SUMMARY REPORTS OF EXCHANGE SCIENTISTS

(1) Shaw Watanabe
Pathology Division
National Cancer Center Research Institute, Tokyo

Sponsor and Host Institution:
Dr. Robert Miller National Cancer Institute National Institutes of Health Bethesda, Maryland
Dates of Visits: May 26, 1983 July 9, 1983

Summary of Activities
I spent the majority (whole June) of my stay in the United States in the Clinical Epidemiology Branch in the National Cancer Institute, National Institutes of Health, and the following activities were performed.
1. Registration system for cancer cases: The strategy of the Surveillance, Epidemiology and End Results Program was studied. Coding system, rate of registration, accuracy and cost were discussed. The efficiency was compared with that of Japan. A seminar on “Double primary malignant neoplasms from autopsy data” was presented in the Epidemiology Branch, NCI.
2. Method and idea of clinical epidemiology: In the United States, the review of clinical charts had been restricted by the Law of Privacy, which was enacted in 1974. Alternative new methods (clinical epidemiology) were elaborated on by Dr. Miller and his associates. The system of study, such as the size of laboratory, researchers, technicians, way of collaboration with outer institutes and the necessary budget, was studied. I joined the weekly case conferences, including clinical examination and clinical interview, and knew the level of interviewer.
3. Genetic aspects of patients with multiple primary cancers were not studied enough, because the Clinical Epidemiology Branch, NCI sends the materials to other institutes under the contract. Visit to Drs. Hecht & Hech in the Genetic Center, Tempi, Arizona, gave some impression on oncogene problems. The way of cell storage, cell registration and their availability were observed.
4. Methods and the way of registration of surgical materials were observed in the Laboratory of Pathology, NCI, University Hospital of Maryland, Cancer Center of Johns Hopkins University, Stanford University Hospital, General Hospital of Los Angeles, and University Hospital of Washington. Methods of data inputs were discussed and compared with that of the National Cancer Center of Japan.
5. The possibility of contribution to cancer research in the field of pathology was discussed with Drs. S. C. Sommer and H. L. Loachim in the Lenox Hill Hospital, New York; Drs. P. Rosen and A. Lieberman in the Memorial Sloan-Kettering Cancer Center; Drs. C. C. Harris, R. Ferrans, E. S. Jaffe and T. Trich. in NCI; Dr. C. W. Berard in St. Jude Children's Hospital; Drs. R. Lukes and C. R. Taylor in the University of Southern California; Drs. R. Dorfman, R. A. Warnke and S. Shiurba in Stanford University; and Dr. Kadin in the Washington University.


(2) Masanao Miwa
Virology Division
National Cancer Center Research Institute, Tokyo

Sponsor and Host Institution:
Dr. Frank H. Ruddle
Department of Biology
Yale University, New Haven, Connecticut
Dates of Visit: June 15, 1983 - August 14, 1983

