(1) NOBUYOSHI SHIMIZU
Department of Cellular and Developmental Biology
The University of Arizona, Tucson, AZ.
Sponsor and Host Institution:
Dr. Takashi Sugimura, Director
National Cancer Center Research Institute, Tokyo
Dates of Visit: June 1, 1981 - August 15, 1981.
Summary of Activities:
Objectives of our study were: (i) to examine effects of various newly found tumor promoters, including teleocidin, on EGF binding to cell surface receptors, cell growth and cell differentiation, (ii) to determine chromosomal localization of the genes involved in diptheria toxin resistance by cell hybridization analysis, and (iii) to initiate somatic cell genetic analysis of analbuminemic rats.
(i) Tumor promoters, such as TPA, have been known to modulate EGF receptors. We have tested the effect of teleocidin on the 125I-EGF binding to human epidermoid carcinoma A431 cells which possess an abnormally high number of EGF receptors. To our surprise, A431 cell's EGF receptor activity was only slightly influenced by teleocidin (10-20% inhibition at 10-100 ng/ml), while mouse 3T3 cell's EGF receptor activity was largely inhibited (80-90% at the above concentration ranges). Using the same A431 cells, we observed that teleocidin stimulated seven-fold orinithine decarboxylase activity but did not enhance phospholipid biosynthesis. We have used human-mouse cell hybrids, which were produced between A431 cells and mouse A9 cells (EGF receptor deficient), expressing A431-derived EGF receptor at different levels and found that inhibition of EGF binding activity by teleocidin appears to be related to retension of certain human chromosomes. These observations may eventually explain the somewhat abnormal properties of EGF receptors in A431 carcinoma cells.
Another well known action of tumor promoters is to stimulate cellular DNA synthesis and proliferation. We have used a mouse mutant cell line, NR-6, that lacks EGF receptors to examine mitogenic activity of various known and suspected tumor promoters. For this, confluent cells were treated with drugs (1 ng-100 ng/ml) for 18 hours and incorporation of 3H-thymidine into DNA was measured and compared with that of control cells. Significant stimulation was observed when cells were treated with TPA (6-8x), phorbol-12, 13, dide-canoate (8x), phorbol-12, 13-dibenzoate (5x), phorbol-12, 13-dibutyrate (6x), teleocidin (9-14x), dihydroteleocidin B (4x), lyngbyatoxin (4x), aplysiatoxin (5-6x) and mezereine (2-5x). No stimulation was observed for 4-0-methyl-TPA (1.1 x), phorbol (1.2x) and A23184 (1.1 x). The ability of drugs to stimulate DNA synthesis in EGF receptor-deficient cells appears to correlate with the tumor-promoting potential of the drugs. Thus, the system may serve as a convenient screening method for tumor promoters.
We have also examined the effects of various tumor promoters on proliferation and the differentiation potential of mouse 3T3-L1 preadipocytes. Differentiation of 3T3-L1 preadipocytes to adipocytes is induced by treating confluent cells with dexamethasone (1 uM) and methylisobutylxanthine (0.5 mM). We have so far examined the effects of three tumor promoters, teleocidin (TC), aplysiatoxin (APT) and 12-0-tetradecanoyl phorbol-13-acetate (TPA). TC, at 1 ng/ml, and APT, at 10 ng/ml, stimulated cell proliferation 2-3 fold and inhibited adipocyte differentiation. TPA, on the other hand, even at 100 ng/ml, did not stimuate 3T3-L1 proliferation but inhibited adipocyte differentiation. Binding of 125I-EGF to its membrane receptors was inhibited by all three tumor promoters, though to different degrees (ID50 was 0.5, 0.8 and 10 ng/ml for TC, APT and TPA, respectively). Thus, tumor promoters appear to antagonize the action of inducers for adipocyte differentiation. While TPA lacks the ability to stimulate growth of 3T3-L1 preadipocytes, it is as efficient as TC and APT at inhibiting their differentiation. This suggests a difference between the post-membrane mechanisms of TPA and the other two tumor promoters. Detailed biochemical analysis will be carried out.
