(1) Naoto Aoki
Tokyo University, Department of Pathology
Sponsor and Host Institution:
Dr. W. S. Robinson
Department of Medicine, Stanford
Dr. W. Mason
Fox Chase Cancer Institute
Dr. J. L. Gerin
Georgetown University
Dates of Visit: October 21 - November 11, 1984
Summary of Activities
At Stanford University, Dr. Robinson told me that hepatitis B virus (HBV) DNA replication forms in the liver of hepatitis B surface antigen-positive (HBsAg) patients. In their study using the Southern blot method, they showed distinct bands at the position expected for linear double-stranded DNA of 3.6 kb and 2.0 kb. These DNA bands were shown to represent relaxed circular and closed circular (supercoiled) HBV DNA, respectively. When infected cell nuclei were isolated and nuclear and cytoplasmic nucleic acid separately analyzed, the nuclear fraction contained the 2.0-kb DNA species. This species was shown to be supercoiled 3.2-kb HBV DNA by electron microscopy, restriction endonuclease digestion, and thermal denaturation. Although they found free and integrated HBV DNA in hepatocellular carcinoma (HC) tissue, our data suggested only the integrated HBV DNA exists in HC. We discussed this difference and agreed that collaborative work was needed. Professor Robinson agreed to give us his strand-specific HBV DNA which was cloned in M13mp8 and M13mp9 vector.
At the Rancho and USC Liver Unit, Professor Peters and I discussed the technical aspects of the extraction of tissue DNA from autopsy and surgical materials. They have some difficulty extracting undamaged long DNA from these materials. I suggested more gentle techniques of tissue homogenization. They also have some false positive bands in Southern blot autoradiography which seemed to be due to the use of plasmid and the HBV DNA complex as a probe. Therefore, I suggested the use of a purified HBV DNA complex as the probe, and I explained how to purify the HBV DNA from the plasmid complex.
At the Fox Chase Cancer Center Dr. Mason and I discussed the mode of duck HBV (DHBV) replication and tissue tropism of DHBV. The presence of DHBV antigen in exocrine and endocrine pancreatic tissue was very interesting because few researchers have reported the existence of HBV antigen in human pancreatic tissue. Dr. Summers agreed to give me cloned woodchuck hepatitis virus (WHV) DNA in E. coli. This will facilitate our research on woodchuck hepatitis WHV and its relation to hepatocarcinogenesis. Dr. DeTolla of Dr. Millman's laboratory asked me to collaborate in making monoclonal antibodies against HBV antigens. I offered him human spleen lymphocytes extracted from HBV marker-positive patients. At the Center I gave a talk on the results of the HBV DNA state in hepatocellular carcinoma, liver cirrhosis, and hepatitis in the Japanese population.
1. The Program assisted me in reaching my research objectives. First, I was given much information which will be helpful as I plan future research. Second, I was given many materials I need for my research, such as cloned WHV DNA and strand-specific HBV DNA. I would be unable to complete my research without these materials. Third, Dr. DeTolla and I developed a new collaborative program.
2. The enhancement of progress in my research effort is contained in the above response.
3. In my visits, my hosts and I recognized the differences in our data, a development that will facilitate mutual understanding and precise analysis of the relation between HBV infection and hepatocarcinogenesis. A new collaborative program was made between our laboratory and Dr. DeTolla's, which could contribute to the progress of the US-Japan Cancer Research Program.
(2) Kazuhiro Sogawa
Cancer Institute, Department of Biochemistry
Sponsor and Host Institution:
Dr. Charles B. Kasper
McArdle Laboratory for Cancer Research
Dates of Visit: March 30 - April 30, 1985
Summary of Activities
(a) I attended the Workshop "P-450 Genes and Their Regulation which was held at Airlie House in a suburb of Washington, D.C. from March 31 to April 3. About 70 people got together at the workshop, and 37 papers were presented. I gave a talk entitled "Structural comparison of two MC-inducible cytochrome P-450 genes in rat."
