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Altered hematopoiesis in mice lacking DNA polymerase mu is due to inefficient double-strand break repair.

Lucas D, Escudero B, Ligos JM, Segovia JC, Estrada JC, Terrados G, Blanco L, Samper E, Bernad A - PLoS Genet. (2009)

Bottom Line: In vivo, Polmicro deficiency results in impaired Vkappa-Jkappa recombination and altered somatic hypermutation and centroblast development.Hematopoietic progenitors were reduced both in number and in expansion potential.Our results show that Polmicro function is required for physiological hematopoietic development with an important role in maintaining early progenitor cell homeostasis and genetic stability in hematopoietic and non-hematopoietic tissues.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Cientificas, Campus Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain.

ABSTRACT
Polymerase micro (Polmicro) is an error-prone, DNA-directed DNA polymerase that participates in non-homologous end-joining (NHEJ) repair. In vivo, Polmicro deficiency results in impaired Vkappa-Jkappa recombination and altered somatic hypermutation and centroblast development. In Polmicro(-/-) mice, hematopoietic development was defective in several peripheral and bone marrow (BM) cell populations, with about a 40% decrease in BM cell number that affected several hematopoietic lineages. Hematopoietic progenitors were reduced both in number and in expansion potential. The observed phenotype correlates with a reduced efficiency in DNA double-strand break (DSB) repair in hematopoietic tissue. Whole-body gamma-irradiation revealed that Polmicro also plays a role in DSB repair in non-hematopoietic tissues. Our results show that Polmicro function is required for physiological hematopoietic development with an important role in maintaining early progenitor cell homeostasis and genetic stability in hematopoietic and non-hematopoietic tissues.

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Polμ−/− mice show increased radiosensitivity.A. Survival of Polμ−/− (open circles) and WT mice (closed circles) after whole-body γ-irradiation (9 Gy); n = 15. B. Percentage survival by γ-irradiated WT (solid bars) and Polμ−/− (open bars) bone marrow CFU-C progenitors; n = 4 mice analyzed in duplicate assays. C. Percentage survival by γ-irradiated WT (closed circles) and Polμ−/− (open circles) mouse embryonic fibroblasts (MEF); The figure shows one experiment with each point assayed in quadruplicate. D. Photomicrographs of formalin-fixed, paraffin-embedded, hematoxylin-eosin-stained sections of liver, lung, kidney and testis from irradiated WT and Polμ−/− mice. Note extensive damage (arrowheads) in Polμ−/− tissues: vacuolar degeneration (liver); inflammation and hemorrhaging (lung) and tubular degeneration (kidney and testis). E. Flow cytometry ROS measurements by DCFDA fluorescence in irradiated and mock-irradiated WT (solid bars) and Polμ−/− bone marrow cells (open bars); n = 3. There was no significant difference in ROS levels between WT and Polμ−/− cells under basal conditions or upon irradiation. All data are means+/−SEM. *: p<0.05; **: p<0.01; ***: p<0.001.
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pgen-1000389-g005: Polμ−/− mice show increased radiosensitivity.A. Survival of Polμ−/− (open circles) and WT mice (closed circles) after whole-body γ-irradiation (9 Gy); n = 15. B. Percentage survival by γ-irradiated WT (solid bars) and Polμ−/− (open bars) bone marrow CFU-C progenitors; n = 4 mice analyzed in duplicate assays. C. Percentage survival by γ-irradiated WT (closed circles) and Polμ−/− (open circles) mouse embryonic fibroblasts (MEF); The figure shows one experiment with each point assayed in quadruplicate. D. Photomicrographs of formalin-fixed, paraffin-embedded, hematoxylin-eosin-stained sections of liver, lung, kidney and testis from irradiated WT and Polμ−/− mice. Note extensive damage (arrowheads) in Polμ−/− tissues: vacuolar degeneration (liver); inflammation and hemorrhaging (lung) and tubular degeneration (kidney and testis). E. Flow cytometry ROS measurements by DCFDA fluorescence in irradiated and mock-irradiated WT (solid bars) and Polμ−/− bone marrow cells (open bars); n = 3. There was no significant difference in ROS levels between WT and Polμ−/− cells under basal conditions or upon irradiation. All data are means+/−SEM. *: p<0.05; **: p<0.01; ***: p<0.001.

