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Low dose radiation induced senescence of human mesenchymal stromal cells and impaired the autophagy process.

Alessio N, Del Gaudio S, Capasso S, Di Bernardo G, Cappabianca S, Cipollaro M, Peluso G, Galderisi U - Oncotarget (2015)

Bottom Line: Individuals may be exposed to low doses of radiation either intentionally for medical purposes or accidentally, such as those exposed to radiological terrorism or those who live near illegal radioactive waste dumpsites.We studied the effects of low dose radiation on human bone marrow mesenchymal stromal cells (MSC), which contain a subpopulation of stem cells able to differentiate in bone, cartilage, and fat; support hematopoiesis; and contribute to body's homeostasis.The main outcome of low radiation exposure, besides reduction of cell cycling, is the triggering of senescence, while the contribution to apoptosis is minimal.We hypothesize that the autophagy prevented radiation deteriorative processes, and its decline contributed to senescence.An increase in ATM staining one and six hours post-irradiation and return to basal level at 48 hours, along with persistent gamma-H2AX staining, indicated that MSC properly activated the DNA repair signaling, though some damages remained unrepaired, mainly in non-cycling cells.This suggested that the impaired DNA repair capacity of irradiated MSC seemed mainly related to the reduced activity of a non-homologous end-joining (NHEJ) system rather than HR (homologous recombination).

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Medicine, Biotechnology and Molecular Biology Section, Second University of Naples, Naples 80138, Italy.

ABSTRACT
Low doses of radiation may have profound effects on cellular function. Individuals may be exposed to low doses of radiation either intentionally for medical purposes or accidentally, such as those exposed to radiological terrorism or those who live near illegal radioactive waste dumpsites.We studied the effects of low dose radiation on human bone marrow mesenchymal stromal cells (MSC), which contain a subpopulation of stem cells able to differentiate in bone, cartilage, and fat; support hematopoiesis; and contribute to body's homeostasis.The main outcome of low radiation exposure, besides reduction of cell cycling, is the triggering of senescence, while the contribution to apoptosis is minimal. We also showed that low radiation affected the autophagic flux. We hypothesize that the autophagy prevented radiation deteriorative processes, and its decline contributed to senescence.An increase in ATM staining one and six hours post-irradiation and return to basal level at 48 hours, along with persistent gamma-H2AX staining, indicated that MSC properly activated the DNA repair signaling, though some damages remained unrepaired, mainly in non-cycling cells. This suggested that the impaired DNA repair capacity of irradiated MSC seemed mainly related to the reduced activity of a non-homologous end-joining (NHEJ) system rather than HR (homologous recombination).

No MeSH data available.


Related in: MedlinePlus

Analysis of senescence, apoptosis, stemness and autophagy in irradiated MSCsPanel (A) – Flow cytometry analysis of apoptosis with Nexin assay. The experiments were carried out six and 48 hours post-irradiation. The assay allows the identification of early (Annexin V + and 7ADD −) and late apoptosis (Annexin V + and 7ADD +). Nevertheless, apoptosis is a continuous process and we calculated the percentage of apoptosis as the sum of early and late apoptotic cells, to avoid a discretional separation between early and late apoptosis. The table shows the global percentage of Annexin V-positive cells. Data are expressed with standard deviation (n = 3, *p < 0.05). Panel (B) – CFU assay. The pictures show representative crystal violet staining of clones obtained after 14 days of incubation, with MSCs plated following irradiation experiments. The mean number of clones per 1,000 cells plated in 100 mm dish (± SD, n = 3, *p < 0.05, **p < 0.01) is indicated below each picture. Panels (C, D) – Senescence assay. Representative microscopic fields of acid beta-galactosidase (blue) in irradiated and control cells are shown. Arrows indicate senescent cells. The graph shows mean percentage value of senescent cells (± SD, n = 3, *p < 0.05).
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Figure 2: Analysis of senescence, apoptosis, stemness and autophagy in irradiated MSCsPanel (A) – Flow cytometry analysis of apoptosis with Nexin assay. The experiments were carried out six and 48 hours post-irradiation. The assay allows the identification of early (Annexin V + and 7ADD −) and late apoptosis (Annexin V + and 7ADD +). Nevertheless, apoptosis is a continuous process and we calculated the percentage of apoptosis as the sum of early and late apoptotic cells, to avoid a discretional separation between early and late apoptosis. The table shows the global percentage of Annexin V-positive cells. Data are expressed with standard deviation (n = 3, *p < 0.05). Panel (B) – CFU assay. The pictures show representative crystal violet staining of clones obtained after 14 days of incubation, with MSCs plated following irradiation experiments. The mean number of clones per 1,000 cells plated in 100 mm dish (± SD, n = 3, *p < 0.05, **p < 0.01) is indicated below each picture. Panels (C, D) – Senescence assay. Representative microscopic fields of acid beta-galactosidase (blue) in irradiated and control cells are shown. Arrows indicate senescent cells. The graph shows mean percentage value of senescent cells (± SD, n = 3, *p < 0.05).

