National Toxicology Program were classified into nongenotoxic and genotoxic compounds, mutagenic carcinogens tended to be more potent carcinogens than nonmutagenic carcinogens. Concerning carcinogenic potency, it was found that trans-species and multi-site carcinogens are more potent carcinogens than carcinogens restricted to single species, single sex, or single tissue. Based on this information, I evaluated heterocyclic amines which are genotoxic, and trans-species and multi-site carcinogens and found them to be potent carcinogens.
During my stay at Case Western Reserve University, I had an opportunity to attend a seminar given by Dr. M. Poirrie. She studied the relationship between AAF-DNA adduct levels and tumor development in the target organs. Levels of AAF-DNA adducts detected by monoclonal antibody were two-to-fourfold higher in bladder than in liver. However, liver is more sensitive to AAF-carcinogenicity. Based on carcinogenicity data for 33-month experiments, it was calculated that liver cancer in Balb/c mice was developed by single hit and bladder cancer by 3 hits. However, the adduct level required for development of liver tumor is different between chemicals. We are analyzing DNA adducts of experimental animals by feeding carcinogenic doses of various heterocyclic amines, and her study is very helpful to me in analyzing our experimental data. However, for determination of number of hits required for cancer development, I thought we should be careful with pathological findings, because the numbers of required hits are affected by the degree of malignancy.
During my stay at the University of Texas, I learned the usefulness of the in vitro micronucleus test. Peripheral blood B-cells were stimulated by PHA and Pokweed and micronuclei, as well as nuclei, can be very elegantly stained by fluorescence. This method is useful for detection of benzene exposure, and no difference was detected between smokers and non-smokers. Thus, the amount of occupational exposure to benzene can be measured without confounding by cigarette smoking. We have been studying food mutagens which were picked up by the Ames Salmonella test. It is well known that benzene is carcmogemc, but not mutagenic in the Ames test. Application of genotoxicity tests other than the Ames test for detection of genotoxic compounds present in food should be important.
We are carrying out a collaborative project with Prof. D. Hsieh on measurement of human exposure to aflatoxin B1, which has also been designated as a human carcinogen. I visited Prof. Hsieh at the University of California, Davis, and discussed details of the ELISA method. Based on the discussion, we revised several aspects of our experimental methods, and were able to determine the characteristics of antiserum which we and Dr. Hsieh have used for ELISA to detect aflatoxin B₁-DNA adduct (Manuscript in preparation).
During my stay at Case Western Reserve University, I had an opportunity to talk with Prof. S. J. Berger, who is working on poly(ADP-ribose) polymerase. We have been studying the effect of poly(ADP-ribose) polymerase inhibitors on oncogenes, and found that the amplified c-myc gene in human promyelocytic leukemia HL-60 was deleted after treatment with poly(ADP-ribose) polymerase inhibitors. Dr. Berger obtained a mutant which has reduced activity of the poly(ADP-ribose) polymerase activity. The mutant was sensitive to the alkylating agent and grew slowly. Although involvement of poly(ADP-ribose) polymerase in DNA repair and also in DNA replication had been suggested in the studies using inhibitors of this enzyme, Berger's mutant experiments more directly indicated involvement of this enzyme in DNA repair and DNA replication. It was a great opportunity to discuss the physiological roles of poly(ADP-ribose) polymerase with Prof. Berger.


(2) Akio Matsukage
Laboratory of Cell Biology
Aichi Cancer Center Research Institute
Chikusa-ku, Nagoya 464, Japan

Sponsors and Host Institutions:
Samuel H. Wilson Laboratory of Biochemistry National Cancer Institute
National Institutes of Health
Bruce Stillman
Cold Spring Harbor Laboratory
Michael Levine
Department of Biological Sciences
Columbia University
Teresa S.-F. Wang
Department of Pathology
School of Medicine
Stanford University
Dates of Visit: August 26 - September 5, 1989

Summary of Activities:
a) Summary and Presentation:
A formal seminar entitled “Genetic control of DNA synthesizing enzymes in cell proliferation and differentiation” was given at the Laboratory of Biochemistry, National Cancer Institute. This seminar dealt with our findings indicating that homeodomain proteins are involved in the regulation of the PCNA (proliferation cell nuclear antigen) gene expression and that the product of c-fos proto-oncogene is one of the components regulating the silencers of DNA polymerase beta gene. I obtained valuable information as a result of the discussion and comments during this presentation. I also presented a paper dealing with the same subject as above at the meeting on “Eukaryotic DNA replication” in Cold Spring Harbor.
b) Cooperations:
Since the US-Japan Cancer Meeting on DNA Synthesis which was held in 1984 at Stanford, Dr. Wilson and I have cooperated by exchanging biological materials and information on research on DNA polymerase genes and succeeded in cloning the DNA polymerase beta gene. During this visit, we discussed extensively the results which were obtained in our two laboratories. We recognized that our progress in research on the genetic regulation mechanisms of this gene and structure-function relationships of DNA polymerase beta are satisfactory and that the cooperation should be continued.
I obtained Dr. Levine’s agreement to support my research on Drosophila PCNA and DNA polymerase alpha gene regulation by supplying me with his recombinants expressing various homeodomain proteins.
At the Cold Spring Harbor Meeting, Dr. Paul Fisher of New York State University and I reached an agreement of cooperation on PCNA research by exchanging biological materials such as antibodies and cloned genes.
c) Other Activities:
From Dr. Stillman, Dr. Wang, and other scientists who attended the meeting at Cold Spring Harbor I obtained much information on the eukaryotic DNA replication and its regulatory mechanisms: enzymes involved in simian virus 40 DNA replications, regulatory mechanism of DNA polymerase alpha gene, involvement in DNA replication of products of protooncogenes such as p53, c-myc, c-jun, c-fos, etc.


