Oxidative damage of DNA, RNA and their metabolites in leukocytes, plasma and urine of Macaca mulatta: 8-oxoguanosine in urine is a useful marker for aging.

The levels of the oxidised forms of guanosine in leukocytes, plasma and urine of Macaca mulatta were determined using a sensitive method based on high-performance liquid chromatography-triple quadruple mass spectrometry (LC-MS/MS). The amounts of 8-oxo-7,8-dihydrodeoxyguanosine (8-oxo-dGsn) and 8-oxo-7,8-dihydroguanosin (8-oxoGsn), derived from DNA and RNA, respectively, increased with age in leukocytes. The measurement of the free forms of oxidised guanosine revealed similar age-dependent increases of 8-oxo-dGsn and 8-oxoGsn in both plasma and urine, which showed considerably larger amounts of 8-oxoGsn than 8-oxo-dGsn. The 8-oxoGsn content of urine could be a useful biomarker for evaluating aging, as age-dependent increases of 8-oxoGsn are more evident in urine compared to plasma and because urine samples are readily available.

Human MTH3 (NUDT18) protein hydrolyzes oxidized forms of guanosine and deoxyguanosine diphosphates: comparison with MTH1 and MTH2.

Most of the proteins carrying the 23-residue MutT-related sequence are capable of hydrolyzing compounds with a general structure of nucleoside diphosphate linked to another moiety X and are called the Nudix hydrolases. Among the 22 human Nudix proteins (identified by the sequence signature), some remain uncharacterized as enzymes without a defined substrate. Here, we reveal that the NUDT18 protein, whose substrate was unknown, can degrade 8-oxo-7,8-dihydroguanine (8-oxo-Gua)-containing nucleoside diphosphates to the monophosphates. Because this enzyme is closely related to MTH1 (NUDT1) and MTH2 (NUDT15), we propose that it should be named MTH3. Although these three human proteins resemble each other in their sequences, their substrate specificities differ considerably. MTH1 cleaves 8-oxo-dGTP but not 8-oxo-dGDP, whereas MTH2 can degrade both 8-oxo-dGTP and 8-oxo-dGDP, although the intrinsic enzyme activity of MTH2 is considerably lower than that of MTH1. On the other hand, MTH3 is specifically active against 8-oxo-dGDP and hardly cleaves 8-oxo-dGTP. Other types of oxidized nucleoside diphosphates, 2-hydroxy-dADP and 8-hydroxy-dADP, were also hydrolyzed by MTH3. Another notable feature of the MTH3 enzyme is its action toward the ribonucleotide counterpart. MTH3 can degrade 8-oxo-GDP as efficiently as 8-oxo-dGDP, which is in contrast to the finding that MTH1 and MTH2 show a limited activity against the ribonucleotide counterpart, 8-oxo-GTP. These three enzymes may function together to help maintain the high fidelity of DNA replication and transcription under oxidative stress.

Greater nucleic acids oxidation in the temporal lobe than the frontal lobe in SAMP8.

Our previous studies have shown that substantial amounts of 8-oxoguanine are present in the DNA and RNA in the hippocampi of old senescence-accelerated mice (SAMP8); however, oxidative damage to DNA and RNA in the other regions of the brain from a month after birth to the onset of aging has not been examined completely. In this study, we analyzed the amount of 8-oxoguanine in DNA and RNA in the temporal and frontal lobes of SAMP8 during aging by the immunohistochemical method. Compared with age-matched control acceleration-resistant mice (SAMR1), 8- and 12-month-old SAMP8 had increased amounts of 8-oxoguanine in the DNA and RNA in the frontal lobe, whereas in the temporal lobe, this trend began to appear as early as 4 months. The levels of 8-oxoguanine in the temporal lobe were significantly higher than those in the frontal lobe. These results indicate that nucleic acid oxidative damage occurs as an age-associated phenomenon, and can occur more easily in the temporal lobe than in the frontal lobe of SAMP8.

