EXCHANGE SCIENTIST REPORTS

Hirota Fujiki, M.D.,
National Cancer Center Research Institute, Section of Studies on Metastasis, Tokyo

Dates of Visit: February 8 to March 6, 1981
Host:
Dr. Richard E. Moore, Professor, Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii

Summary of Activities:
Recently we proved that the compound teleocidin isolated from Strep tomyces is a new potent tumor promoter on mouse skin. We also found that both teleocidin and Lyngbyatoxin A, from the marine blue-green algae Lyngbya majuscula, have biological activities on mouse skin and cultured cells that are similar to those of the phorbol ester tumor promoters. Since the chemical structures of teleocidin and Lyngbyatoxin A are very similar, we suspect that Lyngbyatoxin A will also be a potent tumor promoter. For an in vivo carcinogenicity test, Lyngbyatoxin A must be isolated in rather large amounts from the marine blue-green algae, which is collected on Kahala Beach, Oahu, in Hawaii. During a 2'7-day stay at the Department of Chemistry, University of Hawaii, we successfully isolated 50 mg of Lyngbyatoxin A using six steps of purification. This amount of Lyngbyatoxin A will be enough to perform an in vivo carcinogenicity test on mouse skin, making this visit extremely productive and the cooperative studies at the University of Hawaii very fruitful.
Related publications include the following:

  1. A possible naturally occurring tumor promoter, teleocidin B from Streptomyces (Biochem., Biophys. Res. Comm. 90, 976, 1979).
  2. Aggregation of human lymphoblastoid cells by tumor-promoting phorbol esters and dihydroteleocidin B (Biochem. Biophys. Res. Comm. 95, 842, 1980)
  3. Indole alkaloids: dihydroteleocidin B, teleocidin, and Lyngbyatoxin A, as a new class of tumor promoters (Proc. Natl. Acad. Sci., U.S., in press)
  4. Teleocidin B and the phorbol ester tumor promoters produce similar effects on membranes and membrane receptors (Nature, in press)
  5. Teleocidin: New naturally occurring tumor promoter ("Cocarcinogenesis and Biological Effects of Tumor Promoter, " in press)
  6. Teleocidin, Lyngbyatoxin A, and their hydrogenated derivatives, possible tumor promoters, induce terminal differentiation in HL-60 cells (Cancer Letters, in press)


Dr. Yoshio Okada,
Research Institute for Microbial Diseases, Osaka University, Osaka

Dates of Visit: October 27 to November 22, 1980
Hosts:
Dr. Hidesaburo Hanafusa, Rockefeller University, New York, New York
Dr. Thomas L. Benjamin, Department of Pathology, Harvard University School of Medicine, Boston, Massachusetts

Summary of Activities:
During this exchange with the United States, I visited the following:

This exchange visit achieved its objectives since I received numerous valuable criticisms and suggestions about my work through discussion with the United States scientists. I understand that these scientists also found my work quite informative. My visit provided an opportunity to share ideas related to the problem of introducing DNA, or other macromolecules, into cells via liposome mediated membrane fusion. Collaborative studies were also begun using this important method. In my seminars I demonstrated that macromolecules can be introduced into living cells utilizing cell fusion mediated by HVJ (Sendai virus). Quantitative and selective introduction of macromolecules are possible. This new method may be useful for fundamental studies on cancer biology and also as a novel approach to cancer treatment.


Dr. Takemi Yanagimoto,
The Institute of Statistical Mathematics, Ministry of Education, Science, and Culture, Tokyo

Dates of Visit: September 15 to October 5, 1980
Host:
Dr. William J. Blot, National Cancer Institute, National Institutes of Health, Bethesda, Maryland

Summary of Activities:
The main aim of this visit was to talk with several cancer researchers about statistical methods for the bioassay for carcinogenicity of chemicals in the environment. The discussions with biostatisticians, who are responsible for data analysis in the United States National Toxicology Program, were extremely informative. Numerous excellent approaches were reviewed and this knowledge will be invaluable in my collaborative studies with Dr. T. Kawachi, National Cancer Center Institute, Tokyo. I also had very fruitful discussions with Professor B. Ames and Dr. J. McCann, University of California, on the quantitative relationship between carcinogenic and mutagenic potency. Though their approach is very interesting, certain possible defects in their statistical models were revealed from our discussions. In addition, I had an opportunity to review the use of computer programs in biomedical research, a field that is rapidly expanding in the United States.
When I joined cancer research as a biostatistician, I found that there were very few biostatistical studies in this field in Japan. The situation will be rapidly improved by promoting the type of visit I had to the United States, since Japan has many excellent statisticians. The recent visit was highly informative and has led to potential areas of collaboration. This type of collaboration is a must for biostatisticians engaged in cancer research. During this visit my major effort was focused on data analysis of the quantitative relationship between carcinogenic and mutagenic potency. I intend to pursue this important subject in Japan and look forward to continued contacts with United States biostatisticians.