Mentions: We then analyzed the level of apoptosis and senescence by annexin V and acid-beta-galactosidase assay, respectively (Fig. 2). Six hours post treatment we detected an increase in apoptosis in both experimental conditions, but the apoptosis rate dropped below the control level at 48 hours (Fig. 2A). This suggests that apoptosis is an acute reaction to IR, while long-lasting effects may be associated to other phenomena. Indeed, a huge percentage of cells entered senescence six hours following IR, both for the low and high dose radiation (Fig. 2C, D). This percentage further increased at 48 hours. Senescence may be considered the “preferential answer” of MSC to stress induced by IR. This result is in good agreement with data on cell proliferation and clonogenic properties of MSC, as detected by quick proliferation assay and CFU evaluation, respectively (Suppl. File 1; Fig. 2B). In fact, senescence could greatly affect the “stemness” of MSC cultures.


Low dose radiation induced senescence of human mesenchymal stromal cells and impaired the autophagy process.

Alessio N, Del Gaudio S, Capasso S, Di Bernardo G, Cappabianca S, Cipollaro M, Peluso G, Galderisi U - Oncotarget (2015)

Analysis of senescence, apoptosis, stemness and autophagy in irradiated MSCsPanel (A) – Flow cytometry analysis of apoptosis with Nexin assay. The experiments were carried out six and 48 hours post-irradiation. The assay allows the identification of early (Annexin V + and 7ADD −) and late apoptosis (Annexin V + and 7ADD +). Nevertheless, apoptosis is a continuous process and we calculated the percentage of apoptosis as the sum of early and late apoptotic cells, to avoid a discretional separation between early and late apoptosis. The table shows the global percentage of Annexin V-positive cells. Data are expressed with standard deviation (n = 3, *p < 0.05). Panel (B) – CFU assay. The pictures show representative crystal violet staining of clones obtained after 14 days of incubation, with MSCs plated following irradiation experiments. The mean number of clones per 1,000 cells plated in 100 mm dish (± SD, n = 3, *p < 0.05, **p < 0.01) is indicated below each picture. Panels (C, D) – Senescence assay. Representative microscopic fields of acid beta-galactosidase (blue) in irradiated and control cells are shown. Arrows indicate senescent cells. The graph shows mean percentage value of senescent cells (± SD, n = 3, *p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4480742&req=5

