SUMMARY REPORTS OF EXCHANGE SCIENTISTS
(1) HIROYASHU ESUMI
Virology Division
National Cancer Center Research Institute, Tokyo
Sponsor and Host Institution:
Dr. James D. Watson
Cold Spring Harbor Laboratory
Cold Spring Harbor, New York
Dates of Visit: May 1, 1982-May 25, 1982
Summary of Activities
Objectives:
(1) To study changes in the 2D-gel pattern of cellular proteins induced by tumor promoters teleocidin, lyngbyatoxin, and aplysiatoxin.
(2) To investigate the changes in cytoskeletal architecture induced by tumor promoters probed with fluorescent monoclonal antibodies against cytoskeletal proteins.
The above two subjects were studied in collaboration with Drs. James Garrels, James Feramisco, James Lin, and Fumio Matsumura at Cold Spring Harbor Laboratory. To study these subjects, we took with us two cell lines, REF 52 (rat embryo fibroblast) and chick embryo fibroblast. The tumor promoters teleocidin, dihydroteleocidin, lyngbyatoxin, aplysiatoxin, and debromoaplysiatoxin were generous gifts from Dr. Hirota Fujiki, National Cancer Center Research Institute, Tokyo.
When REF 52 cells were treated with TPA or teleocidin at 10 ng/ml, actin distribution was dramatically changed in approximately 30% of the treated cells. This change in actin distribution was evident about 4 hours after addition of tumor promoters to the culture medium. In untreated REF 52 cells, actin fibers were quite regularly distributed longitudinally, but in about 30% of the treated cells, actin formed granular structures rather than regular fibers. The distributions of beta-actin, myosin, tropomyosin, and fibronectin were also studied in the same cells using a double staining method. Similar changes were observed in the case of alpha-actin, but no effect was observed in the cases of myosin, tropomyosin, and fibronectin. Similar effects were also observed when chick embyro fibroblasts were used.
Because the effects of tumor promoters on the cytoskeletal architecture were evident, systematic studies on the effects of tumor promoters are underway and will continue in collaboration with Cold Spring Harbor Laboratory and the National Cancer Center Research Institute.
(1) This exchange program made available a wide variety of monoclonal antibodies against cytoskeletal proteins to study the effects of tumor promoters on cytoskeletal architecture.
(2) The Cold Spring Harbor Laboratory and the National Cancer Center Research Institute will continue collaborative research on cytoskeletal proteins.
(3) Through my visit to Cold Spring Harbor Laboratory, continuous collaborative research on tumor promoters became possible. This long-standing mutual interaction between U.S. laboratories and Japanese laboratories will contribute to the progress of the NCI-3apan Cancer Program.
(2) HIROSHI HANDA
The Institute of Medical Science
The University of Tokyo, Tokyo
Sponsor and Host Institution:
Dr. Phillip A. Sharp
Massachusetts Institute of Technology
Cambridge, Massachusetts
Dates of Visit: June 12, 1982-September 2, 1982
Summary of Activities
The mechanisms that control gene expression in eukaryotic cells are of great importance, but these processes are only poorly understood. Adenovirus provides a simple but useful model system to investigate eukaryotic gene expression due to dependence on the host's synthetic machinery for synthesis of its mRNA species and polypeptides. Adenovirus-infected cells also offer the unique advantage of efficient suppression of cellular macromolecular synthesis as infection proceeds, facilitating recovery and analysis of virus-specific RNAs. The purpose of the proposed research is to elucidate the mechanisms that regulate adenovirus gene expression.
Six regions of the adenovirus genome encoding early RNA species, defined as those synthesized in the absence of viral DNA replication, were identified and designated E1A, E1B, E2A, E2B, E3, and E4. It was reported that the 72 kd DNA binding protein (Ad-DBP), encoded by E2 mRNA species, had a repressive effect on early mRNA synthesis in adenovirus-infected cells.
