Biomedical subjects

Mutsuo Sekiguchi

Publications and source records attributed to Mutsuo Sekiguchi.

At least 19 recordsLinked to original sources

PubMed2006

Human polynucleotide phosphorylase protein in response to oxidative stress.

8-Oxo-7,8-dihydroguanine (8-oxoGua) is generated in nucleic acids as well as in their precursors due to the actions of oxygen radicals produced through a normal cellular metabolism. Since oxidized guanine can pair with both cytosine and adenine, it causes alterations in the phenotypic expression when it is present in RNA. To prevent such an outcome, organisms must have some mechanism for eliminating such oxidized guanine nucleotides from RNA and its precursors. In mammalian cells, MTH1 and NUDT5 proteins degrade 8-oxoGTP and 8-oxoGDP to 8-oxoGMP, which is an unusable form for RNA synthesis. In a search for proteins functioning at the RNA level, polynucleotide phosphorylase (PNP) protein has been suggested to be a good candidate for such a role. The human PNP protein has an ability to bind specifically to RNA containing 8-oxoGua. When human cells are exposed to agents that induce oxidative stress, such as hydrogen peroxide and menadion, the amounts of PNP protein decrease rapidly while amounts of other proteins in the cells do not change after such treatments. No specific decrease in the PNP protein level is observed when cells are treated with ACNU and cycloheximide at doses sufficient to provide the same degree of growth suppression. These results imply that the PNP protein might thus play a role in excluding oxidized forms of RNA from the translation mechanism.

Base Sequence↗

PubMed2006

A quest to understand molecular mechanisms for genetic stability.

In the midst of the post-war turmoil in Japan, I fortunately followed a path to become a scientist. Sometime at an early stage of my career, I encountered the problem of the cellular response to DNA damage and had the chance to discover a DNA repair enzyme. This event greatly influenced the subsequent course of my research, and I extended my studies toward elucidating the molecular mechanisms of mutagenesis as well as of carcinogenesis. Through these studies I came to understand the importance of mechanisms for dealing with the actions of reactive oxygen species to the living systems. These recollections deal with these endeavors with emphasis on the early part of my scientific career.

DNA Damage↗

PubMed2005

PCNA-MutSalpha-mediated binding of MutLalpha to replicative DNA with mismatched bases to induce apoptosis in human cells.

Modified bases, such as O6-methylguanines, are produced in cells exposed to alkylating agents and cause apoptosis. In human cells treated with N-methyl-N-nitrosourea, we detected a protein complex composed of MutSalpha, MutLalpha and PCNA on damaged DNA by immunoprecipitation method using chromatin extracts, in which protein-protein interactions were stabilized by chemical crosslinking. Time course experiments revealed that MutSalpha, consisting of MSH2 and MSH6 proteins, and PCNA bind to DNA to form an initial complex, and MutLalpha, composed of MLH1 and PMS2, binds to the complex when the DNA is damaged. This sequential mode of binding was further confirmed by the findings that the association of PCNA-MutSalpha complex on chromatin was observed even in the cells that lack MLH1, whereas in the absence of MSH2 no association of MutLalpha with the chromatin was achieved. Moreover, reduction in the PCNA content by small-interfering RNA or inhibition of DNA replication by aphidicolin, an inhibitor of DNA polymerase, significantly reduced the levels of the PCNA-MutSalpha-MutLalpha complex and also suppressed an increase in the caspase-3 activity, a hallmark for the induction of apoptosis. These observations imply that the induction of apoptosis is coupled with the progression of DNA replication through the action of PCNA.

Apoptosis↗

PubMed2005

Mammalian enzymes for preventing transcriptional errors caused by oxidative damage.

