SUMMARY REPORTS OF EXCHANGE SCIENTISTS

(1) Kei Fujinaga
Sapporo Medical College
Cancer Research Institute, Sapporo

Sponsor and Host Institution:
Dr. Maurice Green
St. Louis University Medical Center
Institute for Molecular Virology
Dates of Visit: August 4-23, 1988

Summary of Activities:
The main purpose of the visit was to discuss and exchange scientific information on experimental results obtained by Dr. M. Green's research group and by our research group for further development of studies on the mechanism of viral carcinogenesis.
The recent results obtained by our research group were presented for discussion and information exchange, including the relationship between the structure and function of the adenovirus E1 gene, focusing mainly on the N-terminal half of the adenovirus type 12 E1A coding region. In-frame insertion and deletion mutations were introduced into the N-terminal half of the E1A coding region and the transforming activity and the trans-regulation ability of mutants were measured. The results obtained are summarized as follows. (1) N-terminal region (domain N) and domain 1 play critical roles in cell transformation. (2) Domains N and 1 also affect E3CAT transactivation. (3) Small and local structural changes in domain 1 affect transformation-related function(s). (4) E1B modulates E1A functions associated with domains N and 3. (5) Transactivation (E3 enhancer promoter CAT) and transrepression (polyoma enhancer SV40 promoter CAT) functions are not always associated with transformation function. Cooperative transformation of rat cells with transforming genes including the adenovirus E1A gene were also presented and discussed. Transformation of senescent REabl cells (early-passaged rat embryonal fibroblast cells carrying v-abl gene) in the absence of dexamethasone was tested by transfecting different oncogenes. Among nuclear oncogenes such as Ad 12 E1A, polyoma large T, cellular p53, and c-myc, only Ad12 E1A could induce transformation of Go-arrested REabl cells.
Discussion and information exchange concerning results obtained by Dr. M. Green's research group were held. This includes the mechanism of the trans-acting function of the E1A protein domain 3, which is known as an essential region for transactivation of other adenovirus early genes such as E2 and E3. Dr. M. Green's research group is working very actively on the above using microinjections of syntetic polypeptides. The recent results obtained by his research group are summarized as follows. (1) A synthetic peptide of 49 amino acids, encoding mainly adenovirus E1A protein domain 3 (PD3), efficiently activates an E1A-inducible promoter. This peptide is the smallest known protein fragment functioning as a transcriptional activator. (2) PD3 rapidly transactivates E1A-inducible early viral genes in the presence of inhibitors of protein synthesis, as demonstrated by microinjection-in situ hybridization experiments. (3) PD3 greatly stimulates transcription of E1A-inducible genes in vitro. Mutant PD3 peptides with single amino acid substitutions in conserved cystein residues are defective in transactivation both in vivo and in vitro. (4) These support a model in which E1A modifies the activity of a pre-exising cellular protein(s) involved in transcription activation.
The above discussion and information exchange concerning experimental results and experimental techniques provided an important guide and future perspectives for our research.



(2) Yasushi Yamazoe
Department of Pharmacology
School of Medicine
Keio University

Sponsor and Host Institution:
Dr. Philip S. Guzelian
Medical College of Virginia
Virginia Commonwealth University
Dr. Frank J. Gonzalez
National Cancer Institute
National Institutes of Health
Dr. Fred F. Kadlubar
National Center for Toxicological Research
Food and Drug Administration
Dates of Visit October 1-18, 1988

Summary of Activities:
I visited Dr. Guzelian at Virginia Commonwealth University and learned newly developed techniques for the maintenance of primary hepatocytes including the preparation of a matrigel, which is produced in Engelbreth-Holm-Swarm sarcoma, plating methods of the extract gel and maintenance of hepatocytes. The tumor was kindly supplied to me and transplanted to mice after returning to Keio University. The tumor is now successfully growing and the procedure for the preparation of the matrigel is established in our laboratory. I plan to conduct studies of the expression and regulation of cytochrome P450 and other hepatic enzymes using the matrigel.
At NIH, I met Dr. Gonzalez and discussed the characterization and expression of types of cytochrome P450 induced by steroids in humans. I received very valuable information on the recent progress of the expression of cytochrome P450 using vaccinia. Using this system, we planned to express human cytochrome b₅, which was cloned in Keio University. Drs. Gonzalez and Guzelian asked me to use propoxycoumarin, which was synthesized in our laboratory as a typical substrate of this type of cytochrome P450. I sent the chemical to them when I returned to Japan.
I also visited the National Center for Toxicological Research in Arkansas and discussed the problem of the structural elucidation of arylamine-modified adducts, especially the nucleoside adduct produced in the acetyltransferase-mediated O-acetylation of N-hydroxy-Glu-P-1 in the presence of thiols.



