SUMMARY REPORTS OF EXCHANGE SCIENTISTS

(1) Hiroyuki Tsuda
Fujita Health University, School of Medicine, Tokyo

Sponsor(s) and Host Institution(s):
Dr. James E. Klaunig
Laboratory of Environmental Toxicolgy and Carcinogenesis
Department of Patholog
Medical College of Ohio
Dr. George K. Michalopoulos
Duke University Medical Center
Dr. Curtis C. Harris
Laboratory of Human Carcinogenesis
National Cancer Institute
Dates of Visit: October 16-28, 1990

Summary of Activities:
Objective
Alterations in cellular behavior and morphology, which are associated with tumor progression, have been well described. However, the mechanisms underlying the acquisition by tumor cells of the capacity to show local invasion and remote metastasis are still obscure. These phenomena appeared to develop in a multistep manner possibly due to a complex of alterations in phenotypic expression.
The purpose of the present study was to obtain information regarding factors causing malignant conversion of premalignant or preclinical tumors in man and rodents and to assess potentiai application for the diagnosis and control of progression.
The project topics included (1) analysis of gap-junction expression during progression of hepatocarcinogenesis (Dr. J.E. Klaunig), (2) investigations of tissue distribution of hepatocyte growth factor (HGF) and characterization of its role in local invasion and remote metastasis (Dr. G. K. Michalopoulos), (3) study of tumor suppressor p53 gene point mutations during tumor progression in man and discussion of possible application in animal models (Dr. C. C. Harris).

Achievements
(1) Careful examination of mouse liver slides stained with connexin 32, which is normally localized on the surface of the hepatocytes as small spots, revealed a decrease in the number apparently correlating with progression of hepatocarcinogenesis. This finding has stimulated us to start collaborative work for further investigation of alteration in this parameter during malignant conversion in organs other than the liver.
(2) The organ distribution of anit-rabbit and-rat HGF raised in chickens was discussed. HGF was found positive in GI tract epithelium, pancreas acinar cells, bronchial epithelium, distal tubules and collecting ducts of the kidney, large nerve cells, fibroblasts, cartilage cells, syncytial trophoblasts, thyroid parafollicular cells, and megakaryocytes. The results suggest correspondence to provision by involved cells of HGF, with important implications for metastasis. Collaborative work in this area is planned.
(3) The occurrence of mutations in the p53 gene in organs other than previously reported was discussed. Variable incidences (40 to 70%) of point mutations were found to be largely localized within four regions in carcinoma lesions in the esophagus, pancreas, and liver. Discussions of methodological details and practical problems regarding detection of p53 mutations in rat tumors also will provide the basis for future research on kidney, lung, and thyroid lesions at the home institution.



(2) Hiroshi Hamada
The University of Tokyo, Tokyo

Sponsor(s) and Host Institution(s):
Dr. Phillip A Sharp
Massachusetts Institute of Technology
Dates of Visit: August 27-September 14, 1990