Mentions: To test this in vivo, we irradiated Polμ−/− and wt mice (9 Gy) and analyzed survival over time. Fifteen days post-irradiation all Polμ−/− mice were dead, compared with only 40% of wt mice (p<0.001 Log rank test; Figure 5A). Most Polμ−/− mice died between days 9 and 11 (Figure 5A), and hematologic analysis revealed extreme neutropenia (not shown), indicating hematopoietic failure. The impact of Polμ deficiency on the response to irradiation damage is also illustrated by the higher radiosensitivity of Polμ−/− progenitors (Figure 5B; p<0.01). Bone marrow cells from wt and Polμ−/− mice showed G2 accumulation after irradiation (5Gy), suggesting that the G2/M cell cycle checkpoint is functional in Polμ−/− cells (Figure S4A). Western blot detected increased p21 accumulation in irradiated Polμ−/− splenocytes and even in non-irradiated cells (Figure S4B), suggesting activation of the G1 cell cycle checkpoint. Irradiation of Polμ−/− MEF (2–8 Gy) significantly reduced survival compared with similarly treated wt MEF (2 to 123-fold; p<0.05. Figure 5C). Histological analysis of irradiated mice showed vacuolar degeneration in liver (where we detect intense γ-H2AX accumulation after irradiation; Figure S4C), hemorrhage and congestion in the lung, tubular degeneration in kidney, and tubular atrophy in testis (Figure 5D). An alternative explanation for increased tissue damage in irradiated Polμ−/− mice might be increased production of reactive oxygen species (ROS). We therefore measured intracellular ROS levels in irradiated bone marrow cells (4 and 8 Gy) by staining with DCFDA (Di-cloro-fluorescein diacetate: a marker of intracellular peroxides). ROS accumulation was unaffected by Polμ−/− deficiency (Figure 5E), indicating that ROS production does not contribute to the radiosensitivity of Polμ−/− animals or cells. These data thus show that the requirement for Polμ in DNA repair extends to tissues outside the hematopoietic compartment (Figure 5C,D).


Altered hematopoiesis in mice lacking DNA polymerase mu is due to inefficient double-strand break repair.

Lucas D, Escudero B, Ligos JM, Segovia JC, Estrada JC, Terrados G, Blanco L, Samper E, Bernad A - PLoS Genet. (2009)

