SUMMARY REPORTS OF EXCHANGE SCIENTISTS

(1) Norio Hayashi
First Department of Medicine, Osaka University School of Medicine

SPONSOR AND HOST INSTITUTION :
Dr. Michael A. Gerber/Department of Pathology, Tulane University School of Medicine
DATE OF VISITS: October 31-November 8, 1995

SUMMARY OF ACTIVITIES:
The lack of a small animal model of hepatitis C virus (HCV) infection has impeded elucidation of the pathogenesis of HCV We are developing an HCV-expressing animal model by means of cationic liposome-mediated and receptor-mediated gene transfer. An expression vector composed of the HCV gene complexed with lipofectin was injected retrogradely into common bile duct of adult rats. Immunohistochemical analysis showed the HCV core protein in the hepatic lobules. We could also express the HCV protein in, rats using asialoglycoprotein receptor-mediated gene transfer. Now, Dr. Hiramatsu in my department is studying the expression of the b-galactosidase(b-Gal) gene in monkeys using adenovirus vector-mediated gene transfer at Prof. Gerber’s laboratory. In this visit, we confirmed the expression of the b-Gal in the liver. Then, we decided to express the HCV gene in the monkeys using adenovirus vector-mediated gene transfer.
Also, I gave a lecture about the mechanism of liver cell injury induced by Fas ligand-Fas system in type C hepatitis. The Fas is a cell surface protein that mediates apoptosis with treatment of the Fas ligand. Antigen-specific T cell activation requires the presentation of antigen by MHC molecules and the delivery of costimulatory signals. CD28 is one of the most important molecules which can mediate costimulatory signals and CD80 (B7/BB-1) is proved to be a ligand for CD28. To investigate the role of Fas in type C hepatitis, we examined the correlation between liver cell damage and the expression of Fas antigen. Expression of Fas antigen was found mainly in the cytoplasm of hepatocytes and these positive cells were found especially among infiltrating lymphocytes. High prevalence of Fas antigen expression was shown in liver tissue with more severe inflammation. Also, we isolated a cDNA clone for human Fas ligand and examined the expression of Fas ligand in liver-infiltrating mononuclear cells obtained from patients with chronic hepatitis C. Approximately 1.9 kb of human Fas ligand CDNA was clarified. The amino acids sequence of human Fas ligand indicates 76% and 77% identy with those of rat and mouse ligand, respectively The amplified products (231 bp) derived from Fas ligand transcripts were detected in liver-infiltrating mononuclear cells. However, no signal was detected in liver tissues. CD80 expression was detected in HCV infected liver tissues and there was s positive correlation between activity of inflammation and the degree of CD80 expression. These findings suggest that the Fas ligand -Fas system and CD80 may play an important role in liver cell injury and its control in type C hepatitis. Prof. Gerber was very interested in this mechanism, because the modification of this mechanism may induce the elimination of hepatocytes with HCV infection.



(2) Teruhiko Yoshida
National Cancer Center Research Institute, Section for Studies of Metastasis

SPONSOR AND HOST INSTITUTION:
Dr. Michael Sadelain/Memorial Sloan-Kettering Cancer Center,
Dr. Thea D. Tlsty, Ph.D./University of California San Francisco,
Dr. Yutaka Kawakami, M.D./National Cancer Institute,
Dr. Robert Hoffman, Ph.D./University of California San Diego
DATES OF VISIT: September 5-20, 1995

SUMMARY OF ACTIVITIES:
A. Objectives of Study: Genomic Instability of Cancer: The last 15 years of extensive investigation on genomic abnormalities of various human cancers have formulated a central dogma of molecular oncology; carcinogenesis is a multi-stage process characterized by an accumulation of multiple genomic abnormalities. The first objective of the study is to elucidate the origin of such cumulative genomic abnormalities in cancer. “Genomic instability” is still an enigmatic answer, and here I have specifically focused on gene amplification, a form of genomic instability, and tried to develop a new assay to capture this event on the genome as a whole. Metastasis and Therapy of Cancer: Metastasis mostly remains as a synonym of the therapeutic failure, and a new generation of treatment based on a new biology has been eagerly awaited. The recent advent of gene transfer technology together with the rapidly expanding knowledge on genes has enabled the first series of clinical trials of human gene therapy. The second objective of the study is to examine the various new possibilities of gene therapy and gene transfer strategies.
B. Achievements and How Study Relates to Future Work: Genomic Instability of Cancer: Intensive discussion with Dr. Thea Tlsty at University of California, San Francisco has clarified the following important points: 1) the use of human deployed epithelial primary culture is critical to study genomic instability as an early event in the carcinogenesis cascade. Established cell lines may have certain genetic abnormalities, such as p53 mutation, and rodent cells have inherently less stable genomes. 2) FACS and CGH(Comparative Genomic Hybridization) will become powerful tools to scan the entire genome for amplification event without relying on drug selection, which has many limitations. 3) although p53 mutation is one of the molecular mechanisms of genomic instability, there certainly exists the pathway without p53 involvement , and there is no evidence for the presence of a single master “genomic instability” gene. These notions and ideas clearly gave a framework for my next stage investigation to examine the effect of cyclin D1 deregulation on the genomic stability of the human deployed esophageal epithelial cells. Metastasis and Therapy of Cancer: Immune gene therapy is the current major strategy for metastatic and advanced cancers. Through discussion with many investigators in this field, it seems critical to address the heterogeneity of the patients’ response to the immune therapy. A number of factors, such as altered expression of MHC and related molecules, local immunosuppression by TGF may play a role, but prediction and possible modification of therapeutic responsiveness are the urgent goal of immune gene therapy. On the strategy side, identification of tumor-regression antigens and their presentation by professional antigen presenting cells such as dendritic cells is a promising new approach. Systematic cloning of the tissue-specific genes and activation of the tissue specific auto-immune reaction will be considered in my future study to develop targeted gene therapy. The bone marrow protection strategy based on mdr drug resistance gene seems to be encountered with some difficulty in the expression efficiency of the transduced gene. Genetic modification of the mdr gene to enhance expression, drug resistance capability and more strict specificity for the drug will be a way to overcome the problem. In addition to the mdr gene, a range of other drug resistance genes and drugs are being examined, including dihydrofolate reductase gene and methotrexate, aldehyde dehydrogenase gene and cyclophosphamide. Among the various gene transfer vectors which are currently available, the pseudotyped retroviral vectors seem to suite our in vivo gene transfer of the antisense K-ras expression vector for pancreatic cancer, because of the vector’s high titer, excellent in vivo stability, ease of vector construction and specificity for the proliferating cells. We have started a collaborative work with Drs. Friedmann and Miyanohara at University of California, San Diego, to see if the new vector improves our preclinical data of the antisense K-ras vector to suppress peritoneal dissemination in the pancreatic cancer.