Summary of Activities
The objective of my visit was to study the biological function of poly(ADP-ribose) by microinjection of the monoclonal antibody for poly(ADP-ribose) into cells. I also hoped to initiate some future collaborative work which would be of mutual interest and importance in cancer research by discussions with many of the distinguished scientists at Yale University.
(i) Poly(ADP-ribose) is a biopolymer discovered some 17 years ago in our institute and has thus far been found only in eukaryotic cells. We observed high poly(ADP-ribose) polymerase activity in SV40 transformed mouse and monkey fibroblasts, and also change of the poly(ADP-ribose) polymerase activity according to the growth stage of cultured cells. So, if poly(ADP-ribose) has any effect on cell growth, it might be reasonable to expect some change in DNA synthesis after microinjection of the monoclonal antibody for poly(ADP-ribose).
Swiss 3T3 cells were cultured on coverslips for 5 days in a medium containing 1% serum to inhibit DNA synthesis. Then IgG3 monoclonal antibody for poly(ADP-ribose) at a 10 mg/ml concentration of phosphate buffered saline (PBS) was microinjected into the nuclei of the bells. These cells were then cultured for 18 hours in the presence of a medium containing 10% fetal calf serum and 20 µCi/ml of [3H] dTHd. The cells on the coverslips were fixed and washed with 5% trichloroacetic acid. The number of cells stimulated to perform DNA synthesis after medium renewal was determined by autoradiography.
OBS and the monoclonal antibody for poly(ADP-ribose) were injected into 413 and 484 cells, respectively. 391 and 469 cells remained attached to the coverslips after fixation for PBS- and the monoclonal antibody-injected cells, respectively. 602 uninjected cells served as the control. The percentages of the cells showing incorporation of [3H]dTHd were 62%, 48% and 39%, for uninjected, PBS-injected and monoclonal antibody-injected cells, respectively. These results suggest that there is some inhibitory effect caused by the microinjection of PBS and even more by the microinjection of the monoclonal antibody. But the inhibitory effect by the monoclonal antibody was not very intense. Further experimentation would be needed to check the stability of the monoclonal antibody during the incubation period. During my stay, I was also able to learn the technique of microinjection into mouse embryo cells. These techniques will be invaluable in the near future for studying the function of various c-onc genes and their products. Future collaborative research is planned on chromosomal localization of the gene for poly(ADP-ribose) polymerase which will clarify the function of poly(ADP-ribose).
(ii) A new collaborative project was started to elucidate the chromosomal integration sites of human retrovirus from adult T-cell leukemia (ATL), which is a unique leukemia found in Japan and in very limited parts of the world. We have established, with human T-cell growth factor (TCGF), 10 cell lines from patients with ATL. At this time, three out of the ten cell lines do not require TCGF for growth. One of the above three ceil lines did not express a viral related antigen throughout the culture period of 13 months, but has already integrated ATLV (or HTLV). The project involves using the established culture cell lines and the in situ hybridization technique which has been successfully performed with various genes in the Department of Biology.
(iii) During my stay in the Department of Biology, I gave two seminars: one on poly(ADP-rebose) and the other on the human retrovirus involved in ATL. I think these seminars motivated the scientists of the Department of Biology and prompted the initiation of new collaborative projects, namely, projects involving the chromosomal localization of the gene for poly(ADP-ribose) polymerase and the chromosomal integration site of human retrovirus.
(iv) During my stay, I briefly experienced working on the M13 phage system, which is going to be successfully used in DNA sequencing and also in nucleic acid hybridization. I was able to realize the importance of the M13 phage system in cancer research.
(v) I suggested the usefulness of the established cell lines of EB virus transformed xeroderma pigmentosum (XP) cells to be used in biochemical work to analyze the molecular defect of XP due to the ease of culturing many cells as compared with fibroblasts.
(vi) Although I remained at Yale University throughout my stay, I had many chances to exchange information with various distinguished scientists in the states by telephone. I was impressed by their valuable information and their responsiveness in sending me cell lines. More frequent long distance telephone exchange will promote better understanding and cooperation between U.S. scientists and Japanese scientists.
Overall Assessment of the Program:
1. The program assisted me very much in learning and undertaking the microinjection experiments in a very short period of time.
2. Through discussions with scientists, new collaborative projects have started. These projects are (1) the determination of the chromosomal localization of the gene poly(ADP-ribose) polymerase and (2) the determination of the chromosomal integration site for human retrovirus involved in adult T-cell leukemia.
3. I believe that the research, seminars and discussions which I took part in stimulated scientists in the Department of Biology who are working on cancer research, which was clearly shown in the new collaborative research which emerged during my stay.


(3) Hirota Fujiki
High-risk Study Division
National Cancer Center Research Institute, Tokyo

Sponsor and Host Institution:
Dr. Richard E. Moore
Department of Chemistry
University of Hawaii at Manoa
Honolulu, Hawaii
Dates of Visit: July 4, 1983 - July 19. 1983