(ii) We planned to carry out chromosomal localization of the genes which are involved in determining diptheria toxin sensitivity and dipthamide biosynthesis using several mutants that have been isolated by Dr. Terada. However, these DT resistant mutants did not possess any selectable biochemical markers such as HPRT or TK which are essential for selecting cell hybrids in HAT medium. For this reason, we decided to construct HPRT deficient mutants from hamster CHL cells. We mutagenized 2 X 10 cells with 0.6 ul/ml EMS for 3 hours, collected survivors and grew them to 4 X 107 cells and treated them stepwise with 5 ug/ml and 10 ug/ml 6-thioguanine. We isolated 3 colonies and one of them turned out to be truly resistant to 6-TG and highly sensitive to HAT. This HPRT-minus CHL mutant (CHL-TG I ) is being used for mutagenesis test of carcinogens on DT sensitivity genes from which DT-resistant mutants will be generated. We will continue our collaboration in this line.
(iii) I have advised Dr. Sugimura's colleagues, especially Dr. Esumi, on the somatic cell genetic analysis of analbuminemic rats. We realized the need to establish primary or permanent cell lines from this abnormal rat for cell hybridization analysis.
Overall Assessment of the Program:
1. Certainly the program assisted me in achieving my objectives. Under this program I was able to meet a number of excellent people who are in different disciplines, particularly in clinical cancer research. We could exchange various ideas for the mutual benefit of both, from basic and clinical points of view.
2. During this collaborative venture, my research interest was broadened not only by interaction with Dr. Sugimura's research group, but also by visiting other cancer centers and universities in Japan. It became more obvious to me that a thorough understanding of basic cell growth control mechanisms and its deregulation by extracellular factors is crucial to establish a way to prevent and cure cancer; and that for this goal in vitro cancer cell biology should mimic the in vivo situation as close as possible in relation to the molecular and genetic basis of cell differentiation. I wish to continue our collaborative efforts toward this goal.
3. My participation in this program may have been unique since I was trained originally as a molecular biologist under the Japanese education system and then trained as a somatic cell geneticist under the American postdoctoral training system. For the past five years I have served as a teacher and a researcher at the University of Arizona. This unique career was often helpful to better understand and evaluate the Japanese scientific society for which a number of pros and cons have been realized.
(2) KEI FUJINAGA
Sapporo Medical College, Cancer Research Institute
Sponsor and Host Institution:
St. Louis University Medical Center, Institute for Molecular Virology
Dates of Visit: July 2 - September 3, 1981
Summary of Activities:
Objectives
Deletion, insertion and substitution mutants provide very important tools to study the structure and function of the viral genome. The purpose of the proposed research is to isolate and characterize site-directed mutants of Adenovirus 7 (and/or 12) in order to understand structures and functions of adenovirus genes including viral transforming gene(s).
Results
Adenovirus (Ad) 7, strain Grider, has a single cleavage site for restriction endonuclease EcoRI at map position (mp) 86.6. Viable mutants were isolated by transfection of Ad5-transformed human cell line, 293, with Ad7 DNA-protein complexes which were digested with EcoRI. This procedure yielded mutants lacking the EcoRI cleavage site as well as mutants still retaining an EcoRI site. Four of the mutants were characterized by digestion of viral DNAs with restriction endonucleases as well as by electron microscopic DNA-DNA heteroduplex analysis. Three of them, dl 707, dl 709, and dl 714, were deletion mutants lacking aproximately 770, 2000, and 2800 base pairs around EcoRI site, respectively. One of them, in 721 , was an insertion mutant which had an insertion of approximately 450 base pairs at the EcoRI site.
The insertion mutant in 721 was further characterized by Southern blot hybridization and direct DNA sequencing by the Maxam Gilbert method. The results obtained are as follows. (1) the insertion was not of viral origin but was a part of a human repeated sequence. (2) The repeated sequence was present as a unit length of 319 base pairs in tandemly joined arrays in 293 cells. (3) The total length of the insertion was 455 base pairs, corresponding to 1.4 units of the human repeated sequence. (4) Structures of the viral-cell DNA junction and the inserted sequence resemble those of transposable elements and retrovirus proviruses.
