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Maria Shipkova - One of the best experts on this subject based on the ideXlab platform.
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association between adverse effects under azathioprine therapy and inosine triphosphate pyrophosphatase activity in patients with chronic inflammatory bowel disease
Therapeutic Drug Monitoring, 2011Co-Authors: Maria Shipkova, Jutta Franz, Manabu Abe, Corinne Klett, Eberhard Wieland, Tilo AndusAbstract:Background: Inosine triphosphate pyrophosphatase (ITPA) catalyzes the pyrophosphohydrolysis of inosine triphosphate to inosine monophosphate. Recently, single-nucleotide polymorphisms in the ITPA gene, associated with decreased enzyme activity, have been reported. Some clinical studies have demonstrated that the 94C>A mutation is linked to flu-like symptoms, rash, and pancreatitis during azathioprine (AZA) therapy and to early AZA discontinuation. In this study, we investigated whether the enzyme phenotype is also related to adverse effects (AEs). Methods: Patients suffering from inflammatory bowel disease who were treated with AZA (N = 160; age 43 ± 12 years) were included. Data were categorized into quartiles according to the ITPA activity. Information about the therapeutic regimen, AEs [leucopenia, increased hepatic enzymes (alanine aminotransferase, aspartate aminotrasnferase, gamma-glutamyl transferase), flu-like symptoms, and pancreatitis], cotherapy, and comorbidity was obtained from the responsible clinicians and patients by using a standardized questionnaire. ITPA activity was measured by a validated high-performance liquid chromatography procedure. In patients with decreased ITPA activity, the 94C>A and IVS2 + 21A>C genotypes were determined. Results: AEs were reported significantly more often for patients with low ITPA activity than for patients with high ITPA activity; the highest odds ratio for occurrence of AEs was found to be below a threshold of 59.9 μmol/(gHb·h) [hemoglobin (Hb)]. Decreased ITPA activities [particularly <89.2 μmol/(gHb·h)] were frequently accompanied by leucopenias, whereas very low enzyme activities [<37.3 μmol/(gHb·h)] were associated with a higher incidence of increased liver enzymes. Conclusions: The results demonstrate a relationship between low ITPA activity and AEs and support the idea that the determination of ITPA phenotype might be an appropriate alternative to genotyping.
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analysis of ITPA phenotype genotype correlation in the bulgarian population revealed a novel gene variant in exon 6
Therapeutic Drug Monitoring, 2007Co-Authors: Srebrena Atanasova, Maria Shipkova, Eberhard Wieland, Michael Oellerich, Dobrin Svinarov, Antoaneta Mladenova, Mariana Genova, Nicolas Von AhsenAbstract:Abstract:Mutations in the inosine triphosphate pyrophosphohydrolase (ITPA) gene causing enzyme deficiency were shown to have pharmacogenetic implications in azathioprine-induced adverse drug reactions. The distribution of ITPA activity as well as the types and the frequencies of gene variants associ
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measurement of erythrocyte inosine triphosphate pyrophosphohydrolase ITPA activity by hplc and correlation of ITPA genotype phenotype in a caucasian population
Clinical Chemistry, 2006Co-Authors: Maria Shipkova, Eberhard Wieland, Kristin Lorenz, Michael Oellerich, Nicolas Von AhsenAbstract:Background: Inosine triphosphate (ITP) pyrophosphohydrolase (ITPA) catalyzes the pyrophosphohydrolysis of ITP/dITP and xanthosine triphosphate to prevent incorporation of unusual nucleotides into RNA and DNA. Important mutations leading to enzyme deficiency are 94C>A and IVS2 + 21A>C. An association between ITPA 94C>A and adverse reactions during azathioprine treatment has been shown. To investigate the ITPA phenotype, an HPLC procedure was developed and phenotype-genotype correlations were assessed. Methods: The enzymatic conversion of ITP to inosine monophosphate (IMP) was terminated by perchloric acid and saturated dipotassium hydrogen phosphate. We quantified the IMP at 262 nm after separation on an Aqua perfect C18 column using 20 mmol/L phosphate buffer, pH 2.5. We also genotyped samples for ITPA 94C>A and IVS2 + 21A>C by real-time fluorescence PCR. Results: The assay was linear to 3 mmol/L IMP [∼500 μmol/(g Hb · h)] with a lower limit of quantification of 4 μmol/L [∼0.5 μmol/(g Hb · h)]. With IMP-enriched samples, within- and between-day imprecision was ≤3.6% and ≤4.9%, respectively, and the inaccuracy was ≤5.2%. With pooled erythrocytes, within- and between-day imprecision was 3.8% and 7.5%, respectively. ITPA activity in 130 healthy controls was between A) and 0.131 (IVS2 + 21A>C). When we used a cutoff of 125 μmol IMP/(g Hb · h), phenotyping detected all 94C>A mutant cases, all 94C>A and IVS2 + 21A>C compound heterozygotes, all IVS2 + 21A>C homozygotes, and 6 of 24 IVS2 + 21A>C heterozygote-only cases. A novel IVS2 + 68T>C mutation was also found. Conclusions: The HPLC procedure provides an excellent ITPA phenotype-genotype correlation and led to the discovery of a novel IVS2 + 68T>C mutation. The method could facilitate investigation of the role of ITPA activity for drug toxicity during thiopurine therapy.