Age-dependent increases in the oxidative damage of DNA, RNA, and their metabolites in normal and senescence-accelerated mice analyzed by LC-MS/MS: urinary 8-oxoguanosine as a novel biomarker of aging.

A sensitive and accurate isotope-diluted LC-MS/MS method was developed for determination of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGsn), derived from DNA, and 8-oxo-7,8-dihydroguanosine (8-oxo-Gsn), derived from RNA, in various tissue specimens obtained from normal SAMR1 and senescence-accelerated SAMP8 mice. An age-dependent accumulation of oxidative DNA and RNA damage was observed in all the organs examined, namely, the brain, liver, lungs, heart, kidneys, and testes. Among these, the brain samples exhibited the highest values for DNA damage. These age-related increases in the 8-oxoguanine content in DNA and RNA occurred more rapidly in SAMP8 than in SAMR1 mice. Age-related increases in the contents of 8-oxo-dGsn and 8-oxo-Gsn were also observed in the plasma and urine; however, the ratios of 8-oxo-Gsn to 8-oxo-dGsn in these samples were considerably higher (6 to 13) compared with the values for the samples derived from other tissues (roughly 1), indicating that measurement of 8-oxo-Gsn in urine could be a novel means of evaluating the aging process.

Lowered nudix type 5 (NUDT5) expression leads to cell cycle retardation in HeLa cells.

The molecule 8-oxo-7,8-dihydroguanine (8-oxoGua), an oxidized form of guanine, can pair with adenine or cytosine during nucleic acid synthesis. Moreover, RNA containing 8-oxoGua causes translational errors, thus leading to the production of abnormal proteins. Human NUDT5, a MutT-related protein, catalyzes the hydrolysis of 8-oxoGDP to 8-oxoGMP, thereby preventing misincorporation of 8-oxoGua into RNA. To investigate the biological roles of NUDT5 in mammalian cells, we established cell lines with decreased level of NUDT5 expression. In NUDT5 inhibited cells, the RNA oxidation was not significantly higher than that of normal cells. However, the cell cycle G1 phase was significantly delayed, and cell numbers in both S and G2/M phases were reduced, indicating that cell proliferation was hampered by NUDT5 suppression. Key proteins for preventing the G1-S transition, including p53, p16, and Rb were increased, while the Rb phosphorylation was decreased. These results suggested that the NUDT5 protein may play significant roles in regulating the G1-S transition in mammalian cells.

Diverse substrate recognition and hydrolysis mechanisms of human NUDT5.

Human NUDT5 (hNUDT5) hydrolyzes various modified nucleoside diphosphates including 8-oxo-dGDP, 8-oxo-dADP and ADP-ribose (ADPR). However, the structural basis of the broad substrate specificity remains unknown. Here, we report the crystal structures of hNUDT5 complexed with 8-oxo-dGDP and 8-oxo-dADP. These structures reveal an unusually different substrate-binding mode. In particular, the positions of two phosphates (α and ß phosphates) of substrate in the 8-oxo-dGDP and 8-oxo-dADP complexes are completely inverted compared with those in the previously reported hNUDT5-ADPR complex structure. This result suggests that the nucleophilic substitution sites of the substrates involved in hydrolysis reactions differ despite the similarities in the chemical structures of the substrates and products. To clarify this hypothesis, we employed the isotope-labeling method and revealed that 8-oxo-dGDP is attacked by nucleophilic water at Pß, whereas ADPR is attacked at Pα. This observation reveals that the broad substrate specificity of hNUDT5 is achieved by a diversity of not only substrate recognition, but also hydrolysis mechanisms and leads to a novel aspect that enzymes do not always catalyze the reaction of substrates with similar chemical structures by using the chemically equivalent reaction site.

Molecular actions of Escherichia coli MutT for control of spontaneous mutagenesis.

