(1) Toshikazu Ushijima
National Cancer Center Research Institute
Carcinogenesis Division

Dr. David E. Housman
Center for Cancer Research
Massachusetts Institute of Technology
DATES OF VISIT: February 25-March 1l, 1994

1. Main subject
Two strains of rats, ACI and Buffalo, are sensitive and resistant, respectively, to MNNGinduced glandular stomach carcinogenesis. Different degree of cell proliferation enhancement in the pyloric region by MNNG is supposed to be the cause of the different cancer susceptibility. Using BrdU uptake as a marker, the responsible locus for the different susceptibility is being mapped in I 05 F2 rats. Further identification of the responsible gene in the chromosomal region is the final goal of this study.

2. Achievements

  1. Presentation and discussion of the main subject: A seminar on the main subject was given, and several critical and encouraging suggestions were obtained. They included necessity of a hypothesis on the mechanism of different cell proliferation enhancement, verification of the role of cell proliferation in carcinogenesis, introduction of a scientist who is interested in genome mapping in rats, and some technical comments on linkage analysis. The main subject is being modified according to these suggestions.
  2. Collection of information on the best approach from a locus to a gene: Superiority of YAC libraries to P1 phage libraries was suggested in the case of our main subject. Exon trapping, the routine procedure after covering the chromosomal region of interest by YAC clones, was studied. Computational devices to assist exon trapping was surveyed in several laboratories.
  3. Training in handling of a YAC library: Addressing system of Yac clones by three dimensional axis's was learned. Identification of clones of interest by PCR and also by Southern blot was tried, and merits of demerits of the two methods were discussed. I was trained in the techniques isolate YAC DNA from 96 well plates. These techniques will greatly help me to handle YACS in the near future.

(2) Yoshiaki Ito
Institute for Virus Research
Kyoto University

Dr. Peter J. Gergen
Department of Biochemistry and Cell Biology
State University of New York at Stony Brook
Dr. Tom Curran
Department of Molecular Oncology & Virology
Roche Institute of Molecular Biology
Hoffman-La Roche Inc.
Dr. Céline Gélinas
Center of Advanced Biotechnology and Medicine
Dr. Joseph R. Nevins
Howard Hughes Medical Institute
Department of Genetics
Duke University Medical Center
DATES OF VISITS: September 1-13, 1993