Summary of Activities
During the stay at Dr. Moore’s laboratory I worked to achieve several new research projects in collaboration with Dr. Moore’s group.
1. Isolation of lyngbyatoxin A from Lyngbya mafuscula
Marine blue-green alga was collected on Kahala Beach, Oahu in Hawaii. 60 g of freeze-dried sea weed were processed to isolate lyngbyatoxin A using adsorption chromatography with Florisil and further gel-filtration chromatography with LH-20. The partially purified lyngbyatoxin A was stocked for further purification, since my visit was timely limited. Dr. Moore’s group will continue further purification.
2. Collection of sea hares
To study the existence of 3H-TPA receptor in sea hares, we intended to collect sea hares in Hawaii. Stylocheilus longicauda, which is feeding Lyngbya majuscula containing aplysiatoxin and debromoaplysiatoxin is reported to be present in Hawaii. Drs. Michael Hadfield and Marilyn Dunlap, Pacific Biomedical Research Center, Kewalo Marine Laboratory in Hawaii provided us kindly with 3 strains of sea hares: Stylocheilus longicauda, Dolabrifera dolabrifera and Aplysia juliana. After I return from Hawaii, the receptor binding of 3H-TPA will be studied with these sea hares
3. Experiments of anti-tumor promoting activity with anhydrodebromoaplysiatoxin
We found that debromoaplysiatoxin possessed a strong tumor promoting activity in mouse skin, whereas anhydrodebromoaplysiatoxin did not. However, anhydrodebromoaplysiatoxin binds to the same receptor of TPA and debromoaplysiatoxin. Therefore we suppose that anhydrodebromoaplysiatoxin can prevent the tumor promotion with TPA or debromoaplysiatoxin. Dr. Moore provided us with 335 mg of anhydrodebromoaplysiatoxin. This research is newly started by the occasion of my visit.
We have mutual interests to isolate various promoters and anti-tumor promoters from marine algae. Our collaborative research will be intensively continued in the future.


(4) Kinichiro Oda
The institute of Medical Sciences
The University of Tokyo, Tokyo

Sponsors and Host Institutions:
Dr. Walter Eckhart
Tumor Virus Section, The Salk Institute, San Diego, California
Dr. Hiroto Okayama Laboratory of Molecular Genetics, NICHD
National Institutes of Health Bethesda, Maryland
Dates of Visit: July 7, 1983 - August 10, 1983

Summary of Activities
(a) At the Salk Institute
Dr. W. Eckhart and his colleague succeeded in the purification of polyma virus middle T antigen 2500 fold by immunoaffinity chromatography. I learned the technique of a critical step in the purification procedure; the coupling of anti-peptide IgG from rabbit to CNBr-activated Sepharose 4B. This method will be applied for the purification of adenovirus transforming gene products when enough quantity of anti-peptide serum is prepared.
(b) At National Institutes of Health
A cDNA library from adenovirus type 12 E1A gene-transformed rat 3Y1 cells was constructed by using a cDNA cloning vector of Okayama and Berg. The following experiments were carried out.
(1) The vector primer DNA, tailed with poly(dT) at the KpnI site closest to the polyadenylation signal, was prepared from the plasmid, pcDV1 DNA.
(2) The linker fragment containing Hind III and oligo (dG) cohesive ends was prepared from the plasmid, pL1 DNA.
(3) The cDNA library was constructed with poly(A)-containing mRNA from the E1A-transformed cells by using above components.
(4) After coming back to Japan, E coli strain MC 1016 was transformed by the cDNA library and about 3 x 105 independent transformants were obtained.
The clones which contain the cDNA corresponding to the cellular genes activated by adenovirus E1A gene will be isolated by colony hybridization.


(5) Tadashi Yamamoto
Laboratory of Oncology, The institute of Medical Science The University of Tokyo, Tokyo

Sponsor and Host Institution:
Dr. Ira H. Pastan
Laboratory of Molecular Biology
National Cancer Institute
National Institutes of Health Bethesda, Maryland
Dates of Visit: September 1, 1983 - November 29, 1983