Polμ−/− mice show increased radiosensitivity.A. Survival of Polμ−/− (open circles) and WT mice (closed circles) after whole-body γ-irradiation (9 Gy); n = 15. B. Percentage survival by γ-irradiated WT (solid bars) and Polμ−/− (open bars) bone marrow CFU-C progenitors; n = 4 mice analyzed in duplicate assays. C. Percentage survival by γ-irradiated WT (closed circles) and Polμ−/− (open circles) mouse embryonic fibroblasts (MEF); The figure shows one experiment with each point assayed in quadruplicate. D. Photomicrographs of formalin-fixed, paraffin-embedded, hematoxylin-eosin-stained sections of liver, lung, kidney and testis from irradiated WT and Polμ−/− mice. Note extensive damage (arrowheads) in Polμ−/− tissues: vacuolar degeneration (liver); inflammation and hemorrhaging (lung) and tubular degeneration (kidney and testis). E. Flow cytometry ROS measurements by DCFDA fluorescence in irradiated and mock-irradiated WT (solid bars) and Polμ−/− bone marrow cells (open bars); n = 3. There was no significant difference in ROS levels between WT and Polμ−/− cells under basal conditions or upon irradiation. All data are means+/−SEM. *: p<0.05; **: p<0.01; ***: p<0.001.
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pgen-1000389-g005: Polμ−/− mice show increased radiosensitivity.A. Survival of Polμ−/− (open circles) and WT mice (closed circles) after whole-body γ-irradiation (9 Gy); n = 15. B. Percentage survival by γ-irradiated WT (solid bars) and Polμ−/− (open bars) bone marrow CFU-C progenitors; n = 4 mice analyzed in duplicate assays. C. Percentage survival by γ-irradiated WT (closed circles) and Polμ−/− (open circles) mouse embryonic fibroblasts (MEF); The figure shows one experiment with each point assayed in quadruplicate. D. Photomicrographs of formalin-fixed, paraffin-embedded, hematoxylin-eosin-stained sections of liver, lung, kidney and testis from irradiated WT and Polμ−/− mice. Note extensive damage (arrowheads) in Polμ−/− tissues: vacuolar degeneration (liver); inflammation and hemorrhaging (lung) and tubular degeneration (kidney and testis). E. Flow cytometry ROS measurements by DCFDA fluorescence in irradiated and mock-irradiated WT (solid bars) and Polμ−/− bone marrow cells (open bars); n = 3. There was no significant difference in ROS levels between WT and Polμ−/− cells under basal conditions or upon irradiation. All data are means+/−SEM. *: p<0.05; **: p<0.01; ***: p<0.001.
Mentions: To test this in vivo, we irradiated Polμ−/− and wt mice (9 Gy) and analyzed survival over time. Fifteen days post-irradiation all Polμ−/− mice were dead, compared with only 40% of wt mice (p<0.001 Log rank test; Figure 5A). Most Polμ−/− mice died between days 9 and 11 (Figure 5A), and hematologic analysis revealed extreme neutropenia (not shown), indicating hematopoietic failure. The impact of Polμ deficiency on the response to irradiation damage is also illustrated by the higher radiosensitivity of Polμ−/− progenitors (Figure 5B; p<0.01). Bone marrow cells from wt and Polμ−/− mice showed G2 accumulation after irradiation (5Gy), suggesting that the G2/M cell cycle checkpoint is functional in Polμ−/− cells (Figure S4A). Western blot detected increased p21 accumulation in irradiated Polμ−/− splenocytes and even in non-irradiated cells (Figure S4B), suggesting activation of the G1 cell cycle checkpoint. Irradiation of Polμ−/− MEF (2–8 Gy) significantly reduced survival compared with similarly treated wt MEF (2 to 123-fold; p<0.05. Figure 5C). Histological analysis of irradiated mice showed vacuolar degeneration in liver (where we detect intense γ-H2AX accumulation after irradiation; Figure S4C), hemorrhage and congestion in the lung, tubular degeneration in kidney, and tubular atrophy in testis (Figure 5D). An alternative explanation for increased tissue damage in irradiated Polμ−/− mice might be increased production of reactive oxygen species (ROS). We therefore measured intracellular ROS levels in irradiated bone marrow cells (4 and 8 Gy) by staining with DCFDA (Di-cloro-fluorescein diacetate: a marker of intracellular peroxides). ROS accumulation was unaffected by Polμ−/− deficiency (Figure 5E), indicating that ROS production does not contribute to the radiosensitivity of Polμ−/− animals or cells. These data thus show that the requirement for Polμ in DNA repair extends to tissues outside the hematopoietic compartment (Figure 5C,D).

Bottom Line: In vivo, Polmicro deficiency results in impaired Vkappa-Jkappa recombination and altered somatic hypermutation and centroblast development.Hematopoietic progenitors were reduced both in number and in expansion potential.Our results show that Polmicro function is required for physiological hematopoietic development with an important role in maintaining early progenitor cell homeostasis and genetic stability in hematopoietic and non-hematopoietic tissues.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Cientificas, Campus Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain.

ABSTRACT
Polymerase micro (Polmicro) is an error-prone, DNA-directed DNA polymerase that participates in non-homologous end-joining (NHEJ) repair. In vivo, Polmicro deficiency results in impaired Vkappa-Jkappa recombination and altered somatic hypermutation and centroblast development. In Polmicro(-/-) mice, hematopoietic development was defective in several peripheral and bone marrow (BM) cell populations, with about a 40% decrease in BM cell number that affected several hematopoietic lineages. Hematopoietic progenitors were reduced both in number and in expansion potential. The observed phenotype correlates with a reduced efficiency in DNA double-strand break (DSB) repair in hematopoietic tissue. Whole-body gamma-irradiation revealed that Polmicro also plays a role in DSB repair in non-hematopoietic tissues. Our results show that Polmicro function is required for physiological hematopoietic development with an important role in maintaining early progenitor cell homeostasis and genetic stability in hematopoietic and non-hematopoietic tissues.

Show MeSH
Related in: MedlinePlus