Mutsuo Sekiguchi
Department of Biology and Frontier Research Center, Fukuoka Dental College, Fukuoka 814-0193, JAPAN
(Tel & Fax: 81-92-801-0685 E-mail: sekim1 @college.fdcnet.ac.jp)
The frequency of errors during DNA replication is very low. This high accuracy is achieved by appropriate execution of several consecutive reactions, including (i) selection of a base complementary to the template DNA, (ii) removal of a noncomplementary base by an editing nuclease, and (iii) correction of a misincorporated base by the post-replicational repair system. A defect in any one of these steps would increase the frequency of spontaneous alteration of genes, hence yielding high incidences of tumors (1,2). This has been shown with mutations in DNA polymerase and mismatch recognition genes. Here we show that discrimination of unfavorable precursors in the nucleotide pool is also important.
Oxygen radicals are produced through normal cellular metabolism and generate various modified bases in DNA. Among them, 8-oxo-7, 8-dihydroguanine (8-oxoG) is the most abundant, and appears to play critical roles in carcinogenesis and in aging. 8-oxo-dGTP is produced in normally growing cells and can be incorporated into cellular DNA. Human cells contain an enzyme that hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, thereby preventing misincorporation of 8-oxoguanine into DNA. When the CDNA for human 8-oxo-dGTPase was expressed in Escherichia coli mutT- mutant cells devoid of self 8-oxo-dGTPase activity, the elevated level of spontaneous A:T to C:G mutation frequency reverted to normal. We isolated the corresponding gene from human and mouse and named MTHI (3). By means of gene targeting, we established MTHI gene-knockout cell lines and mice. When examined 18 months after birth, a greater number of tumors were formed in the lungs, livers and stomachs of MTH1-deficient mice, as compared with wild-type mice (4).
When the template DNA is damaged by externally administered agents, mutations and tumors are induced at much higher frequencies, as compared with spontaneous one. To prevent such outcomes, cells possess elaborate mechanisms for repairing DNA damage and executing apoptosis. We have analyzed these processes with the use of simple alkylating agents, such as methylnitrosourea (MNU). Among many methylated bases produced by MNU, O6-methylguanine is the most responsible for induction of mutation as well as cancer. O6-Methylguanine can pair with thymine as well as cytosine during DNA replication, leading to G:C to A:T transition, and such mutations are frequently found in DNA sequences of oncogenes derived from alkylation-induced tumors.
Gene targeting was used to obtain mice defective in the MGMT gene, encoding O6-methylguanine-DNA methyltransferase that repairs O6-methylguanine lesions in DNA. These MGMT-/- mice were most sensitive to alkylating carcinogens; LD50s of MGMT+/+ and MGMT-/- mice to MNU were 240 and 20 mg/kg of body weight, respectively. A large number of thymic lymphomas, as well as lung adenomas, occurred in MGMT -/- mice exposed to a low dose of MNU, which produced only few tumors in MGMT +/+ mice. Thus, mouse lines deficient in methyltransferase activity are hypersensitive to both the killing and to the tumorigenic effects of MNU, and similar effects were observed with chemotherapeutic agents, such as dacarbazine (5). These dual effects of alkylating agents can be dissociated by introduction of an additional defect in the MLH1 gene, whose product is involved in recognition of mismatched bases. MGMT -/- MLH1 -/- and even MGMT -/- MLH1 +/- mice are resistant to the killing effect of MNU, but produce numerous tumors after receiving MNU (6). It seems that the MLH1-involved system functions to eliminate cells with potentially mutation-evoking DNA damage through apoptosis.
The MGMT -/- MLH1 -/- status is frequently observed in human tumors and tumor-derived cell lines. This may be achieved by turning off of gene expression, due to hypermethylation of the promoter region. Elucidation of the regulatory mechanism for this phenomenon is important for our understanding of genomic instability and carcinogenesis.
References:
(1) L. Loeb (1996). Many mutations in cancers. Genetic Instability in Cancer.
Cancer Surveys 28:329-342
(2) D. L. Stoler, N. Chen, M. Basik, M. S. Kahlenberg, M. A. Rodriguez-Bigas, N. J. Petrelli, and G. R. Anderson (1999). The onset and extent of genomic instability in sporadic colorectal tumor progression. Proc. Natl. Acad. Sci. U.S.A. 96:15121-15126
(3) H. Igarashi, T. Tsuzuki, T. Kakuma, Y. Tominaga, and M. Sekiguchi (1997).
Organization and expression of the mouse MTH1 gene for preventing transversion mutation. J. Biol. Chem. 272:3766-3772
(4) T. Tsuzuki, A. Egashira, H. Igarashi, T. Iwakuma, Y. Nakatsuru, Y. Tominaga, H. Kawate, K. Nakao, K. Nakamura, F. Ide, S. Kura, Y. Nakabeppu, M. Katsuki, T. Ishikawa, and M. Sekiguchi (2001). Spontaneous tumorigenesis in mice defective in the MTH1 gene encoding 8-oxo-dGTPase.
(5) A. Shiraishi, K. Sakumi, and M. Sekiguchi (2000). Increased susceptibility to chemotherapeutic alkylating agents of mice deficient in DNA repair methyltransferase. Carcinogenesis 21: 1879-1883
(6) H. Kawate, K. Sakumi, T. Tsuzuki, Y. Nakatsuru, T. Ishikawa, S. Takahashi, H. Takano, T.Noda, and M. Sekiguchi (1988). Separation of killing and tumorigenic effects of an alkylating agent in mice defective in two of the DNA repair genes.
Proc. Natl. Acad. Sci. U.S.A. 95:51116-5120
Mutsuo Sekiguchi 1955 BSc, Osaka University, Japan
1960 PhD. Osaka University, Japan
1961-1965 Research Associate, University of Pennsylvania and Purdue University, U.S.A.
1965-1969 Associate Professor, Kyushu University. Japan
1969-1996 Professor, Kyushu University. Japan
1966 Professor, Fukuoka Dental College, Japan
Speciality and Special Interest:
Molecular mechanism for high fidelity of DNA replication, DNA repair mechanism, control of mutagenesis and carcinogenesis, apoptosis