Figure 2: Analysis of senescence, apoptosis, stemness and autophagy in irradiated MSCsPanel (A) – Flow cytometry analysis of apoptosis with Nexin assay. The experiments were carried out six and 48 hours post-irradiation. The assay allows the identification of early (Annexin V + and 7ADD −) and late apoptosis (Annexin V + and 7ADD +). Nevertheless, apoptosis is a continuous process and we calculated the percentage of apoptosis as the sum of early and late apoptotic cells, to avoid a discretional separation between early and late apoptosis. The table shows the global percentage of Annexin V-positive cells. Data are expressed with standard deviation (n = 3, *p < 0.05). Panel (B) – CFU assay. The pictures show representative crystal violet staining of clones obtained after 14 days of incubation, with MSCs plated following irradiation experiments. The mean number of clones per 1,000 cells plated in 100 mm dish (± SD, n = 3, *p < 0.05, **p < 0.01) is indicated below each picture. Panels (C, D) – Senescence assay. Representative microscopic fields of acid beta-galactosidase (blue) in irradiated and control cells are shown. Arrows indicate senescent cells. The graph shows mean percentage value of senescent cells (± SD, n = 3, *p < 0.05).
Mentions: We then analyzed the level of apoptosis and senescence by annexin V and acid-beta-galactosidase assay, respectively (Fig. 2). Six hours post treatment we detected an increase in apoptosis in both experimental conditions, but the apoptosis rate dropped below the control level at 48 hours (Fig. 2A). This suggests that apoptosis is an acute reaction to IR, while long-lasting effects may be associated to other phenomena. Indeed, a huge percentage of cells entered senescence six hours following IR, both for the low and high dose radiation (Fig. 2C, D). This percentage further increased at 48 hours. Senescence may be considered the “preferential answer” of MSC to stress induced by IR. This result is in good agreement with data on cell proliferation and clonogenic properties of MSC, as detected by quick proliferation assay and CFU evaluation, respectively (Suppl. File 1; Fig. 2B). In fact, senescence could greatly affect the “stemness” of MSC cultures.

Bottom Line: Individuals may be exposed to low doses of radiation either intentionally for medical purposes or accidentally, such as those exposed to radiological terrorism or those who live near illegal radioactive waste dumpsites.We studied the effects of low dose radiation on human bone marrow mesenchymal stromal cells (MSC), which contain a subpopulation of stem cells able to differentiate in bone, cartilage, and fat; support hematopoiesis; and contribute to body's homeostasis.The main outcome of low radiation exposure, besides reduction of cell cycling, is the triggering of senescence, while the contribution to apoptosis is minimal.We hypothesize that the autophagy prevented radiation deteriorative processes, and its decline contributed to senescence.An increase in ATM staining one and six hours post-irradiation and return to basal level at 48 hours, along with persistent gamma-H2AX staining, indicated that MSC properly activated the DNA repair signaling, though some damages remained unrepaired, mainly in non-cycling cells.This suggested that the impaired DNA repair capacity of irradiated MSC seemed mainly related to the reduced activity of a non-homologous end-joining (NHEJ) system rather than HR (homologous recombination).

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Medicine, Biotechnology and Molecular Biology Section, Second University of Naples, Naples 80138, Italy.

ABSTRACT
Low doses of radiation may have profound effects on cellular function. Individuals may be exposed to low doses of radiation either intentionally for medical purposes or accidentally, such as those exposed to radiological terrorism or those who live near illegal radioactive waste dumpsites.We studied the effects of low dose radiation on human bone marrow mesenchymal stromal cells (MSC), which contain a subpopulation of stem cells able to differentiate in bone, cartilage, and fat; support hematopoiesis; and contribute to body's homeostasis.The main outcome of low radiation exposure, besides reduction of cell cycling, is the triggering of senescence, while the contribution to apoptosis is minimal. We also showed that low radiation affected the autophagic flux. We hypothesize that the autophagy prevented radiation deteriorative processes, and its decline contributed to senescence.An increase in ATM staining one and six hours post-irradiation and return to basal level at 48 hours, along with persistent gamma-H2AX staining, indicated that MSC properly activated the DNA repair signaling, though some damages remained unrepaired, mainly in non-cycling cells. This suggested that the impaired DNA repair capacity of irradiated MSC seemed mainly related to the reduced activity of a non-homologous end-joining (NHEJ) system rather than HR (homologous recombination).

No MeSH data available.


Related in: MedlinePlus