(1) Kunitada Shimotohno
Virology Division
National Cancer Center Research Institute, Tokyo
Sponsors and Host Institutions:
Dr. David W. Golde
University of California, Los Angeles
Dr. M. Yamada
Cold Spring Harbor Laboratory, New York
Dr. Howard M. Temin
University of Wisconsin, Madison
Dates of Visit: May 21 - June 10, 1984
Summary of Activities
Subject of Research: Gene structure and expression of human T cell leukemia virus.
Human T cell leukemia viruses are thought to be closely related to some human diseases with T cell malignancies. Nucleotide sequence analysis of human T cell leukemia virus type-I (HTLV-I) revealed that the genome has an extra sequence, termed pX, between env and 3' LTR with an, as yet, unknown function. A similar virus, but distinct from HTLV-I, was discovered from a patient with hairy cell leukemia. A provirus of this virus was molecularly cloned from the virus-producing cell line. This virus was named HTLV-II.
In the case of the viral infection, envelope gene product plays an important role in absorption by target cells. Therefore, it is important to characterize env gene product to elucidate the mechanism of viral absorption by the cell surface and also to search for the ways of preventing viral infection. I was interested in analyzing env gene product which was obtained in quantity using the eukaryotic expression vector system. It is known that env protein in virion is glycosylated and such modification may not be achieved properly by the prokaryotic-expressing system. At Cold Spring Harbor Laboratory, I discussed the viral gene expression using the eukaryotic system with Dr. M. Yamada who has developed a eukaryotic-expressing vector using adenovirus late promoter. Since this system uses a potentially strong promoter and a 5' non-coding region, which increases the efficiency of translation, the efficiency of expression of a gene which is under the control of this promoter was found to be very high. The vector carrying the env gene is now under construction in collaboration with Dr. M. Yamada.
Discussion of gene structure and expression of human T cell leukemia virus was carried out with Dr. Howard M. Temin at The McArdle Laboratory for Cancer Research, University of Wisconsin. The discussion focused on whether or not the gag open reading frame in HTLV has enough information to code all gag-related proteins. Avian or murine retroviruses have five domains of a gag-related protein, including a protease. However, in HTLV only three major domains have been identified so far. This discussion was very helpful in discovering an open reading frame located between gag and pol in HTLV-II after completion of sequencing of the total nucleotide sequence of the provirus. I suspect this as the frame for a protease.
To clarify the function of the pX region, I have searched for the proteins in cells infected with HTLV-I or HTLV-II, in collaboration with Dr. Golde's group at UCLA. I was confident that pX protein was expressed in the viral-infected cells according to the result of the analysis of the nucleotide sequence of HTLV-I and HTLV-II which was carried out by our group. The search was continued after returning to Japan, and we were able also to find the viral proteins in the virus-infected cells.
I believe that the research concerning the molecular biology of HTLV reported here could not have progressed quickly without the support of the US-Japan Cooperative Cancer Research Program.
(2) Yoichi Taya
National Cancer Center Research Institute, Tokyo
Sponsor and Host Institution:
Dr. Fuyuhiko Tamanoi
Cold Spring Harbor Laboratory
Cold Spring Harbor, New York
Dates of Visit: August 1-31, 1984
Summary of Activities
We recently found the activation of a novel combination of oncogenes in a human lung giant cell carcinoma (LGCC) Lu-65. Amplifications of the myc and K-ras oncogenes are accompanied by a point mutation in the twelfth amino acid of the K-ras gene product in this cancer cell. The major objective was the collaboration with Dr. Tamanoi to make clear whether the mutated form of K-ras gene product p21 is really increased in large quantities and how mutated p21 interacts with other cellular components in this LGCC.
For this purpose, Lu-65 cells and control cells were labeled with [35S]methionine, and the cell lysates were immunoprecipitated with a monoclonal antibody against ras proteins. Analysis of the precipitated proteins clearly showed the increase (~10-fold) of K-ras protein in Lu-65 where K-ras gene is amplified. The increased K-ras protein was also suggested to be the one point mutated by this analysis. Such findings give advantage to this LGCC Lu-65 in the current work on the interaction between ras protein and other cellular substances.