Similar procedures of site-directed mutagenesis as mentioned above can be applied for future work isolating of Ad7 (and/or) Ad12 mutants which have mutation(s) within and around the transforming genes.
The above works were carried out partly at Sapporo Medical College and partly at St. Louis University and completed by the US-Japan Cooperative Cancer Research Program. The results will be published soon as a collaborative research.
Overall Assessment of the Program:
1. By the Program, Isolations and characterizations of Ad7 mutants were performed, and detailed experimental results were discussed.
2. On the basis of the results obtained, experiments for isolating Ad7 and/or Ad12 mutants which have mutations within and around the transforming genes can be planned as collaborative works.
3. The results obtained will be published in the very near future as a collaborative work and provide backgrounds to the future project which can be carried out as one of the NCI-Japan Cancer Programs.
(3) KANJI HIRAI
Tokai University School of Medicine
Bohseidai, Isehara
Sponsor and Host Institution:
Dr. Meihan Nonoyama
University of South Florida, Medical Center, College of Medicine, Department of Medical Microbiology
Dates of Visit: July 20, 1981 to August 26, 1981.
Summary of Activities:
Subject of Research; Detection of integrated viral genomes in cell lines established from Marek's disease (MD) tumors.
It has been reported that most of latent Marek's disease virus (MDV) genomes in the virus-nonproducer cell lines established from MD tumors exist as circular episomal DNA. However, a portion of the viral DNA in the transformed cells could be integrated into the cellular DNA. We have examined for the possibility of integration of MDV DNA into cellular DNA by Southern's blotting hybridization. Recently, this reporter and his coworkers have found that MDV genome contains virulent strain specific DNA fragments in the restriction endonuclease-digested products. To analyse the structure of the specific DNA fragment, we have isolated the cloned viral DNA fragments in plasmids pBR 322 and pACYC 184.
It was found by experiments with Southern blot hybridization that the Bam HI-cleavage patterns of MDV DNA from MD lymphoma cell lines showed a high degree of similarity with ones of MDV DNA in the virions. These cell line possess the virulent strain specific fragments, Bam HI D and H. However, the sizes of the terminal fragments, A, D and H, were different among the cell lines. Even the sizes of these fragments were different among MDV strains. Therefore, most of the viral DNA in the transformed cell lines may exist as a plasmid circular DNA. Otherwise the size differences could be due to integration of MDV DNA into the cellular DNA at the terminal of the viral DNA. We will continue this work by cloning the recombinant DNA with viral and cellular DNA sequences in the plasmid.
We have isolated several cloned MDV DNA in the plasmids, pBR 322 and pACYC184, including the virulent strain-specific DNA fragments, Bam HI D and H. We will use these cloned viral DNA as a probe DNA for DNA-DNA hybridization and blot hybridization to analyze further the structure of virulent strain-specific DNA.
Overall Assessment of the Program:
1. I have obtained many important information and I could isolate the virulent strain-specific cloned DNAs in the plasmids.
2. Especially the techniques used for my research were improved very much through this program.
3. Recently, molecular biological studies on MDV and MD lymphomas have been done mainly by Dr. Nonoyama's laboratory and us. I am sure, therefore, that the informational exchange between both laboratories has contributed to the progress.
(4) AKINORI ISHIMOTO
Department of Biophysics, Institute for Virus Research, Kyoto University
Sponsor and Host Institution:
Dr. James W. Gautsch
Scripps Clinic and Research Foundation
Dates of Visit:Scripps Clinic and Research Foundation (Aug. 20-Sep. 21, 1981)
Stanford University, Dr. H. Kaplan (Sep. 22, 1981)
NIH, NIAID, Dr. W. P. Rowe (Sep. 23-Sep. 26, 1981)
Summary of Activities:
Dr. Gautsch proposed that some of the xenotropic murine leukemia viruses are B-tropic as their cryptic form from the p-30 finger print analysis of the viruses, and that a B-tropic ecotropic virus is a recobinant virus between a N-tropic ecotropic and xenotropic virus. They have already isolated a B-tropic ecotropic virus from cells infected with a N-tropic ecotropic and a xenotropic viruses. We have been trying to show whether some of the xenotropic viruses are B-tropic as their cryptic form by a biological assay system using the phenotypically mixed virus. However all the xenotropic viruses, so far examined, including the virus which was shown to be B-tropic xenotropic by Dr. Gautsch at his laboratory seem to be N-tropic or NB-tropic.
