At high exposure levels ionizing radiation is a carcinogen. activation of homologous recombination. Also, proliferation status may significantly affect the biological outcome, since homologous repair is not activated in resting MSCs. (R2=0.98), where is a number of H2AX foci and is radiation dose in mGy. This result was consistent with our previous observations showing linear H2AX dose responses in human fibroblasts , as well as with the results reported by others for this cell type . Similar results were obtained for 53BP1 foci, another marker frequently used for quantification of DNA DSBs (Figure ?(Figure1b).1b). For prolonged irradiation, a different dose-response Aciclovir (Acyclovir) relationship was observed in that the initial linear portion of the curve turned TNFAIP3 into a plateau at around 1 Gy (Figure ?(Figure1c).1c). A statistically significant difference between acute and prolonged irradiation was found for doses of 1350 mGy (for H2AX, p=0.0082; for 53BP1, p=0.0417) and 1620 mGy (for H2AX, p=0.0009; for 53BP1, p=0.0229). Open in a separate window Figure 1 H2AX and 53BP1 foci formation in MSCs exposed to either acute or prolonged X-ray irradiation(a) Representative microphotographs of immunofluorescently stained irradiated MSCs showing H2AX (red) and 53BP1 (green) foci. DAPI counterstaining is shown in blue. (b) Quantification of H2AX and 53BP1 foci, as well as their colocalization,in MSC exposed to acute (5400 mGy/h) or prolonged (270 mGy/h) (c) X-ray irradiation. Mean foci numbers derived from at least three independent experiments are shown. Error bars show SE. Rad51 foci formation during prolonged irradiation We examined the position of homologous DNA restoration by quantifying Rad51 foci in cells subjected to long term X-ray irradiation. Shape ?Shape2a2a shows consultant pictures of Rad51 foci in MSCs subjected to irradiation. Quantification of Rad51 foci can be presented in Shape ?Shape2b.2b. As opposed to H2AX foci dosage responses (Shape ?(Shape1b),1b), considerable raises in Rad51 foci weren’t found until about 2 h of prolonged irradiation (cumulative Aciclovir (Acyclovir) dose of 540 mGy). This finding suggests a threshold for homologous repair activation upon prolonged 270 mGy/h X-ray irradiation of MSC cultures. Between 2 and 6 h of irradiation, Aciclovir (Acyclovir) Rad51 foci accumulated linearly and the overall dose response Aciclovir (Acyclovir) could be fit by a linear regression (R2=0.95), where is a number of RAD51 foci and is radiation dose in mGy. There was a dose overlap between the linear portion of Rad51 foci dose-response curve and the plateau portion of the H2AX foci curve, suggesting that linear activation of homologous DNA repair may explain the plateau. Open in a separate window Figure 2 RAD51 foci formation in MSCs exposed to prolonged X-ray irradiation(a) Representative microphotographs of immunofluorescently stained irradiated MSCs showing Rad51 foci (red). DAPI counterstaining is shown in blue. (b) Quantification of Rad51 in MSC exposed to prolonged (270 mGy/h) X-ray irradiation. Mean foci numbers derived from at least three independent experiments are shown. Error bars show SE. H2AX foci formation in Ki67+ vs. Ki67- cell subpopulations during prolonged irradiation To further characterize H2AX foci formation upon prolonged irradiation, we measured the responses in proliferating vs. non-proliferating cells. We used Ki67 as a marker of the proliferation status and scored H2AX foci in Ki67 negative (Ki67-) G0 cells vs. Ki67 positive (Ki67+) interphase and mitotic cells (Figure ?(Figure3a).3a). First, we observed a statistically significant difference between the two subpopulations of control non-irradiated cells for each time point: 2.29 0.36 for Ki67+ vs. 0.35 0.08 for Ki67- cells (Figure ?(Figure3b).3b). Similarly, for irradiated cells for all of the time points examined the number of H2AX foci was higher for Ki67+ subpopulation compared to Ki67- Aciclovir (Acyclovir) cells. We also constructed H2AX histograms for each time point for these two subpopulations (Figure ?(Figure3c)3c) to examine heterogeneity of cells for H2AX foci numbers. This data indicates that proliferating cells tend to have higher numbers of H2AX foci. However, the shape of the dose-response curves did not differ between Ki67+ and Ki67- cells in that.