The results of my research objective were obtained efficiently and quickly through this program. In addition, during my stay, I received much information on oncogenes, especially on ras gene products, which convinced me of the importance of characterization of oncogene proteins. I am sure our collaboration has contributed to the progress of the NCI-Japan Cancer Program. We are continuing our collaboration.
I hope many more researchers are accepted for this useful exchange program.
(3) Yoshinari Ohnishi
Department of Bacteriology, School of Medicine
The University of Tokushima, Tokushima
Sponsor and Host Institution:
Dr. Frederick A. Beland
Division of Carcinogenesis Research
National Center for Toxicological Research
Jefferson, Arkansas
Dates of Visit: August 8 - September 7, 1984
Summary of Activities
1. Introduction and Research Objectives
We have reported that many nitroarenes that are possibly present in the environment are mutagenic. In addition, we have shown that mutagenic nitro-pyrenes are present in diesel exhaust particles, wastewater from gasoline stations, used crankcase oil, indoor air particles, and "yakitori" grilled chicken, and that 1,6-dinitropyrene is carcinogenic in mice. In other studies we have reported that 1-aminopyrene (1-AP) is a metabolite of 1-nytropyrene (1-NP) in intestinal microflora and that a particular anaerobic bacterium, Bacteroides fragilis, contained four nitroreductases capable of reducing 1-NP to a mutagen. These nitroreductases were purified, and one was shown to be capable of catalyzing the reduction of 1-NP to a DNA binding derivative.
As part of these investigations, we examined the in vivo metabolism of 1-NP in conventional and germ-free rats. High pressure liquid chromatographic (HPLC) profiles indicated that substantial differences existed in the metabolism of 1-NP by these two types of rats. In order to identify these metabolites, I proposed to travel to the National Center for Toxicological Research and work with Dr. Beland, since he had the necessary standards and equipment to facilitate identification of these metabolites. In addition, I planned to see if there were differences in the extent of DNA binding by 1-NP in hepatic and other tissues by using a 32P-postlabeling technique.
2. Achievements and Results
Based on preliminary data, it became apparent that two additional standards were necessary: 1-acetylamine-6- and -8-hydroxypyrenes (1-AA-6-HP and 1-AA-8-HP). These were prepared from the selective acetylation of 1-amino-6- and -8-hydroxypyrene, respectively.
The following metabolites were detected in the feces of conventional rats treated orally with [3H]1-NP: 1-AP, 1-AA-6-HP, and 1-AA-8-HP, with each metabolite being formed in nearly an equal quantity. Urinary metabolites were analyzed after treatment with!!
!-glucuronidase. Two metabolites were detected, 1-AA-6-HP and 1-AA-8-HP, with 6-hydroxylation occurring to a greater extent than 8-hydroxylation. In each instance, the metabolites were separated by reversed phase HPLC using a Waters µBondapak C18 column, and their identify was confirmed by coelution with the standards by their UV spectra as obtained with a Hewlett-Packard 1040A detector and by their mass spectra as obtained with a Finnigan 4023 instrument. Metabolites from the germ-free rats were analyzed in the same manner. The major products detected in both the urine and feces were 1-nitro-3-hydroxypyrene (1-N-3-HP), 1-nitro-4, 5-dihydro-4, 5-dihydroxypyrene (NDHP), 1-N-6-NP, and 1-N-8-HP. 1-N-6-NP could not be resolved from 1-N-8-HP by HPLC but could be separated by thin layer chromatography. The concentration of metabolites in the feces was 1-N-3-HP > NDHP > 1-N-6-HP > 1-N-8-HP, while in the urine the order was NDHP > 1-N-3-HP > 1-N-6-HP > 1-N-8-HP. 1-aminopyrene was not found as a metabolite in germ-free rats.
DNA was extracted from the liver of both conventional and germ-free rats that had been treated with [3H]1-NP. Since the specific activity of this compound was low (ca. 3 mCi/mmol), in order to detect binding to DNA a 32P-postlabeling technique was used. The profile obtained from both types of rats was essentially the same. Since 1-AP was not detected as a metabolite in germ-free rats, this suggested that reductive metabolism was not essential for the formation of the DNA adducts that were detected. The identity of these adducts and the pathways leading to their formation remained to be determined. However, since oxidative metabolism predominated in both types of rats, they may arise from epoxidation.