Summary of Activities
My main project was to isolate monoclonal antibodies against the erbB gene product of the avian erythroblastosis virus-H (AEV-H) and to learn the technique to dissect the cellular localization of the erbB protein or its target protein(s) immunocytochemically. The works I have performed for this purpose are as follows:
(1) As a purer antigen to immunize mice, Dr. Pastan and I have decided to use erbB protein made in E. coli. An expression vector pJL6, which is described by Dr. T. Papas et al. (Gene 23, 75 1983), was chosen to construct a plasmid pJerb13 that carries most of the erbB gene sequence. The erbB gene sequence in the plasmid pJerb 13 is placed under the control of the PL Promoter from!!!phage. Therefore, when E. coli 6405, a lysogen of!!!phage that carries a temperature sensitive mutation in the CI gene, is used as a host cell, transcription of the erbB gene sequence from the PL Promoter is induced at high temperature (42°C). In fact we observed that the rate of synthesis of the erbB gene product was high at 42°C. Although the accumulation of the erbB gene products was not as high as we expected, we are now planning to prepare the protein from mass culture of the E. coli containing the pJerb13 and to use the protein to immunize mice.
(2) Since several projects to isolate monoclonal antibodies was going on at Dr. Pastan’s laboratory, I have followed the procedure such as preparation of mouse spleen feeder layers and fusions of spleen cells of the immunized mice and NS-1 myeloma cells.
I was also involved in several projects shown below.
(i) Characterization of the mutant KB cells that were made resistant to several drugs such as colchicin, adriamycin, vinbrastin, vincristin sulfate, actinomycin D or puromycin. The work I have done for this project was to examine whether any genes that direct the resistancy against the drugs are amplified or not using newly developed hybridization technique combined with S1 digestion (described by I. B. Robinson, Nucleic Acid Research 11, 5413 1983). With this technique, any genes which are amplified more than 50 times are possible to be identified and I found that the drug-resistant cells that I have examined do not seem to carry any genes amplified more than 50 times.
(ii) Characterization of the EGF gene of A431 carcinoma cell that is known to overproduce the EGF receptor. Since the erbB gene, lately, was found to be related to the EGF receptor gene, the collaboration between Dr. Pastan and I has been continuing and we have found that the EGF receptor gene (or c-erbB gene) is amplified about 20 times in A43 1 cells. The results were published in Science (G. T. Merlino et al., Science 224, 417 1984). Beside the activities shown above, I gave a talk about the structure and function of the v-erbB ene at the National Cancer Institute.
Overall Assessment of the Program:
1. First, I could construct a recombinant plasmid that can direct the synthesis of the erbB related protein. This protein, after purification, can be used as purer antigen to immunize mice. Second, I could learn the technique to isolate monoclonal antibodies.
2. This program helped me to collaborate with Dr. Pastan on the project to study the function and structure of the c-erbB gene. This collaboration has been continuing.
3. Since our collaboration fruited the publication of the paper on the EGF receptor (c-erbB) gene, this NCI-JAPAN Cancer Program was revealed to be very effective for the research in this field.


(6) Stuart A. Aaronson
Laboratory of Cellular and Molecular Biology
National Cancer Institute, National Institutes of Health
Bethesda. Maryland
Sponsor and Host Institute: Dr. Takashi Sugimura
National Cancer Center Research Institute, Tokyo
Dates of Visit: December 4. 1983 - December 11, 1983

Summary of Activities
In addition to participating in a UICC course on Carcinogenesis, I visited two Institutes: the Institute of Medical Science. University of Tokyo; and the National Cancer Center Research Institute in Tokyo.
The institute of Medical Science at the University of Tokyo has a number of investigators whose studies of RNA and DNA tumor viruses are at the forefront of research in their respective areas. However, their facilities, although adequate, are not as good as those of academic institutions of comparable reputation and quality in the United States. Thus, it appears that the actual research support for investigators may be less than in the United States.
The National Canter Center Institute is a modern, recently constructed facility headed by Dr. Sugimura. The facilities at this institution are very impressive, comparable to or exceeding in standard those of most cancer research facilities in the United States. I met heads of a number of departments at the Institute and was extremely impressed with the variety and the quality of investigations being performed. This institution appears to be tremendously benefited by the strong and modern leadership of its director.
In each of these high level institutes, a number of faculty members have spent time in the United States as postdoctoral workers or on sabbaticals. We personally have hosted several postdoctorals from these and other Japanese institutions and have found them to be exceedingly well trained and well read. There is no question that continued close liaison and collaboration between research groups in Japan and the United States will be of enormous value to both.


(7) Joseph E. De Larco
Laboratory of Chemoprevention
National Cancer Institute, National Institutes of Health
Bethesda, Maryland

Sponsor and Host Institution: Dr. Takashi Sugimura
National Cancer Center Research Institute, Tokyo
Dates of Visit: November 29, 1983 - December 11, 1983