(3) Osamu Katoh
Genetics Division
National Cancer Center Research Institute.

Sponsors and Host Institutions:
Dr. Peter M. Howley
Laboratory of Tumor Virus Biology
National Cancer Institute National Institutes of Health
Dr. Paul A. Marks
Memorial Sloan-Kettering Cancer Center
Dates of Visit: September 23 - October 13, 1989

Summary of Activities:
I have been working on heparin-binding secretory transforming factor 1 (HST-1)and HST-2 genes, a novel family of growth factor genes, related to FGF genes. The HST-1 gene product was shown to have a potent mitogenic activity towards endothelial cells and fibroblasts. However, it is not known whether HST-1 and HST-2 gene products and fibroblast growth factors (FGFs) have any effects on normal epithelial cells of human origin. Therefore, I have been planning to clarify the biological activities of these genes and products on human epithelial cells. Dr. Peter Howley and his colleagues have been studying molecular mechanisms involved in human papillomavirus (HPV)-induced transformation by using a new transfection method, cationic liposome-mediated transfection. This method works more efficiently than other protocols for human keratinocytes. Liposome-mediated transfection is an extremely simple and fast way of delivering nucleic acids into tissue culture cells. Transfection efficiencies with this method tend to be one or more orders of magnitude higher than with other transfection methods for some specific cell lines. I learned this procedure and discussed the expected effects of these growth factors and growth factor genes such as HST-1 and FGFs On human epithelial cells using this liposome-mediated transfection.
I visited Dr. Paul Marks and his colleagues who have been studying cellular and molecular mechanisms implicated in the differentiation of a hematopoietic cell lineage. They have shown that the virus-transformed murine erythroleukemia cell (MELC) can be induced by a chemical agent, hexamethylene bisacetamide (HMBA) to initiate a virtually normal developmental program. We exchanged information on growth factor effects on the differentiation of hematopoietic cells.


(4) Masabumi Shibuya
Institute of Medical Science University of Tokyo

Sponsors and Host Institutions:
Dr. Hidesaburo Hanafusa The Rockefeller University, New York
Dates of Visit: November 26 - December 10, 1989

Summary of Activities:
The major purpose of this U.S. visit were: (1) to discuss the recent progress in oncogene research, and (2) to discuss research on “Human Gene Therapy”
(1) Oncogene research
I had an opportunity to attend, as a formal speaker, an exciting science symposium called “Retroviruses, Oncogenes and Growth Control,” held at Rockefeller University in New York (November 30-December 1, 1989), where I presented the results of our recent work on a unique activation of the EGF receptor gene in human glioblastoma multiform and on a novel protein kinase gene specifically expressed in testis. During and after this symposium, I met with many pioneering researchers in the oncogene field such as Dr. H. Hanafusa (Rockefeller University), Dr. L.-H. Wang (Mount Sinai Medical College), Dr. W. S. Hayward (Sloan-Kettering Memorial Research Center), Dr. G. Calothy (Curie Institute, France), Dr. J. Samarut (Lyon, France), etc.
From these discussions, my understanding is as follows: a number of oncogenes have been characterized in the levels of primary structure, oncogenic domains within the structure, and activation patterns in human malignancies. However, many quite important problems remain to be solved: (1) identification of unknown oncogenes involved in carcinogenesis, since in more than half of human tumors there is no clear evidence for activation of known oncogenes. (2) Understanding the mechanisms (molecular process) of oncogene activation, such as specific chromosomal translocation, gene amplification and structural alteration will be crucial for cancer prevention in the future. (3) The signal transduction pathway starting from activated oncongenes or inactivated anti-oncogenes to abnormal cell growth has not been characterized yet because no one has been able to clearly show the key target substrates for any oncogene product. (4) Very little information has been obtained about the physiological functions of proto-oncogenes.
To solve these problems two approaches appear to be quite effective, and I wish to introduce them into my research projects: (1) Large scale preparation of oncogene products --- Protein-protein interaction or protein-high MW substrate interaction appear to be very important for signal transduction of cell growth within the cell. Thus, direct purification of the "target" substrate(s) using an oncogene product-affinity column chromatography may be a useful method to isolate such an important molecule. For this purpose, the Baculovirus system is quite effective for preparation of considerable amounts of oncogene products. These oncogene products could also be used for crystallization to characterize the 3-dimensional structure of these molecules. (2) Genetic analysis of oncogenes --- To isolate the gene(s) located downstream from an oncogene, cellular mutants affecting transformation phenotype appear to be very important. Several mutants have already been isolated from mammalian culture cell lines; however, these mutated genes have not yet been identified. These problems might be overcome by using other simpler organisms such as Drosophila and yeast.
During this U,S. visit, I studied the Baculovirus system in Dr: Hanafusa’s laboratory in the Rockefeller University, and would like to introduce this system to our projects.
(2) Human Gene Therapy
In the U.S., a clinical trial is in progress at NIH by Drs. F. Anderson, S. Rosenberg and coworkers. I discussed the future of human gene therapy with Dr. Rosenberg. From discussions with Dr. Rosenberg and with other researchers in the field of retroviruses and oncogenes, I understood that many problems must be solved before application of “Gene Therapy” to clinical patients. We need to know much more about the mechanism of regulation of gene expression, the integration process of retrovirus genome into chromosomes, growth and differentiation factors acting on keratinocytes, bone marrow stem cells and many other types of cells. Furthermore, to utilize another procedure, i.e. homologous recombination, an improvement in the efficiency of DNA transfer seems critical. The experimental animal model system is also quite important. Therefore, in Japan we need to determine how gene therapy can be applied to basic and clinical research on each disease.



