Limits...
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.

Show MeSH

Related in: MedlinePlus

Increased DNA content in ITPA deficient immortalized 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 immortalized 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 = 0.00015. P-values are shown following a Bonferroni post hoc test.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2875033&req=5

Figure 5: Increased DNA content in ITPA deficient immortalized 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 immortalized 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 = 0.00015. P-values are shown following a Bonferroni post hoc test.

Mentions: To obtain established cell lines with ITPA deficiency, spontaneously-immortalized MEFs were isolated after 30–40 passages of each primary MEF line. We noticed that each immortalized MEF population showed the same proliferation rate, irrespective of their genotype (Figure 4A). The extent of chromosomal abnormalities (Figure 4B), and both levels of dI (2.49 ± 0.24 dI residues per 106 nucleosides) and ssDNA (positive in 6.17 ± 0.68% of cells) in nuclear DNA (Figure 7E and F) were similarly decreased in immortalized Itpa−/– MEFs. Furthermore, the G1 phase fraction increased significantly in immortalized Itpa−/– MEFs (Figure 5A) compared with primary MEFs (Figure 2A). Because the percentages of tetraploids were significantly higher in both primary and immortalized Itpa−/– MEFs than Itpa+/+ or Itpa+/− MEFs (Figures 2B and 5B), the G2/M fraction in immortalized Itpa−/– MEFs was apparently less than in primary Itpa−/– MEFs.


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 immortalized 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 immortalized 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 = 0.00015. P-values are shown following a Bonferroni post hoc test.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875033&req=5

Figure 5: Increased DNA content in ITPA deficient immortalized 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 immortalized 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 = 0.00015. P-values are shown following a Bonferroni post hoc test.
Mentions: To obtain established cell lines with ITPA deficiency, spontaneously-immortalized MEFs were isolated after 30–40 passages of each primary MEF line. We noticed that each immortalized MEF population showed the same proliferation rate, irrespective of their genotype (Figure 4A). The extent of chromosomal abnormalities (Figure 4B), and both levels of dI (2.49 ± 0.24 dI residues per 106 nucleosides) and ssDNA (positive in 6.17 ± 0.68% of cells) in nuclear DNA (Figure 7E and F) were similarly decreased in immortalized Itpa−/– MEFs. Furthermore, the G1 phase fraction increased significantly in immortalized Itpa−/– MEFs (Figure 5A) compared with primary MEFs (Figure 2A). Because the percentages of tetraploids were significantly higher in both primary and immortalized Itpa−/– MEFs than Itpa+/+ or Itpa+/− MEFs (Figures 2B and 5B), the G2/M fraction in immortalized Itpa−/– MEFs was apparently less than in primary Itpa−/– MEFs.

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