p21 is Responsible for Ionizing Radiation-induced Bypass of Mitosis

Objective To explore the role of p21 in ionizing radiation-induced changes in protein levels during the G2/M transition and long-term G2 arrest.Methods Protein expression levels were assessed by western blot in the human uveal melanoma 92-1 cells after treatment with ionizing radiation.Depletion of p21 was carried out by employing the siR NA technique.Cell cycle distribution was determined by flow cytometry combined with histone H3 phosphorylation at Ser28,an M-phase marker.Senescence was assessed by senescenceassociated-β-galactosidase（SA-β-gal） staining combined with Ki67 staining,a cell proliferation marker.Results Accompanying increased p21,the protein levels of G2/M transition genes declined significantly in 92-1 cells irradiated with 5 Gy of X-rays.Furthermore,these irradiated cells were blocked at the G2 phase followed by cellular senescence.Depletion of p21 rescued radiation-induced G2 arrest as demonstrated by the upregulation of G2/M transition kinases,as well as the high expression of histone H3 phosphorylated at Ser28.Knockdown of p21 resulted in entry into mitosis of irradiated 92-1 cells.However,cells with serious DNA damage failed to undergo cytokinesis,leading to the accumulation of multinucleated cells.Conclusion Our results indicated that p21 was responsible for the downregulation of G2/M transition regulatory proteins and the bypass of mitosis induced by irradiation.Downregulation of p21 by siR NA resulted in G2-arrested cells entering into mitosis with serious DNA damage.This is the first report on elucidating the role of p21 in the bypass of mitosis.

Irradiation Response of Adipose-derived Stem Cells under Three-dimensional Culture Condition

Objective Adipose tissue distributes widely in human body. The irradiation response of the adipose cells in vivo remains to be investigated. In this study we investigated irradiation response of adipose-derived stem cells (ASCs) under three-dimensional culture condition. Methods ASCs were isolated and cultured in low attachment dishes to form three-dimensional (3D) spheres in vitro. The neuronal differentiation potential and stem-liked characteristics was monitored by using immunofluoresence staining and flow cytometry in monolayer and 3D culture. To investigate the irradiation sensitivity of 3D sphere culture, the fraction of colony survival and micronucleus were detected in monolayer and 3D culture. Soft agar assays were performed for measuring malignant transformation for the irradiated monolayer and 3D culture. Results The 3D cultured ASCs had higher differentiation potential and an higher stem-like cell percentage. The 3D cultures were more radioresistant after either high linear energy transfer (LET) carbon ion beam or low LET X-ray irradiation compared with the monolayer cell. The ASCs’ potential of cellular transformation was lower after irradiation by soft agar assay. Conclusion These findings suggest that adipose tissue cell are relatively genomic stable and resistant to genotoxic stress.