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NUDT16 and ITPA play a dual protective role in maintaining chromosome stability and cell growth by eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals.

Abolhassani N, Iyama T, Tsuchimoto D, Sakumi K, Ohno M, Behmanesh M, Nakabeppu Y - Nucleic Acids Res. (2010)

Bottom Line: Mammalian inosine triphosphatase encoded by ITPA gene hydrolyzes ITP and dITP to monophosphates, avoiding their deleterious effects.Itpa(-) mice exhibited perinatal lethality, and significantly higher levels of inosine in cellular RNA and deoxyinosine in nuclear DNA were detected in Itpa(-) embryos than in wild-type embryos.We thus conclude that NUDT16 and ITPA play a dual protective role for eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals.

View Article: PubMed Central - PubMed

Affiliation: Division of Neurofunctional Genomics, Department of Immunobiology and Neuroscience, Medical Institute of Bioregulation, Kyushu University, Fukuoka, 812-8582, Japan.

ABSTRACT
Mammalian inosine triphosphatase encoded by ITPA gene hydrolyzes ITP and dITP to monophosphates, avoiding their deleterious effects. Itpa(-) mice exhibited perinatal lethality, and significantly higher levels of inosine in cellular RNA and deoxyinosine in nuclear DNA were detected in Itpa(-) embryos than in wild-type embryos. Therefore, we examined the effects of ITPA deficiency on mouse embryonic fibroblasts (MEFs). Itpa(-) primary MEFs lacking ITP-hydrolyzing activity exhibited a prolonged doubling time, increased chromosome abnormalities and accumulation of single-strand breaks in nuclear DNA, compared with primary MEFs prepared from wild-type embryos. However, immortalized Itpa(-) MEFs had neither of these phenotypes and had a significantly higher ITP/IDP-hydrolyzing activity than Itpa(-) embryos or primary MEFs. Mammalian NUDT16 proteins exhibit strong dIDP/IDP-hydrolyzing activity and similarly low levels of Nudt16 mRNA and protein were detected in primary MEFs derived from both wild-type and Itpa(-) embryos. However, immortalized Itpa(-) MEFs expressed significantly higher levels of Nudt16 than the wild type. Moreover, introduction of silencing RNAs against Nudt16 into immortalized Itpa(-) MEFs reproduced ITPA-deficient phenotypes. We thus conclude that NUDT16 and ITPA play a dual protective role for eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals.

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ITPA-deficient primary MEFs exhibit various cellular dysfunctions. (A) ITPA deficiency caused a significantly increased accumulation of inosine in cellular RNA. Inosine level was determined by LC–MS/MS analysis of cellular RNA prepared from embryos (N3). Result of non-repeated measures ANOVA (two-tailed), P = 1.69 × 10−7. Student–Newman–Keuls (SNK) post hoc test, **P < 0.01 (versus Itpa+/+ and Itpa+/−). Data are shown as the mean ± SD (n = 3 independent embryos). (B) ITPA deficiency caused a significantly increased accumulation of deoxyinosine (dI) in nuclear DNA. Deoxyinosine (dI) level was determined by LC–MS/MS analysis of nuclear DNA prepared from embryos (N3). Result of non-repeated measures ANOVA (two-tailed), P = 0.00038. SNK post hoc test, **P < 0.01 (versus Itpa+/+ and Itpa+/−). Data are shown as the mean ± SD (n = 3 independent embryos). (C) ITPA deficiency impairs normal cell proliferation. Primary MEFs (Passage 2) isolated from four separate Itpa−/– embryos showed significant prolonged doubling time in comparison to those from Itpa+/+ and Itpa+/− embryos. Result of repeated measures ANOVA (two-tailed), P = 0.0005. Bonferroni/Dunn post-hoc test, *P < 0.05 (versus Itpa+/−), **P < 0.01 (versus Itpa+/+). Data are shown as the mean ± SD (n = 4 independent MEFs). (D) ITPA deficiency causes G2/M arrest. Primary MEFs (Passage 5) were subjected to flow cytometry analysis and the percentages of Cell-cycle phases in each MEF set were determined. Result of non-repeated measures ANOVA (two-tailed), P = 1.74 × 10−8. Bonferroni post hoc test, **P < 0.01 (versus Itpa+/+ and Itpa+/−). Data are shown as the mean ± SD (n = 3 independent isolates).
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Figure 1: ITPA-deficient primary MEFs exhibit various cellular dysfunctions. (A) ITPA deficiency caused a significantly increased accumulation of inosine in cellular RNA. Inosine level was determined by LC–MS/MS analysis of cellular RNA prepared from embryos (N3). Result of non-repeated measures ANOVA (two-tailed), P = 1.69 × 10−7. Student–Newman–Keuls (SNK) post hoc test, **P < 0.01 (versus Itpa+/+ and Itpa+/−). Data are shown as the mean ± SD (n = 3 independent embryos). (B) ITPA deficiency caused a significantly increased accumulation of deoxyinosine (dI) in nuclear DNA. Deoxyinosine (dI) level was determined by LC–MS/MS analysis of nuclear DNA prepared from embryos (N3). Result of non-repeated measures ANOVA (two-tailed), P = 0.00038. SNK post hoc test, **P < 0.01 (versus Itpa+/+ and Itpa+/−). Data are shown as the mean ± SD (n = 3 independent embryos). (C) ITPA deficiency impairs normal cell proliferation. Primary MEFs (Passage 2) isolated from four separate Itpa−/– embryos showed significant prolonged doubling time in comparison to those from Itpa+/+ and Itpa+/− embryos. Result of repeated measures ANOVA (two-tailed), P = 0.0005. Bonferroni/Dunn post-hoc test, *P < 0.05 (versus Itpa+/−), **P < 0.01 (versus Itpa+/+). Data are shown as the mean ± SD (n = 4 independent MEFs). (D) ITPA deficiency causes G2/M arrest. Primary MEFs (Passage 5) were subjected to flow cytometry analysis and the percentages of Cell-cycle phases in each MEF set were determined. Result of non-repeated measures ANOVA (two-tailed), P = 1.74 × 10−8. Bonferroni post hoc test, **P < 0.01 (versus Itpa+/+ and Itpa+/−). Data are shown as the mean ± SD (n = 3 independent isolates).

