SUMMARY REPORTS OF EXCHANGE SCIENTISTS

(1) Yusuke Wataya
Faculty of Pharmaceutical Sciences
Okayama University, Okayama

Sponsor and Host Institution: Dr. Daniel Santi
Department of Biochemistry and Biophysics
University of California
San Francisco
Dates of Visit: January 20-March 6, 1987

Summary of Activities:
I worked on “Metabolism of Mutagenic Nucleoside-Analogs in Mammalian Cells” at the School of Medicine at the University of California, San Francisco, during the period indicated above.
Nucleoside analogs, either given as such to cells or generated by actions of reagents on monomeric or polymeric nucleic acid components in vivo, can cause mutations of the cells. On the other hand, modified nucleosides are sometimes useful as therapeutic agents. For example, 5-fluorouracil and 6-mercaptopurine are anticancer drugs, 5-bromovinyldeoxyuridine (BVdU) is an antiherpes agent, and azidothymidine is an anti-AIDS drug.
During this visit, I studied two specific subjects related to these biological activities of nucleoside analogs. The first was an investigation of the cellular metabolism of 2’-deoxy-2-(p-nitrophenyl)-adenosine, which we had found in our home institute to be strongly mutagenic towards various cells, and the second was a study on the mechanism of BVdU.
In the first subject, I used mouse S49 cells in culture which were available in Dr. Santi’s laboratory. A series of this line of cells that are deficient in individual enzymes in the nucleotide metabolism had been established in his institute. The availability of these mutant strains should facilitate the elucidation of the metabolic pathway for nucleoside analogs. The S49 cells were incubated in the presence of 2’-deoxy-2-(p-nitrophenyl)-adenosine. The cells were then collected, and the low molecular weight components were extracted from the cells by use of trichloroacetic acid precipitation of polymeric substances. The extract was fractionated by high performance liquid chromatography to detect metabolites of this nucleoside analog. Unfortunately, no metabolites of this drug were detected. This negative result was attributed to the low solubility of 2’-deoxy-2-(p-nitrophenyl)-adenosine in the culture medium: during the incubation, a large proportion of the agent apparently precipitated. I discussed with Dr. Santi the prospects of this line of study, and our tentative conclusions are as follows. (1) A derivative of this nucleoside having greater solubility should be synthesized and tested in this system. A good candidate for this purpose would be 2’-deoxy2-(-nitrophenyl)-adenosine 5’-phosphate. Also, Labeling of this compound with ¹⁴C or ³H may be necessary for identifying the metabolites. (2) Since this new class of nucleoside analogs is strongly mutagenic both in prokaryotes and eukaryotes, it is important to elucidate the mechanism of their actions.
For the study of the second subject, Dr. Santi and I decided to test the possibility of BVdU-mediated inhibition of cellular and/or viral thymidylate synthetase. This possibility had never been investigated, which appeared rather surprising to me. I have a long experience in biochemical studies of thymidylate synthetase, especially in kinetic analysis of the action of its inhibitors. This experience was, as a matter of fact, obtained during my previous stay in Dr. Santi’s laboratory as a postdoctorate. I incubated thymidylate synthetase of Lactobacillus casei with BVdU 5’-phosphate in the presence of methylene tetrahydrofolate. Quite interestingly, an inactivation of the enzyme was observed as a function of the time of incubation. This experiment was done in the last stage of my stay; therefore, we decided to pursue this new discovery further, after my return to Okayama.



(2) Jun Yokota
Section of Studies on Metastasis
National Cancer Center Research Institute, Tokyo

Sponsors and Host Institutions:
Dr. Mortimer L. Mendelsohn Lawrence Livermore National Laboratory
University of California, Livermore
Dr. Raymond L. White
Howard Hughes Medical Institute
University of Utah, Salt Lake City
Dr. William F. Benedict
Clayton Molecular Biology Program
Children’s Hospital of Los Angeles
Dates of Visit: February 16-March 6, 1987