(b) During the stay at McArdle Laboratory (April 3 to April 30), I worked on several new research projects concerning the genetic engineering of the enzyme components in the electron pathway of liver microsomes.
1. Expression of cytochrome P-450 oxidoreductase in E. coli. An expression plasmid containing cytochrome P-450 oxidoreductase cDNA had been constructed under the control of!!
!PR promoter when I visited the laboratory. However, the cytochrome P-450 oxidoreductase was not detected in the extracts from bacteria harboring the plasmid by the analysis of SDS slab gel electrophoresis. Dr. Kasper and I discussed the possible causes of the barely detectable expression of the cDNA in the bacteria. We had an agreement that one of the causes is the membrane-bound property of the protein. Therefore, I constructed an expression plasmid from!!!PR promoter and the cDNA with deletion of the DNA sequence coding for membrane-binding domain of the oxidoreductase. I also constructed another expression plasmid from the oxidoreductase cDNA and Trp-lac (tac) promoter. The expression activity of the two recombinant plasmids is currently being assayed in Dr. Kasper's laboratory on a collaborative basis. 2. Isolation of human cytochrome b5 reductase cDNA clones. I also worked on the cloning of cDNA for b5 reductase from a human cDNA library which was constructed from a cell line (MCF-7 cells) using!!
!gt11 as the cloning vector. About 750,000 plaques were screened according to new procedures developed by Wood et al. (Proc. Natl. Acad. Sci. USA 82:1585-1588, 1985) using synthetic oligonucleotide as the probe. Under these hybridization conditions using tetramethyl ammonium chloride, the effect of the GC content of the oligonucleotide on the hybrid formation can be neglected so that the washing temperature may be determined only by the length of the oligonucleotide probe. Eight plaques have been selected as the candidates for recombinant phages carrying b5 reductase cDNA. Further confirmation will be continued in Dr. Kasper's laboratory. (3) Yoshinori Urano
Department of Pathology, Faculty of Medicine
University of Tokyo
Sponsor and Host Institution:
Professor G. N. Stemmermann, M.D.
Kuakini Medical Center
Dates of Visit: September 8-18, 1984
Summary of Activities
1. Objectives of this study consist in the research for US-Japan comparative cancer epidemiology from the autopsy data.
2. Achievements
In order to accomplish the US-Japan geographical comparative cancer epidemiology, two databases are necessary, i.e., that of Japan and that of the United States. We have already the database of autopsy records of Tokyo University (31,000 cases from 1883 to 1982) and that of the registered autopsy cases all over Japan (270,000 cases from 1974 to 1981). We are going to make a new database of the autopsy records of Kuakini Medical Center in Hawaii with courtesy of Professor G. N. Stemmermann. I have gathered the records of 1683 cases which were autopsied in Kuakini Medical Center from 1970 to 1983. Kuakini Medical Center was established in 1900 by the Japanese Benevolent Society and has been called the Hospital for Japanese. So the most of the patients of this medical center were Japanese. For example, among 101 cases which were autopsied in this hospital in 1983, 28 cases were Japanese-Isseis and 45 cases were Japanese-Nisseis, i.e., 72.3 of the autopsy cases were Japanese. So the autopsy data of the Kuakini Medical Center were very precious for the analysis of a role of environmental factors in carcinogenesis in comparing the cancers of Japanese in Japan and those of Japanese in Hawaii. They were different from each other in that they have lived in different environments although belonging to the same race. For this study, the first step was gathering autopsy data of the Kuakini Medical Center. It was accomplished with the aid of US-Japan Cooperative Cancer Research Program.
Now we are making the database from these autopsy data. This work will be finished in a few months. Then our research concerning US-Japan comparative cancer epidemiology will be started. We are looking forward to doing the analytical epidemiological study of the cancer cases by using a new database. We hope that fruitful results will be produced from this study.
Until now, little has been known about the comparative geographical cancer epidemiology from the autopsy data. Our efforts therefore would be a starting point for this kind of study.