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association of inosine triphosphatase 94c a and thiopurine s methyltransferase deficiency with adverse events and study drop outs under azathioprine therapy in a prospective crohn disease study
Clinical Chemistry, 2005Co-Authors: Nicolas Von Ahsen, Victor W Armstrong, Christoph Behrens, Christian Von Tirpitz, A Stallmach, Hans H Herfarth, Jurgen Stein, Peter Bias, Guido Adler, Maria ShipkovaAbstract:Background: Azathioprine (aza) therapy is beneficial in the treatment of inflammatory bowel disease, but 10%–30% of patients cannot tolerate aza therapy because of adverse drug reactions. Thiopurine S -methyltransferase (TPMT) deficiency predisposes to myelotoxicity, but its association with other side effects is less clear. Inosine triphosphatase (ITPA) mutations are other pharmacogenetic polymorphisms possibly involved in thiopurine metabolism and tolerance. Methods: We analyzed data from a 6-month prospective study including 71 patients with Crohn disease undergoing first-time aza treatment with respect to aza intolerance. Patients were genotyped for common TPMT and ITPA mutations and had pretherapy TPMT activity measured. Results: Early drop-out (within 2 weeks) from aza therapy was associated with ITPA 94C>A [ P = 0.020; odds ratio (OR), 4.6; 95% confidence interval (95% CI), 1.2–17.4] and low TPMT activity [ A or TPMT A ( P = 0.002; OR = 7.8; 95% CI, 2.1–29.1). Time-to-event analysis over the 24-week study period revealed a significant association ( P = 0.031) between the time to drop-out and ITPA 94C>A mutant allele carrier status. Conclusions: Patients with ITPA 94C>A mutations or low TPMT activity constitute a pharmacogenetic high-risk group for drop-out from aza therapy. ITPA 94C>A appears to be a promising marker indicating predisposition to aza intolerance.
Nicolas Von Ahsen - One of the best experts on this subject based on the ideXlab platform.
