Endogenous ROS production in early differentiation state suppresses endoderm differentiation via transient FOXC1 expression.

Oxidative stress plays a pivotal role in the differentiation and proliferation of cells and programmed cell death. However, studies on the role of oxidative stress in differentiation have mainly employed the detection of reactive oxygen species (ROS) during differentiation or generated by ROS inducers. Therefore, it is difficult to clarify the significance of endogenous ROS production in the differentiation of human cells.

PCBP1 and PCBP2 both bind heavily oxidized RNA but cause opposing outcomes, suppressing or increasing apoptosis under oxidative conditions.

PCBP1, a member of the poly(C)-binding protein (PCBP) family, has the capability of binding heavily oxidized RNA and therefore participates in the cellular response to oxidative conditions, helping to induce apoptosis. There are four other members of this family, PCBP2, PCBP3, PCBP4, and hnRNPK, but it is not known whether they play similar roles. To learn more, we first tested their affinity for an RNA strand carrying two 8-oxoguanine (8-oxoG) residues at sites located in close proximity to each other, representative of a heavily oxidized strand or RNA with one 8-oxoG or none.

SMARCAD1-mediated recruitment of the DNA mismatch repair protein MutLα to MutSα on damaged chromatin induces apoptosis in human cells.

The mismatch repair (MMR) complex is composed of MutSα (MSH2-MSH6) and MutLα (MLH1-PMS2) and specifically recognizes mismatched bases during DNA replication. -Methylguanine is produced by treatment with alkylating agents, such as -methyl--nitrosourea (MNU), and during DNA replication forms a DNA mismatch ( an -methylguanine/thymine pair) and induces a G/C to A/T transition mutation. To prevent this outcome, cells carrying this DNA mismatch are eliminated by MMR-dependent apoptosis, but the underlying molecular mechanism is unclear.

Two ways of escaping from oxidative RNA damage: Selective degradation and cell death.

Reactive oxygen species (ROS) are produced during normal cellular metabolism, and various oxidized compounds are formed by the ROS attack. Among oxidized bases, 8-oxo-7,8-dihydroguanine (8-oxoG) is most abundant and seems important with respect to the maintenance and transfer of genetic information. The accumulation of 8-oxoG in messenger RNA may cause errors during codon-anticodon pairing in the translation process, which may result in the synthesis of abnormal proteins.

ROS control in human iPS cells reveals early events in spontaneous carcinogenesis.

Reactive oxygen species (ROS) generated during cellular respiration oxidize various cellular constituents, which cause carcinogenesis. Because most studies on the role of ROS in carcinogenesis have mainly been performed using tumor-derived cell lines, which harbor various types of mutation, it has been difficult to determine the molecular details that lead to cancer formation. To overcome this difficulty, we established human-induced pluripotent stem cell lines in which the intracellular ROS levels are controlled at various differentiation stages by manipulating the ROS-yielding mitochondria.

Specific binding of PCBP1 to heavily oxidized RNA to induce cell death.

In aerobically growing cells, the guanine base of RNA is oxidized to 8-oxo-7,8-dihydroguanine (8-oxoG), which induces alteration in their gene expression. We previously demonstrated that the human AUF1 protein binds to 8-oxoG in RNA to induce the selective degradation of oxidized messenger RNA. We herein report that the poly(C)-binding protein PCBP1 binds to more severely oxidized RNA to activate apoptosis-related reactions.

Transcriptional mutagenesis mediated by 8-oxoG induces translational errors in mammalian cells.

Reactive oxygen species formed within the mammalian cell can produce 8-oxo-7,8-dihydroguanine (8-oxoG) in mRNA, which can cause base mispairing during gene expression. Here we found that administration of 8-oxoGTP in MTH1-knockdown cells results in increased 8-oxoG content in mRNA. Under this condition, an amber mutation of the reporter luciferase is suppressed.

The roles of human MTH1, MTH2 and MTH3 proteins in maintaining genome stability under oxidative stress.

The hydrolysis of nucleotides containing 8-oxo-7,8-dihydroguanine (8-oxoG) is important in the maintenance of genome stability. Human cells possess three types of proteins, MTH1 (NUDT1), MTH2 (NUDT15) and MTH3 (NUDT18), which have the potential to hydrolyze deoxyribonucleoside di- and triphosphates containing 8-oxoG to the monophosphate, the form of which is unusable for DNA synthesis. To elucidate the physiological roles of these enzymes, we constructed single knockout (KO) cell lines for each of the MTH1, MTH2 and MTH3 genes and MTH1 and MTH2-double KO cell lines from the human HeLa S3 line using CRISPR/Cas9.

MutT-related proteins are novel progression and prognostic markers for colorectal cancer.

Background: MutT-related proteins, including MTH1, MTH2, MTH3 and NUDT5, can effectively degrade 8-oxoGua-containing nucleotides. The MTH1 expression is elevated in many types of human tumors and MTH1 overexpression correlates with the tumor pathological stage and poor prognosis. However, the expression of other MutT-related proteins in human cancers remains unknown.