To elucidate the mechanism and regulation of gene expression at a molecular level, it was necessary to develop an in vitro transcriptional system. The system was originally developed by Massachusetts Institute of Technology (MIT) researchers. They have used this modified system, in which the amount of specific DNA template required for accurate transcription can be about ten-fold lower than that previously required. This allows the detection of transcriptional regulation by a specific DNA-binding protein at a ten-fold lower concentration of the protein. They also have developed an excellent technique to analyze the in vitro RNA products. I therefore hope to achieve my research objectives in collaboration with the MIT group.
The study was designed to examine the repressive effect of Ad-DBP, purified from adenovirus-infected cells, on early and late transcriptions of adenovirus with the use of in vitro transcriptional systems for RNA polymerase II and to elucidate the mechanism of the regulation. I found that Ad-DBP selectively repressed E4 transcription and had no effect on E1A, E1B, E3, and late transcription in in vitro transcriptional reactions. The repressive effect was reduced by addition of single-stranded DNA, to which Ad-DBP had higher affinity than to double-stranded DNA, and by heating Ad-DBP at 60°C for 5 minutes before addition to a reaction mixture, indicating that Ad-DBP itself had a repressive effect on E4 transcription. Our initial objective was achieved by showing the selective repression of E4 transcription by purified Ad-DBP in the in vitro transcriptional system. The results of these studies will be submitted to Nature for publication.
Future studies that will attempt to elucidate the mechanisms of repression of E4 transcription by Ad-DBP include the following objectives:
1) To determine the specific binding site of Ad-DBP to E4 promoter or initiation site;
2) To elucidate what level of transcription is regulated by Ad-DBP with the use of preincubation pulse-chase experiments in an in vitro transcriptional system;
3) To make deletion or base-changed mutants of E4 promoter site, and examine the effect of Ad-DBP on them with an in vitro transcriptional system;
4) To examine the binding activity of Ad-DBP to E4 RNA; and
5) To examine whether the repressive effect of Ad-DBP would be changed under different stages of its phosphorylation, because Ad-DBP is more phosphorylated at a late phase.
We will continue our collaboration on the experiments mentioned above. The elucidation of the mechanism and regulation of adenovirus gene expression will facilitate understanding of the intricate regulation of gene expression in transformed cells.
(3) OHTSURA NIWA
Department of Experimental Radiology
Faculty of Medicine
Kyoto University, Kyoto
Sponsor and Host Institution:
Dr. James W. Gautsch
Scripps Clinic and Research Foundation
La Jolla, California
Dates of Visit: August 2, 1982-September 16, 1982
Summary of Activities
The objective of the present study was to elucidate the mechanism of repression of Moloney leukemia virus genome in undifferentiated cells. I have already found that endogenous murine leukemia virus is suppressed by DNA modification in fibroblasts (Niwa & Sugahara, PNAS 78, 6290, 1981). Suppression of Moloney leukemia virus genome by DNA modification was also observed in differentiated teratocarcinoma cells carrying the viral genome. However, in undifferentiated teratocarcinoma cells, a mechanism other than DNA modification seemed to be responsible for the repression of the viral genome, although integrated Moloney virus genomes were extensively methylated.
Through discussions with several scientists during my stay in the United States, it became clear that the mechanism of suppression of viral gene expression in undifferentiated cells may involve recognition of a particular promoter sequence of the genome. This possibility will be tested in the near future by investigating the expression of Moloney leukemia virus genome carrying promoter sequences from housekeeping genes, such as dihydrofolate reductase.
Through collaboration with Dr. Gautsch, it was found that modification of Moloney leukemia virus genome in undifferentiated teratocarcinoma cells occurs within 15 days after infection. Moloney leukemia virus genome was not transcribed in undifferentiated cells, regardless of the state of its modification. Another joint experiment with Dr. Gautsch involved in vitro modification of a cloned Moloney leukemia virus genome and its expression in cultured fibroblasts. HpaII methylase and HhaI methylase, which methylate the internal cytosine residue of CCGG and GCGC sequences, respectively, were isolated and purified from corresponding Haemophilus species. Recombinant DNA carrying integrated Moloney leukemia virus genome was methylated to completion with these methylases and introduced into NIH3T3 and SC-1 cells. The efficiency of transfection was too low to obtain a significant difference in activity of methylated DNA. The same experiment is currently in progress in both Dr. Gautsch's laboratory in the United States and my laboratory in Japan.
