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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 deficiency reduces the proliferation potential of hematopoietic progenitors.A. Left panel: estimated size (planar surface) of CFU-PreB and CFU-C colonies derived from WT BM (solid bar) and Polμ−/− BM (open bar). Right panel: Micrographs of representative CFU-PreB colonies derived from WT and Polμ−/− BM, showing the reduced colony size of Polμ−/− colonies. B. Numbers of myeloid (left) and erythroid (right) progenitors recovered after 4 days expansion of WT BM (solid bars, (n = 7) and Polμ−/− BM (open bars, (n = 7) in IL3 and SCF supplemented medium. C. Left: Absolute cell numbers, per culture, recovered after a 2 week expansion of WT (solid, (n = 5) and Polμ−/− (open, (n = 4) long-term bone marrow cell cultures (LTBMC). Right: representative micrographs of stroma generated in WT and Polμ−/− LTBMC (3 weeks). Data are means+/−SEM. *: p<0.05; **: p<0.01; ***: p<0.001.
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pgen-1000389-g003: Polμ deficiency reduces the proliferation potential of hematopoietic progenitors.A. Left panel: estimated size (planar surface) of CFU-PreB and CFU-C colonies derived from WT BM (solid bar) and Polμ−/− BM (open bar). Right panel: Micrographs of representative CFU-PreB colonies derived from WT and Polμ−/− BM, showing the reduced colony size of Polμ−/− colonies. B. Numbers of myeloid (left) and erythroid (right) progenitors recovered after 4 days expansion of WT BM (solid bars, (n = 7) and Polμ−/− BM (open bars, (n = 7) in IL3 and SCF supplemented medium. C. Left: Absolute cell numbers, per culture, recovered after a 2 week expansion of WT (solid, (n = 5) and Polμ−/− (open, (n = 4) long-term bone marrow cell cultures (LTBMC). Right: representative micrographs of stroma generated in WT and Polμ−/− LTBMC (3 weeks). Data are means+/−SEM. *: p<0.05; **: p<0.01; ***: p<0.001.

Mentions: The low progenitor numbers in Polμ−/− mice could result either from impaired differentiation or from defective progenitor proliferation or self renewal capacity. To investigate this, we examined the proliferation of Polμ−/− hematopoietic stem and progenitor cells in vitro. In clonogenic differentiation assays, colonies of Polμ−/− progenitors were significantly smaller (50% smaller for CFU-C, p<0.05; 60% smaller for CFU-PreB, p<0.05) and contained fewer cells than wt colonies, indicating defective self-renewal or proliferative capacity (Figure 3A). This was confirmed by growing BMNC in conditions that promote myeloid progenitor expansion (see methods). After 4 days, wt BMNC cultures contained 2.3-fold more myeloid progenitors (p<0.001) and 2.8-fold more erythroid progenitors (p<0.01) than Polμ−/− cultures (Figure 3B). We then assayed BMNC in long-term bone marrow cell cultures (LTBMC, see methods). Two weeks after initiation, Polμ−/− cultures contained 35% fewer cells than wt (p<0.01, Figure 3C). Cell cycle analysis revealed an increased incidence of cell death in Polμ−/− cultures (Figure S3). Together with the bone marrow reconstitution experiments, these results suggest that Polμ−/− bone marrow stromal cells fail to efficiently support hematopoiesis and that Polμ−/− hematopoietic progenitor numbers do not expand in vitro as efficiently as WT progenitors.


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 deficiency reduces the proliferation potential of hematopoietic progenitors.A. Left panel: estimated size (planar surface) of CFU-PreB and CFU-C colonies derived from WT BM (solid bar) and Polμ−/− BM (open bar). Right panel: Micrographs of representative CFU-PreB colonies derived from WT and Polμ−/− BM, showing the reduced colony size of Polμ−/− colonies. B. Numbers of myeloid (left) and erythroid (right) progenitors recovered after 4 days expansion of WT BM (solid bars, (n = 7) and Polμ−/− BM (open bars, (n = 7) in IL3 and SCF supplemented medium. C. Left: Absolute cell numbers, per culture, recovered after a 2 week expansion of WT (solid, (n = 5) and Polμ−/− (open, (n = 4) long-term bone marrow cell cultures (LTBMC). Right: representative micrographs of stroma generated in WT and Polμ−/− LTBMC (3 weeks). Data are means+/−SEM. *: p<0.05; **: p<0.01; ***: p<0.001.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2638008&req=5

pgen-1000389-g003: Polμ deficiency reduces the proliferation potential of hematopoietic progenitors.A. Left panel: estimated size (planar surface) of CFU-PreB and CFU-C colonies derived from WT BM (solid bar) and Polμ−/− BM (open bar). Right panel: Micrographs of representative CFU-PreB colonies derived from WT and Polμ−/− BM, showing the reduced colony size of Polμ−/− colonies. B. Numbers of myeloid (left) and erythroid (right) progenitors recovered after 4 days expansion of WT BM (solid bars, (n = 7) and Polμ−/− BM (open bars, (n = 7) in IL3 and SCF supplemented medium. C. Left: Absolute cell numbers, per culture, recovered after a 2 week expansion of WT (solid, (n = 5) and Polμ−/− (open, (n = 4) long-term bone marrow cell cultures (LTBMC). Right: representative micrographs of stroma generated in WT and Polμ−/− LTBMC (3 weeks). Data are means+/−SEM. *: p<0.05; **: p<0.01; ***: p<0.001.
Mentions: The low progenitor numbers in Polμ−/− mice could result either from impaired differentiation or from defective progenitor proliferation or self renewal capacity. To investigate this, we examined the proliferation of Polμ−/− hematopoietic stem and progenitor cells in vitro. In clonogenic differentiation assays, colonies of Polμ−/− progenitors were significantly smaller (50% smaller for CFU-C, p<0.05; 60% smaller for CFU-PreB, p<0.05) and contained fewer cells than wt colonies, indicating defective self-renewal or proliferative capacity (Figure 3A). This was confirmed by growing BMNC in conditions that promote myeloid progenitor expansion (see methods). After 4 days, wt BMNC cultures contained 2.3-fold more myeloid progenitors (p<0.001) and 2.8-fold more erythroid progenitors (p<0.01) than Polμ−/− cultures (Figure 3B). We then assayed BMNC in long-term bone marrow cell cultures (LTBMC, see methods). Two weeks after initiation, Polμ−/− cultures contained 35% fewer cells than wt (p<0.01, Figure 3C). Cell cycle analysis revealed an increased incidence of cell death in Polμ−/− cultures (Figure S3). Together with the bone marrow reconstitution experiments, these results suggest that Polμ−/− bone marrow stromal cells fail to efficiently support hematopoiesis and that Polμ−/− hematopoietic progenitor numbers do not expand in vitro as efficiently as WT progenitors.

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