Hiroyuki Shimizu M.D.,
Aichi Cancer Center Research Institute, Division of Epidemiology, Nagoya

Dates of Visit: October 1, 1979 to September 30, 1980 (The period from April I to September 30, 1980 was supported by a grant from the University of Southern California (USC) School of Medicine.
Host:
Brian E. Henderson, M.D., Professor and Chairman, Department of Community and Family Medicine, University of Southern California, Los Angeles, California

Summary of Activities:
Descriptive Epidemiology of Lung Cancer in Los Angeles County. There have been few epidemiological studies of lung cancer by histologic type. The demographic characteristics of lung cancer for individual histologic types still remain to be explored. For example, some previous data indicate that adenocarcinoma of the lung is unrelated to smoking, while others suggest that adenocarcinoma is associated with smoking but to a lesser degree than squamous cell carcinoma of the lung.
To analyze the characteristics of lung cancer by histologic type, the descriptive epidemiology of adenocarcinoma of the lung was compared to that of squamous cell carcinoma, using data from the population-based cancer registry in Los Angeles County from 1972 to 1976. Several differences in the demographic characteristics between these two types of lung cancer were observed, including low male/female ratios and lack of social class gradients for adenocarcinoma of the lung. Incidence rates of lung cancer after subtraction of the proportion of the incidence due to smoking were also studied by using data from a previously conducted case-control study in Los Angeles County. These rates for adenocarcinoma of the lung were almost the same between men and women across all ages, and were higher than those for squamous cell carcinoma in the older age groups for both sexes. These findings, combined with descriptive data, indicate that smoking may be etiologically less important for adenocarcinoma than for squamous cell carcinoma, that other major etiologic factors exist for adenocarcinoma, and that these factors are age-correlated and affect men and women equally.
Further investigation by specific histologic type should be undertaken to identify major causes other than cigarette smoking. A new case-control study of adenocarcinoma of the lung is being conducted at the USC School of Medicine, based on the results mentioned above. I am planning to study the relationship between smoking and lung cancer by histologic type and by site of the primary tumor using data from the Aichi Cancer Center Hospital. (A manuscript describing the above results is in preparation.)
A Case-Control Stud of Prostate Cancer. To elucidate etiologic factors related to prostate cancer, I conducted a case-control study of this disease among Japanese in Los Angeles County. The latter show a higher incidence rate than Japanese in Japan, but the rate is still low compared to that for blacks and whites in the United States. The questionnaire, which was newly designed for this study, was sent to each living Japanese case with prostate cancer registered in the population-based cancer registry in Los Angeles County from 1972. Chinese and Philippino patients with prostate cancer were added to increase the number of the cases. The total number of responses from the patients was about fifty when I left the United States (September 1980). The controls were matched with cases by age, sex, ethnic group, and residence area, using telephone directories and telephone interviews. The same questionnaire was mailed to all of the Japanese controls. The collection of the questionnaire from the controls in two other ethnic groups is being continued.
Other Aspects. The U.S.-Japan Cooperative Cancer Research Program gave me a chance to meet with several excellent epidemiologists and biostatisticians at the University of Southern California (USC). Their friendliness helped me to carry out the studies easier than I expected. Furthermore, I could learn several epidemiological methods and ways of thinking through direct discussions, so the Program indirectly trained me to think more logically.
One year was too short for me to complete the epidemiologic studies, but I believe that the findings from the study on lung cancer indicate several interesting questions for the next stage in the study of this disease. I would like to continue the study on prostate cancer in collaboration with researchers at the USC School of Medicine.
Both studies made me realize the importance of U.S.-Japan cooperative studies. Epidemiology may be one of the most important fields for such cooperation.
I learned about problems specific to cancer epidemiology in the United States, and I informed young American researchers about several problems specific to epidemiologic studies in Japan. For example, in Japan we sometimes do not obtain cooperation from family members of patients with malignancy, especially in rural areas, because they are very sensitive to the question of familial disease" or genetic factors in cancer.


Dr. Nobuo Yamaguchi,
The Institute of Medical Science, The University of Tokyo, Tokyo.