8-Oxo-7,8-dihydroguanine (8-oxoGua) is produced in cells by reactive oxygen species normally formed during cellular metabolic processes. This oxidized base can pair with both adenine and cytosine, and thus the existence of this base in messenger RNA would cause translational errors. The MutT protein of Escherichia coli degrades 8-oxoGua-containing ribonucleoside di- and triphosphates to the monophosphate, thereby preventing the misincorporation of 8-oxoGua into RNA. Here, we show that for human the MutT-related proteins, NUDT5 and MTH1 have the ability to prevent translational errors caused by oxidative damage. The increase in the production of erroneous proteins by oxidative damage is 28-fold over the wild-type cells in E.coli mutT deficient cells. By the expression of NUDT5 or MTH1 in the cells, it is reduced to 1.4- or 1.2-fold, respectively. NUDT5 and MTH1 hydrolyze 8-oxoGDP to 8-oxoGMP with V(max)/K(m) values of 1.3 x 10(-3) and 1.7 x 10(-3), respectively, values which are considerably higher than those for its normal counterpart, GDP (0.1-0.5 x 10(-3)). MTH1, but not NUDT5, possesses an additional activity to degrade 8-oxoGTP to the monophosphate. These results indicate that the elimination of 8-oxoGua-containing ribonucleotides from the precursor pool is important to ensure accurate protein synthesis and that both NUDT5 and MTH1 are involved in this process in human cells.

DNA Repair Enzymes↗

PubMed2005

Multiple enzyme activities of Escherichia coli MutT protein for sanitization of DNA and RNA precursor pools.

8-OxoGua (8-oxo-7,8-dihydroguanine) is produced in nucleic acids as well as in nucleotide pools of cells, by reactive oxygen species normally formed during cellular metabolic processes. MutT protein of Escherichia coli specifically degrades 8-oxoGua-containing deoxyribo- and ribonucleoside triphosphates to corresponding nucleoside monophosphates, thereby preventing misincorporation of 8-oxoGua into DNA and RNA, which would cause mutation and phenotypic suppression, respectively. Here, we report that the MutT protein has additional activities for cleaning up the nucleotide pools to ensure accurate DNA replication and transcription. It hydrolyzes 8-oxo-dGDP to 8-oxo-dGMP with a K(m) of 0.058 microM, a value considerably lower than that for its normal counterpart, dGDP (170 microM). Furthermore, the MutT possesses an activity to degrade 8-oxo-GDP to the related nucleoside monophosphate, with a K(m) value 8000 times lower than that for GDP. These multiple enzyme activities of the MutT protein would facilitate the high fidelity of DNA and RNA syntheses.

DNA Replication↗

PubMed2005

Two modes of microsatellite instability in human cancer: differential connection of defective DNA mismatch repair to dinucleotide repeat instability.

Microsatellite instability (MSI) is associated with defective DNA mismatch repair in various human malignancies. Using a unique fluorescent technique, we have observed two distinct modes of dinucleotide microsatellite alterations in human colorectal cancer. Type A alterations are defined as length changes of < or =6 bp. Type B changes are more drastic and involve modifications of > or =8 bp. We show here that defective mismatch repair is necessary and sufficient for Type A changes. These changes were observed in cell lines and in tumours from mismatch repair gene-knockout mice. No Type B instability was seen in these cells or tumours. In a panel of human colorectal tumours, both Type A MSI and Type B instability were observed. Both types of MSI were associated with hMSH2 or hMLH1 mismatch repair gene alterations. Intriguingly, p53 mutations, which are generally regarded as uncommon in human tumours of the MSI+ phenotype, were frequently associated with Type A instability, whereas none was found in tumours with Type B instability, reflecting the prevailing viewpoint. Inspection of published data reveals that the microsatellite instability that has been observed in various malignancies, including those associated with Hereditary Non-Polyposis Colorectal Cancer (HNPCC), is predominantly Type B. Our findings indicate that Type B instability is not a simple reflection of a repair defect. We suggest that there are at least two qualitatively distinct modes of dinucleotide MSI in human colorectal cancer, and that different molecular mechanisms may underlie these modes of MSI. The relationship between MSI and defective mismatch repair may be more complex than hitherto suspected.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Crystallization and preliminary X-ray analysis of Escherichia coli MutT in binary and ternary complex forms.

