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

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.

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).

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.

Figure 3.

ITPA deficiency increases chromosome abnormalities with increased accumulation of SSBs in nuclear DNA. (A) Various chromosome structural abnormalities were observed in Itpa^−/– primary MEFs (Passage 2). (B) ITPA deficiency increases chromosome abnormalities. The frequency of chromosomal abnormality was significantly increased in Itpa^−/– MEFs in comparison to Itpa^+/+ and Itpa^+/− MEFs. Result of non-repeated measures ANOVA (two-tailed), P = 0.0149. Bonferroni post hoc test, P < 0.01. Data are shown as pie charts with the mean ± SD (n = 4 independent isolates). (C) Detection of immunoreactivity against ssDNA in Itpa^−/– primary MEFs. Immunofluorescence microscopy with anti-ssDNA antibody (green) revealed significantly increased ssDNA immunoreactivity in nuclei (DAPI, blue) of Itpa^−/– primary MEFs (Passage 2) compared with the wild-type (Itpa^+/+). (D) Significant Increase in the ssDNA-positive population in Itpa^−/– primary MEF tested by unpaired Student’s t-test (two-tailed): **P < 0.01. Data are shown in a bar graph (mean ± SD, n = 4 independent isolates).

Figure 4.

ITPA-deficient phenotypes are reversed during immortalization. (A) Immortalized Itpa^−/– MEFs (triangle) showed the same proliferation rate as did immortalized Itpa^+/+ (circle) and Itpa^+/− (square) MEFs. Error bars represent the SD (n = 3 independent isolates). (B) The frequency of chromosomal abnormalities was decreased in immortalized Itpa^−/– MEFs to the levels seen in immortalized Itpa^+/+ MEFs. Data are shown as the mean ± SD (n = 3 independent isolates).

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.

Figure 6.

NUDT16 with strong dIDP/IDP-hydrolyzing activity is the back-up enzyme responsible for the cancellation of ITPA-deficient phenotypes during immortalization. (A) Expression of Nudt16 mRNA. Quantitative real-time RT–PCR was performed to compare Nudt16 mRNA levels between primary (passage 3) and immortalized MEFs. Levels of Nudt16 mRNA were normalized to those of Gapdh mRNA. Values relative to the highest level of Nudt16 mRNA in immortalized Itpa^−/– MEFs (isolate No. 3) are shown. Unpaired Student’s t-test (two-tailed) showed P < 0.01 between primary and immortalized Itpa^−/– MEFs. Data are shown in a bar graph (mean ± SD), with fold changes between primary and immortalized MEFs (n = 3 independent isolates). Open and black bars show primary MEFs; shaded bars indicate immortalized MEFs. (B) Expression of NUDT16 protein. Western blotting was performed for crude cell extracts (40 µg) prepared from primary (Passage 2) and immortalized MEFs, using anti-NUDT16 antibody (top). GAPDH was detected as an internal control (middle). Membranes were stained with Gel Code Blue in order to confirm the loading and transfer (bottom). Intensities of NUDT16 bands were measured and relative levels normalized to GAPDH are shown in a bar graph. Open and black bars show primary MEFs; shaded bars indicate immortalized MEFs. Result of non-repeated measures ANOVA (two-tailed), P < 0.019. Bonferroni post hoc test, **P < 0.01 (versus others). Error bars represent the SD (n = 3 independent isolates).

Figure 7.

Knockdown of Nudt16 mRNA suppressed ITPA-deficient phenotypes in immortalized Itpa^−/– MEFs. (A) Expression of Nudt16 mRNA. To knock down the expression of Nudt16, two different siRNAs (80, 82 or 80 + 82) against Nudt16 mRNA or control siRNA were introduced into immortalized Itpa^−/– MEFs. Forty-eight hours after the introduction, total RNA was prepared and quantitative real-time RT–PCR was performed. Levels of Nudt16 mRNA were normalized to those of Gapdh mRNA and their relative values are shown. Data are shown in a bar graph (mean ± SD, n = 3). (B) Expression of NUDT16 protein. Western blotting was performed for crude cell extracts (40 µg) prepared from immortalized Itpa^−/– MEFs treated with two Nudt16 siRNAs (80 + 82) or control siRNA, using anti-NUDT16. GAPDH was detected as an internal control. (C) Knockdown of Nudt16 mRNA suppressed proliferation of immortalized Itpa^−/– MEFs. Expression of Nudt16 mRNA was blocked using a mix of two different Nudt16 siRNAs and cell proliferation was examined. Circles, Itpa^+/+; triangles, Itpa^−/–; open marks, control siRNA; closed marks, Nudt16 siRNAs. Result of repeated measures ANOVA, two-tailed, P < 0.0001; Bonferroni/Dunn post hoc test, **P < 0.01 (versus other three measures). Error bars represent the SD (n = 4). (D) Knockdown of Nudt16 mRNA significantly increased the accumulation of inosine in RNA of immortalized Itpa^−/– MEFs. Level of inosine in RNA was determined by LC–MS/MS analysis of RNA prepared from immortalized Itpa^−/– MEFs treated with control or Nudt16 siRNAs. Result of unpaired Student’s t-test (two-tailed), **P < 0.01. Data are shown as a bar graph with the mean ± SD (n = 3). (E) Knockdown of Nudt16 mRNA significantly increased the accumulation of dI in nuclear DNA of immortalized Itpa^−/– MEFs. Level of dI in nuclear DNA was determined by LC–MS/MS analysis of nuclear DNA prepared from immortalized Itpa^−/– MEFs treated with control or Nudt16 siRNAs. Result of unpaired Student’s t-test (two-tailed), **P < 0.01. Data are shown as a bar graph with the mean ± SD (n = 3). (F) Increased immunoreactivity against ssDNA in immortalized Itpa^−/– MEFs after Nudt16 knockdown. Immunofluorescence microscopy with anti-ssDNA antibody revealed significantly increased immunoreactivity after Nudt16 knockdown. Result of unpaired Student’s t-test (two-tailed), **P < 0.01. Data are shown as a bar graph with the mean ± SD (n = 4). (G) Knockdown of Nudt16 mRNA increased chromosomal abnormalities in immortalized Itpa^−/– MEFs. The frequency of chromosomal abnormalities was increased significantly in immortalized Itpa^−/– MEFs after Nudt16 knockdown. Result of unpaired Student’s t-test (two-tailed), P < 0.05 (versus control siRNA). Data are shown as pie charts with the mean ± SD (n = 4).