(3) Katsumi Imaida
First Department of Pathology, Nagoya City University Medical School

SPONSOR AND HOST INSTITUTION:
Dr. Frank J. Gonzalez, Laboratory of Molecular Carcinogenesis
DATES OF VISIT: March 17-24, 1996

SUMMARY OF ACTIVITIES:
Transgenic mouse and knockout mouse have been used to investigate mechanisms of cancer development in vivo at molecular level. These animals can also be used to determine carcinogenic potential of test chemicals for a relatively short period, instead of using conventional mouse strain, which is required for a long term period. Since I have been involved in carcinogenesis studies using transgenic mice, the purposes of my visit were to learn what kind of transgenic or knockout animals are used by American cancer researchers. What is their purpose to use? Do they think these animals can be used for bioassay instead of using conventional animals? Before starting the visit, I selected following questions for each researchers:
1. What kind of transgenic animals did you use? Transgenic animals? Knockout animals?
2. Do you use double transgenic animals? If so, please list bellow.
3. What is the purpose to use transgenic animals? (mechanistically approach, bioassay, etc.)
4. Do you think that bioassay model using transgenic mice can be replaced conventional long term mouse carcinogenicity assay?
5. Other comments
Dr. Gonzalez, Chief, Laboratory of Molecular Carcinogenesis, NCI has used human peroxisomal proliferator-activate receptor (PPAR) or human CYP1A2, CYP2E1 transgenic mice for carcinogenesis studies of heterocyclic amines and WY14643 (a peroxisomal proliferator). In his laboratory, they also used PPAR knockout mouse. The purpose for using these animals for them was to investigate basic role of receptors of drug metabolizing enzyme on the development of cancer in vivo. I had a chance to discuss with Dr. Fernandoz-Salguero, who introduced dioxin-binding aryl hydrocarbon receptor knockout mouse. Dr. S. Thorgeirsson, Chief, Laboratory of Experimental Carcinogenesis, NCI, explained in detail of his studies using TGF sgenic mouse and bc12 knockout mouse. Organ abnormalities were observed in these mice. Furthermore, double transgenic (TFG and c-myc) mouse study has been already done in his laboratory. Dr. J. M. Ward, Chief, Veterinary and Tumor Pathology Section, Office of Laboratory Animal Science, NCI, is involved in many studies in NIH regarding transgenic and knockout mice as a pathologist. Liver, pancreas and stomach carcinogenesis in TGF transgenic mice and prostate and mammary gland carcinogenesis in SV40 large T antigen transgenic mice studies have been published by him and his colleagues. Furthermore, p53 mice were also used in his laboratory. TGF immunoglobulin gene double transgenic mice and p53 and rb gene double transgenic mice have also been used. His main purpose to use transgenic animals is to investigate mechanisms of carcinogenesis and also to use as a tool of bioassay. In NIEHS, I visited The Chemical Industry Institute of Toxicology (CIIT) to see Dr. T. L. Goldworthy. In CIIT, they have several transgenic animal experiments with Dr. Tennant or Dr. Maronpot, NIEHS. Dr. L. A. Hansen and Dr. J. Spalding, collaborators of Dr. Tennant, explained their studies regarding bioassay system using TG.AC transgenic mice and p53 knockout mice for bioassay. Their collaborating basic studies has been already started including Japanese researchers. In Dr. Maronpot laboratory, I had a seminar entitled “Biological differences tumors in transgenic and non-transgenic animals.” We discussed about biological characteristics of tumors found in transgenic mice. I greatly appreciate to the US-JAPAN Program for giving me the opportunity to meet and to discuss with researchers of this field in the U.S.A. This experience will be very beneficial for me to our future research using these interesting animals.