(5) Ken-ichi Yamamura
Institute for Medical Genetics
Kumamoto University Medical School

Sponsors and Host Institutions:
Dr. Francis V. Chisari
Research Institute of Scripps Clinic
Dates of Visit: January 8-21, 1990

Summary of Activities:
The purpose of this study was to discuss the current status of the application of the transgenic mouse system for cancer research in the U.S. and Japan. For this purpose I chose to visit four places to discuss these issues, with special emphasis on tumors of lymphoid cell lineage, hepatitis B virus related diseases and gene targeting. At the University of Washington School of Medicine, I discussed with Dr. Roger M. Perlmutter the oncogenesis of T cell lineage and the usefulness of the transgenic mouse system for these type of studies. At the University of Utah, I met Dr. Mario R. Capecchi and obtained useful information on gene targeting and its possible use for cancer research. At the University of California, Berkeley, I visited Dr. Hitoshi Sakano’s laboratory and discussed the molecular mechanism of immunoglobulin rearrangement and its implication for oncogenesis of B lymphocytes. At the Research Institute of Scripps Clinic, I met Dr. Francis V. Chisari and discussed molecular mechanisms of the development of HBV-related diseases, such as hepatitis and hepatocellular carcinoma. One fruitful result of this visit is the establishment of cooperative work on HBV transgenic mice between Chisari’s laboratory and us.
Apart from the discussion on the subject mentioned above, I gave seminars at the University of Washington, the University of Utah, and the Research Institute of Scripps Clinic. All of them agreed that the scientific level of transgenic studies in Japan is quite good and is on the same level as the U.S.
Based on these results the initial purpose of my visit to the U.S. is almost accomplished. However, I would like to make a special comment on the difference of scientific situation with a special emphasis on transgenic experiments between the U.S. and Japan. The technique of gene targeting is one of the most powerful means for biomedical research, including cancer research, and approximately 1000 laboratories in the U.S. are working on this project. However, in Japan only a few (probably four or five) laboratories are working on this. This situation is the same as in the production of the transgenic mouse. Not many Japanese laboratories can carry out this transgenic experiment. However, in the U.S. it is easy to start this type of experiment because of the ease of establishing the system needed. How could it be possible in the U.S. but not in Japan? Because the U.S. has many advantages over Japan in terms of scientific systems. For example they can expand the mouse facility, or they can employ technicians to take care of these mice. I discussed this subject with Dr. Richard D. Palmiter, University of Washington, and Dr. Chisari. Dr. Palmiter, who is a famous scientist in this field and is a member of the National Academy of Science, USA. established a system for transgenic experiment. Other scientists can ask him to produce transgenic mice by paying $500 per 100 fertilized eggs. A similar system was established by the Research Institute of Scripps Clinic. In the United States, investigators can pay for the production of transgenic mice from their grants. On the contrary, we in Japan have great difficulty in finding mouse rooms and it is very difficult to employ such technicians. I have been working on transgenic projects and have been maintaining a system for the manipulation of mouse embryos for many years. But the effort is enormous and we have to spend 60 to 70% of labor and time just for handling and maintaining mouse facilities.
From my own experience as well as the experience obtained during this visit to the U.S., I would like to make a suggestion concerning the system for mouse embryonic manipulation. I would like to propose the establishment of a Research Center for Recombinant DNA Experiment on Mouse where we can ask for the manipulation of mouse embryos including microinjection of DNA and gene targeting, Judging from the number of scientists engaged in this type of work, I do not think that every university needs such a center. Instead, one center for each district (for example, one center in Kyushu) will be enough. I would say that the establishment of such a unique system will result in a great advance of science in biomedical research in Japan. But if not, we will be far behind the progress of science in this field within the next decade.