Mentions: Primary or immortalized MEFs were seeded at 1 × 105 cells (Figures 1C and 4A) or 0.5 × 105 cells (Figure 7C) per well in six-well plates (Nalge Nunc International K.K., Tokyo, Japan). Cells were harvested every 2 days or every day, respectively, and the numbers of cells were counted using a hemocytometer.Figure 1.


NUDT16 and ITPA play a dual protective role in maintaining chromosome stability and cell growth by eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals.

Abolhassani N, Iyama T, Tsuchimoto D, Sakumi K, Ohno M, Behmanesh M, Nakabeppu Y - Nucleic Acids Res. (2010)

ITPA-deficient primary MEFs exhibit various cellular dysfunctions. (A) ITPA deficiency caused a significantly increased accumulation of inosine in cellular RNA. Inosine level was determined by LC–MS/MS analysis of cellular RNA prepared from embryos (N3). Result of non-repeated measures ANOVA (two-tailed), P = 1.69 × 10−7. Student–Newman–Keuls (SNK) post hoc test, **P < 0.01 (versus Itpa+/+ and Itpa+/−). Data are shown as the mean ± SD (n = 3 independent embryos). (B) ITPA deficiency caused a significantly increased accumulation of deoxyinosine (dI) in nuclear DNA. Deoxyinosine (dI) level was determined by LC–MS/MS analysis of nuclear DNA prepared from embryos (N3). Result of non-repeated measures ANOVA (two-tailed), P = 0.00038. SNK post hoc test, **P < 0.01 (versus Itpa+/+ and Itpa+/−). Data are shown as the mean ± SD (n = 3 independent embryos). (C) ITPA deficiency impairs normal cell proliferation. Primary MEFs (Passage 2) isolated from four separate Itpa−/– embryos showed significant prolonged doubling time in comparison to those from Itpa+/+ and Itpa+/− embryos. Result of repeated measures ANOVA (two-tailed), P = 0.0005. Bonferroni/Dunn post-hoc test, *P < 0.05 (versus Itpa+/−), **P < 0.01 (versus Itpa+/+). Data are shown as the mean ± SD (n = 4 independent MEFs). (D) ITPA deficiency causes G2/M arrest. Primary MEFs (Passage 5) were subjected to flow cytometry analysis and the percentages of Cell-cycle phases in each MEF set were determined. Result of non-repeated measures ANOVA (two-tailed), P = 1.74 × 10−8. Bonferroni post hoc test, **P < 0.01 (versus Itpa+/+ and Itpa+/−). Data are shown as the mean ± SD (n = 3 independent isolates).
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Related In: Results  -  Collection

