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Ionizing radiation-induced adaptive response in fibroblasts under both monolayer and 3-dimensional conditions.

Zhao Y, Zhong R, Sun L, Jia J, Ma S, Liu X - PLoS ONE (2015)

Bottom Line: Trypan blue staining was used to detect the cell death.Priming low dose radiation induced AR both under monolayer and 3-D condition.The expression of p53 and p21 was changed and indicated that they might participate in the regulation of AR.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Radiobiology (Ministry of Health), School of Public Health, Jilin University, Changchun, China; Dept. Nuclear Medicine, 2nd Hospital Jilin University, Changchun, China.

ABSTRACT
To observe the adaptive response (AR) induced by ionizing radiation in human fibroblasts under monolayer and 3-dimensional (3-D) condition. Three kinds of fibroblasts were cultured under both monolayer and 3-D condition. Immunofluorescent staining was used to detect the γ-H2AX foci and the morphological texture. Trypan blue staining was used to detect the cell death. Western blot was used to detect the expressions of γ-H2AX, p53 and CDKN1A/p21 (p21). We found that DNA damage increased in a dose-dependent and time-dependent manner after high doses of radiation. When cells were pretreated with a priming low dose of radiation followed by high dose radiation, DNA damage was attenuated under both monolayer and 3-D condition, and the adaptive response (AR) was induced. Additionally, the morphology of cells under monolayer and 3-D conditions were different, and radiation also induced AR according to morphological texture analysis. Priming low dose radiation induced AR both under monolayer and 3-D condition. Interestingly, 3-D microenvironment made cells more sensitive to radiation. The expression of p53 and p21 was changed and indicated that they might participate in the regulation of AR.

No MeSH data available.


Related in: MedlinePlus

Radiation-induced AR in RMP-4 cells under 2-D condition.For priming dose (also named as D1), 25, 50, 75, 100 mGy of radiation were used. 2 Gy was used as challenging dose (D2), with a 6-hour interval. (A) The changes of γ-H2AX in RMP-4 cells by immunofluorescent staining after 2 Gy radiation in different time points. (B) The changes of γ-H2AX in RMP-4 cells by immunofluorescent staining at 1h after different doses of radiation. (C) The radiation-induced AR based on γ-H2AX changes. (D) The changes of γ-H2AX, p53, and p21 expression in radiation-induced AR by western blot. (E) The statistical analysis of γ-H2AX, p53 and p21 expression from Western blot. (F) The statistical analysis of γ-H2AX from immunofluorescent staining based on the foci numbers per cell after different doses of radiation. (G)The statistical analysis of γ-H2AX from immunofluorescent staining based on the foci numbers per cell after different dose rate of radiation.* p<0.05 vs control; # p<0.05 vs D2. The bar corresponds to 25 μm.
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pone.0121289.g001: Radiation-induced AR in RMP-4 cells under 2-D condition.For priming dose (also named as D1), 25, 50, 75, 100 mGy of radiation were used. 2 Gy was used as challenging dose (D2), with a 6-hour interval. (A) The changes of γ-H2AX in RMP-4 cells by immunofluorescent staining after 2 Gy radiation in different time points. (B) The changes of γ-H2AX in RMP-4 cells by immunofluorescent staining at 1h after different doses of radiation. (C) The radiation-induced AR based on γ-H2AX changes. (D) The changes of γ-H2AX, p53, and p21 expression in radiation-induced AR by western blot. (E) The statistical analysis of γ-H2AX, p53 and p21 expression from Western blot. (F) The statistical analysis of γ-H2AX from immunofluorescent staining based on the foci numbers per cell after different doses of radiation. (G)The statistical analysis of γ-H2AX from immunofluorescent staining based on the foci numbers per cell after different dose rate of radiation.* p<0.05 vs control; # p<0.05 vs D2. The bar corresponds to 25 μm.

Mentions: Ionizing radiation is one of the most important DNA damage inducers. The DSB is the mechanism. DSB maybe induced directly by ionizing energy or indirectly by secondary radicals [20]. Phosphorylation of H2AX at Ser 139 (γ-H2AX) is the most sensitive marker that can be used to examine the DNA damage and the subsequent repair of the DNA lesion [12, 13]. In these experiments, the γ-H2AX was used as the marker of DNA damage induced by radiation. The rationale was based on reports that H2AX is phosphorylated after exposure to X rays in a linear dose-dependent manner and is correlated with the presence of DSBs [14]. For priming dose (also named as D1), 25, 50, 75, 100 mGy of radiation were used, while 2 Gy was used for challenging dose (D2) with a 6-hour interval. Fig. 1A showed the time-course changes of γ-H2AX after 2 Gy radiation, the increase of γ-H2AX could be seen at 0.5 h after radiation and reached the maximum at 1 h, then decreased rapidly, but at 4 h after radiation the γ-H2AX was still visible.


