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Autophagy confers DNA damage repair pathways to protect the hematopoietic system from nuclear radiation injury.

Lin W, Yuan N, Wang Z, Cao Y, Fang Y, Li X, Xu F, Song L, Wang J, Zhang H, Yan L, Xu L, Zhang X, Zhang S, Wang J - Sci Rep (2015)

Bottom Line: In vivo autophagy activation improved bone marrow cellularity following nuclear radiation exposure.Strikingly, in vivo defective autophagy caused an absence or reduction in regulatory proteins critical to both homologous recombination (HR) and non-homologous end joining (NHEJ) DNA damage repair pathways, as well as a failure to induce these proteins in response to nuclear radiation.In contrast, in vivo autophagy activation increased most of these proteins in hematopoietic cells.

View Article: PubMed Central - PubMed

Affiliation: Hematology Center of Cyrus Tang Medical Institute, Jiangsu Institute of Hematology, Collaborative Innovation Center of Hematology, Jiangsu Key Laboratory for Stem Cell Research, Soochow University School of Medicine, Suzhou 215123, China.

ABSTRACT
Autophagy is essentially a metabolic process, but its in vivo role in nuclear radioprotection remains unexplored. We observed that ex vivo autophagy activation reversed the proliferation inhibition, apoptosis, and DNA damage in irradiated hematopoietic cells. In vivo autophagy activation improved bone marrow cellularity following nuclear radiation exposure. In contrast, defective autophagy in the hematopoietic conditional mouse model worsened the hematopoietic injury, reactive oxygen species (ROS) accumulation and DNA damage caused by nuclear radiation exposure. Strikingly, in vivo defective autophagy caused an absence or reduction in regulatory proteins critical to both homologous recombination (HR) and non-homologous end joining (NHEJ) DNA damage repair pathways, as well as a failure to induce these proteins in response to nuclear radiation. In contrast, in vivo autophagy activation increased most of these proteins in hematopoietic cells. DNA damage assays confirmed the role of in vivo autophagy in the resolution of double-stranded DNA breaks in total bone marrow cells as well as bone marrow stem and progenitor cells upon whole body irradiation. Hence, autophagy protects the hematopoietic system against nuclear radiation injury by conferring and intensifying the HR and NHEJ DNA damage repair pathways and by removing ROS and inhibiting apoptosis.

No MeSH data available.