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analysis of ITPA phenotype genotype correlation in the bulgarian population revealed a novel gene variant in exon 6
Therapeutic Drug Monitoring, 2007Co-Authors: Srebrena Atanasova, Maria Shipkova, Eberhard Wieland, Michael Oellerich, Dobrin Svinarov, Antoaneta Mladenova, Mariana Genova, Nicolas Von AhsenAbstract:Abstract:Mutations in the inosine triphosphate pyrophosphohydrolase (ITPA) gene causing enzyme deficiency were shown to have pharmacogenetic implications in azathioprine-induced adverse drug reactions. The distribution of ITPA activity as well as the types and the frequencies of gene variants associ
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measurement of erythrocyte inosine triphosphate pyrophosphohydrolase ITPA activity by hplc and correlation of ITPA genotype phenotype in a caucasian population
Clinical Chemistry, 2006Co-Authors: Maria Shipkova, Eberhard Wieland, Kristin Lorenz, Michael Oellerich, Nicolas Von AhsenAbstract:Background: Inosine triphosphate (ITP) pyrophosphohydrolase (ITPA) catalyzes the pyrophosphohydrolysis of ITP/dITP and xanthosine triphosphate to prevent incorporation of unusual nucleotides into RNA and DNA. Important mutations leading to enzyme deficiency are 94C>A and IVS2 + 21A>C. An association between ITPA 94C>A and adverse reactions during azathioprine treatment has been shown. To investigate the ITPA phenotype, an HPLC procedure was developed and phenotype-genotype correlations were assessed. Methods: The enzymatic conversion of ITP to inosine monophosphate (IMP) was terminated by perchloric acid and saturated dipotassium hydrogen phosphate. We quantified the IMP at 262 nm after separation on an Aqua perfect C18 column using 20 mmol/L phosphate buffer, pH 2.5. We also genotyped samples for ITPA 94C>A and IVS2 + 21A>C by real-time fluorescence PCR. Results: The assay was linear to 3 mmol/L IMP [∼500 μmol/(g Hb · h)] with a lower limit of quantification of 4 μmol/L [∼0.5 μmol/(g Hb · h)]. With IMP-enriched samples, within- and between-day imprecision was ≤3.6% and ≤4.9%, respectively, and the inaccuracy was ≤5.2%. With pooled erythrocytes, within- and between-day imprecision was 3.8% and 7.5%, respectively. ITPA activity in 130 healthy controls was between A) and 0.131 (IVS2 + 21A>C). When we used a cutoff of 125 μmol IMP/(g Hb · h), phenotyping detected all 94C>A mutant cases, all 94C>A and IVS2 + 21A>C compound heterozygotes, all IVS2 + 21A>C homozygotes, and 6 of 24 IVS2 + 21A>C heterozygote-only cases. A novel IVS2 + 68T>C mutation was also found. Conclusions: The HPLC procedure provides an excellent ITPA phenotype-genotype correlation and led to the discovery of a novel IVS2 + 68T>C mutation. The method could facilitate investigation of the role of ITPA activity for drug toxicity during thiopurine therapy.
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association of inosine triphosphatase 94c a and thiopurine s methyltransferase deficiency with adverse events and study drop outs under azathioprine therapy in a prospective crohn disease study
Clinical Chemistry, 2005Co-Authors: Nicolas Von Ahsen, Victor W Armstrong, Christoph Behrens, Christian Von Tirpitz, A Stallmach, Hans H Herfarth, Jurgen Stein, Peter Bias, Guido Adler, Maria ShipkovaAbstract:Background: Azathioprine (aza) therapy is beneficial in the treatment of inflammatory bowel disease, but 10%–30% of patients cannot tolerate aza therapy because of adverse drug reactions. Thiopurine S -methyltransferase (TPMT) deficiency predisposes to myelotoxicity, but its association with other side effects is less clear. Inosine triphosphatase (ITPA) mutations are other pharmacogenetic polymorphisms possibly involved in thiopurine metabolism and tolerance. Methods: We analyzed data from a 6-month prospective study including 71 patients with Crohn disease undergoing first-time aza treatment with respect to aza intolerance. Patients were genotyped for common TPMT and ITPA mutations and had pretherapy TPMT activity measured. Results: Early drop-out (within 2 weeks) from aza therapy was associated with ITPA 94C>A [ P = 0.020; odds ratio (OR), 4.6; 95% confidence interval (95% CI), 1.2–17.4] and low TPMT activity [ A or TPMT A ( P = 0.002; OR = 7.8; 95% CI, 2.1–29.1). Time-to-event analysis over the 24-week study period revealed a significant association ( P = 0.031) between the time to drop-out and ITPA 94C>A mutant allele carrier status. Conclusions: Patients with ITPA 94C>A mutations or low TPMT activity constitute a pharmacogenetic high-risk group for drop-out from aza therapy. ITPA 94C>A appears to be a promising marker indicating predisposition to aza intolerance.
Youri I Pavlov - One of the best experts on this subject based on the ideXlab platform.