MutT protein of Escherichia coli hydrolyzes oxidized guanine nucleotides, 8-oxo-dGTP and 8-oxoGTP, to the corresponding monophosphates, thereby preventing misincorporation of 8-oxoguanine into DNA and RNA, respectively. Although the biological significance of the MutT has been established, how MutT protein actually works in vivo remains to be elucidated. The current study shows the molecular behavior of the MutT protein in vivo and in vitro with special reference to control of spontaneous mutagenesis. A single E. coli cell carries about 70-75 molecules of the MutT protein and that this number does not change even when the cells were cultured in anaerobic and hyper-oxidative conditions. Conditional gene silencing analyses revealed that about a half number of MutT molecules are needed for keeping the spontaneous mutation frequency at the normal level. The MutT functions are not needed under anaerobic condition, yet the level of the MutT protein in cell is kept constant, probably for preparing for sudden changes of oxygen pressure. There is a possibility that MutT functions in close association with other proteins, and evidence is presented that MutT protein can interact with some proteins in vivo.

Neurite elongation from Drosophila neural BG2-c6 cells stimulated by 20-hydroxyecdysone.

Neurite elongation is a critical process in the formation of nerve systems from neural cells. During metamorphosis, the holometabolous insect Drosophila melanogaster reorganizes its central nervous system (CNS) under the influence of the steroid molting hormone 20-hydroxyecdysone (20E). A neural cell line that responds to 20E treatment is therefore desired in order to analyze its signal transduction process. Here, we show that cells of the Drosophila neural cell line BG2-c6 extended long projections of over 30 microm in length after being stimulated with 20E. Most of these projections contained both actin filaments and microtubules. Since microtubules are structural markers of neurites, the projections were considered to be neurites. Live imaging of cells expressing GFP tagged alpha-tubulin showed that the neurites did not have a lamellipodial structure at their tips. Under an electron microscope, microtubules were found to run alongside the actin filaments in the neurite shaft but did not reach the tip, where the actin filaments were loosely bundled rather than being arranged into a meshwork as in lamellipodia. These results indicate that BG2-c6 cells project neurites without the typical growth-corn structure at their tips after 20E stimulation.

Age-related alterations in the expression of MTH2 in the hippocampus of the SAMP8 mouse with learning and memory deterioration.

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. MTH2 (Mut T homolog 2), which belongs to this family of proteins, possesses 8-oxo-7,8-dihydro-2'-deoxyguanosine triphosphatase (8-oxo-dGTPase) activity and appears to function in the protection of the genetic material from the untoward effects of endogenous oxygen radicals. To examine the roles of MTH2 in the aging process, we used the senescence-accelerated prone mouse 8 (SAMP8), which exhibits early aging syndromes and declining abilities of learning and memory. Immunohistochemical and western blot analysis revealed that the level of MTH2 protein in the hippocampus of the SAMP8 mouse progressively decreases beginning from four months after birth, whereas no such change was observed in the control senescence-accelerated resistant mouse 1 (SAMR1). Under these conditions, 8-oxoguanine accumulates in the nuclear DNA in the CA1 and CA3 subregions of the hippocampus of SAMP8 in an age-dependent manner. In SAMR1 mice, accumulation of 8-oxoguanine in the DNA was not observed. These results suggest that the MTH2 deficiency might be one of the causative factors for accelerated aging.

Polymerase chain reaction detection of bacterial 16S rRNA gene in human blood.

Bacterial 16S ribosomal RNA genes (rDNA) were detected in blood samples from two healthy individuals by PCR under conditions involving 30 cycles that did not produce any visible products from negative control saline. Even from control samples, PCR involving 35-40 cycles yielded visible bands. Major clones detected in the blood samples, but not in control, were the Aquabacterium subgroup, Stenotrophomonas subgroup, Budvicia subgroup, Serratia subgroup, Bacillus subgroup and Flavobacteria subgroup. No clone was located within the bacteroides-clostridium-lactobacillus cluster, which is indigenous to gastrointestinal flora.

Different initial steps of apoptosis induced by two types of antineoplastic drugs.