Summary of Activities
The objective of this trip was to discuss, with my Japanese hosts, the work that I have done on the effects of transforming growth factors (TGF’s) on the expression of the transformed phenotype, as well as the discussion of their data and goals in this area. During this period I visited with several laboratories to present our data and discuss with them the possible future directions in this area of research.
On December 1, the day was spent at Kyowa Hakko’s research laboratories in Tokyo as a guest of Dr. Fusao Tomita. While there is presented a seminar on the TGF’s and their possible role in the expression of the transformed phenotype of the cells producing them. At this time we discussed their research in the area of TGF’s and possible utility of reagents to these factors for the treatment or diagnosis of cancer. Since the visit I sent them a small aliquot of antisera raised to a peptide produced to a portion of known sequence to TGF.
December 2 was spent at the National Cancer Center Research Institute with Dr. Sugimura and his staff discussing their research and then I presented a seminar of the TGF’s work performed at the NCI. With Drs. Nagao, Makino and Sato there was a discussion on the effects of chemicals in the environment and their roles in the rate of “spontaneous transformations” in man. We also discussed the possibility of certain of the tumors expressing specific “ectopic” peptides that can either stimulate growth or modulate the state of differentiation of the transformed cells producing them. There was also a discussion with Drs. Abe and Yamaguchi concerning the peptides, they were finding, expressed abnormally in the endocrine tumors they are studying. I spoke briefly with Drs. Miwa and Hoshino on their research data on the human retrovirus. Dr. M. Terada discussed his work on the oncogene transfection and the problems he is having with spontaneous transformation of the NIH 3T3 cells and the lack of the “appropriate” indicator cells for the detection of the yet unknown oncogenes in most human tumors. With respect to the spontaneous transformation, it was suggested that a decrease of the sera concentration in the transformation assay from 10 to 5% would decrease the background of spontaneous tansformants. I also suggested that an epithelioid cell might be better for the detection of certain oncogenes since many of the human malignancies are of epithelioid origin. We have since sent him a pair of untransformed NRK cells for his assay: one is fibroblastic (49F) and the other is epithelioid (52E).
The week-end of December 3 and 4 was spent with Dr. S. Nakagawa of the Department of Biochemistry of the Nihon University School of Medicine.
Monday was spent at the Cancer Institute giving a lecture and speaking with Dr. Sugano and his staff. Much of the time at the Cancer Institute was spent speaking with Dr. Yoichi Kato conferring with him on his work on the transforming growth factors. Tuesday and Wednesday, December 6 and 7 were spent with Dr. Toshi Kuroki and the students of the UICC Training Course in Cancer Research. This period allowed the students and the staff to interact with one another. There was one student from India, Rita Mulgerkar, who spent time asking me questions on the meaning of the research that she was doing on a growth factor receptor. Since then her Director, Dr. M. G. Deo, Research Director Cancer Research Institute of Bombay, has come to the laboratory in Bethesda to inform me of their results and discuss the possible meaning of them. I also spent time discussing Dr. Kuroki’s experiments with him.
Early on December 8, I left Tokyo for Osaka. That afternoon I presented a seminar on TGF’s to Dr. Fujii’s group at the Institute of Protein Chemistry of Osaka University. While there I discussed their results on EGF and wound healing. The next day was spent with several members of the Institute.


(8) Motohisa Kaneko
Biophysics Division
National Cancer Center Research Institute, Tokyo

Sponsor and Host Institution:
Dr. Philip C. Hanawalt
Department of Biological Science
Stanford University, Stanford, California
Dates of Visit: March 3, 1984 - March 20, 1984

Summary of Activities
The distinct parallelism between the carcinogenicity of naphthylamines or aminoazobenzenes and the amount of the free radical formation was previously found by Dr. Nagata and his group in our Division. They also found the generation of active oxygen species along with the formation of free radicals, I checked the possibility of the formation of DNA damages induced by the active oxygen species by the Method of alkaline elution when cultured cells were treated with a radical generating carcinogen (N-hydroxy-2-naphthylamine) and found that nuclear DNA was actually injured indirectly by the active oxygen species. Thus, we were able to confirm that nuclear DNA was damaged both directly by binding with the carcinogen and indirectly by the active oxygen species. For understanding the role of such indirect DNA damages in chemical carcinogenesis, it is very important to know the molecular nature of such damages. Thymine glycol (5, 6-dihydroxy-5, 6-dihydrothymine) is one of the main DNA base damages induced indirectly when cells are irradiated with X-rays. Then, it is very probable to assume that thymine glycol is produced in DNA when cells are treated with the carcinogen. Recently, a monoclonal antibody specific for thymine glycol was prepared by the group of Professor Hanawalt in Stanford University and was used to detect thymine glycol in DNA of X-irradiated cells.
The purpose of this project was to learn the technique of the assay of thymine glycol in DNA by using the monoclonal antibody specific for it and to apply this method to detect thymine glycol in DNA treated with N-hydroxy-2-naphthylamine (NOH-2-NA), both in vitro and in clutured normal human fibroblasts.
As a result, thymine glycol was actually detected in DNA treated with NOH-2-NA both in vitro and in cultured cells. This damage was produced in a dose dependent manner when calf thymus DNA was treated at 37°C with 2-20 µM NOH-2-NA and when normal human fibroblasts were treated at 15°C with 50-500 µM. The presence of catalase during the exposure to NOH-2-NA inhibited the production of thymine glycol. Superoxide dismutase had little or no effect on the production of thymine glycol in DNA treated in vitro with NOH-2-NA in the presence of Fe2+ but produced and 85% reduction in the amount of this damage when present during the exposure of the cells. These results show that hydrogen peroxide and superoxide anion radicals generated during the conversion of NOH-2-NA to nitroxide radical are involved in the formation of thymine glycol in DNA. This is the first direct proof of the presence of a specific indirectly induced DNA base damage when DNA or cells are treated with an active form of a chemical carcinogen.


