Tsuyoshi Kakefuda, M.D., Ph.D.

Tsuyoshi Kakefuda received his M.D. and Ph.D. from Tokyo University School of Medicine. Dr. Kakefuda studied pathology in the Dept. of Pathology of this school, worked as clinical pathologist at the 2nd Tokyo National Hospital, and moved to the City of Hope Medical Center, California in 1960. Dr. Kakefuda then moved to the National Cancer Institute where he worked as senior scientist and Acting Section Chief of the Nucleic Acid Section, LMC. During his basic research career he worked in the NCI's Office of International Affairs (1986-1991), and has returned to the LMC where he continues basic research in the laboratory. Dr. Kakefuda is the Executive Secretary of the U.S.-Japan Cooperative Cancer Research Program (1986-present), and was nominated as an Honorary Member of the Japanese Cancer Association in 1993. 

RESEARCH

Effect of Carcinogen Binding on DNA Replication: Colicin E1 plasmid can undergo a round of semiconservative replication in vitro in the system we established in corroboration with Dr. J. Tomizawa. The replication requires de novo RNA synthesis and proper origin, but not protein synthesis. Modification of the parent DNA by covalent binding of benzo[a]pyrene diol- epoxide (BP-DE) blocks the elongation of daughter strand in a dose depended manner in this in vitro assay system. No possibility of bypass, but removal of the binding site during replication was observed.

 Plasmid Mediated Mutation Assay: Plasmid consisting of two dominant genes, beta-lactamase (amp-r) and galactokinase (gal-K) was first modified by carcinogen in vitro and mutation in gal-K was assayed in amp-r transformants. Mutation frequency, up to three percent in the transformants, was linearly related to the BP-DE bindings per plasmid DNA in the uvrA- strain of E. Coli. This plasmid mediated mutation assay which we developed is an outstandingly sensitive and accurate assay system. Another plasmid designed for transcription termination signal inserted in the upstream of gal-K coding region allowed us to determine sequence alteration of the DNA directly related to the failure of signal termination.

 DNA Conformation and Immunological Visualization of Carcinogen Binding: A novel two dimensional gel electrophoresis technique to determine the conformational changes of double-stranded DNA helix was developed. Effects of heat, dehydration, cations, and carcinogen binding were studied. Based on this study, an advanced method for immunochemical detection of BP-DE DNA adducts was developed. Unwinding of the DNA helix by dehydration allowed the binding sites to open to outside of the helix, leading to antibody recognition and more efficient binding to the adducts.

 Involvement of Cell Cycle Alteration in Human Ovarian Cancer Cell Drug Resistance: To explore the cellular and genetic factors that are associated with multi-drug resistance, special efforts are focused on the diminishing effect of the intracellular drug (taxol) content by a combination of alteration of cell cycle, metabolic conversion and drug transport (efflux). The results at the present experimental stage suggest that the accelerated cell cycle, which is sustained by an enhanced salvage pathway of nucleotide biosynthesis in the taxol resistant cells, contributes dilution and diminishing effects of the intracellular drug content, which is enhanced when coordinated with the drug transport (efflux) mechanism.

U.S.-Japan Cooperative Cancer Research Program: Since its inception in May 1974, the Program has been instrumental in establishing a close relationship between researchers in the Unites States and Japan. The Program provides an important means of exchanging scientific information, modern technology, and materials -- significantly contributing to the marked progress which has been made in recent years in cancer research and treatment. The Bilateral Agreement was renewed by the National Cancer Institute and the Japan Society for the Promotion of Science (JSPS) for another five-year period extending from April 1, 1994 to March 31, 1999. The Progress Reports of this program have been published every year, and is available by request.

 

1. Kakefuda, T. and Yamamoto, H.: Modification of DNA by the benzo[a]pyrene metabolite diol-epoxide r-7, t-8 dihydroxy-t-9,10-oxy-7,8,9,10,-tetrahydrobenzo[a]pyrene. Proc. Nat. Acad. Sci. USA. 75: 415-419, 1978.
2. Tomizawa, J., Sakakibara, Y., Kakefuda, T.: Replication of colicine E1 plasmid DNA added to cell extracts. Proc. Nat. Acad. Sci. USA. 72:1050-1054, 1975.
3. Mizusawa, H. and Kakefuda, T.: Inhibition of DNA synthesis in vitro by the binding to DNA of benzo[a]pyrene metabolite diol-epoxide I. Nature. 279: 75-78, 1979.
4. Mizusawa, H., Lee, C-H. R., Kakefuda, T., McKenney, K., Shimatake, H., and Rosenberg, M.: Base insertion and deletion mutations induced in an E. coli plasmid by benzo[a]pyrene-7-8-dihydrodiol-9-10-oxide. Proc. Nat. Acad. Sci. USA 78: 6817-6820, 1981.
5. Lee, C-H., Mizusawa, H. and Kakefuda, T.: Unwinding of double-stranded DNA by dehydration. Proc. Nat. Acad. Sci. USA. 78: 2838-2842, 1981.
6. Slor, H., Bynum, G. D., Lee, C-H., Mizusawa, H. and Kakefuda. T.: Effect of DNA conformation of the binding of antibodies to benzo(a)pyrene diol-epoxide DNA adducts. Jpn. J. Cancer Res. 76: 488-495, 1985.

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