During replication, Escherichia coli MutT prevents the misincorporation of mutagenic 8-oxoguanine into nascent DNA strands opposite adenine by hydrolyzing 8-oxo-dGTP in nucleotide pools to 8-oxo-dGMP. E. coli MutT is the most widely investigated member of the Nudix hydrolase family, which is large and found in all organisms. By co-crystallization of MutT with 8-oxo-dGMP, a reaction product, crystals of the binary complex were obtained using ammonium sulfate as a precipitant. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 37.9, b = 56.0, c = 59.4 A. Assuming the presence of one protein-nucleotide complex in the asymmetric unit, the Matthews coefficient V(M) is 2.1 A(3) Da(-1). Crystals of the ternary complex were prepared by soaking crystals of the binary complex in 1 mM MnCl(2) solution. They diffracted to 1.96 and 2.56 A resolution, respectively.

Crystallization↗

PubMed2004

Killing and mutagenic actions of dacarbazine, a chemotherapeutic alkylating agent, on human and mouse cells: effects of Mgmt and Mlh1 mutations.

Among various types of drugs designed for use in cancer chemotherapy, some have the potential for alkylation. After metabolic activation, these chemicals attack DNA and alkylate their bases, thereby preventing multiplication of rapidly growing tumor cells. Some of alkylated bases cause mutations, leading to untoward induction of tumors. To search for the rationale to separate lethal and mutagenic effects of alkylation drugs, we investigated actions of dacarbazine, a monofunctional triazene, on mouse and human cell lines defective in the Mgmt and/or the Mlh1 gene, the former encoding a DNA repair methyltransferase and the latter a protein involved in mismatch repair and induction of apoptosis. Mgmt-deficient cells are hypersensitive to the killing action of dacarbazine. On the other hand, cells defective in both Mgmt and Mlh1 genes are as resistant to the drug as are wild-type cells, in terms of survival, but do have many mutations after dacarbazine treatment. Thus, the killing and mutagenic actions of dacabazine can be dissociated by manipulating actions of these gene products.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Silencing effect of CpG island hypermethylation and histone modifications on O6-methylguanine-DNA methyltransferase (MGMT) gene expression in human cancer.

O6-methylguanine-DNA methyltransferase (MGMT) repairs the cytotoxic and mutagenic O6-alkylguanine produced by alkylating agents such as chemotherapeutic agents and mutagens. Recent studies have shown that in a subset of tumors, MGMT expression is inversely linked to hypermethylation of the CpG island in the promoter region; however, how the epigenetic silencing mechanism works, as it relates to hypermethylation, was still unclear. To understand the mechanism, we examined the detailed methylation status of the whole island with bisulfite-sequencing in 19 MGMT non-expressed cancer cell lines. We found two highly methylated regions in the island. One was upstream of exon 1, including minimal promoter, and the other was downstream, including enhancer. Reporter gene assay showed that methylation of both the upstream and downstream regions suppressed luciferase activity drastically. Chromatin immunoprecipitation assay revealed that histone H3 lysine 9 was hypermethylated throughout the island in the MGMT negative line, whereas acetylation on H3 and H4 and methylation on H3 lysine 4 were at significantly high levels outside the minimal promoter in the MGMT-expressed line. Furthermore, MeCP2 preferentially bound to the CpG-methylated island in the MGMT negative line. Given these results, we propose a model for gene silencing of MGMT that is dependent on the epigenetic state in cancer.

CpG Islands↗

PubMed2003

Roles of MGMT and MLH1 proteins in alkylation-induced apoptosis and mutagenesis.