3. Future Work
These studies have provided insight into the importance of intestinal microflora in the metabolism of 1-NP. Through a combination of HPLC and various spectroscopic techniques, a number of metabolites were identified, but a number of questions remain. What pathways lead to the metabolites that were identified? Does reduction only occur in the intestine, catalyzed by intestinal microflora? Does hydroxylation precede N-acetylation? Do the bacteria acetylate metabolites or does this only occur in the liver? What is the identity of the DNA adducts that were formed and what was the pathway that led to them? What is the nature of the conjugates that were resistant to!!
!-glucuronidase? Are they glutathione conjugates, and if so, what do they contribute to the overall metabolism of 1-NP? (4) William Haseltine
Laboratory of Biochemical Pharmacology
Dana-Farber Cancer Institute, Boston, Massachusetts
Sponsor and Host Institution:
Dr. Yohei Ito
Faculty of Medicine, Kyoto University, Kyoto
Dates of Visit: August 8 - September 8, 1984
Summary of Activities
1. Purpose
The purpose of the visit was to establish collaborative research programs with Japanese scientists working on human T cell leukemia virus.
2. Rationale
Japan is the focus of HTLV infection and HTLV-induced disease. The disease is well studied from the epidemiological point of view and medical consequences. The Japanese also have a number of first-rate laboratories that are studying the biology of HTLV as well as the molecular biology of the infected cells, of the tumor cells, and of the virus itself.
3. The Visit
Visit Tokyo August 9 and 10
Discussions were held with members of the National Cancer Center Research Institute, Tokyo, on the subject of HTLV. The visit to Tokyo was coordinated by the Director of the Institute, Dr. Sugimura. I presented a seminar and spent time discussing with researchers in Dr. Sugimura's group, in particular Drs. Miwa and Harada, as well as Dr. Shimotohno, their current work on HTLV. This group has recently sequenced the genome of HTLV-II and is working on virally encoded proteins.
At the National Cancer Center Research Institute, I arranged for an exchange of reagents in the future. Furthermore, I discussed Drs. Miwa's and Harada's work on the p42 protein. I discussed with Dr. Shimotohno details on HTLV-II sequencing. We discussed possible collaborative arrangements in the future on the HTLV subject. I also helped to plan a symposium in Japan on HTLV for November 1984.
Arrive Kyoto August 10
In Kyoto I was provided with an office and laboratory at the Kyoto Medical School in the laboratory of Dr. Yohei Ito.
Except for short visits to other laboratories in Japan and for attendance at the Sendai meeting of the International Congress of Virology, September 2-7, I remained in Kyoto for the rest of the visit.
The program in Kyoto included:
1. Lectures. I delivered four 2-hour lectures to researchers at Kyoto Medical School on the subject of my research. The lectures emphasized new discoveries in the area of virology and HTLV biology.
2. Discussion of HTLV with members of the Kyoto faculty. These included:
• Dr. Ito -- epidemiology and cofactors of HTLV-related disease. Dr. Ito's work focuses on the possible role of naturally occurring tumor promoters in HTLV-II-induced disease.
• Dr. Hinuma -- These discussions focused on the epidemiology of HTLV and on diagnostic and vaccine methods for controlling HTLV infection.
• Dr. Sugimura -- Discussions with Dr. Sugimura on mechanism of action of HTLV virus. We have agreed to exchange data and reagents for several experiments.
• Dr. Yodoi -- Discussion of factors produced by HTLV-infected cells that stimulate IL-2 receptor proteins.
• Dr. Honjo -- Discussions of T4 cell regulation and his current work on the IL-2 receptor gene.
I also discussed DNA damage and repair problems with a group of researchers in the Kyoto area who work on radiation biology and carcinogenesis. This group was coordinated by Dr. Takabe and his coworkers at the radiation laboratory. A special day was arranged for a seminar on this topic by me, and scientists from Kobe, Osaka, and Kyoto Universities attended this meeting. This constituted a small workshop.
Dr. Ito also sponsored a meeting on papilloma viruses held in Kyoto during my visit there. I presented an introductory lecture on HTLV to the scientists working on papilloma viruses with the notion that this might be of some relevance to their studies on mechanisms of action.