(3) Hiroyasu Esumi
Biochemistry Division
National Cancer Center Research Institute, Tokyo

Sponsor and Host Institution:
Dr. Curtis Harris
Laboratory of Human Carcinogenesis
National Cancer Institute, Bethesda
Dr. Bernard Weinstein
Comprehensive Cancer Center
Columbia University, New York
Dr. Ruth Sager
Dana-Farber Cancer Institute
Harvard Medical School, Boston
Dates of Visit: October 10-29, 1988

Summary of Activities:
The main purpose of this visit to the United States was both to collect information about research on environmental carcinogenesis and to discuss future cooperative research between our laboratory and those of the host scientists.
At the National Cancer Institute, Bethesda, I discussed with Dr. Curtis Harris the way to evaluate risk posed by chemical carcinogens. We discussed the importance of molecular dosimetry by using specific monoclonal antibody against chemical carcinogen-DNA adducts and carcinogen-protein adducts. In addition to this technique, the 32P-postlabeling method is quite important. We decided to exchange our samples and monoclonal antibodies. We also discussed possible new methods of evaluating the risk from specific chemical carcinogens with, for example, in vitro carcinogenesis experiments using primary human epithelial cell culture.
At the Comprehensive Cancer Center, Columbia University, I visited Dr. Bernard Weinstein and again discussed the matter of molecular dosimetry. His group has developed a monoclonal antibody against DNA-benzopyrene adducts and applied it to human samples for assaying exposure to benzopyrene. I gave a seminar entitled "Possible use of analbuminemic rats in the study of risk estimation" and discussed the ways in which risk estimation should be done. We agreed on possible future sample exchange and technical transfer. In addition to this, we also agreed to some collaborative research on colon cancer.
At the Dana-Farber Cancer Institute, I visited Dr. Ruth Sager and discussed recessive oncogene identification and cloning. She and her colleagues have identified several candidates for recessive oncogenes involved in skin and mammary cancer. We discussed a possible exchange of cell lines and probes, and we agreed on future collaborative work.
I Iearned many things during my stay in the U.S. and agreed on several collaborative research projects. I am sure that those agreements will be a big help in studying environmental carcinogenesis in Japan.



(4) Yutaka Hattori
Genetics Division,
National Cancer Center Research Institute,
Tokyo

Sponsor and Host Institution:
Dr. Stuart A. Aaronson
Laboratory of Cellular
and Molecular Biology
National Cancer Institute, Bethesda
Dr. Igor B. Roninson
Center for Genetics
College of Medicine
University of lllinois, Chicago
Dates of Visit: February 20- March 12, 1989

Summary of Activities:
There has been increasing evidence showing that growth factors and their corresponding receptors play important roles in the control of proliferation of normal and cancer cells. Yet, little information is available on the mechanisms involved in the regulation of proliferation exerted by growth factors. We have isolated and characterized a new transforming growth factor gene, HST1. Recently we also identified a novel oncogene, K-sam, encoding a receptor type tyrosine-kinase gene. The receptor for the HST1 protein and the ligand for the K-sam gene product have yet to be determined.
Dr. Aaronson's laboratory has been a world-leading laboratory in the field of research on growth factors and oncogenes. KGF (keratinocyte-derived growth factor) has recently been discovered in his laboratory. Unlike many other growth factors, it renders growth promotive activity specifically on epithelial cells. Sequence analysis reveals that the gene encoding KGF shares sequence homology with HST1 and other FGF family genes. KGF has a putative signal sequence and a heparin binding site as does HST1. Identification of the KGF receptor is now one of the main projects in Dr. Aaronson's laboratory. The elucidation of the KGF receptor will bring us important information on understanding the biological significance of HST1. To understand the role of ligand-receptor interaction at the molecular level, Dr. Aaronson's laboratory has extensively studied the role of the EGF receptor-related genes other than erbB-2 and a new PDGF receptor. We also cloned a new receptor type tyrosine-kinase gene, tentatively designated "K-sam." Discussions with the researchers in Dr. Aaronson's laboratory and exchange of information on the characteristics of HST1 and K-sam were fruitful. Some possible future collaborative work was also discussed. Furthermore, discussion on new techniques required for identification of receptors or ligands was quite rewarding even if the period of stay was only several days.
PCR (polymerase chain reaction) is now one of the most appreciated methods in molecular biology. Dr. Roninson and his staff members conducted extensive research on MDR (multidrug-resistant gene) analysis in various tumor cells. Quantitation of mRNA by PCR and sequencing of DNA fragments obtained by PCR are suitable applications of this method. Histoblotting is being tried by direct transfer and fixation of the tissue on nitrocellulose filter, which is one of the most exciting projects in Dr. Roninson's laboratory. A novel K-sam gene was identified as an amplified gene in poorly differentiated stomach cancer by the DNA renaturation method in gel in our laboratory. This method was originally developed by Dr. Roninson's laboratory. It was extremely fruitful to discuss with the people in his laboratory the technical aspects of this method and the significance of amplified sequence in cancer in general.
Through this program I was able to have extensive discussion and information exchange. I believe this experience will be of great help in my future research on cancer.