SUMMARY REPORTS OF EXCHANGE SCIENTISTS

(1) Nam-ho Huh
Institute of Medical Science
University of Tokyo

Sponsors and Host Institutions:
Dr. Anthohy E. Pegg
The Milton S. Hershey Medical Center,
The Pennsylvania State University
Dr. Curtis C. Harris
National Cancer Institute
Dr. Eliezer Huberman
Argonne National Laboratory
Dates of Visit: August 12 - September 3, 1987

Summary of Activities:
The aim for the visit to Dr. Pegg’s laboratory was to learn a new methodology for an enzymological approach of O⁶-alkylguanine removal in mammalian cells, and to discuss the strategy to isolate a gene of mammalian O⁶-alkylguanine repair enzyme. During my stay in Hershey, I learned one of the most reliable assay methods of O⁶-alkylguanine DNA alkyltransferase in detail as well as a newly established, most sensitive assay, which has not yet been published. Combination of the enzyme assay with our presently available sensitive method to quantitate alkylated DNA adducts will lead to a better understanding of the relevance of cellular repair mechanisms for alkylated DNA adducts to carcinogenesis.
In Dr. Harris’ laboratory, NCI, I discussed with a number of staff scientists present problems and future perspectives of molecular epidemiology. Through the discussion, several technical problems became clear, which need to be improved further.
In Dr. Hubermans’ laboratory, I had a number of opportunities to talk about our ongoing experimental projects, including detection of O⁴-ethyldeoxythymidine in human liver DNA, removal of O⁶-ethyldeoxyguanosine from rat liver mitochondrial DNA, and sensitization of mammalian cells by O⁶-methylguanine to cytotoxicity of genotoxic substances. Critical comments as well as appreciation of our study encouraged me to further intensify my effort in this direction.



(2) Wolfgang Zacharias, Ph D.
Dept. of Biochemistry, Schools of Medicine and Dentistry
University of Alabama at Birmingham
University Station, Birmingham, AL 35294

Sponsor and Host Institution:
Dr. Hikoya Hayatsu
Faculty of Pharmaceutlcal Sciences, Okayama University
Tsushima, Okayama 700, Japan
Dates of Visit: September 5 September 26, 1987

Summary of Activities
Objective:
To determine whether KMnO_⁴ can be use as a chemical probe for the detection and analysis of DNA secondary structures, especially junction regions between right-handed B-DNA and left-handed Z-DNA, in supercoiled plasmids.

Achievements:
It could be demonstrated that KMnO₄ (at 4°C, pH 4.3, 0.5-1.0 mM KMnO₄, reaction times 10 to 60 min.) specifically modifies bases within B-Z junction regions, as detected by the inhibition of cleavage of restriction sites located within these B-Z junction regions. It was shown that this effect was: (a) dependent on the negative superhelical density of the recombinant plasmid; (b) observed for two different Z-DNA-forming sequences (dC-dG inserts as well as dT-dG inserts); (c) observed for two different restriction sites (BamHI and EcoRI); (d) not observed for restriction sites located outside of the B-Z junctions.

Relation to future work:
After appropriate adjustments of reaction conditions, it may be possible to use KMnO₄ as a probe for altered DNA secondary structures in general. Application of a variety of such probes is essential for our ongoing investigations on correlations between DNA secondary structure formation and the regulation of gene expression.
After finalizing the study and improving some of the data obtained so far in Okayama, we are planning to submit this work for publication in a refereed journal.



(3) Dr. Katsuyuki Yaginuma
Department of Gene Research, Cancer Institute,
Japanese Foundation for Cancer Research
Kami-Ikebukuro, Toshima-ku, Tokyo 170, Japan

Sponsor and Host Institution:
Dr. Jesse W. Summers
Institute for Cancer Research,
Fox Chase Cancer Center
Philadelphia, Pennsylvania 19111. USA
Dates of Visit: September 27 - October 30, 1987