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measuring deaminated nucleotide surveillance enzyme ITPA activity with an atp releasing nucleotide chimera
Nucleic Acids Research, 2017Co-Authors: Youri I Pavlov, Elena I Stepchenkova, Jian Cui, Miriam R Menezes, Eric T KoolAbstract:Nucleotide quality surveillance enzymes play important roles in human health, by detecting damaged molecules in the nucleotide pool and deactivating them before they are incorporated into chromosomal DNA or adversely affect metabolism. In particular, deamination of adenine moiety in (deoxy)nucleoside triphosphates, resulting in formation of (d)ITP, can be deleterious, leading to DNA damage, mutagenesis and other harmful cellular effects. The 21.5 kDa human enzyme that mitigates this damage by conversion of (d)ITP to monophosphate, ITPA, has been proposed as a possible therapeutic and diagnostic target for multiple diseases. Measuring the activity of this enzyme is useful both in basic research and in clinical applications involving this pathway, but current methods are nonselective and are not applicable to measurement of the enzyme from cells or tissues. Here, we describe the design and synthesis of an ITPA-specific chimeric dinucleotide (DIAL) that replaces the pyrophosphate leaving group of the native substrate with adenosine triphosphate, enabling sensitive detection via luciferase luminescence signaling. The probe is shown to function sensitively and selectively to quantify enzyme activity in vitro, and can be used to measure the activity of ITPA in bacterial, yeast and human cell lysates.
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ITPA inosine triphosphate pyrophosphatase from surveillance of nucleotide pools to human disease and pharmacogenetics
Mutation Research-reviews in Mutation Research, 2013Co-Authors: Peter D Simone, Youri I Pavlov, Gloria E. O. BorgstahlAbstract:Abstract Cellular nucleotide pools are often contaminated by base analog nucleotides which interfere with a plethora of biological reactions, from DNA and RNA synthesis to cellular signaling. An evolutionarily conserved inosine triphosphate pyrophosphatase (ITPA) removes the non-canonical purine (d)NTPs inosine triphosphate and xanthosine triphosphate by hydrolyzing them into their monophosphate form and pyrophosphate. Mutations in the ITPA orthologs in model organisms lead to genetic instability and, in mice, to severe developmental anomalies. In humans there is genetic polymorphism in ITPA. One allele leads to a proline to threonine substitution at amino acid 32 and causes varying degrees of ITPA deficiency in tissues and plays a role in patients’ response to drugs. Structural analysis of this mutant protein reveals that the protein is destabilized by the formation of a cavity in its hydrophobic core. The Pro32Thr allele is thought to cause the observed dominant negative effect because the resulting active enzyme monomer targets both homo- and heterodimers to degradation.
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Pivotal role of inosine triphosphate pyrophosphatase in maintaining genome stability and the prevention of apoptosis in human cells
PloS one, 2012Co-Authors: Miriam R. Menezes, Irina S.-r. Waisertreiger, Hernando Lopez-bertoni, Xu Luo, Youri I PavlovAbstract:Pure nucleotide precursor pools are a prerequisite for high-fidelity DNA replication and the suppression of mutagenesis and carcinogenesis. ITPAses are nucleoside triphosphate pyrophosphatases that clean the precursor pools of the non-canonical triphosphates of inosine and xanthine. The precise role of the human ITPAse, encoded by the ITPA gene, is not clearly defined. ITPA is clinically important because a widespread polymorphism, 94C>A, leads to null ITPAse activity in erythrocytes and is associated with an adverse reaction to thiopurine drugs. We studied the cellular function of ITPA in HeLa cells using the purine analog 6-N hydroxylaminopurine (HAP), whose triphosphate is also a substrate for ITPA. In this study, we demonstrate that ITPA knockdown sensitizes HeLa cells to HAP-induced DNA breaks and apoptosis. The HAP-induced DNA damage and cytotoxicity observed in ITPA knockdown cells are rescued by an overexpression of the yeast ITPAse encoded by the HAM1 gene. We further show that ITPA knockdown results in elevated mutagenesis in response to HAP treatment. Our studies reveal the significance of ITPA in preventing base analog-induced apoptosis, DNA damage and mutagenesis in human cells. This implies that individuals with defective ITPAse are predisposed to genome damage by impurities in nucleotide pools, which is drastically augmented by therapy with purine analogs. They are also at an elevated risk for degenerative diseases and cancer.