Related in: MedlinePlus

Rapamycin protects ex vivo bone marrow cells from radiation damage.(A) Rapamycin improves cell proliferation under radiation exposure. Bone marrow cells were isolated from wild-type B6/C57 mice and cultured in Iscove’s Modified Dulbecco’s Media with carrier (DMSO), rapamycin (50 nM), bafilomycin A1 (5 nM), and rapamycin together with bafilomycin A1 for 24 h. After irradiation with 3 Gy, each group of cells was seeded at a density of 5 × 103 cells/well in 96-well culture plates for growth recovery. The cells were counted with a hemocytometer at the indicated postirradiation times. A proliferation advantage was observed in irradiated bone marrow cells pretreated with rapamycin, which was blocked by bafilomycin A1 (right panel). Neither rapamycin nor bafilomycin A1 altered the overall proliferation of total non-irradiated bone marrow cells (left panel). (B). Rapamycin inhibited the apoptosis of irradiated bone marrow cells, which was blocked by bafilomycin A1. The above irradiated cells were labeled with propidium iodide and FITC-conjugated annexin V for cytometric analysis. Significantly reduced cell apoptosis was observed in cells treated with rapamycin, and bafilomycin A1 reduced the reduction in apoptosis (right panel). Neither rapamycin nor bafilomycin A1 altered the obvious apoptosis level of total non-irradiated bone marrow cells (left panel). (C) Rapamycin reduced the DNA damage of irradiated bone marrow cells, which was reversed by bafilomycin A1 (right panel). Neither of the two drugs altered DNA damage level of the non-irradiated bone marrow cells (left panel). The DNA damage response was measured with specific marker γ-H2A.X in the above irradiated or non-irradiated cells. Shown are a representative flow histogram and statistical data for γH2A.X–positive cells. (D) Representative immunofluorescence microscopic data for γH2A.X foci formation in the same groups of bone marrow cells as in C. Data are mean ± SD from at least three independent experiments. n ≥ 5, *p < 0.05 and **p < 0.01.
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f1: Rapamycin protects ex vivo bone marrow cells from radiation damage.(A) Rapamycin improves cell proliferation under radiation exposure. Bone marrow cells were isolated from wild-type B6/C57 mice and cultured in Iscove’s Modified Dulbecco’s Media with carrier (DMSO), rapamycin (50 nM), bafilomycin A1 (5 nM), and rapamycin together with bafilomycin A1 for 24 h. After irradiation with 3 Gy, each group of cells was seeded at a density of 5 × 103 cells/well in 96-well culture plates for growth recovery. The cells were counted with a hemocytometer at the indicated postirradiation times. A proliferation advantage was observed in irradiated bone marrow cells pretreated with rapamycin, which was blocked by bafilomycin A1 (right panel). Neither rapamycin nor bafilomycin A1 altered the overall proliferation of total non-irradiated bone marrow cells (left panel). (B). Rapamycin inhibited the apoptosis of irradiated bone marrow cells, which was blocked by bafilomycin A1. The above irradiated cells were labeled with propidium iodide and FITC-conjugated annexin V for cytometric analysis. Significantly reduced cell apoptosis was observed in cells treated with rapamycin, and bafilomycin A1 reduced the reduction in apoptosis (right panel). Neither rapamycin nor bafilomycin A1 altered the obvious apoptosis level of total non-irradiated bone marrow cells (left panel). (C) Rapamycin reduced the DNA damage of irradiated bone marrow cells, which was reversed by bafilomycin A1 (right panel). Neither of the two drugs altered DNA damage level of the non-irradiated bone marrow cells (left panel). The DNA damage response was measured with specific marker γ-H2A.X in the above irradiated or non-irradiated cells. Shown are a representative flow histogram and statistical data for γH2A.X–positive cells. (D) Representative immunofluorescence microscopic data for γH2A.X foci formation in the same groups of bone marrow cells as in C. Data are mean ± SD from at least three independent experiments. n ≥ 5, *p < 0.05 and **p < 0.01.

Mentions: To explore a possible role for autophagy in protecting the hematopoietic system against nuclear radiation exposure, we first isolated bone marrow cells from mice and treated with or without rapamycin, an autophagy inducer. The results show that rapamycin protected ex vivo bone marrow cell proliferation from nuclear irradiation exposure, whereas treatment with bafilomycin A1, an autophagy inhibitor, reduced the rapamycin-induced protection of bone marrow cell proliferation. In contrast, as compared with the carrier DMSO, neither rapamycin nor bafilomycin A1 at the same concentrations caused an obvious decrease in cell proliferation without radiation exposure (Fig. 1A), suggesting that rapamycin or bafilomycin A1 at such concentration does not cause detectable change of overall numbers of bone marrow hematopoietic cells. In line with the above results, autophagy inducer rapamycin reduced the apoptotic death of irradiated bone marrow cells, but autophagy inhibitor bafilomycin A1 increased the apoptosis of bone marrow cells exposed to the radiation, whereas .rapamycin or bafilomycin A1 at the same concentrations neither activate nor inhibit apoptosis of bone marrow cells without nuclear radiation exposure (Fig. 1B). Furthermore, rapamycin decreased the radiation-induced DNA damage of bone marrow cells, and bafilomycin A1 caused the opposite effect, as seen by examination of the DSB DNA damage marker γH2AX by flow cytometry (Fig. 1C, right panel) and confocal microscopy (Fig. 1D, lower panel), whereas the control groups not exposed to the radiation did not show detectable changes in DNA damage response (Fig. 1C left panel, 1D upper panel). The above data from autophagy inducer or inhibitor treatment suggest a possible role of autophagy in ex vivo protection of hematopoietic cells.