The results of the visit to Kyoto were:
1. Establishment of a good exchange of information on an ongoing basis with Japanese scientists working on HTLV.
2. Collaborative studies with Japanese scientists in Kyoto, including ex-change of reagents such as cell lines and immunological reagents. This is to be coordinated by Dr. Ito.
3. Visits to Other Laboratories
Visit to the University of Kobe, September 22. My hosts at the University of Kobe were Drs. Fujiwara and Nakao. At the University of Kobe, I presented a seminar on my work on HTLV viruses and established collaborative programs in discussions with Dr. Nakao and exchange of HTLV-related sera.
Visit to the Cancer Institute, Tokyo, August 7-8. Dr. Sugano was my host. I presented a seminar on my work on HTLV and other viruses. Dr. Sugano coordinates a research group that includes Drs. Yoshida, Seiki, and Mikami working on HTLV. This group is one of the leaders in HTLV biology, including DNA sequence analysis of HTLV-I and primate HTLV viruses. They are also working on mechanisms of action of HTLV transformation.
We discussed possible collaborations on primate HTLV work.
I attended the National Congress on Virology, Sendai, Japan, on September 2-7. I presented three papers at this meeting on my work on RNA tumor viruses and discussed with colleagues from all over the world progress on HTLV research.
In summary, the visit was productive both in terms of exchange of information both from my laboratory and the Japanese laboratories. It was also productive in the establishment of a number of collaborative research programs between my laboratory and various Japanese laboratories.
(5) Thomas L. Benjamin
Harvard University, Boston, Massachusetts
Sponsor and Host Institution:
Dr. Hiroto Shimojo
Department of Enteroviruses
National Institute of Health, Tokyo
Dates of Visit: August 25 - September 13, 1984
Summary of Activities
During my trip to Japan, several meetings and exchanges occurred with professional colleagues.
I visited the Institute of Medical Science, Tokyo University, where I presented a seminar on August 27. Informal discussions were held on August 27 and 28 with Drs. Nobuo Yamaguchi and Kinichiro Oda and members of their group. These discussions concerned approaches to identify and analyze the multiple functions of SV40 large T antigen and the use of the MMTV-LTR to regulate expression of E-1a and E-1b proteins of human adenoviruses.
I also attended the Fourth Sapporo Cancer Symposium from August 28 to August 31 which was organized by Dr. Kei Fujinaga. This was a broadly based meeting including DNA and RNA tumor viruses, human genetics, oncogenes, and HTLV. From September 1 to 7, I attended the Sixth International Virology Congress in Sendai and presented a workshop/seminar entitled "Phosphorylation Events in Productive Infection and Transformation by Polyoma Virus."
I visited several other institutes from September 10 to 13, including the Virus Institute, University of Kyoto (hosted by Dr. Masakazu Hatanaka), and the Institute of Applied Microbiology (hosted by Dr. Michio Oishi) where I presented a seminar. Dr. Oishi has recently taken up work on polyoma, and we discussed several projects, including his recent findings of possible altered episomal polyoma DNAs Carried in some strains of L cells. This episomal state related to our work on free viral DNA found in some kinds of polyoma-induced primary tumors of epithelial origin in the mouse.
The visit to Japan was pleasant and the exchanges and visits to the various institutes were mutually beneficial.