Summary of Activities:
Chronic infection of hepatitis B virus (HBV) is known to be closely related to the occurrence of human hepatocellular carcinoma. Furthermore, it was experimentally revealed that chronic infection of other hepadna viruses, woodchuck hepatitis virus (WHV) and ground squirrel hepatitis virus (GSHV), caused liver tumors at a high rate. However, the lack of an in vitro culture system of HBV has impeded the understanding of the gene expression of HBV in the viral life cycle and its role in hepatocarcinogenesis.
The first report in the establishment of a culture system was published by Dr. Summers’ group. They established an in vitro infection system of duck hepatitis B virus (DHBV) by using primary-cultured liver cells of duck. However, it was not suitable for an infection cycle because of changes in the cell phenotypes, suggesting that the phenotypes of normal liver cells were necessary for the infection of DHBV. On the other hand, the infection of HBV to cultured human liver cells have long been unsuccessful. Therefore, DNA transfection experiments of the cloned HBV DNA were conducted to overcome the barrier for virus infection to cultured cells. Dr. M. Essex’s group was the first to succeed in selecting the stable transformant of the human hepatoblastoma cell, HepG2. In their system, the HBV genome was integrated into the cellular DNA and HBV particles were produced constitutively. In contrast, we first reported an in vitro system for HBV production by the transient expression of transfected HBV DNA using the human hepatocellular carcinoma cell, HuH-7, as a recipient. The advantage of our system was that the genetic information of HBV was easily manipulated by the in vitro mutagenesis of template HBV DNA. Additionally, this transient expression system made it possible to examine whether other hepadna viruses could propagate in the human cells. In this cooperative cancer research program, therefore, I planned the experiment to try to produce other hepadna viruses, woodchuck hepatitis virus (WHV) and ground squirrel hepatitis virus (GSHV), caused liver tumors at a high rate. However, the lack of an in vitro culture system of HBV has impeded the understanding of the gene expression of HBV in the viral life cycle and its role in hepatocarcinogenesis.
The first report in the establishment of a culture system was published by Dr. Summers’ group. They established an in vitro infection system of duck hepatitis B virus (DHBV) by using primary-cultured liver cells of duck. However, it was not suitable for an infection cycle because of changes in the cell phenotypes, suggesting that the phenotypes of normal liver cells were necessary for the infection of DHBV. On the other hand, the infection of HBV to cultured human liver cells have long been unsuccessful. Therefore, DNA transfection experiments of the cloned HBV DNA were conducted to overcome the barrier for virus infection to cultured cells. Dr. M. Essex’s group was the first to succeed in selecting the stable transformant of the human hepatoblastoma cell, HepG2. In their system, the HBV genome was integrated into the cellular DNA and HBV particles were produced constitutively. In contrast, we first reported an in vitro system for HBV production by the transient expression of transfected HBV DNA using the human hepatocellular carcinoma cell, HuH-7, as a recipient. The advantage of our system was that the genetic information of HBV was easily manipulated by the in vitro mutagenesis of template HBV DNA. Additionally, this transient expression system made it possible to examine whether other hepadna viruses could propagate in the human cells. In this cooperative cancer research program, therefore, I planned the experiment to try to produce DHBV particles by DNA transfection of the DHBVS genome to HuH-7 cells in the laboratory of Dr. Summers at Fox Chase Cancer Center.
As a template DNA of DHBV, the plasmid pSP65DHBV 5.2 X 2 was used, which had a tandem repeat of two DHBV genomes. Both supercoiled and linearized forms of the template plasmid were transfected to HuH-7 cells (5 X 10⁶ cells/o 10cm) by the calcium phosphate procedure. The vector plasmid was used as a negative control. After 5 days incubation, transfected cells were collected for the preparation of core particles from the cytoplasm. The culture medium was pooled for the preparation of secreted virus particles. The fractions of core and virus particles were treated with SDS and Pronase, and subjected to agarose gel electrophoresis. After Southern blotting to a nitrocellulose filter, the hybridization with ³²P-labeled DHBV probe was carried out to detect replicative intermediate DNAs. As a result, hybridization signals of replicative intermediates were clearly observed in both cases of supercoiled and linearized templates, indicating that DHBV was successfully produced in HuH-7 cells by the transient expression of transfected DHBV DNA. The efficiency of DHBV production in this transient expression system was observed to be almost the same level as that in the HBV production.
In addition, I also tried to establish stable transformants of HuH-7 cells, which contain integrated DHBV DNA in the cellular DNA and produce virus particles constitutively, by the cotransfection of two plasmids, pSP65DHBV 5.2 X 2 and pRSVneo. The plasmid pRSVneo was the expression plasmid, in which the neomycin-resistant gene was ligated to the Raus sarcoma virus (RSV) promoter sequence. After three weeks’ culture with the medium containing G418, many G418-resistant colonies were obtained. However, because my stay was limited, I left them to Dr. Summers for the selection of virus-producing cells.
On the way back, I visited Dr. D. Ganem at the University of California San Francisco, San Francisco, and Dr. P. Marion and Dr. W. Robinson at Stanford University to exchange information with them. Dr. Ganem has also been trying experiments to produce virus particles of other hepadna viruses by the transient expression in HuH-7 cells, which were previously sent to him from our laboratory. Interestingly, he also observed the virus production of DHBV in HuH-7 cells, but the productions of WHV and GSHV were not detected in the same system. Dr. Marion investigated the integrated structures of GSHV DNA obtained from the cellular DNA of ground squirrel hepatoma tissues. She told me that her data could be explained well by our model for the mechanism of HBV DNA integration, which we previously proposed. Dr. Robinson emphasized that it was necessary to produce the intact protein of the X gene by genetic engineering techniques and to characterize its function, because the presence of X gene in the viral genome had been suggested to be related to the occurrence of liver tumors in animal models; e.g., woodchucks and ground squirrels.
Since I was able to establish the in vitro culture system of DHBV production in human HuH-7 cells, my project under the U.S.-Japan Cooperative Cancer Research Program was successfully performed. Hereafter, the combination of this in vitro production system with the in vitro infection system of DHBV will give us a good model system to investigate the pathogenicity of hepadna viruses.