I coworked with Dr. Gautsch at his laboratory to show by my biological method whether some of the xenotropic viruses in his laboratory are really B-tropic or N-tropic as a cryptic form.
In our experiments at his laboratory, when NB-tropic ecotropic Friend virus-chronically infected mink cells were superinfected with a xenotropic virus (18081), the phenotypically mixed xenotropic virus with B-tropic ecotropic host range was harvested from the mink cells. This data suggests that the xenotropic virus (18081) has a B-tropic determinant in the virion. In another xenotropic virus (8959), the presence of N-tropic determinant was also demonstrated by the same methods.
Now it was shown by our coworks that N- or B-tropic Fv-1 determinant is expressed in some xenotropic viruses. So next questions are whether B-tropic virus is really recombinant virus between ecotropic and xenotropic virus.
The exchange program made it possible for me to demonstrate the presence of Fv-l determinant in the xenotropic virus by the biological methods. We are planning to continue to detect the Fv-1 determinant in the other xenotropic MuLVs which were used for p-30 analysis in Scripps Clinic and Research Foundation.
I believe my efforts to show the Fv-1 determinant in xenotropic virus by the biological method have much contributed to the progress of the NCI-Japan Cancer Program, since B-tropic or N-tropic Fv-1 activity in the xenotropic virus has not been shown biologically by any researcher in United States.
(5) AKIO MATSUKAGE
Laboratory of Biochemistry, Aichi Cancer Center
Research Institute, Nagoya
Sponsor and Host Institution:
(1) Tsuyoshi Kakefuda, Laboratory of Molecular Carcinogenesis, National Cancer Institutes, National Institutes of Health, Bethesda, Maryland, U.S.A.
(2) Samuel H. Wilson, Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, U.S.A.
Dates of Visit: January 10, 1982 - March 6, 1982
Summary of Activities:
(1) Collaboration with Dr. Kakefuda
The present collaboration dealt with the initial step of experiment to clarify the effect with the modification of DNA structures, which might induce mutations. On the other hand, it is recently recognized to be important how exactly DNA molecule is replicated by DNA polymerase, an enzyme involved in DNA replication process. Chemical carcinogens may directly interact with DNA polymerase so that they affect the fidelity of DNA replication by this enzyme.
The present collaboration dealt with the initial step of experiment to clarify the effect of carcinogens on the activities of animal cell DNA polymerase!
!!,!!
!and!!
!.DNA polymerase!!
!was inactivated by treatment with benzo(a)pyrene diolepoxide (DE-BP), but DNA polymerases! and!!!were not. Result indicates that DE-BP interact directly to DNA polymerase!!!. The rate of fault in DNA replication by the carcinogen-treated DNA polymerase!!!was measured by the incorporation of dGTP depending on poly (dA) template, on which dTTP was normally incorporated. The rate of the fault, dGTP incorporation/dTTP incorporation, by the enzyme treated with 40 µg/ml DE-BP was 4.0 X 10-3, which is significantly higher than that by the untreated enzyme, 1.7 X 10-3. Thus, the result suggests DE-BP interacts with DNA polymerase!!!, a replicative enzyme, so that it induced the DNA replication fault which may cause the mutation and carcinogenesis. These studies are only at the initial step. More extensive and careful experiments are necessary in future to obtain the conclusive evidence.
(2) Collaboration with Dr. Wilson
DNA replication is one of the essential reactions for the cell growth. The molecular mechanism of regulation of DNA replication is, therefore, important to clarify the uncontrolled growth of tumor cells. The present project deals with the structures and functions of DNA polymerases, DNA replicating enzymes, in order to find out the role of these enzymes in DNA replication and its regulation.
