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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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Increased DNA content in ITPA deficient primary MEFs. (A) Flow cytometric analysis of the cell cycle was performed and the sub G1 fraction (M1), diploid fraction (M2) and a fraction with an increased DNA content (M3) were determined. (B) ITPA deficiency increased chromosomal ploidy in primary MEFs. Percentages of diploid, tetraploid and others are shown in pie charts with the mean ± SD (three independent isolates). The frequency of tetraploidy was increased significantly in Itpa−/– MEFs. Results show non-repeated measures ANOVA (two-tailed): P = 0.094. P-value is shown following a Bonferroni post hoc test.
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Figure 2: Increased DNA content in ITPA deficient primary MEFs. (A) Flow cytometric analysis of the cell cycle was performed and the sub G1 fraction (M1), diploid fraction (M2) and a fraction with an increased DNA content (M3) were determined. (B) ITPA deficiency increased chromosomal ploidy in primary MEFs. Percentages of diploid, tetraploid and others are shown in pie charts with the mean ± SD (three independent isolates). The frequency of tetraploidy was increased significantly in Itpa−/– MEFs. Results show non-repeated measures ANOVA (two-tailed): P = 0.094. P-value is shown following a Bonferroni post hoc test.

Mentions: To examine the nature of the cellular dysfunction caused by ITPA deficiency, we isolated embryos (at E13.5 and E14.5) from intercrosses of Itpa+/− littermates (N10, one pair; N11, two pairs) and determined their genotypes (Supplementary Table S1). Among 20 embryos, five were found to be Itpa−/–, 10 were Itpa+/− and the remaining five were Itpa+/+. Then we isolated primary MEFs independently from four embryos of each genotype and their genotypes and the expression levels of ITPA protein were confirmed (Supplementary Figure S1A and B). All Itpa−/– primary MEFs at the second passage showed significantly longer doubling time (132.0 ± 14.6 h) than those from Itpa+/+ (78.6 ± 10.1 h) and Itpa+/− (87.5 ± 14.3 h) embryos (Figure 1C). There was no obvious difference in their morphology under phase contrast microscopy (Supplementary Figure S1C). In Passage 5, we observed essentially the same proliferation deficiency in Itpa−/– MEFs and slightly increased numbers of senescence-associated β-galactosidase (SA-β-Gal)-positive cells in Itpa−/– MEFs (Itpa+/+ versus Itpa−/–, 3.64% versus 7.74%; Supplementary Figure S2A and B). Flow cytometry analysis of the cell cycle revealed that Itpa−/– MEFs exhibited a significant increase in the G2/M phase (Itpa+/+ versus Itpa−/–, 28.4% versus 77.4%; Figure 1D) and a slight increase in the sub G1 fraction (Itpa+/+ versus Itpa−/–, 1.61% versus 5.51%; fraction M1 in Figure 2A). There was an apparent increase in cells with an abnormally increased DNA content in Itpa−/– MEFs compared with Itpa+/+ MEFs (fraction M3 in Figure 2A), indicating that the G2/M phase shown in Figure 1D might have contained some tetraploid cells at the G1 phase (Figure 2B). There was no increase in dead cells detected as propidium iodide (PI)/Hoechst-double positive cells (Supplementary Figure S2C). These results indicate that ITPA deficiency caused delay or arrest in cell-cycle progression. We further observed that exposure of Itpa−/–, but not Itpa+/+, Itpa+/− MEFs, to sodium nitrite (NaNO2), which causes predominant deamination of purine bases (18), resulted in growth suppression without inducing cell death (Supplementary Figure S2C).


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)

Increased DNA content in ITPA deficient primary MEFs. (A) Flow cytometric analysis of the cell cycle was performed and the sub G1 fraction (M1), diploid fraction (M2) and a fraction with an increased DNA content (M3) were determined. (B) ITPA deficiency increased chromosomal ploidy in primary MEFs. Percentages of diploid, tetraploid and others are shown in pie charts with the mean ± SD (three independent isolates). The frequency of tetraploidy was increased significantly in Itpa−/– MEFs. Results show non-repeated measures ANOVA (two-tailed): P = 0.094. P-value is shown following a Bonferroni post hoc test.
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Figure 2: Increased DNA content in ITPA deficient primary MEFs. (A) Flow cytometric analysis of the cell cycle was performed and the sub G1 fraction (M1), diploid fraction (M2) and a fraction with an increased DNA content (M3) were determined. (B) ITPA deficiency increased chromosomal ploidy in primary MEFs. Percentages of diploid, tetraploid and others are shown in pie charts with the mean ± SD (three independent isolates). The frequency of tetraploidy was increased significantly in Itpa−/– MEFs. Results show non-repeated measures ANOVA (two-tailed): P = 0.094. P-value is shown following a Bonferroni post hoc test.
Mentions: To examine the nature of the cellular dysfunction caused by ITPA deficiency, we isolated embryos (at E13.5 and E14.5) from intercrosses of Itpa+/− littermates (N10, one pair; N11, two pairs) and determined their genotypes (Supplementary Table S1). Among 20 embryos, five were found to be Itpa−/–, 10 were Itpa+/− and the remaining five were Itpa+/+. Then we isolated primary MEFs independently from four embryos of each genotype and their genotypes and the expression levels of ITPA protein were confirmed (Supplementary Figure S1A and B). All Itpa−/– primary MEFs at the second passage showed significantly longer doubling time (132.0 ± 14.6 h) than those from Itpa+/+ (78.6 ± 10.1 h) and Itpa+/− (87.5 ± 14.3 h) embryos (Figure 1C). There was no obvious difference in their morphology under phase contrast microscopy (Supplementary Figure S1C). In Passage 5, we observed essentially the same proliferation deficiency in Itpa−/– MEFs and slightly increased numbers of senescence-associated β-galactosidase (SA-β-Gal)-positive cells in Itpa−/– MEFs (Itpa+/+ versus Itpa−/–, 3.64% versus 7.74%; Supplementary Figure S2A and B). Flow cytometry analysis of the cell cycle revealed that Itpa−/– MEFs exhibited a significant increase in the G2/M phase (Itpa+/+ versus Itpa−/–, 28.4% versus 77.4%; Figure 1D) and a slight increase in the sub G1 fraction (Itpa+/+ versus Itpa−/–, 1.61% versus 5.51%; fraction M1 in Figure 2A). There was an apparent increase in cells with an abnormally increased DNA content in Itpa−/– MEFs compared with Itpa+/+ MEFs (fraction M3 in Figure 2A), indicating that the G2/M phase shown in Figure 1D might have contained some tetraploid cells at the G1 phase (Figure 2B). There was no increase in dead cells detected as propidium iodide (PI)/Hoechst-double positive cells (Supplementary Figure S2C). These results indicate that ITPA deficiency caused delay or arrest in cell-cycle progression. We further observed that exposure of Itpa−/–, but not Itpa+/+, Itpa+/− MEFs, to sodium nitrite (NaNO2), which causes predominant deamination of purine bases (18), resulted in growth suppression without inducing cell death (Supplementary Figure S2C).

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