O6-Methylguanine and O6-chloroethylguanine are primary DNA lesions produced by two types of antineoplastic drugs, 8-carbamoyl-3-methylimidazo[5,1-d]-1,2,3,5-tetrazin-4(3H)-one (temozolomide, TMZ) and 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), respectively. They can be repaired by O6-methylguanine-DNA methyltransferase, coded by the Mgmt gene. Otherwise, these two types of lesions induce apoptosis in different ways. O6-Chloroethylguanine blocks DNA replication thereby inducing apoptosis. On the other hand, O6-methylguanine does not block DNA replication and the resulting O6-methylguanine-thymine mispair is recognized by mismatch repair-related proteins, including MLH1, thereby inducing apoptosis. Reflecting this, mouse cells lacking both MGMT and MLH1 are resistant to TMZ, but not to ACNU. The translocation of phosphatidylserine in cell membrane as well as a change of mitochondrial transmembrane potentials occurred in an MLH1-dependent manner after treatment with TMZ, but no such MLH1 dependency was observed in the case of ACNU treatment. By using cell lines defective in both APAF-1 and MGMT, it was revealed that the APAF-1 function is required for execution of apoptosis induced by either TMZ or ACNU. There is almost 12h delay in occurrence of apoptosis-related mitochondrial depolarization in TMZ-treated cells in comparison to those of ACNU-treated cells, reflecting the fact that at least one cycle of DNA replication is required to trigger apoptosis in the former case, but not in the latter.

Modes of actions of two types of anti-neoplastic drugs, dacarbazine and ACNU, to induce apoptosis.

O(6)-Methylguanine and O(6)-chloroethylguanine, which are the primary cytotoxic DNA lesions produced by 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (dacarbazine) and 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), respectively, can be repaired by O(6)-methylguanine-DNA methyltransferase (MGMT), coded by the MGMT gene. However, the two types of drugs exhibit different effects on cells defective in both MGMT and MLH1 functions, the latter being related to the cellular activity to recognize mismatched bases of DNA for inducing apoptosis. Human cells deficient in both MGMT and MLH1 are resistant to the killing effect of dacarbazine and exhibit an increased mutant frequency after treatment with dacarbazine. On the other hand, these doubly deficient cells are sensitive to the killing action of ACNU and there is no significant increase in ACNU-induced mutant frequency. A mismatch recognition complex, composed of MSH2, MSH6, MLH1, PMS2 and PCNA, is formed after exposing MGMT-deficient cells to dacarbazine, but not in cells treated with ACNU. In contrast, the phosphorylation of Chk1 efficiently occurs in cells treated with dacarbazine as well as with ACNU, the former being in MLH1-dependent manner, whereas the latter in MLH1-independent manner. Therefore, the signals delivered from different sources would merge at the step of Chk1 activation or at an earlier step, and the subsequent process leading to apoptosis appears to be common.

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.

Active sites in the carboxyl-terminal region of the laminin alpha chain in Drosophila neuronal cell spreading.

An established Drosophila neuronal cell line (BG2-c6) proved to be useful to analyze laminin-mediated cell spreading and signal transduction [Takagi et al. (2000) Biochem Biophys Res Commun 270:482-487]. Here, we report, in addition to the whole molecule, the truncated alpha chain of Drosophila laminin (containing the entire carboxyl-terminal globular domain) and two dodecapeptides corresponding to the cell-binding sites identified in the alpha1 chain of mouse laminin were also active to stimulate BG2-c6 cell spreading. Our previous study [Takagi et al. (1996) J Biol Chem 271:18074-18081] revealed that these recombinant protein and synthetic peptides promoted neurite outgrowth in the primary cell culture system prepared from Drosophila embryo. Therefore, the similar effects by these proteins and peptides suggest the presence of a common mechanism of laminin and neuronal cell interaction working in both primary and established cells. One of the two active peptides contains the sequence SIKVGV. Its murine counterpart carries the sequence SIKVAV by which the interaction of laminin and cells is mediated. Furthermore, laminin-dependent BG2-c6 cell spreading was inhibited by heparin. This observation suggests that cell surface glycoproteins participate in the interaction of laminin and BG2-c6 cells.

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.

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.

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.