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Pivotal Role of Inosine Triphosphate Pyrophosphatase in Maintaining Genome Stability and the Prevention of
2012Co-Authors: Apoptosis In Human Cells, Miriam R. Menezes, Irina S.-r. Waisertreiger, Xu Luo, O Lopez-bertoni, Youri I PavlovAbstract:Pure nucleotide precursor pools are a prerequisite for high-fidelity DNA replication and the suppression of mutagenesis and carcinogenesis. ITPAses are nucleoside triphosphate pyrophosphatases that clean the precursor pools of the non-canonical triphosphates of inosine and xanthine. The precise role of the human ITPAse, encoded by the ITPA gene, is not clearly defined. ITPA is clinically important because a widespread polymorphism, 94C.A, leads to null ITPAse activity in erythrocytes and is associated with an adverse reaction to thiopurine drugs. We studied the cellular function of ITPA in HeLa cells using the purine analog 6-N hydroxylaminopurine (HAP), whose triphosphate is also a substrate for ITPA. In this study, we demonstrate that ITPA knockdown sensitizes HeLa cells to HAP-induced DNA breaks and apoptosis. The HAP-induced DNA damage and cytotoxicity observed in ITPA knockdown cells are rescued by an overexpression of the yeast ITPAse encoded by the HAM1 gene. We further show that ITPA knockdown results in elevated mutagenesis in response to HAP treatment. Our studies reveal the significance of ITPA in preventing base analog-induced apoptosis, DNA damage and mutagenesis in human cells. This implies that individuals with defective ITPAse are predisposed to genome damage by impurities in nucleotide pools, which is drastically augmented by therapy with purine analogs. They are also at an elevate
Gloria E. O. Borgstahl - One of the best experts on this subject based on the ideXlab platform.
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ITPA inosine triphosphate pyrophosphatase from surveillance of nucleotide pools to human disease and pharmacogenetics
Mutation Research-reviews in Mutation Research, 2013Co-Authors: Peter D Simone, Youri I Pavlov, Gloria E. O. BorgstahlAbstract:Abstract Cellular nucleotide pools are often contaminated by base analog nucleotides which interfere with a plethora of biological reactions, from DNA and RNA synthesis to cellular signaling. An evolutionarily conserved inosine triphosphate pyrophosphatase (ITPA) removes the non-canonical purine (d)NTPs inosine triphosphate and xanthosine triphosphate by hydrolyzing them into their monophosphate form and pyrophosphate. Mutations in the ITPA orthologs in model organisms lead to genetic instability and, in mice, to severe developmental anomalies. In humans there is genetic polymorphism in ITPA. One allele leads to a proline to threonine substitution at amino acid 32 and causes varying degrees of ITPA deficiency in tissues and plays a role in patients’ response to drugs. Structural analysis of this mutant protein reveals that the protein is destabilized by the formation of a cavity in its hydrophobic core. The Pro32Thr allele is thought to cause the observed dominant negative effect because the resulting active enzyme monomer targets both homo- and heterodimers to degradation.
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Structure of the orthorhombic form of human inosine triphosphate pyrophosphatase
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2006Co-Authors: Jason Porta, Carol Kolar, Stanislav G. Kozmin, Gloria E. O. BorgstahlAbstract:The structure of human inosine triphosphate pyrophosphohydrolase (ITPA) has been determined using diffraction data to 1.6 A resolution. ITPA contributes to the accurate replication of DNA by cleansing cellular dNTP pools of mutagenic nucleotide purine analogs such as dITP or dXTP. A similar high-resolution unpublished structure has been deposited in the Protein Data Bank from a monoclinic and pseudo-merohedrally twinned crystal. Here, cocrystallization of ITPA with a molar ratio of XTP appears to have improved the crystals by eliminating twinning and resulted in an orthorhombic space group. However, there was no evidence for bound XTP in the structure. Comparison with substrate-bound NTPase from a thermophilic organism predicts the movement of residues within helix α1, the loop before α6 and helix α7 to cap off the active site when substrate is bound.
Yusaku Nakabeppu - One of the best experts on this subject based on the ideXlab platform.