Nucleotide excision repair of oxidised genomic DNA is not a source of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine.

Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) is a widely measured biomarker of oxidative stress. It has been commonly assumed to be a product of DNA repair, and therefore reflective of DNA oxidation. However, the source of urinary 8-oxodGuo is not understood, although potential confounding contributions from cell turnover and diet have been ruled out.

Function of high-mobility group A proteins in the DNA damage signaling for the induction of apoptosis.

O(6)-Methylguanine produced in DNA can pair with thymine during DNA replication, thus leading to a G-to-A transition mutation. To prevent such outcomes, cells harboring O(6)-methylguanine-containing mispair undergo apoptosis that requires the function of mismatch repair (MMR) protein complex. To identify the genes involved in the induction of apoptosis, we performed gene-trap mutagenesis and isolated a clone of mouse cells exhibiting an increased resistance to the killing effect of an alkylating agent, N-methyl-N-nitrosourea (MNU).

Role of Auf1 in elimination of oxidatively damaged messenger RNA in human cells.

In aerobically growing cells, in which reactive oxygen species are produced, the guanine base of RNA is oxidized to 8-oxo-7,8-dihydroguanine, which induces alterations in gene expression. Here we show that the human Auf1 protein, also called HNRNPD, binds specifically to RNA containing this oxidized base and may be involved in cellular processes associated with managing the problems caused by RNA oxidation. Auf1-deficient cells were constructed from human HeLa and Nalm-6 lines using two different targeting procedures.

Search for proteins required for accurate gene expression under oxidative stress: roles of guanylate kinase and RNA polymerase.

In aerobically growing cells, in which reactive oxygen species are produced, the guanine base is oxidized to 8-oxo-7,8-dihydroguanine, which can pair with adenine as well as cytosine. This mispairing causes alterations in gene expression, and cells possess mechanisms to prevent such outcomes. In Escherichia coli, 8-oxo-7,8-dihydroguanine-related phenotypic suppression of lacZ amber is enhanced by mutations in genes related to the prevention of abnormal protein synthesis under oxidative stress.

Age-dependent accumulation of 8-oxoguanine in the DNA and RNA in various rat tissues.

The relationship between the oxidative damage of nucleic acids and aging of animals was investigated by analyzing the nucleic acids derived from various tissue specimens of naturally aged Sprague-Dawley (SD) rats. For this purpose, we established an accurate and sensitive isotope-diluted LC-MS/MS method to determine the levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGsn) in DNA and 8-oxo-7,8-dihydroguanosine (8-oxo-Gsn) in RNA. An age-dependent increase in oxidative DNA and RNA damage was observed in the various organs examined, including the brain, liver, kidneys, and testes.

Elimination and utilization of oxidized guanine nucleotides in the synthesis of RNA and its precursors.

Reactive oxygen species are produced as side products of oxygen utilization and can lead to the oxidation of nucleic acids and their precursor nucleotides. Among the various oxidized bases, 8-oxo-7,8-dihydroguanine seems to be the most critical during the transfer of genetic information because it can pair with both cytosine and adenine. During the de novo synthesis of guanine nucleotides, GMP is formed first, and it is converted to GDP by guanylate kinase.

Stabilization of MAPO1 by specific binding with folliculin and AMP-activated protein kinase in O⁶-methylguanine-induced apoptosis.

When DNA is damaged by alkylating agents, apoptosis is induced to exclude cells carrying DNA lesions in order to prevent mutations and cancer. MAPO1, identified as a component involved in the induction of apoptosis, interacts with AMP-activated protein kinase (AMPK) and folliculin (FLCN). We herein report that MAPO1 is stabilized during the course of apoptosis, triggered by alkylation-induced O(6)-methylguanine in DNA.

The identification of a novel gene, MAPO2, that is involved in the induction of apoptosis triggered by O⁶-methylguanine.

O⁶-Methylguanine, one of alkylated DNA bases, is especially mutagenic. Cells containing this lesion are eliminated by induction of apoptosis, associated with the function of mismatch repair (MMR) proteins. A retrovirus-mediated gene-trap mutagenesis was used to isolate new genes related to the induction of apoptosis, triggered by the treatment with an alkylating agent, N-methyl-N-nitrosourea (MNU).

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.

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.

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.

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.

Activation of AMP-activated protein kinase by MAPO1 and FLCN induces apoptosis triggered by alkylated base mismatch in DNA.

O₆-methylguanine produced in DNA by the action of simple alkylating agents, such as N-methyl-N-nitrosourea (MNU), causes base-mispairing during DNA replication, thus leading to mutations and cancer. To prevent such outcomes, the cells carrying O⁶-methylguanine undergo apoptosis in a mismatch repair protein-dependent manner. We previously identified MAPO1 as one of the components required for the induction of apoptosis triggered by O⁶-methylguanine.

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.