Dates of Visit: August 7 to October 18, 1980
Hosts:
Dr. David M. Livingston, Division of Medical Oncology, Sidney Farber Cancer Institute, Boston, Massachusetts
Dr. Thomas L. Benjamin, Department of Pathology, Harvard University School of Medicine, Boston, Massachusetts

Summary of Activity:
Recent studies by Dr. Livingston and his coworkers suggested that one of the SV40 T antigen-binding sites on SV40 DNA is located on the "late" side, near the origin of DNA replication. This site includes a portion of the two 55 base pair tandem repeat sequences present in SV40 DNA. To elucidate the function of SV40 T antigen, and also the role of the 55 base pair tandem repeat sequence in SV40 transcription, we planned to make mutants of SV40 in which a part of the tandem repeat sequence is deleted. Our strategy was as follows: Any restriction enzyme having one cleavage site in the 55 base pair repeated sequence can remove 55 pairs from the repeated sequence by cleaving the site in each repeated sequence. Because restriction enzymes have many recognition sites on SV40 DNA outside the repeated sequence, we planned to eliminate such recognition sites by making the regions single-stranded and thus resistant to the action of these restriction enzymes. The plan was to make partial double-stranded SV40 DNA with a restriction enzyme-cleaved SV40 DNA fragment containing the tandem repeat sequence.
During my visit we achieved the following. (1) Strand separation of SV40 DNA. We first tried to separate SV40 DNA strands by electrophoresis in many different conditions, but were unsuccessful. Then SV40 DNA was cloned in the DNA of a single-stranded DNA phage, M13 mp6. (2) Isolation of a restriction enzyme-cleaved fragment. A fragment containing the 55 base pair repeat sequence was isolated by electrophoresis in agarose gel after cleaving SV40 DNA with restriction enzymes (3) Restriction enzyme. A restriction enzyme, BstNI. was tested and proved to be double-stranded DNA specific. These achievements indicated the possibility of site-directed mutagenesis using a restriction enzyme, BstNI, in the region of the 55 base pair repeat sequence. At this stage of the experiments my period of stay was over. The rest of the experiments are now being continued in Japan in collaboration with Dr. Livingston's and Dr. Benjamin's laboratories.
Drs. Livingston and Benjamin kindly provided me with enough facilities and materials to achieve my research objectives. Their discussions and suggestions have assisted me greatly in achieving these objectives. Technically, I have learned recombinant DNA technique, which will be very valuable in my research. In addition, I was psychologically stimulated through intense discussions with young researchers in highly active laboratories. This stimulation will also contribute greatly to the progress of my research. Drs. Livingston and Benjamin kindly provided me with phage DNA in which SV40 DNA had been cloned. In collaboration with them, I have been continuing to make SV40 mutants using this recombinant DNA. Unfortunately, I could not fully complete my research objective, to make specific deletion mutants, during the period of this visit. Since the research has been continued in Japan, the specific mutants will soon be isolated and used in both the United States and Japan. Hopefully this approach will enhance our understanding of the mechanism of carcinogenesis. Besides this short-term contribution, I gained a better understanding of the advantages and disadvantages in the way that research is conducted in both countries.


Hiroshi Yasue,
Laboratory of Viral Oncology, Aichi Cancer Center, Nagoya

Dates of Visit: August 28 to November 27, 1980
Host:
Dr. Eiichi Ohtsubo, Associate Professor, Department of Microbiology, School of Basic Health Sciences, State University of New York at Stony Brook, New York