Autophagy confers DNA damage repair pathways to protect the hematopoietic system from nuclear radiation injury.

Lin W, Yuan N, Wang Z, Cao Y, Fang Y, Li X, Xu F, Song L, Wang J, Zhang H, Yan L, Xu L, Zhang X, Zhang S, Wang J - Sci Rep (2015)

Rapamycin protects ex vivo bone marrow cells from radiation damage.(A) Rapamycin improves cell proliferation under radiation exposure. Bone marrow cells were isolated from wild-type B6/C57 mice and cultured in Iscove’s Modified Dulbecco’s Media with carrier (DMSO), rapamycin (50 nM), bafilomycin A1 (5 nM), and rapamycin together with bafilomycin A1 for 24 h. After irradiation with 3 Gy, each group of cells was seeded at a density of 5 × 103 cells/well in 96-well culture plates for growth recovery. The cells were counted with a hemocytometer at the indicated postirradiation times. A proliferation advantage was observed in irradiated bone marrow cells pretreated with rapamycin, which was blocked by bafilomycin A1 (right panel). Neither rapamycin nor bafilomycin A1 altered the overall proliferation of total non-irradiated bone marrow cells (left panel). (B). Rapamycin inhibited the apoptosis of irradiated bone marrow cells, which was blocked by bafilomycin A1. The above irradiated cells were labeled with propidium iodide and FITC-conjugated annexin V for cytometric analysis. Significantly reduced cell apoptosis was observed in cells treated with rapamycin, and bafilomycin A1 reduced the reduction in apoptosis (right panel). Neither rapamycin nor bafilomycin A1 altered the obvious apoptosis level of total non-irradiated bone marrow cells (left panel). (C) Rapamycin reduced the DNA damage of irradiated bone marrow cells, which was reversed by bafilomycin A1 (right panel). Neither of the two drugs altered DNA damage level of the non-irradiated bone marrow cells (left panel). The DNA damage response was measured with specific marker γ-H2A.X in the above irradiated or non-irradiated cells. Shown are a representative flow histogram and statistical data for γH2A.X–positive cells. (D) Representative immunofluorescence microscopic data for γH2A.X foci formation in the same groups of bone marrow cells as in C. Data are mean ± SD from at least three independent experiments. n ≥ 5, *p < 0.05 and **p < 0.01.
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f1: Rapamycin protects ex vivo bone marrow cells from radiation damage.(A) Rapamycin improves cell proliferation under radiation exposure. Bone marrow cells were isolated from wild-type B6/C57 mice and cultured in Iscove’s Modified Dulbecco’s Media with carrier (DMSO), rapamycin (50 nM), bafilomycin A1 (5 nM), and rapamycin together with bafilomycin A1 for 24 h. After irradiation with 3 Gy, each group of cells was seeded at a density of 5 × 103 cells/well in 96-well culture plates for growth recovery. The cells were counted with a hemocytometer at the indicated postirradiation times. A proliferation advantage was observed in irradiated bone marrow cells pretreated with rapamycin, which was blocked by bafilomycin A1 (right panel). Neither rapamycin nor bafilomycin A1 altered the overall proliferation of total non-irradiated bone marrow cells (left panel). (B). Rapamycin inhibited the apoptosis of irradiated bone marrow cells, which was blocked by bafilomycin A1. The above irradiated cells were labeled with propidium iodide and FITC-conjugated annexin V for cytometric analysis. Significantly reduced cell apoptosis was observed in cells treated with rapamycin, and bafilomycin A1 reduced the reduction in apoptosis (right panel). Neither rapamycin nor bafilomycin A1 altered the obvious apoptosis level of total non-irradiated bone marrow cells (left panel). (C) Rapamycin reduced the DNA damage of irradiated bone marrow cells, which was reversed by bafilomycin A1 (right panel). Neither of the two drugs altered DNA damage level of the non-irradiated bone marrow cells (left panel). The DNA damage response was measured with specific marker γ-H2A.X in the above irradiated or non-irradiated cells. Shown are a representative flow histogram and statistical data for γH2A.X–positive cells. (D) Representative immunofluorescence microscopic data for γH2A.X foci formation in the same groups of bone marrow cells as in C. Data are mean ± SD from at least three independent experiments. n ≥ 5, *p < 0.05 and **p < 0.01.
Mentions: To explore a possible role for autophagy in protecting the hematopoietic system against nuclear radiation exposure, we first isolated bone marrow cells from mice and treated with or without rapamycin, an autophagy inducer. The results show that rapamycin protected ex vivo bone marrow cell proliferation from nuclear irradiation exposure, whereas treatment with bafilomycin A1, an autophagy inhibitor, reduced the rapamycin-induced protection of bone marrow cell proliferation. In contrast, as compared with the carrier DMSO, neither rapamycin nor bafilomycin A1 at the same concentrations caused an obvious decrease in cell proliferation without radiation exposure (Fig. 1A), suggesting that rapamycin or bafilomycin A1 at such concentration does not cause detectable change of overall numbers of bone marrow hematopoietic cells. In line with the above results, autophagy inducer rapamycin reduced the apoptotic death of irradiated bone marrow cells, but autophagy inhibitor bafilomycin A1 increased the apoptosis of bone marrow cells exposed to the radiation, whereas .rapamycin or bafilomycin A1 at the same concentrations neither activate nor inhibit apoptosis of bone marrow cells without nuclear radiation exposure (Fig. 1B). Furthermore, rapamycin decreased the radiation-induced DNA damage of bone marrow cells, and bafilomycin A1 caused the opposite effect, as seen by examination of the DSB DNA damage marker γH2AX by flow cytometry (Fig. 1C, right panel) and confocal microscopy (Fig. 1D, lower panel), whereas the control groups not exposed to the radiation did not show detectable changes in DNA damage response (Fig. 1C left panel, 1D upper panel). The above data from autophagy inducer or inhibitor treatment suggest a possible role of autophagy in ex vivo protection of hematopoietic cells.