Dr. Peter Gergen: The transcription factor, PEBP2, that we are studying has two subunits,!!!and!!!. cDNA cloning revealed that the!!!subunit is highly homologous to two other proteins in a 128 amino acid region: the products of Drosophila segmentation gene, runt, and the human gene, AML1, detected at the breakpoint of 8 and 21 chromosome translocation, which is a characteristic karyotypic abnormality of the FAB M2 subtype of acute myeloid leukemia (AML). Available evidence suggests that AMLI is indeed involved in leukemogenesis. Recently, the gene encoding the human homologue of the!!!subunit was also found at another subtype (M4Eo) of AMLI , indicating that PEBP2 is a critically important factor in the transcriptional regulation in the cells in myeloid lineage and myeloid leukemogenesis.
Dr. Gergen has isolated cDNA clones of the Drosophila runt gene. In order to accelerate our research on Runt/AML1/PEBP2, we agreed to collaborate on characterization of the genes and their products. Earlier, our joint effort revealed that the Drosophila Runt protein forms a heterodimer with the mousee!!!protein and binds to the PEBP2 site (Kagoshima et al., Trends in Genetics, 8:338-341, 1993). Since the!!!-like gene and protein are not known in Drosophila, our mouse studies have given a valuable information to the Drosophila researchers. We are jointly trying to identify Drosophila!!!-like protein and its gene. Once we detected it, the Drosophila system will be powerful tool to analyze the function of the !!!protein, since the Drosophila system has well-established genetics.
With three other researchers, we jointly applied for a Human Frontier Science Program Research Grant at the end of August. During my visit, we discussed the detailed plans for this proposed research project. We will broadly study the structure and function of our transcription factors, especially from the point of view of developmental regulation and leukemogenesis.
Dr. Tom Curran: We have an ongoing collaboration on the role of c-Jun and c-Fos in polyomavirus DNA replication. In my laboratory in Kyoto, we are analyzing this in the in vitro polyomavirus DNA replication system. Dr. Curran is supplying us E. coli-produced purified c-Jun and c-Fos. We discussed the current status and the future plan. I requested deletion derivatives of the proteins for the purpose of examining a possible interaction between these proteins and Replication Protein A (RPA), which was described recently to interact with transcription factors containing the acidic activation domains y
Dr. Céline Gélinas: We have a long-standing collaboration on the role of Rel oncoprotein in polymovirus DNA replication. Contrary to popular belief, we found that there is a clear dissociation between the domain responsible for stimulation of transcription and the one for stimulation of replication. The evolutionarily conserved DNA binding and dimerization domain at the N-terminal region, referred to as the Rel homology region (RHR), retains the activity to stimulate DNA replication, while the transcription activation domain is located at the C-terminal end (Ishikawa et al. Oncogene, 8:2889-2896, 1993).
Dr. Gélinas's lab is primarily responsible for generating mutants of Rel proteins, while DNA replication assay is mainly performed in my laboratory. Jointly, we found at least one mutant which retains the DNA binding ability but lacks the ability to stimulate DNA replication. Which biological activity is missing in this mutant is an extremely interesting question. We discussed about our plan to answer this question.
Dr. Joseph R. Nevins: We have a lot of common interests on transcriptional regulation. I gave a seminar on PEBP2 transcription factor and its involvement in leukemia. We exchanged information on cell cycle regulation, especially from the point of view of the involvement of pRb, E2F, cyclins and cyclin-dependent kinases and their possible role on the initiation of DNA replication. In addition to visiting these scientists, I attended two meetings in Cold Spring Harbor: "Transcription of eukaryotic mRNAs" and "Eukaryotic DNA replication." I exchanged information throughout the meeting with many scientists.

(3) Takahiro Ochiya
National Cancer Center Research Institute
Genetics Division

Dr. Stuart A. Aaronson
Director, Derald H. Ruttenberg Cancer Center
The Mount Sinai Medical Center
DATE OF VISIT: February 22-March 2, 1 994

Dr. Stuart A. Aaronson (The Mount Sinai Medical Center, New York): I have been working on the effect of HST- 1 on mouse development. HST-1 is first identified as a transforming gene and also known as fibroblast factor (FGF4). By using antisense oligonucleotides, I found HST-1 expression is required for limb development. Dr. Aaronson is extensively studying the role of FGFs and their receptors on tumor growth as well as cell differentiation. After I presented my new data on HST-1, we discussed much about the significant role of FGFS in the field of interests. Among them he gave me very important information on the HST-1 detection system. Identification of the exact amount of HST-1 protein is highly significant in our system. His suggestions will be of great help in furthering my analysis of HST-1.
Dr. Elliot Kieff (Harvard Medical School, Boston): I am trying to use viral vectors for introducing a couple of interesting genes into the animals to see their in vivo effect. Dr. Kieff has been working on EB virus and also is interested in the use of viral vectors for human genes therapy. It was a good opportunity to discuss viral vectors and I received much information about adenovirus vectors on human gene therapy which has done recently in the United States.
Dr. William B. Stallcup (La Jolla Cancer Research Foundation, San Diego): Recently, a novel method to produce chimeric mice from ES-morula aggregates was developed. This method is an easy and rapid method compared to the injection system. Therefore, if it works, it will save time and money for producing knock out mice. This method is working well in Dr. Stallcup's lab. He kindly showed me a lot of his excellent techniques for making good aggregates, and I had a chance to make them by myself. I have learned many things from Dr. Stallcup on this new method, and I am sure that they will contribute to making HST-1 knock out mice in my lab.