Summary of Activities.
Using the nucleic acid blotting and hybridization technique of Southern, I analyzed several rat and hamster tumor cell lines transformed by the adenovirus designated CELO virus. These studies led to the following hypothesis. A few viral DNA molecules are integrated into the DNA of the original cell infected with this virus and these copies form amplification units with flanking cellular sequences. The units are amplified during the course of events by which the original cell progresses to a transformed colony or a visible tumor.
In order to examine the above "amplification hypothesis," I decided to work on the following projects in Dr. Ohtsubo's laboratory: (1) Cloning of the junction fragments between viral and cellular DNA in the vectors PBR322 or Charon 4A (or!!!gtwes!!!B); (2) Base-sequencing of cloned junction fragments and of viral DNA fragments relevant to the junctions; and (3) Location of junction points between viral and cellular DNA by comparing the sequences of the junction fragments with those of viral DNA fragments.
To begin with, I tried to clone a junction fragment of the EcoRI-F side of viral DNA in RC13, a rat tumor cell line. Since the junction fragment had been found to be 15 kilo-base (KB) long, the DNA fraction with a molecular weight of about 15KB (supposed to contain the junction) was eluted from agarose gel, in which EcoRI-generated fragments of RC13 DNA had been electro-phoresed. Using an in vitro packaging system, the fragment DNAs in this fraction were cloned in Charon 4A. A junction fragment on the EcoRI-A side (the opposite side of ECORI-F) in RC13 were also cloned in!!!gtwes!!!B.
Charon 4A containing the 15KB junction was purified through two cycles of plaque isolation. The DNA extracted from the Charon 4A will be subjected to base-sequencing analysis.
All the EcoRI-fragments of virion DNA, except for the terminal fragments EcoRI-F and -H, were cloned in the ECORI site of PBR322, since it would otherwise be difficult to obtain the amounts of virion DNA fragments necessary for base-sequencing. I attempted to clone the terminal fragment EcoRI-F in the Pst I site of PBR322 by the GC-tailing method, but this method did not work well. I did not have enough time to determine the base sequence of the junction DNA and the viral DNA fragment relevant to the junction. However, I did sequence the EcoRI-H fragment, since the EcoRI-H fragment has only 83 base pairs and is a terminal fragment of virion DNA. Thus this information is relevant to junctions other than the junction fragments described above.
As a future project, we will identify the junction point between viral and cellular DNA by determining the base sequence of the junction and of the viral DNA fragment relevant to the junction. Thus, we will be able to determine whether or not there are specific sequences which are involved in recombination and amplification of viral DNA in tumor cells. In addition, we will examine the "amplification hypothesis" by measuring the copy number of cellular DNA sequences flanking the viral DNA, both in normal cells and the tumor cells RC13. This will be done by Cot analysis using the flanking cellular DNA sequence of the cloned junction fragment as a probe.
This visit allowed me to achieve my research objectives. I accomplished about one-half of the projects which I proposed to do during my stay in the United States. Project I was completed, and project 2 is under way. Project 3 has not been started. Through this visit, I obtained Charon 4A DNA containing a junction between viral and cellular DNA, using the gene cloning method of in vitro packaging. This will facilitate project 3. In addition, as described above, we can now examine the "amplification hypothesis" more directly.
Through contacts with Dr. Ohtsubo and other scientists in the United States, I also learned a good deal of information that is not yet published. For example, I became familiar with a modified method of nick-translation which is much more rapid than the conventional method. Another aspect which enhanced the advancement of my research was the facilities available for gene cloning in the United States. I plan to continue my collaboration with Dr. Ohtsubo. In addition, my exchange visit to the United States gave an opportunity for host scientists to become familiar with my investigations focusing on the amplification of viral DNA.


Dr. Thomas L. Benjamin,
Department of Pathology, Harvard University School of Medicine, Boston, Massachusetts

Dates of Visit: June 3 to June 17, 1981
Hosts:
Dr. Hiroto Shimojo, Institute of Medical Science, University of Tokyo, Tokyo
Dr. Geuki Kimura, Kyushu University, Kyushu
Dr. Takagi, Kyushu University, Kyushu
Dr. Yohei Ito, Kyoto University, Kyoto
Dr. Yoshio Okada, Osaka University, Osaka
Dr. Igarashi, Takeda Laboratories, Osaka

Summary of Activities:
During the two weeks I spent in Japan, I visited and consulted with researchers at four universities and the Takeda Laboratories, and attended a June 12 and 13 conference in Osaka on "Recent Topics in Cancer Research." In chronological order, my visits included:

  1. Tokyo: I learned of the most recent work on adenovirus type 12 and its transforming genes, which is surely among the most interesting and important work in this field, not just in Japan but worldwide. Dr. Shimojo kindly arranged a group of "mini-seminars" for my benefit, following my own seminar. I heard of ongoing work on human papovaviruses by Drs. Yogo and Kato from Dr. Uchida's group, and also Dr. Miyamura (formerly with Dr. Takemoto at NCI). Dr. Soeda, from the Genetics Institute at Mishima, presented his work on comparative sequencing of papovavirus DNAs. I also had the opportunity to discuss with Drs. Kinichi Oda and Nobuo Yamaguchi their interesting work on SV40 chromatin.
  2. Kyushu University: I heard about SV40 cell interactions and cosmid vectors and gene transfer experiments.
  3. Kyoto: I learned of the recent work on Shope papilloma virus, and on the epidemiology and molecular biology of EBV. I also had a chance to renew acquaintances with Drs. Yura and Kawade, and to hear the latest on their work on genetics and biochemistry of E. coli RNA polymerase and interferon, respectively.
  4. Osaka University: In addition to attending the conference in Osaka, I spent a half day at the University visiting Dr. Yoshio Okada. I met with a group of Dr. Okada's young colleagues and was most impressed by the range of important work on cell fusion and on delivery systems for macromolecules into cells. I also met Dr. Hakura whose work on polyoma and SV40 1 have known for many years.
  5. Osaka: Dr. Igarashi invited me to the Takeda Laboratories. At the Takeda Laboratories I gave a seminar and discussed various aspects of their work, including that on bovine adenovirus (Dr. Igarashi and others in Dr. Sugino's group) and on host genetic factors in disease.