Summary of Activities:
Recent advances in molecular biology have made it possible to analyze the abnormalities of cancer cells at the DNA level. Until now, various types of DNA abnormalities in cancer cells have been reported, which are thought to be causally related to the development of cancer. DNA abnormalities are often accompanied by chromosomal alterations. Thus, chromosome analysis of cancer cells gives us much information to identify the DNA abnormalities which would be the cause of cancer. Using techniques of molecular biology, rapid progress has been made in chromosome analysis and soon it will be possible to sequence whole chromosomal DNA. To obtain the most recent information on the analysis of the chromosome at the DNA level, I visited three different laboratories in which chromosome abnormalities in cancer have been analyzed extensively at the DNA level.
At the Lawrence Livermore National Laboratory, a project to produce a chromosome-specific human gene library has been proceeding. Dr. Mendelsohn’s group has already produced complete digest (4 kb average insert size) libraries from each of the 24 human chromosomal types purified by flow sorting. They have begun the construction of libraries with large inserts in a bacteriophage vector (about 20 kb inserts) or in a cosmid vector (about 40 kb inserts). These libraries will make a great contribution not only for the basic studies of gene structure and function, but also for the studies of cancer and genetic disease.
Recently, rapid progress has been made in chromosome analysis by using probes which detect DNA polymorphisms of the human gene. Dr. White is one of the pioneers in this field, applying restriction fragment length polymorphism (RFLP) analysis to do chromosome mapping and linkage analysis. When I visited his laboratory, his group had just successfully isolated a huge number of DNA fragments which detect RFLPs located on various loci of the chromosome. Linkage analysis of most genetic diseases will soon be completed using these RFLP probes. Using these DNA fragments, it is now possible to analyze, in more detail, the chromosome abnormalities in cancer.
Dr. Benedict has proposed that there is anti-oncogene(s) in normal cells and that inactivation of the anti-oncogene is one of the causes of cancer, since chromosomal deletion occurs frequently in childhood cancers. He applied RFLP analysis to detect the specific lesion which is deleted in retinoblastoma, and recently his group has cloned the genes which are frequently missing in retinoblastoma cells. I discussed with him the possibility that the same mechanism might be involved in the genesis of adult cancer, and we decided to collaborate on a project to detect anti-oncogenes in adult cancer.
I learned a lot about recent studies on molecular analysis of human chromosomes during this visit and this experience will help me to expand our research work in Japan,



(3) Katsumi Yamashita
Biochemistry Division
National Cancer Center Research Institute, Tokyo

Sponsor and Host Institution:
Dr. Kurt Randerath
Department of Pharmacology
Baylor College of Medicine
Houston, Texas
Dates of Visit: February 20-March 19, 1987

Summary of Activities:
The ³²P-postlabeling method was applied to detect DNA adduct formed by cancer therapeutic agents such as adriamycin, cis-platinum, bleomycin, neocarzinostatin and chromomycin A3 which are supposed to form DNA adducts, for the estimation of cancer risks caused by these chemicals in humans. For the detection of DNA adducts formed by these chemicals, a highly sensitive nuclease P1 digestion method, which has recently been developed by the host laboratory, was used. DNA samples which were extracted from the liver and kidney of F344 male rats administered by these chemicals were analyzed with various chromatographic conditions. Of these five types of therapeutic agents, only cis-platinum-treated DNA demonstrated adduct formation and the adduct level was one adduct per 10⁸ nucleotides. The expected adduct level of the sample was about one adduct per 10⁶ nucleotides which was a 100 times higher level than that obtained by the experiment. This might mean that the major adducts formed by cis-platinum would not be detected by conventional techniques because of the hydrophilicity of the compound which confers the similar chromatographic mobility to the normal nucleotides. Taking advantage of the chemical character of platinum moiety of this chemical, cis-platinum-adducted nucleotides might be extracted selectively from the normal nucleotides for the enrichment of adducts. Such a modified procedure would be necessary for the detection of the adducts.
Other compounds, whose adducts were not detected by this experiment using the ³²P-postlabeling method, might exert their mutagenicities or carcinogenicities through manners of DNA damaging other than the formation of adducts, such as DNA strand breaks. For instance, adriamycin is thought to induce DNA strand breaks by intercalating between DNA bases and forming oxygen radicals. Our results may support the hypothesis of this mode of action of adriamycin.
I learned much during my stay in the host laboratory. I would like to express my sincere thanks to this program and especially to the Japan Society for the Promotion of Science for providing me with this opportunity.