Summary of Activities:
Oncogenic transformation of cells is accompanied by many biological changes. Each change is perhaps caused, at the fundamental level, by regulation of transcription from subgroups of genes. Furthermore, it has recently been elucidated that most of the oncogenes have activities which signal changes in gene expression. Some oncogenes can specify growth factors, hormone receptors, and intracellular signal transduction proteins. Some nuclear oncogenes are transcription factors and others may regulate the activity of transcription factors. Therefore, elucidation of regulation of transcription in eukaryotes is important for cancer research.
I discussed some topics relating to factors involved in transcription initiation, oncogenic transformation, cell growth control, differentiation and signal transduction with host scientists in the United States.
From August 27 to August 29, I visited Dr. A J. Berk (University of California, Los Angeles). We each discussed our current research and I learned his group's progress. They have successfully cloned a transcriptionally active human TATA binding factor (TFIID). The carboxyl-terminal 181 amino acids of the human TFIID (339 amino acids) shares 80% identity with the TFIID protein from Saccharomyces cerevisiae. The amino terminus contains an unusual repeat of 38 consecutive glutamine residues and the X-Thr-Pro repeat. They have purified the proteins using the cloned cDNA and tried to elucidate the interaction between TFIID and adenovirus E1A proteins. We are now studying the interactions of TFIID with either E4TF1 or E4TF3, identified and purified in our laboratory, in collaboration with his group.
From August 29 to September 1, I visited Dr. M.-C. Hung (University of Texas) who has been working on factors responsible for transcription of the c-erbB-2 gene. We have recently developed sequence-specific DNA affinity latex particles to purify DNA-binding transcription factors. Using this method, we purified two mammalian transcription factors, E4TF1 and E4TF3, to homogeneity from HeLa cell extracts and characterized them. E4TF1 is composed of two distinct subunits identified as 60 kd and 53 kd polypeptides, which interact in order for E4TF1 to function as a transcription factor. We are now cloning cDNAs of the 60 kd and 53 kd proteins on the basis of their partial amino acid sequences Similar sequences to the E4TF1-binding site are present in the promoter region of cellular oncogenes, including the c-erbB gene. Both of us are interested in the regulation of transcription from the c-erbB-2 gene by E4TP1. We exchanged information about our current research.
From September 1 to September 9, I mainly visited with Dr. J. L. Manley (Columbia University) and discussed our current data with him. He gave me information about a protein factor, ASF, responsible for cell-specific alternative splicing of SV40 early pre-mRNA, and a multisubunit factor, CstF, required for polyadenylation of mammalian pre-mRNAs in addition to Drosophila Gap proteins which either positively or negatively regulated expression of the engrailed gene. They are trying to establish an in vitro Drosophila transcription system. I also visited Dr. C. Prives (Columbia University). She gave me information about assembly of functional U1 and U2 human-amphibial hybrid snRNPs in Xenopus laevis oocytes, and several topics relating antioncogene products including p53 and Rb proteins. She also showed me the functional interaction of SV40 T antigen with p53 using an in vitro SV40 DNA replication. During my stay in New York, I visited Dr. R. G. Roeder (Rockefeller University) and exchanged information about basic transcription factors including RFIID and RFIIG, and our recent data. They have also cloned human RFIID and are now trying to isolate all cDNA clones of the basic transcription factors.
From September 9 to September 13, I mainly visited Dr. P. A Sharp (Massachusetts Institute of Technology) and discussed our current research with him. He discussed information about regulation of transcription and RNA splicing of HIV and HSV. I also held discussions with Dr. M. Green (University of Massachusetts Medical Center) who has developed a transcription factor, ATF, and ioslated several ATF cDNA clones. We ha purified E4TF3 from HeLa cell extracts using the affinity latex particles. The binding site of E4TF3 is the same as ATF, indicating that E4TF3 is identical to ATF. The g affinity-purified E4TF3 represented at least eight polypeptides of molecular weights: 36 kd, 39 kd, 43 kd, 45 kd, 47 kd, 60 kd, 66 kd and 80 kd. Two of them, the 43 kd and 47 kd polypeptides were eluted from preparative SDS polyacrylamide gels, denatured, and then renatured. Each renatured protein has the activities of both DNA binding and stimulation of transcription in vitro. Each purified protein formed a homodimer. These two homodimers were easily altered into a heterodimer when mixed together in the absence of the specific DNA sequence, whereas not in the presence of it. Differential binding and transcriptional activation activities of these different dimers were observed. It therefore, is interesting to elucidate the regulation of dimer formation of ATF/E4TF3. Since ATF/E4TF3 is included in the transcription factors whose activities are regulated by the adenovirus oncogene, E1A it is also interesting to know whether E1A proteins regulate the dimer formation of ATF/E4TF3. I discussed the function of myc protein with Dr. R. E Kingston (Massachusetts General Hospital) and also discussed the factor required for SV40 late transcription with Dr. U. Hansen (Dana Farber Cancer Institute). During my stay in Boston, I visited Dr. R. J. Kaufman (G. I. Institute) and exchanged information about eIF2, eIF4B and adenovirus VA RNA, which are involved in regulation of mammalian translation. I also discussed our current research.



(3) Masao Yamada
Genetics Laboratory
National Children's Medical Research Center, Tokyo

Sponsor and Host Institution:
Dr. Terri Grodzicker
Cold Spring Harbor Laboratory
Dates of Visit: December 1-14, 1990

Summary of Activities:
From December 1 to December 10, I visited several research institutes and universities in the New York area as described below, and then visited the Massachusetts Institute of Technology in Boston on December 11 and 12.
(1) Cold Spring Harbor Laboratory (CSHL), New York. During my stay, I presented our recent work on cell type conversion by artificial amplication of the human N-myc gene. I also met with several researchers individually to discuss our and their research results as well as broader aspects of gene regulation in mammalin cells and oncogene research. In addition, I participated in several seminars held at CSHL, joined group discussions concerning ongoing research and spent a couple of days conducting experiments to learn new methods that are currently being used in the laboratory.
(2) Professor Frederick W. Alt, College of Physicians and Surgeons of Columbia University, New York. I visited the University and gave a seminar on our recent results entitled "Conversion of HeLA Cell to Neurogenic Cells by N-myc Amplication." Before and after my seminar, I met with several researchers in the University, including Assistant Professor Mitchell Goldfarb, who is an oncogene researcher and used to work at CSHL, to discuss our and their research projects. Since Professor Alt is one of the discoverers of the N-myc oncogene and is a leading scientist in the field of nuclear oncogenes, his comments on our results as well as discussions with his fellows were very useful for me to continue this research project.
(3) Dr. Jack Silver, Chief, Division of Molecular Medicine, North Shore University Hospital, New York. I visited Dr. Silver's laboratory to discuss expression regulation of the Thy-1 gene as well as our results. He expressed his interest in our results and promised to help us in the analysis of Thy-1 expression in our engineered HeLa cells.
(4) I met with Dr. Yakov Gluzman at Lederle Laboratory, who used to work at CSHL where he studied SV40 and an adenovirus vector. We discussed our and their recent research results.
(5) Dr. David E. Housman, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts. I met with Dr. Housman and Dr. Daniel Haber to discuss our results of analyses of Wilms' tumor in Japanese patients using his clone.
(6) Dr. Earl Ruley, Center for Cancer Research, Massachusetts Institute of Technology Cambridge, Massachusetts. I visited Dr. Ruley's laboratory to discuss our and their recent results.

ACKNOWLEDGEMENTS:
This exchange program enabled me to visit several leading laboratories in the U.S. and touch the cutting edge of science. I thank the Japan Society for the Promotion of Science Drs. T. Sugimura and S. Nishimura, Coordinator and Adviser of this program respectively, for giving me this wonderful opportunity.