(4) KUNITO YOSHIIKE
Department of Enteroviruses
National Institute of Health, Tokyo
Sponsor and Host Institution:
Dr. Kenneth K. Takemoto
National Institute of Allergy and Infectious Diseases
Laboratory of Molecular Microbiology
National Institutes of Health
Bethesda, Maryland
Dates of Visit: August 10, 1982-October 19, 1982
Summary of Activities
I have been studying the genomic structure of primate polyomaviruses (simian virus 40, human polyomaviruses BKV and JCV, and monkey B-lymphotropic papovavirus LPV) to analyze their tumorigenic and transforming capacities. During the past few years, Dr. K. K. Takemoto and I have been collaborating in studies on JCV and LPV by visiting each other's laboratories. On my present visit (supported by the Program) to his laboratory, we decided to concentrate our efforts on the study of LPV, as we believe that this study is urgent in our mutual projects and important in the field of DNA tumor virus research.
LPV, isolated from a B-lymphoblastoid cell line of an African green monkey by zur Hausen and Gissman (1979), shows an interesting host range in vitro which is distinct from those of the other polyomaviruses. Its growth is restricted to certain continuous lines of monkey or human B-lymphoblasts. LPV has a number of characteristics common to polyomaviruses, but its transforming capacity has not been proven and its pathogenicity is unknown at present. Serological surveys have shown that antibody against monkey LPV is detectable in humans, apes, and monkeys, providing strong evidence that unidentified viruses antigenically related to LPV occur widely among primates.
Previously, we molecularly cloned LPV DNA in Escherichia coli, as LPV DNA grown in a human cell line, BJA-B, was heterogeneous in size. We characterized various cloned DNAs and constructed the restriction endonuclease cleavage maps of the longest molecule (5.1 kilobases), which was found to be nondefective. For future biological and biochemical studies, it was necessary to correlate the physical and functional maps of the LPV genome.
In the present study we located the origin of LPV DNA replication by analyzing the newly synthesized form I DNA. With the replication origin as a reference point, the LPV genome was aligned to the genomes of simian virus 40 and BKV from DNA homology between specific fragments hybridized under low stringency conditions. From the results of these experiments, it was possible to deduce the correlation of the physical to functional maps of the LPV genome. (A paper entitled "Alignment of the Genome of Monkey B-Lymphotropic Papovavirus to the Genomes of SV40 and BK Virus," by T. Kanda, K. Yoshiike, and K. K. Takemoto, has been submitted for publication to journal of Virology.)
Results obtained during my Program-supported visit will provide a basis for further studies of this interesting virus, both in my laboratory in Japan and in Dr. Takemoto's laboratory in the United States. During my stay, in addition to the research activities, I engaged in stimulating discussions with a number of scientists at the National Institutes of Health, Bethesda. I am grateful for having been given the productive and stimulating opportunity to participate in the Program.
(5) TETSUYA KAMATAKI
Department of Pharmacology
School of Medicine, Keio University, Tokyo
Sponsor and Host Institution:
Dr. F. P. Guengerich
Vanderbilt University Medical School
Nashville, Tennessee
Dates of Visit: February 28, 1983-March 26, 1983
Summary of Activities
I spent the majority (20 days) of my stay in the United States at Vanderbilt University in order to purify cytochrome P-450 from human liver microsomes. At the Chemical Industry Institute of Toxicology, I met with several scientists to discuss their research projects and I presented a seminar. At the National Heart, Lung, and Blood Institute, I met Drs. J. R. Gillette, H. A. Sasame, and H. Sato; I also had discussions with Drs. M. R. Boyd, S. S. Thorgeirsson, and T. Fujino at the National Cancer Institute. In chronological order, my visit included—
1) Vanderbilt University: I brought microsomes of human livers that I had prepared in our laboratory. Following the procedure that they developed, I purified two forms of cytochrome P-450 to near homogeneity as judged by SDS-polyacrylamide gel electrophoresis. One form showed a peak at 447 nm in the carbon monoxide-binding difference spectrum; this form of cytochrome P-450 was assumed to be different from those they have purified. Also this form was expected to be significant in the activation of promutagens and carcinogens, since forms of cytochrome P-450 with similar spectral properties present in liver microsomes of experimental animals have been known to be efficient catalysts in the metabolic activation of many promutagens involving benzo(a)pyrene, Trp-P-2, Glu-P1, and IQ. I discussed current problems on cytochrome P-450 with Drs. F. P. Guengerich, P. Wang, G. A. Dannan, and others in the same department. Further, I had a chance to meet Dr. F. Chytil and his associates and hear their ideas on the significance of retinol- and retinoic acid-binding protein in cancer.