(9) Takeshi Seno
Department of Immunology and Virology
Saitama Cancer Center Research Institute, Saitama

Sponsors and Host Institution:
Dr. Mario R. Capecchi
Dr. Karl G. Lark
Department of Biology, University of Utah Salt Lake City, Utah
Dates of Visit: March 4, 1984 - March 31, 1984

Summary of Activities
Accumulating knowledge on the genetic basis of cancer suggests that genetic recombination and chromosome nondisjunction play an important role in triggering cancer. It is then urged to establish a defined experimental genetic system with which such genetic changes is assayed qualitatively as well as quantitatively, and can be distinguished from point-mutations. As a candidate for such systems, we have recently discovered an inducible genetic recombination in the human thymidylate synthase gene in a mouse transformant cell. In this system, recombination is induced when the cells are deprived of thymidylate. We have cloned genomic fragments and a complete cDNA of human thymidylate synthase gene whose structure is now being determined.
From necessity of developing this system to meet the study of carcinogenesis in molecular level, I visited with Drs. Capecchi and Lark in Utah and successfully discussed the problems with them and planned the future collaborative program as follows:
(1) I have learned the microinjection technique from Dr. Capecchi. For establishing a refined genetic system for recombination, it is necessary to characterize first the kind of mutations in the genetic marker to be used. For this, microinjection to the mutant cells of nonsense-suppressor tRNA genes, which Drs. Capecchi and RajBhandary established, should be decisive.
(2) Dr. Lark and his colleagues have recently cloned a 3.25 kilobase DNA from kangaro rat. It contains highly repeated sequence and interspersed in the genome. The 3.25 kilobase DNA sequence is rearranged to produce a deletion by a prokaryotic recombination system (Red or RecE). We planned to see if this is also true in somatic cells by introducing the 3.25 kilobase DNA into our mouse cell system. Concerning our recombination system inducible by thymidylate stress, Drs. Lar, Capecchi and I further discussed on the way to analyze the base sequence specificity of the double strand breaks in nuclear DNA occurring at the replicon origins.
(3) My visits at the National Institute of Environmental Health Sciences and Yale University were fruitful and successful. In the former institute, I discussed with Drs. P. Nettesheim, J. C. Barrett and M. Oshimura about a possible application of our recombination system to their study on the induction of autosomal recessive genetic changes in mammalian cells, and with Drs. J. Drake, B. Glickman and T. Kunkel about the comparison in bacteria and mammalian cells of mechanisms of thymineless death and its genetic consequences. At Yale University, I discussed with Dr. F. H. Ruddle and his colleagues about the chromosomal mapping of human thymidylate synthase gene with our cloned DNA and a possible collaboration with them on the mapping on the mouse chromosome. I also discussed with Dr. J. Bertino and his group about the mechanisms of 5-fluorouracil resistant genetic changes in the treated tumor cells and a possible use of our cloned DNA for the analysis.
(4) I also participated in a conference, entitled “Eukaryotic DNA Replication and Repair”, which was held on March 16-18 at Stanford University, sponsored by the U.S.-Japan Cooperative Cancer Research Program and organized by Drs. D. Korn, E. Friedberg and K. Koike. My colleagues and I thank the National Cancer Institute and the Japan Society for the Promotion of Science for their full support of my fruitful and successful visit this time in the United States, which enhanced our research and enabled us to continue the collaborations with above mentioned people in the States.