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Figure 1: ITPA-deficient primary MEFs exhibit various cellular dysfunctions. (A) ITPA deficiency caused a significantly increased accumulation of inosine in cellular RNA. Inosine level was determined by LC–MS/MS analysis of cellular RNA prepared from embryos (N3). Result of non-repeated measures ANOVA (two-tailed), P = 1.69 × 10−7. Student–Newman–Keuls (SNK) post hoc test, **P < 0.01 (versus Itpa+/+ and Itpa+/−). Data are shown as the mean ± SD (n = 3 independent embryos). (B) ITPA deficiency caused a significantly increased accumulation of deoxyinosine (dI) in nuclear DNA. Deoxyinosine (dI) level was determined by LC–MS/MS analysis of nuclear DNA prepared from embryos (N3). Result of non-repeated measures ANOVA (two-tailed), P = 0.00038. SNK post hoc test, **P < 0.01 (versus Itpa+/+ and Itpa+/−). Data are shown as the mean ± SD (n = 3 independent embryos). (C) ITPA deficiency impairs normal cell proliferation. Primary MEFs (Passage 2) isolated from four separate Itpa−/– embryos showed significant prolonged doubling time in comparison to those from Itpa+/+ and Itpa+/− embryos. Result of repeated measures ANOVA (two-tailed), P = 0.0005. Bonferroni/Dunn post-hoc test, *P < 0.05 (versus Itpa+/−), **P < 0.01 (versus Itpa+/+). Data are shown as the mean ± SD (n = 4 independent MEFs). (D) ITPA deficiency causes G2/M arrest. Primary MEFs (Passage 5) were subjected to flow cytometry analysis and the percentages of Cell-cycle phases in each MEF set were determined. Result of non-repeated measures ANOVA (two-tailed), P = 1.74 × 10−8. Bonferroni post hoc test, **P < 0.01 (versus Itpa+/+ and Itpa+/−). Data are shown as the mean ± SD (n = 3 independent isolates).
Mentions: Primary or immortalized MEFs were seeded at 1 × 105 cells (Figures 1C and 4A) or 0.5 × 105 cells (Figure 7C) per well in six-well plates (Nalge Nunc International K.K., Tokyo, Japan). Cells were harvested every 2 days or every day, respectively, and the numbers of cells were counted using a hemocytometer.Figure 1.

Bottom Line: Mammalian inosine triphosphatase encoded by ITPA gene hydrolyzes ITP and dITP to monophosphates, avoiding their deleterious effects.Itpa(-) mice exhibited perinatal lethality, and significantly higher levels of inosine in cellular RNA and deoxyinosine in nuclear DNA were detected in Itpa(-) embryos than in wild-type embryos.We thus conclude that NUDT16 and ITPA play a dual protective role for eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals.

View Article: PubMed Central - PubMed

Affiliation: Division of Neurofunctional Genomics, Department of Immunobiology and Neuroscience, Medical Institute of Bioregulation, Kyushu University, Fukuoka, 812-8582, Japan.

ABSTRACT
Mammalian inosine triphosphatase encoded by ITPA gene hydrolyzes ITP and dITP to monophosphates, avoiding their deleterious effects. Itpa(-) mice exhibited perinatal lethality, and significantly higher levels of inosine in cellular RNA and deoxyinosine in nuclear DNA were detected in Itpa(-) embryos than in wild-type embryos. Therefore, we examined the effects of ITPA deficiency on mouse embryonic fibroblasts (MEFs). Itpa(-) primary MEFs lacking ITP-hydrolyzing activity exhibited a prolonged doubling time, increased chromosome abnormalities and accumulation of single-strand breaks in nuclear DNA, compared with primary MEFs prepared from wild-type embryos. However, immortalized Itpa(-) MEFs had neither of these phenotypes and had a significantly higher ITP/IDP-hydrolyzing activity than Itpa(-) embryos or primary MEFs. Mammalian NUDT16 proteins exhibit strong dIDP/IDP-hydrolyzing activity and similarly low levels of Nudt16 mRNA and protein were detected in primary MEFs derived from both wild-type and Itpa(-) embryos. However, immortalized Itpa(-) MEFs expressed significantly higher levels of Nudt16 than the wild type. Moreover, introduction of silencing RNAs against Nudt16 into immortalized Itpa(-) MEFs reproduced ITPA-deficient phenotypes. We thus conclude that NUDT16 and ITPA play a dual protective role for eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals.

Show MeSH
Related in: MedlinePlus