To examine involvement of mismatch repair system in alkylation-induced apoptosis and mutagenesis, cell lines defective in the Mgmt gene encoding a DNA repair enzyme, O(6)-methylguanine-DNA methyltransferase, and/or the Mlh1 gene encoding a protein involved in mismatch repair were established from gene-targeted mice. Mgmt(-/-) cells are hypersensitive to the killing effect of N-methyl-N-nitrosourea (MNU) and this effect of MNU was overcome by introducing an additional mutation in the Mlh1 gene. Mgmt(-/-)Mlh1(-/-) cells are more resistant to MNU than are wild-type cells. When the human Mgmt cDNA sequence with a strong promoter was introduced, the wild-type cells acquired the same high level of resistance to MNU as that of Mgmt(-/-)Mlh1(-/-) cells. Although no apparent increase in MNU-induced mutant frequency was observed in such methyltransferase-overproducing wild-type cells, mutant frequency of Mgmt(-/-)Mlh1(-/-) cells became 10-fold higher after being treated with MNU. Mgmt(-/-)Mlh1(+/-) cells carrying approximately half the normal level of MLH1 protein showed a normal level of spontaneous mutant frequency, yet were still highly responsive to the mutagenic effect of the alkylating carcinogen. This haploinsufficient character of Mlh1 mutation was also observed in cell survival assays; Mgmt(-/-)Mlh1(+/-) cells were as resistant to MNU as were Mgmt(-/-)Mlh1(-/-) cells. While caspase-3 was induced in Mgmt(-/-)Mlh1(+/+) cells after treatment with MNU, no induction occurred in Mgmt(-/-)Mlh1(+/-) cells or in Mgmt(-/-)Mlh1(-/-) cells. The cellular content of MLH1 protein seems to be critical for determining if damaged cells enter into either a death or mutation-inducing pathway. The haploinsufficient phenotype of Mlh1-heterozygous cells may be explained by competition in heterodimer formation between MLH1 homologues.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Mouse MTH2 protein which prevents mutations caused by 8-oxoguanine nucleotides.

MutT-related proteins degrade 8-oxo-7,8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP), a mutagenic substrate for DNA synthesis, in the nucleotide pool, thereby preventing DNA replication errors. During a search of GenBank EST database, we found a new member of MutT-related protein, MTH2, which possesses the 23-amino acid MutT module. The cloned mouse MTH2 (mMTH2) cDNA was expressed in Escherichia coli mutT(-) cells and the protein was purified. mMTH2 protein hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, with Km of 32 microM. Expression of cDNA for mMTH2 reduced significantly the elevated level of spontaneous mutation frequency of E. coli mutT(-) cells. Thus, MTH2 has a potential to protect the genetic material from the untoward effects of endogenous oxygen radicals. MTH2 could act as an MTH1 redundancy factor.

Amino Acid Sequence↗

PubMed2003

Regulation of RecA protein binding to DNA by opposing effects of ATP and ADP on inter-domain contacts: analysis by urea-induced unfolding of wild-type and C-terminal truncated RecA.

RecA protein requires ATP and its hydrolysis to ADP to complete the DNA strand-exchange reaction. We investigated how the nucleotides activate RecA by examining their effect on urea-induced unfolding, which could reflect domain-domain contact of protein. RecA is folded into three continuous domains: the N-terminal, central and C-terminal domains. The fluorescence of tyrosine residues, which lie mainly in the central domain, was modified in 1-3 M urea, while the red shift of fluorescence peak of the tryptophan residues located in the C-terminal domain occurred only in 3-6 M urea. Thus, the C-terminal domain of RecA is unfolded after the central part unfolds. The change in intensity of tryptophan fluorescence without a large shift in the peak at low concentrations of urea suggests that there are weak interactions between the central and C-terminal domains. This is supported by our observation that RecA protein lacking the C-terminal tail unfolded at lower concentrations of urea than the entire RecA, and with clear transitions, unlike the entire RecA. ATP and its unhydrolyzable analog (ATPgammaS), which enhance the binding of RecA to DNA, facilitated the urea-induced change in RecA tryptophan fluorescence, while ADP, an antagonist of ATP, prevented the change. ATP probably weakens the domain-domain contact and facilitates the DNA binding, while ADP stabilizes the contact and inhibits it. Supporting this conclusion, the binding of RecA lacking the C-terminal tail to DNA was not inhibited by ADP, while that of the intact RecA was.