Bottom Line: In vivo autophagy activation improved bone marrow cellularity following nuclear radiation exposure.Strikingly, in vivo defective autophagy caused an absence or reduction in regulatory proteins critical to both homologous recombination (HR) and non-homologous end joining (NHEJ) DNA damage repair pathways, as well as a failure to induce these proteins in response to nuclear radiation.In contrast, in vivo autophagy activation increased most of these proteins in hematopoietic cells.

View Article: PubMed Central - PubMed

Affiliation: Hematology Center of Cyrus Tang Medical Institute, Jiangsu Institute of Hematology, Collaborative Innovation Center of Hematology, Jiangsu Key Laboratory for Stem Cell Research, Soochow University School of Medicine, Suzhou 215123, China.

ABSTRACT
Autophagy is essentially a metabolic process, but its in vivo role in nuclear radioprotection remains unexplored. We observed that ex vivo autophagy activation reversed the proliferation inhibition, apoptosis, and DNA damage in irradiated hematopoietic cells. In vivo autophagy activation improved bone marrow cellularity following nuclear radiation exposure. In contrast, defective autophagy in the hematopoietic conditional mouse model worsened the hematopoietic injury, reactive oxygen species (ROS) accumulation and DNA damage caused by nuclear radiation exposure. Strikingly, in vivo defective autophagy caused an absence or reduction in regulatory proteins critical to both homologous recombination (HR) and non-homologous end joining (NHEJ) DNA damage repair pathways, as well as a failure to induce these proteins in response to nuclear radiation. In contrast, in vivo autophagy activation increased most of these proteins in hematopoietic cells. DNA damage assays confirmed the role of in vivo autophagy in the resolution of double-stranded DNA breaks in total bone marrow cells as well as bone marrow stem and progenitor cells upon whole body irradiation. Hence, autophagy protects the hematopoietic system against nuclear radiation injury by conferring and intensifying the HR and NHEJ DNA damage repair pathways and by removing ROS and inhibiting apoptosis.

No MeSH data available.


Related in: MedlinePlus