(2) Chemical Industry Institute of Toxicology: Dr. R. A. Neal gave me a tour of the facility and explained the workings of the Institute. After I gave a seminar, I met Drs. D. Rickert, M. Dyroff, R. White, and T. R. Skopek to discuss their research on the mechanisms involved in the metabolic activation of chemical compounds and on the molecular approach with genetic techniques.
(3) National Heart, Lung, and Blood Institute: I met Drs. J. R. Gillette, H. A. Sasame, and H. Sato. Two major problems discussed included the application of monoclonal antibody to drug metabolism studies and the purification of a specific form of cytochrome P-450. To attain technical agreement, we spent an entire day performing one experiment.
4) National Cancer Institute: Dr. M. R. Boyd discussed the ongoing research being conducted in his laboratory. Since Dr. S. S. Thorgeirsson was specifically interested in our research on human cytochrome P-450 and on the high spin form of cytochrome P-450, we discussed the toxicological significance of these hemoproteins. Dr. T. Fujino was conducting research for several projects, and we exchanged a variety of information concerning these research projects; this was an opportunity to discuss our research in Japanese.
I experienced several busy days that were more than enjoyable and fruitful for me. I resolved many technical and theoretical problems through experimentation and by discussing approaches and techniques with several scientists.
Staying for 20 days in an apartment at the Vanderbilt campus as a roommate of a German professor was among my most impressive experiences. This opportunity allowed us to talk from morning to night on several subjects, including our research. In North Carolina, Dr. R. A. Neal put my baggage in a room at his home and said "This is your room." It was my first experience staying at an American home. In Bethesda, I stayed at Dr. M. R. Boyd's house. In addition to discussions about research, through these experiences I enjoyed the best humanity of these scientists.
(6) YUZO HAYASHI
Division of Pathology
National Institute of Hygienic Sciences, Tokyo
Sponsor and Host Institution:
Dr. Ronald W. Moch
Food and Drug Administration
Bureau of Foods
Washington, D.C.
Dates of Visit: March 28, 1983-April 1, 1983
Summary of Activities
The object of my visit to the United States, from March 17 to April 1, 1983, was to exchange data on carcinogen tests performed in the United States and Japan, and to discuss the principles and methods for carcinogenicity risk assessment of chemicals. Four main institutions in the field of chemical carcinogenesis were selected for this purpose:
Results of 57 animal testings on 29 compounds performed in Japan through a subsidy from the Ministry of Health and Welfare were presented and discussed at each institution. It was agreed that, for carcinogenicity risk assessment of chemicals, scientific data is needed to indicate (D whether the test compound has a potential of inducing tumors in animals; (2) the mechanism by which the test compound induces tumors in animals, or to which type of carcinogens the test compound belongs (primary carcinogen, secondary carcinogen, or tumor promoter); and (3) how intensely the test compound induces tumors in animals.
At the NIEHS, there was an opportunity to participate in the quality assessment group that has been established as a system in the U.S. National Cancer Institute bioassay program, which is now a part of the National Toxicology Program, to review and check the data and histological slides submitted to NIEHS from each contract laboratory. This system, although somewhat time consuming, is effective both for assessing the quality of data submitted and for training junior pathologists and toxicologists.
At the PDA, I had a chance to discuss with pathologists and other scientists the design, problems, and conclusions of a Japanese study on BHA showing that oral administration of this food additive was associated with high incidence of forestomach cancer in rats.