Adenosine Diphosphate↗

PubMed2003

Ogg1 knockout-associated lung tumorigenesis and its suppression by Mth1 gene disruption.

Using Mth1 and Ogg1 knockout mice, we evaluated the roles of these enzymes to prevent tumorigenesis and the accumulation of 8-oxoguanine (8-oxoG) in DNA. We found that lung adenoma/carcinoma spontaneously developed in Ogg1 knockout mice approximately 1.5 years after birth in which 8-oxoG was found to accumulate in their genomes. The mean number of tumors/mouse was 0.71 for the Ogg1 knockout mice, which was five times higher than that observed in wild-type mice (0.14). Although the accumulation of 8-oxoG was also confirmed in the Ogg1, Mth1 double knockout mice, we found no tumor in the lungs of these mice. This observation suggests that Mth1 gene disruption resulted in a suppression of the tumorigenesis caused by an Ogg1 deficiency.

Adenocarcinoma↗

PubMed2003

Combined loss of expression of O6-methylguanine-DNA methyltransferase and hMLH1 accelerates progression of hepatocellular carcinoma.

BACKGROUND AND OBJECTIVES: O(6)-methylguanine-DNA methyltransferase (MGMT) and human Mut L homologue 1 (hMLH1) are proteins that play an important role in DNA repair. No reports have yet described whether deficient MGMT and hMLH1 expression correlates with tumor progression and the prognosis of patients with hepatocellular carcinoma (HCC). METHODS: Using immunohistochemical analysis, we evaluated the expression status of MGMT and hMLH1 protein in 60 paraffin-embedded samples from consecutive patients with curatively resected HCC. RESULTS: The lack of expression of both MGMT and hMLH1 in HCCs (n = 7) correlated with advanced pTNM stage (P = 0.039), as compared with HCCs expressing both proteins (n = 25). The absence of both MGMT and hMLH1 was a significant indicator of malignant potential. The expression status of both MGMT and hMLH1 was a predictive factor for overall survival in patients with HCC (P < 0.001). CONCLUSIONS: HCC lacking both MGMT and hMLH1 is correlated with an advanced stage and a poor prognosis. The expression status of both repair proteins is a predictive prognostic marker in patients with HCC after surgical resection.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Loss of expression of DNA repair enzymes MGMT, hMLH1, and hMSH2 during tumor progression in gastric cancer.

BACKGROUND: Disorders of the DNA repair system that protects against alkylating mutagens are known to play an important role in carcinogenesis. METHODS: We investigated the expression of the DNA repair enzyme that protects against alkylating mutagens, O(6)-methylguanine DNA methyltransferase (MGMT), and the mismatch repair (MMR) enzymes, hMLH1 and hMSH2, in 135 gastric cancer specimens by immunohistochemical means. RESULTS: The immunoreactivity of MGMT and MMR proteins correlated significantly with several clinicopathologic factors. The survival curve in 116 patients showed that a loss of MGMT or hMLH1, but not of hMSH2, correlated with a poor prognosis. Combined evaluation of MGMT and hMLH1 revealed that the survival of patients with negative status for both MGMT and hMLH1 was shortest. However, this significant association between patient survival and MGMT or hMLH1 expression disappeared when early and advanced cancers were separately analyzed, indicating that synchronous losses of MGMT and hMLH1 increase during tumor progression and stage. Further evaluation according to histologic type revealed that loss of MGMT, hMLH1, and hMSH2 expression significantly differed between early and advanced cancer in differentiated-type cancers. In contrast, in undifferentiated-type cancer, loss of MGMT and MMR expression was frequently found even in intramucosal (m) cancer, and no significant difference was found in loss of hMLH1 and hMSH2 between early and advanced cancer. CONCLUSION: These findings demonstrate that the reduced expression of MGMT, hMLH1, and hMSH2 in differentiated-type cancer may play an important role during tumor progression between the early and advanced stage. On the other hand, in undifferentiated-type cancer, loss of MGMT and the MMR proteins appears to be an important event at carcinogenesis or at an earlier step of tumor progression.