(4) Masao Hirose
Nagoya City University
Medical School

Sponsor and Host Institution:
Dr. H. Witschi
Laboratory of Energy Related Health Research
University of California
Davis, California
Dates of Visit: November 4-November 13, 1987

Summary of Activities:
The objective of this visit was to exchange information on the carcinogenicity of antioxidants, modification of antioxidants on chemical carcinogenesis, and mechanism of action of antioxidants on chemical carcinogenesis. I discussed with Dr. Witschi at the University of California the correlation between antioxidant-induced cell proliferation and tumor promotion. Dr. Witschi showed that BHT induced cell proliferation in the lung and promoted urethane-induced mouse lung tumor formation when it was given after urethane exposure. But, when BHT was given together with SKF 525A or piperonyl butoxide, which inhibited cell proliferation in the lung, after urethane exposure, promotion was still apparent. I showed a similar result that 4-methoxyphenol, which induced hyperplasia in the forestomach, inhibited MNNG-induced forestomach carcinogenesis. Therefore, we shared the same concept that antioxidant-induced cell proliferation does not necessarily correlate with tumor promotion. Further investigations on the mechanisms of difference between these phenomena will be important.
I was advised by Dr. Witschi that we should be very careful when conducting hamster experiments, because hamsters are less sensitive to chemical stimuli when hamsters are given chemicals during the fall and winter season.
I gave a seminar at the American Health Foundation titled “Antioxidants-Carcinogenicity and Modification of Carcinogenesis.” The seminar dealt with carcinogenicity of BHA and caffeic acid on rat forestomach, that of catechol on rat glandular stomach, and promotion of catechol and some other antioxidants on rat forestomach or glandular stomach. Dr. Welsburger said that our animal models will be very useful in the field of non-genotoxic carcinogenesis and tumor promotion. Drs. Hecht and Melikian think that either the carcinogenicity or promotion effect of these antioxidants may be related to free radical reaction. It is very important and timely to study the formation of 8-OH-guanine in vivo in the forestomach or glandular stomach of rats treated with antioxidants which may act as carcinogens or promoters.
This visit, which was supported by the US-Japan Cooperative Cancer Research Program, was very fruitful for me and the discussions with other researchers will be beneficial for my future work.
I greatly appreciate JSPS for giving me an opportunity to visit the US to discuss antioxidants with many researchers.



(5) Yukihito Ishizaka
National Cancer Center Research Institute Tokyo

Sponsor and Host Institution:
Dr. Anthony V. Carrano
Lawrence Livermore National Laboratory
Livermore, California
Date of Visit: March 1-April 4, 1988

Summary of Activities:
I worked on two subjects at Lawrence Livermore National Laboratory (LLNL). One was to establish the cloning method for large molecular weight DNA fragments using an artificial yeast chromosome vector. The other was to determine the chromosome localization of ret oncogene by in situ hybridization.
Since Burke D. T. et al. reported the cloning method of large molecular weight DNA (~800 kb), many laboratories have been trying to construct the library using yeast artificial chromosome. The LLNL has worked on human chromosome 19 and tried to construct a physical map on chromosome 19 using sorted human chromosome DNA from Chinese hamster ovarian hybrid cells which contain only chromosome 19 as human DNA. I tried to establish a method to construct the genomic library of chromosome 19 having ~ 800 kb inserts using an artificial yeast chromosome vector.
During my stay in LLNL, in collaboration with LLNL staff, I discovered that this method should be improved on several points. Yeasts were transformed using the spheroplasting method by which the yeast cell walls were digested. However, the spheroplasting method which we used was not appropriate and most of the cells died after treatment. The optimal condition for spheroplasting should be studied. Furthermore, several yeast strains, other than AB1380 which was used this time, should be checked for their transforming efficiency.
I have learned, however, all the techniques which are necessary for yeast chromosome cloning. After improvement of the points discussed above, I will be able to apply the method for analyzing chromosome aberration in various human tumor cells.
Next the chromosome localization of ret oncogene was determined by in situ hybridization using a cosmid clone which contained proto-ret oncogene as a probe. The cosmid clone was labeled with biotinated dCTP by nick translation. The cosmid hybridized to both 10q11,1 10q11,2 and 12q21 12q23. The cosmid contained about a 40 kb fragment as an insert and proto-ret oncogene localized in a 5’ half of the insert. To exclude the possibility that the gene in a 3’ half of the insert had hybridized to the two spots, we are studying in situ hybridization using a 5’ half of the cosmid which is specific only for proto-ret oncogene.