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Autophagy prevents irradiation injury and maintains stemness through decreasing ROS generation in mesenchymal stem cells.

Hou J, Han ZP, Jing YY, Yang X, Zhang SS, Sun K, Hao C, Meng Y, Yu FH, Liu XQ, Shi YF, Wu MC, Zhang L, Wei LX - Cell Death Dis (2013)

Bottom Line: Stem cells were characterized by their stemness: self-renewal and pluripotency.Autophagy induced by starvation or rapamycin can reduce ROS accumulation-associated DNA damage and maintain stemness in MSCs.Further, inhibition of autophagy leads to augment of ROS accumulation and DNA damage, which results in the loss of stemness in MSCs.

View Article: PubMed Central - PubMed

Affiliation: 1] Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, the Second Military Medical University, Shanghai, China [2] Department of Pharmacy, Changhai Hospital, The Second Military Medical University, Shanghai, China.

ABSTRACT
Stem cells were characterized by their stemness: self-renewal and pluripotency. Mesenchymal stem cells (MSCs) are a unique type of adult stem cells that have been proven to be involved in tissue repair, immunoloregulation and tumorigenesis. Irradiation is a well-known factor that leads to functional obstacle in stem cells. However, the mechanism of stemness maintenance in human MSCs exposed to irradiation remains unknown. We demonstrated that irradiation could induce reactive oxygen species (ROS) accumulation that resulted in DNA damage and stemness injury in MSCs. Autophagy induced by starvation or rapamycin can reduce ROS accumulation-associated DNA damage and maintain stemness in MSCs. Further, inhibition of autophagy leads to augment of ROS accumulation and DNA damage, which results in the loss of stemness in MSCs. Our results indicate that autophagy may have an important role in protecting stemness of MSCs from irradiation injury.

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Autophagy decreases ROS generation and alleviates DNA damage in irradiated MSCs. (a) Irradiated MSCs pretreated with starvation or rapamycin were stained with DCF-DA to determine ROS levels measured by immunofluorescence. Cell nucleus was stained with Hoechst 33258. (b) MSCs were stained with γ-H2A.X antibody to determine DNA damage. Cell nucleus was stained with DAPI. Images were captured with fluorescence microscope, magnification × 100. (c and d) Mitochondrial ROS staining with MitoSOX was measured by FACS and immunofluorescence. (e and f) MMP staining with rhodamine 123 was measured by FACS and immunofluorescence
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fig4: Autophagy decreases ROS generation and alleviates DNA damage in irradiated MSCs. (a) Irradiated MSCs pretreated with starvation or rapamycin were stained with DCF-DA to determine ROS levels measured by immunofluorescence. Cell nucleus was stained with Hoechst 33258. (b) MSCs were stained with γ-H2A.X antibody to determine DNA damage. Cell nucleus was stained with DAPI. Images were captured with fluorescence microscope, magnification × 100. (c and d) Mitochondrial ROS staining with MitoSOX was measured by FACS and immunofluorescence. (e and f) MMP staining with rhodamine 123 was measured by FACS and immunofluorescence

Mentions: We next explored the underlying mechanism that autophagy may protect MSCs against irradiation injury. Irradiation induces ROS generation that leads to DNA damage. However, autophagy has been shown to be regulated by ROS.23, 24, 25 The loss of autophagy can induce ROS accumulation and DNA damage.26 It is possible that the elevated autophagy in irradiated MSCs may also serve as an adaptation to prevent the accumulation of ROS; hence, we detected the intracellular ROS level by using fluorescent probe dichlorofluorescin diacetate (DCF-DA). Irradiation increased the intracellular ROS level in MSCs. However, the intracellular ROS level decreased in MSCs pretreated with starvation or rapamycin, suggesting that autophagy contributes to minimize the accumulation of ROS (Figure 4a). The increase of ROS induced by irradiation was completely reversed by incubation with the antioxidant N-acetylcysteine (NAC, 2 mM). In contrast, MSCs that treated with H2O2 (100 μM) showed an increased ROS accumulation (Figure 4a). The excessive ROS would result in numerous forms of DNA damage, including DNA double-strand breaks (DSBs). γ-H2A.X has a key role in recruitment of repair proteins to the sites of DNA DSBs.27γ-H2A.X in MSCs was examined by immunofluorescence to investigate whether DSBs were induced in MSCs exposed to irradiation. As shown in Figure 4b, the intensity of γ-H2A.X was increased in irradiated MSCs, which indicated that more DNA damage occurred in MSCs when exposed to irradiation. However, γ-H2A.X levels decreased in irradiated MSCs pretreated with starvation or rapamycin. Further, treatment with NAC could abrogate the effect of oxidative DNA damage on MSCs (Figure 4b), confirming that irradiation-induced DNA damage is mediated by ROS generation.