Overall, this was a most beneficial trip for me scientifically. It was also a most fascinating and pleasurable time for me, due to the very kind hospitality of my many Japanese hosts.
I discussed future activities of the Program with Dr. Shimojo. We both felt that the sponsoring of individual scientist exchanges would be mutually beneficial, particularly for senior United States scientists to go to Japan for seminar tours and informal discussions at selected universities and institutes (2-3 week visits), and for Japanese investigators to come to the United States for longer visits (2-3 months) for carrying out specific collaborative research projects. We will attempt to arrange such visits in the succeeding years.
Dr. Livingston and I look forward to hosting the visit of Dr. N. Yamaguchi, who will work for a ten-week period in our laboratories beginning in August. Dr. Hanafusa and I expect to host a visit from Dr. Okada to New York and Boston this fall.



Dezider Grunberger Ph.D.,
Division of Environmental Sciences and Institute of Cancer Research, Columbia University, New York, New York

Dates of Visit: March 19 to March 29, 1981
Host:
Dr. Susumu Nishimura, National Cancer Center Research Institute, Tokyo

Summary of Activities:
I visited Japan to (1) participate in the seminar entitled "Interspecies Correlations in Chemical Carcinogenesis," (2) finalize our collaboration with Dr. S. Nishimura on tumor-specific phenylalanine tRNA, and (3) deliver invited lectures at several institutes in Japan.
At the seminar, I presented a lecture on "Carcinogen-DNA Adducts in Human and Experimental Animals." In my presentation, I reviewed studies on the role of specific carcinogen-DNA adducts in the process of carcinogenesis in different species and tissues, emphasizing that chemical characterization of the specific types of adducts is extremely important. In the case of benzo(a)pyrene it was possible in collaboration with Drs. Poirier, Yuspa, Santella, and Weinstein to prepare antibodies against DNA-benzo(a)pyrene-7,8-diol-9,10 epoxide adducts. This antibody is highly specific and sensitive and should be useful in epidemiologic studies of individuals heavily exposed to benzo(a)pyrene.
In the area of tumor-specific phenylalanine tRNA, our collaboration with Dr. S. Nishimura began during my stay in Tokyo in 1978. The objective of this study was to find out whether the differences in structure between phenylalanine tRNAs of certain tumors and normal tissues is due to expression of a distinct gene or due to under-modification of a specific base in this tRNA designated as the Y base. For this reason, phenylalanine tRNA was isolated and purified from neuroblastoma cells. The primary sequence of this tRNA turned out to be the same as that from the normal liver. The only difference is a deficit in the post-transcriptional modification of the Y base in the tumor tRNA. The tumor-specific phenylalanine tRNA contains in place of the Y base a l-methyl guanosine residue. Therefore, the tumor tRNA differs from the normal tRNA because of a failure to synthesize the complete Y base. This is one of the few cases in which a specific difference between tRNAs Of normal and tumor cells has been elucidated at the molecular level. These findings are now being prepared for publication.
The third aspect of my visit to Japan related to the presentations of lectures at various institutions. During my lecture at the National Cancer Center Research Institute, Tokyo, I spoke on "Tissue Specificity and Alternative Conformations of DNA-Carcinogen Adducts." 'In a lecture at the Cancer Institute of the Japanese Foundation for Cancer Research, Tokyo, I spoke on a similar subject. I also lectured on "Molecular Mechanisms in Chemical Carcinogenesis" at the Suntory Institute for Bioorganic Research located in Wakayamadai, between Kyoto and Osaka. The director of the Institute is Dr. K. Nakanishi from Columbia University. Stimulating discussions followed each of my lectures, and these helped to strengthen friendships and collaborations with my Japanese colleagues.
In general, the entire trip was extremely productive. The collaboration with Dr. S. Nishimura enabled us to complete important studies that would otherwise not have been possible.