(6) Ikuo Abe
Department of Experimental Oncology
Research Institute for Tuberculosis and Cancer
Tohoku University, Sendai
Sponsor and Host Institutions:
Dr. Gary M. Williams
Naylor-Dana Institute of Disease Prevention
Valhalla, New York
Dr. Bruce Chin
School of public Health University of Michigan, Ann Arbor, Michigan
Drs. Gary A. Boorman and Joseph K. Haseman
National Institute of Environmental Health Sciences
Research Triangle Park, North Carolina
Dr. Jerrold M. Ward
National Cancer Institute
Frederick, Maryland
Dates of Visit: October 7-23, 1984
Summary of Activities
The objective of the present study was to discuss methodology and assessment of carcinogenicity screening in vivo with the leading scientists of this field in the United States. The discussion with Dr. Williams concerned "systematic decision point approach," which was proposed for carcinogenicity screening by him and Dr. J. H. Weisburger. They unified the chemicals that gave positive results in animal experiments for carcinogenicity as carcinogens and classified them into two categories -- genotoxic and epigenetic -- depending on their reactivity with DNA. Their concept seems acceptable, at least in principle, though sometimes reluctantly, because of its simplicity. They stressed the importance of handling carcinogens of different categories differently. One of the current problems, i.e., development of the screening method for epigenetic carcinogens including promoters, was discussed with Dr. Williams, Dr. Chin, Dr. Ward, and Dr. James A. Popp of the Chemical Industry Institute of Toxicology, who have been trying to establish this method. Dr. Ward has shown that promoter activity can be detected following single exposure of some promoters. Discussion with Dr. Boorman and Dr. Haseman was valuable and suggestive. They have been conducting chronic animal experiments and accumulating experience. Their opinion is that only chronic animal experiments are reliable for carcinogenicity screening so far. The NTP has changed the protocols; test chemicals are given at three dose levels. Dr. Haseman recommends use of low background strains of animals in carcinogenicity testing. Dr. Elizabeth Weisburger, Dr. William Lijinsky, and Dr. Lionel A. Poirier of the National Cancer Institute, USA, and Professor I. A. Bernstein of the University of Michigan were among the scientists with whom I talked about general problems in carcinogenicity risk assessment. This visit was very valuable and has encouraged my future work using animal experiments for carcinogenicity screening. Finally, I would like to express my hearty thanks to all the persons concerned with the US-Japan exchange program.
(7) Kazuo Yanagi
Institute of Basic Medical Sciences
University of Tsukuba, Ibaragi
Sponsor and Host Institution:
Dr. Bernard Roizman
Marjorie Kovler Viral Oncology Laboratory
University of Chicago, Chicago, Illinois
Dates of Visit: October 10 - December 8, 1984
Summary of Activities
It has been known for many years that herpes simplex virus (HSV) gene expression is tightly regulated. The first set of genes expressed after infection is the!!
!gene.!!!gene products are required for the expression of!!!(!!!1 and!!!2) genes, and!!!genes are required for!!
!(!!!1 and!!!2) gene expression in the lytic infection in which synthesis of host cellular macromolecules is shut off. However, all of the evidence suggests that the genes retained and possibly expressed in HSV-transformed cells are!!!or!!!genes (e.g., biochemical transformation to HSV TK gene). To date, there is no evidence that!!!genes are either expressed or retained in biochemically or morphologically transformed cells. In the case of transformation where no inhibition of synthesis of host cell polypeptides is known, a host cell factor(s) may take the place of a gene products to allow the expression of!!!and!!!genes, and/or the tight regulation of!!!and!!!genes in an HSV genome organization may be lost upon integration of the genes in cellular DNA. On the other hand, it is known that no HSV gene product is required for the maintenance of morphological transformation, and it seems that little HSV DNA sequence was detected in most HSV-transformed cells. Thus, one of the major questions raised as to the mechanism of morphological transformation by HSV is whether it is induced by insertion of a segment of HSV DNA which contains a unique regulatory sequence of HSV DNA into the host cell DNA. Taking such a possibility into consideration, we decided to determine the level of expression of!!!and!!!genes. A series of studies, in which construction of HSV-derived vectors carrying chimeric genes consisting of structural sequences of a foreign gene fused to the promoter-regulatory regions of HSV genes, have been carried out in the past several years in Dr. Roizman’s laboratory. The specific plan of our study was the expression of a chicken ovalbumin gene under control of promoters and regulatory regions of!!!,!!!, !!!2, !!!1, and!!!2 genes. I succeeded in constructing and expressing the chimeric genes of!!!1,!!!2, and!!!1 regulation, but I could not complete construction of!!!2 promoter-regulated chimeric gene before I left Chicago. We are continuing this work at the present and will publish results this year. As described above, this program gave me a chance to learn and apply the most advanced experimental system of genetic engineering of HSV to my research objectives. We have promised to continue our collaboration. I believe that my efforts in my experiments on human herpes viruses in Chicago and visits to many investigators, and discussions with them as listed below, have contributed to the progress of the NCI-Japan Cancer Program.