Dr. Wilson and myself have been studied on the basic enzymology of DNA polymerases since 1972, and recently succeeded to prepare antibodies against DNA polymerases including monoclonal antibodies. Using these antibodies, various techniques are now available to analyse structures and functions of DNA polymerases. The first purpose of the collaboration is to exchange these techniques which were established by either of both laboratories. Following methods were presently owned by both laboratories;
1) Purification methods of all three DNA polymerases!!
!,!!!and!!!. 2) Tryptic peptide mapping to compare the structures polypeptides which are components of DNA polymerases.
3) A method of renuturation of DNA polymerase activity after electrophoresis in sodium dodecylsulfate-polyacrylamide gel containing DNA, which made it possible to find out the polypeptide responsible to the enzyme activity.
4) Protein blotting from polyacrylamide gel to nitrocellulose membrane, after which DNA polymerase polypeptide can be visualized by the peroxidase-antibody binding method.
5) Immunofluorescence technique to see the distribution of DNA polymerase in cell and its change depending on the physiological change in cells.
By those methods, we found that DNA polymerase!!!is basically composed of two kinds of polypeptides and these polypeptides might be modified in various modes including proteolytic processing. It will be important project in future to clarify the relationship between the modifications of the DNA polymerase and the regulation of DNA replication.
Nuclear localizations of DNA polymerase ?? and ?? were confirmed in both mammalian and avian cells in culture by an immunofluorescence technique.
Overall Assessment of the Program:
1. The Program assisted my research very much. The collaboration with Dr. Kakefuda gave me a chance to open new field in approach to DNA polymerase and mutagenesis by chemical carcinogen. The collaboration with Dr. Wilson helped to exchange the information and methodology for the research in DNA polymerase and DNA replication.
2. Through the Program, I was able to have chances for collaborations and discussions with leading scientists of the United States. New research field, new methodologies and up-to-date information encouraged and improved my research very much. The collaboration will be continued by exchanging materials and information. It will be ideal if I can have chance for the short-term collaboration once every two or three years.
3. You can support the publication of the results in project by the Program or arrange a symposium for the scientists supported by the Program.
(6) TAKAO SEKIYA
National Cancer Center Research Institute
Sponsor and Host Institution:
National Cancer Institute, NIH and Rockefeller University
Dates of Visit:February 2 - February 15, 1982
National Institute of Health
Visiting with Drs. G.F. Vande Woude (host Scientist), T. Robinson, E.M. Scolnick, C.J. Sherr, R.C. Gallo, S.A. Aaronson and M.A. Martin.
During the period, also visiting with Drs. D. Söll and J. Steitz (Yale University), A. Landy (Brown University), H.G. Khorana (MIT), M. McCoy and J.Y.J. Wang (Dr. R.A.Weinberg's and D. Baltimore's Laboratory, respectively, Center for Cancer Research, MIT) and G.M. Cooper (Sidney-Farber Cancer Institute, Harvard University)
February 16 - February 20
Rockefeller University
Visiting with Drs. H. Hanafusa (host scientist) and W.S. Hayward
A seminar was given at NIH, Yale University, Brown University and MIT on the title of "Structure and expression of tRNA genes in mammalian cells".
Summary of Activity:
Analysis of onc genes in human cells using v-onc DNA clones as a probe is undertaken in our laboratory. However, it is difficult to evaluate whether it is useful approach for identifying cause of cancer in human and for establishment of new technique for cancer diagnosis or not. Therefore, my purpose under this exchange program is to visit with leading scientists in the States with the intention of knowing the possibilities and limits of application of v-onc genes for studies on human onc genes and getting an idea of the onc gene search.
I was able to discuss on onc genes with more than fifty scientists during my visit. I was deeply impressed with great progresses in onc gene studies in the States. I also very much appreciated the kindness of the scientists to show me their up-to-date results. Through the discussion, I learned that the technique of DNA transfection to tissue cultured-cells, especially NIH3T3 cells, was extensively in use and at present might be the only and best way to find out functional onc genes in various kinds of tumor cells. I realized that we must introduce this technique to our project. The up-to-date results and techniques that I learned should be very helpful for our future research works. More importantly, friendship with outstanding scientists that I could make under the program is inestimable.