Adaptor Proteins, Signal Transducing↗

PubMed2003

A novel mechanism for preventing mutations caused by oxidation of guanine nucleotides.

MutT-related proteins, including the Escherichia coli MutT and human MutT homologue 1 (MTH1) proteins, degrade 8-oxo- 7,8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP) to a monophosphate, thereby preventing mutations caused by the misincorporation of 8-oxoguanine into DNA. Here, we report that human cells have another mechanism for cleaning up the nucleotide pool to ensure accurate DNA replication. The human Nudix type 5 (NUDT5) protein hydrolyses 8-oxo-dGDP to monophosphate with a K(m) of 0.77 microM, a value considerably lower than that for ADP sugars, which were originally identified as being substrates of NUDT5. NUDT5 hydrolyses 8-oxo-dGTP only at very low levels, but is able to substitute for MutT when it is defective. When NUDT5 is expressed in E. coli mutT(-) cells, the increased frequency of spontaneous mutations is decreased to normal levels. Considering the enzymatic parameters of MTH1 and NUDT5 for oxidized guanine nucleotides, NUDT5 might have a much greater role than MTH1 in preventing the occurrence of mutations that are caused by the misincorporation of 8-oxoguanine in human cells.

Amino Acid Sequence↗

PubMed2002

Oxidative nucleotide damage: consequences and prevention.

8-Oxoguanine (8-oxo-7,8-dihydroguanine) is produced in DNA, as well as in nucleotide pools of cells, by reactive oxygen species normally formed during cellular metabolic processes. 8-Oxoguanine nucleotide can pair with cytosine and adenine nucleotides with an almost equal efficiency, then transversion mutation ensues. MutT protein of Escherichia coli and related mammalian protein MTH1 specifically degrade 8-oxo-dGTP to 8-oxo-dGMP, thereby preventing misincorporation of 8-oxoguanine into DNA. The bacterial and mammalian enzymes are close in their size and share a highly conserved region consisting of 23 residues with 14 identical amino acids. Following saturation mutagenesis of this region, most of these residues proved to be essential to exert 8-oxo-dGTPase activity. Gene targeting was done to establish MTH1-deficient cell lines and mice for study. When examined 18 months after birth, a greater number of tumors were formed in the lungs, livers, and stomachs of MTH1(-/-) mice, as compared with findings in wild-type mice. These proteins protect genetic information from untoward effects of threats of endogenous oxygen.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Gene silencing in phenomena related to DNA repair.

DNA methylation is essential for embryonic development and important for transcriptional repression, as observed in several biological phenomena. These include genomic imprinting, X-inactivation and carcinogenesis. The basic mechanism by which DNA methylation silences transcription is generally understood, but there is still much to be learned about how DNA methyltransferase is targeted to a specific region of the gene. Silencing by DNA methylation occurs at an early stage of carcinogenesis, when the DNA repair genes, MGMT and hMLH1, are frequently inactivated, resulting in mutations in key cancer-related genes in cells. Mice defective in Mgmt and/or Mlh1 gave clear evidence of the significant roles of these proteins in carcinogenesis. Recently, it has been demonstrated that DNA methylation is linked to histone methylation in fungi and plants, although it remains unknown whether this mechanism occurs in mammalian systems.

Adaptor Proteins, Signal Transducing↗