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Neural stem cell–specific ITPA deficiency causes neural depolarization and epilepsy
'American Society for Clinical Investigation', 2020Co-Authors: Yuichiro Koga, Yasuto Yoneshima, Nona Abolhassani, Kunihiko Sakumi, Daisuke Tsuchimoto, Yoshinori Hayashi, Hiroshi Nakanishi, Shinya Toyokuni, Yusaku NakabeppuAbstract:Inosine triphosphate pyrophosphatase (ITPA) hydrolyzes inosine triphosphate (ITP) and other deaminated purine nucleotides to the corresponding nucleoside monophosphates. In humans, ITPA deficiency causes severe encephalopathy with epileptic seizure, microcephaly, and developmental retardation. In this study, we established neural stem cell–specific ITPA–conditional KO mice (ITPA-cKO mice) to clarify the effects of ITPA deficiency on the neural system. The ITPA-cKO mice showed growth retardation and died within 3 weeks of birth. We did not observe any microcephaly in the ITPA-cKO mice, although the female ITPA-cKO mice did show adrenal hypoplasia. The ITPA-cKO mice showed limb-clasping upon tail suspension and spontaneous and/or audiogenic seizure. Whole-cell patch-clamp recordings from entorhinal cortex neurons in brain slices revealed a depolarized resting membrane potential, increased firing, and frequent spontaneous miniature excitatory postsynaptic current and miniature inhibitory postsynaptic current in the ITPA-cKO mice compared with ITPA-proficient controls. Accumulated ITP or its metabolites, such as cyclic inosine monophosphates, or RNA containing inosines may cause membrane depolarization and hyperexcitability in neurons and induce the phenotype of ITPA-deficient mice, including seizure
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deoxyinosine triphosphate induces mlh1 pms2 and p53 dependent cell growth arrest and dna instability in mammalian cells
Scientific Reports, 2016Co-Authors: Yasuto Yoneshima, Nona Abolhassani, Teruaki Iyama, Kunihiko Sakumi, Naoko Shiomi, Masahiko Mori, Tadahiro Shiomi, Tetsuo Noda, Daisuke Tsuchimoto, Yusaku NakabeppuAbstract:Deoxyinosine (dI) occurs in DNA either by oxidative deamination of a previously incorporated deoxyadenosine residue or by misincorporation of deoxyinosine triphosphate (dITP) from the nucleotide pool during replication. To exclude dITP from the pool, mammals possess specific hydrolysing enzymes, such as inosine triphosphatase (ITPA). Previous studies have shown that deficiency in ITPA results in cell growth suppression and DNA instability. To explore the mechanisms of these phenotypes, we analysed ITPA-deficient human and mouse cells. We found that both growth suppression and accumulation of single-strand breaks in nuclear DNA of ITPA-deficient cells depended on MLH1/PMS2. The cell growth suppression of ITPA-deficient cells also depended on p53, but not on MPG, ENDOV or MSH2. ITPA deficiency significantly increased the levels of p53 protein and p21 mRNA/protein, a well-known target of p53, in an MLH1-dependent manner. Furthermore, MLH1 may also contribute to cell growth arrest by increasing the basal level of p53 activity.
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Deoxyinosine triphosphate induces MLH1/PMS2- and p53-dependent cell growth arrest and DNA instability in mammalian cells.
Scientific reports, 2016Co-Authors: Yasuto Yoneshima, Nona Abolhassani, Teruaki Iyama, Kunihiko Sakumi, Naoko Shiomi, Masahiko Mori, Tadahiro Shiomi, Tetsuo Noda, Daisuke Tsuchimoto, Yusaku NakabeppuAbstract:Deoxyinosine (dI) occurs in DNA either by oxidative deamination of a previously incorporated deoxyadenosine residue or by misincorporation of deoxyinosine triphosphate (dITP) from the nucleotide pool during replication. To exclude dITP from the pool, mammals possess specific hydrolysing enzymes, such as inosine triphosphatase (ITPA). Previous studies have shown that deficiency in ITPA results in cell growth suppression and DNA instability. To explore the mechanisms of these phenotypes, we analysed ITPA-deficient human and mouse cells. We found that both growth suppression and accumulation of single-strand breaks in nuclear DNA of ITPA-deficient cells depended on MLH1/PMS2. The cell growth suppression of ITPA-deficient cells also depended on p53, but not on MPG, ENDOV or MSH2. ITPA deficiency significantly increased the levels of p53 protein and p21 mRNA/protein, a well-known target of p53, in an MLH1-dependent manner. Furthermore, MLH1 may also contribute to cell growth arrest by increasing the basal level of p53 activity.