Autophagy prevents irradiation injury and maintains stemness through decreasing ROS generation in mesenchymal stem cells.

Hou J, Han ZP, Jing YY, Yang X, Zhang SS, Sun K, Hao C, Meng Y, Yu FH, Liu XQ, Shi YF, Wu MC, Zhang L, Wei LX - Cell Death Dis (2013)

Autophagy decreases ROS generation and alleviates DNA damage in irradiated MSCs. (a) Irradiated MSCs pretreated with starvation or rapamycin were stained with DCF-DA to determine ROS levels measured by immunofluorescence. Cell nucleus was stained with Hoechst 33258. (b) MSCs were stained with γ-H2A.X antibody to determine DNA damage. Cell nucleus was stained with DAPI. Images were captured with fluorescence microscope, magnification × 100. (c and d) Mitochondrial ROS staining with MitoSOX was measured by FACS and immunofluorescence. (e and f) MMP staining with rhodamine 123 was measured by FACS and immunofluorescence
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3824648&req=5

fig4: Autophagy decreases ROS generation and alleviates DNA damage in irradiated MSCs. (a) Irradiated MSCs pretreated with starvation or rapamycin were stained with DCF-DA to determine ROS levels measured by immunofluorescence. Cell nucleus was stained with Hoechst 33258. (b) MSCs were stained with γ-H2A.X antibody to determine DNA damage. Cell nucleus was stained with DAPI. Images were captured with fluorescence microscope, magnification × 100. (c and d) Mitochondrial ROS staining with MitoSOX was measured by FACS and immunofluorescence. (e and f) MMP staining with rhodamine 123 was measured by FACS and immunofluorescence
Mentions: We next explored the underlying mechanism that autophagy may protect MSCs against irradiation injury. Irradiation induces ROS generation that leads to DNA damage. However, autophagy has been shown to be regulated by ROS.23, 24, 25 The loss of autophagy can induce ROS accumulation and DNA damage.26 It is possible that the elevated autophagy in irradiated MSCs may also serve as an adaptation to prevent the accumulation of ROS; hence, we detected the intracellular ROS level by using fluorescent probe dichlorofluorescin diacetate (DCF-DA). Irradiation increased the intracellular ROS level in MSCs. However, the intracellular ROS level decreased in MSCs pretreated with starvation or rapamycin, suggesting that autophagy contributes to minimize the accumulation of ROS (Figure 4a). The increase of ROS induced by irradiation was completely reversed by incubation with the antioxidant N-acetylcysteine (NAC, 2 mM). In contrast, MSCs that treated with H2O2 (100 μM) showed an increased ROS accumulation (Figure 4a). The excessive ROS would result in numerous forms of DNA damage, including DNA double-strand breaks (DSBs). γ-H2A.X has a key role in recruitment of repair proteins to the sites of DNA DSBs.27γ-H2A.X in MSCs was examined by immunofluorescence to investigate whether DSBs were induced in MSCs exposed to irradiation. As shown in Figure 4b, the intensity of γ-H2A.X was increased in irradiated MSCs, which indicated that more DNA damage occurred in MSCs when exposed to irradiation. However, γ-H2A.X levels decreased in irradiated MSCs pretreated with starvation or rapamycin. Further, treatment with NAC could abrogate the effect of oxidative DNA damage on MSCs (Figure 4b), confirming that irradiation-induced DNA damage is mediated by ROS generation.

Bottom Line: Stem cells were characterized by their stemness: self-renewal and pluripotency.Autophagy induced by starvation or rapamycin can reduce ROS accumulation-associated DNA damage and maintain stemness in MSCs.Further, inhibition of autophagy leads to augment of ROS accumulation and DNA damage, which results in the loss of stemness in MSCs.

View Article: PubMed Central - PubMed

Affiliation: 1] Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, the Second Military Medical University, Shanghai, China [2] Department of Pharmacy, Changhai Hospital, The Second Military Medical University, Shanghai, China.

ABSTRACT
Stem cells were characterized by their stemness: self-renewal and pluripotency. Mesenchymal stem cells (MSCs) are a unique type of adult stem cells that have been proven to be involved in tissue repair, immunoloregulation and tumorigenesis. Irradiation is a well-known factor that leads to functional obstacle in stem cells. However, the mechanism of stemness maintenance in human MSCs exposed to irradiation remains unknown. We demonstrated that irradiation could induce reactive oxygen species (ROS) accumulation that resulted in DNA damage and stemness injury in MSCs. Autophagy induced by starvation or rapamycin can reduce ROS accumulation-associated DNA damage and maintain stemness in MSCs. Further, inhibition of autophagy leads to augment of ROS accumulation and DNA damage, which results in the loss of stemness in MSCs. Our results indicate that autophagy may have an important role in protecting stemness of MSCs from irradiation injury.

Show MeSH
Related in: MedlinePlus