Ionizing radiation-induced adaptive response in fibroblasts under both monolayer and 3-dimensional conditions.

Zhao Y, Zhong R, Sun L, Jia J, Ma S, Liu X - PLoS ONE (2015)

Radiation-induced AR in RMP-4 cells under 2-D condition.For priming dose (also named as D1), 25, 50, 75, 100 mGy of radiation were used. 2 Gy was used as challenging dose (D2), with a 6-hour interval. (A) The changes of γ-H2AX in RMP-4 cells by immunofluorescent staining after 2 Gy radiation in different time points. (B) The changes of γ-H2AX in RMP-4 cells by immunofluorescent staining at 1h after different doses of radiation. (C) The radiation-induced AR based on γ-H2AX changes. (D) The changes of γ-H2AX, p53, and p21 expression in radiation-induced AR by western blot. (E) The statistical analysis of γ-H2AX, p53 and p21 expression from Western blot. (F) The statistical analysis of γ-H2AX from immunofluorescent staining based on the foci numbers per cell after different doses of radiation. (G)The statistical analysis of γ-H2AX from immunofluorescent staining based on the foci numbers per cell after different dose rate of radiation.* p<0.05 vs control; # p<0.05 vs D2. The bar corresponds to 25 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4373882&req=5

pone.0121289.g001: Radiation-induced AR in RMP-4 cells under 2-D condition.For priming dose (also named as D1), 25, 50, 75, 100 mGy of radiation were used. 2 Gy was used as challenging dose (D2), with a 6-hour interval. (A) The changes of γ-H2AX in RMP-4 cells by immunofluorescent staining after 2 Gy radiation in different time points. (B) The changes of γ-H2AX in RMP-4 cells by immunofluorescent staining at 1h after different doses of radiation. (C) The radiation-induced AR based on γ-H2AX changes. (D) The changes of γ-H2AX, p53, and p21 expression in radiation-induced AR by western blot. (E) The statistical analysis of γ-H2AX, p53 and p21 expression from Western blot. (F) The statistical analysis of γ-H2AX from immunofluorescent staining based on the foci numbers per cell after different doses of radiation. (G)The statistical analysis of γ-H2AX from immunofluorescent staining based on the foci numbers per cell after different dose rate of radiation.* p<0.05 vs control; # p<0.05 vs D2. The bar corresponds to 25 μm.
Mentions: Ionizing radiation is one of the most important DNA damage inducers. The DSB is the mechanism. DSB maybe induced directly by ionizing energy or indirectly by secondary radicals [20]. Phosphorylation of H2AX at Ser 139 (γ-H2AX) is the most sensitive marker that can be used to examine the DNA damage and the subsequent repair of the DNA lesion [12, 13]. In these experiments, the γ-H2AX was used as the marker of DNA damage induced by radiation. The rationale was based on reports that H2AX is phosphorylated after exposure to X rays in a linear dose-dependent manner and is correlated with the presence of DSBs [14]. For priming dose (also named as D1), 25, 50, 75, 100 mGy of radiation were used, while 2 Gy was used for challenging dose (D2) with a 6-hour interval. Fig. 1A showed the time-course changes of γ-H2AX after 2 Gy radiation, the increase of γ-H2AX could be seen at 0.5 h after radiation and reached the maximum at 1 h, then decreased rapidly, but at 4 h after radiation the γ-H2AX was still visible.

Bottom Line: Trypan blue staining was used to detect the cell death.Priming low dose radiation induced AR both under monolayer and 3-D condition.The expression of p53 and p21 was changed and indicated that they might participate in the regulation of AR.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Radiobiology (Ministry of Health), School of Public Health, Jilin University, Changchun, China; Dept. Nuclear Medicine, 2nd Hospital Jilin University, Changchun, China.

ABSTRACT
To observe the adaptive response (AR) induced by ionizing radiation in human fibroblasts under monolayer and 3-dimensional (3-D) condition. Three kinds of fibroblasts were cultured under both monolayer and 3-D condition. Immunofluorescent staining was used to detect the γ-H2AX foci and the morphological texture. Trypan blue staining was used to detect the cell death. Western blot was used to detect the expressions of γ-H2AX, p53 and CDKN1A/p21 (p21). We found that DNA damage increased in a dose-dependent and time-dependent manner after high doses of radiation. When cells were pretreated with a priming low dose of radiation followed by high dose radiation, DNA damage was attenuated under both monolayer and 3-D condition, and the adaptive response (AR) was induced. Additionally, the morphology of cells under monolayer and 3-D conditions were different, and radiation also induced AR according to morphological texture analysis. Priming low dose radiation induced AR both under monolayer and 3-D condition. Interestingly, 3-D microenvironment made cells more sensitive to radiation. The expression of p53 and p21 was changed and indicated that they might participate in the regulation of AR.

No MeSH data available.


Related in: MedlinePlus