Microgravity is a major stress factor that astronauts have to face in space. In the past, the effects of microgravity on genomic DNA damage were studied, and it seems that the effect on genomic DNA depends on cell types and the length of exposure time to microgravity or simulated microgravity (SMG). In this study we used mouse embryonic stem (MES) and mouse embryonic fibroblast (MEF) cells to assess the effects of SMG on DNA lesions. To acquire the insight into potential mechanisms by which cells resist and/or adapt to SMG, we also included Rad9-deleted MES and Mdc1-deleted MEF cells in addition to wild type cells in this study. We observed significant SMG-induced DNA double strand breaks (DSBs) in Rad9-/- MES and Mdc1-/- MEF cells but not in their corresponding wild type cells. A similar pattern of DNA single strand break or modifications was also observed in Rad9-/- MES. As the exposure to SMG was prolonged, Rad9-/- MES cells adapted to the SMG disturbance by reducing the induced DNA lesions. The induced DNA lesions in Rad9-/- MES were due to SMG-induced reactive oxygen species (ROS). Interestingly, Mdc1-/- MEF cells were only partially adapted to the SMG disturbance. That is, the induced DNA lesions were reduced over time, but did not return to the control level while ROS returned to a control level. In addition, ROS was only partially responsible for the induced DNA lesions in Mdc1-/- MEF cells. Taken together, these data suggest that SMG is a weak genomic DNA stress and can aggravate genomic instability in cells with DNA damage response (DDR) defects.
Fig1-A Neutral comet assay MES cell
Fig. 1. Effects of SMG on DNA damage and apoptosis in Rad9+/+ and Rad9-/- MES cells (A) Evaluation of DNA double strand break by neutral comet assay in Rad9+/+ and Rad9-/- MES cells cultured under 1G or SMG condition. Time points were 1, 2, 3, 4 and 5 days. At least 50 cells for each datum were scored for comet tail moment.
Fig1-B
Flow cytometric analysis of γ-H2AX formation in Rad9+/+ and Rad9-/- mMES cells cultured under 1G or SMG condition for 1 or 5 days
Fig1-C
Evaluation of DNA damage by alkaline comet assay in Rad9+/+ and Rad9-/- MES cells cultured under 1G or SMG condition for 1 or 5 days. At least 50 cells of each datum were scored for comet tail moment.
Fig1-D
Evaluation of DNA damage by alkaline comet assay in Rad9-/- MES cells with ectopic expression of Rad9 (Rad9-/-+Rad9 MES cells) cultured under 1G or SMG condition for 1 or 5 days. At least 50 cells of each datum were scored for comet tail moment.
Fig1-E
Flow cytometric analysis of Rad9+/+ and Rad9-/- MES cells cultured under 1G or SMG condition for 1 day to assess apoptosis using Annexin V labeling. Experiments were performed thrice and representative analyzes are shown (upper). The lower part is the quantitative comparison of apoptosis between the 1G Group and the SMG Group.
Fig2
DNA double strand break by neutral comet assay in Mdc1+/+ and Mdc1-/- MEF cells cultured under 1G or SMG condition for 1 or 5 days. At least 50 cells of each datum were scored for comet tail moment.
Fig3 a,b,c
(A) Flow cytometric analysis of ROS activity in Rad9+/+ and Rad9-/- MES cells exposed to 1G or SMG condition for 1 or 5days. (B) Flow cytometric analysis of ROS activity in Rad9-/-+Rad9 MES cells exposed to 1G or SMG condition for 1 or 5days. (C) N-acetylcysteine inhibited SMG-induced increase of ROS formation in Rad9-/- MES cells. Rad9-/- MES cells were mock-treated or treated with 0.05, 0.1 or 0.5 mM N-acetylcysteine under SMG for 1 day.
Fig3 A,B,C.xls
Fig3 d e
(D) Evaluation of DNA damage by alkaline comet assay in Rad9-/- MES cells mock-treated or treated with 0.5 mM N-acetylcysteine under 1G of SMG condition for 1 day. (E) Evaluation of DNA damage by neutral comet assay in Rad9-/- MES cells mock-treated or treated with 0.5 mM N-acetylcysteine under 1G of SMG condition for 1 day.
Fig3 D,E.xls
Fig4
(A) Flow cytometric analysis of ROS activity in Mdc1+/+ and Mdc1-/- MEF cells exposed to 1G or SMG condition for 1 or 5days.(B) N-acetylcysteine inhibited SMG-induced increase of ROS formation in Mdc1-/- MEF cells. Mdc1-/- MEF cells were mock-treated or treated with 0.05, 0.1 or 0.5 mM N-acetylcysteine under SMG for 1 day. (C) Evaluation of DNA damage by neutral comet assay in Mdc1-/- MEF cells mock-treated or treated with 0.5 mM N-acetylcysteine under 1G of SMG condition for 1 day.
Fig5
(A) Quantitative real-time PCR analysis of Nox2 mRNA expression in Rad9+/+ and Rad9-/- MES cells exposed to 1G or SMG condition for 1 or 5days. The expression levels of Nox2 were normalized to the endogenous control GAPDH expression. (B) Quantitative real-time PCR analysis of Nox4 mRNA expression in Rad9+/+ and Rad9-/- MES cells exposed to 1G or SMG condition for 1 or 5days. The expression levels of Nox4 were normalized to the endogenous control GAPDH expression. (D) Quantitative comparison of Nox2 expression. Data were derived from three independent experiments . The expression levels of Nox2 protein were normalized to the endogenous control GAPDH protein expression.
Fig6-A
Histograms of superoxide dismutase enzyme activity
Fig6.A.xls
Fig6-B
Histograms of catalase enzyme activity
FIG6.B.xls
Fig6-C
Histogram of Glutahione peroxidase
Fig6.C.xls
Fig7
Quantitative comparison of Nox2 expression. Data were derived from three independent experiments The expression levels of Nox2 were normalized to the endogenous control GAPDH expression
Fig8A
(A) The initial MES cells seeding number was 3×104. The doubling generation curve was generated by dividing the cell number with 104 and then transferring the quotient to the logarithm to the base2.
Fig8-A.xls
Fig8B
The initial MEF cells seeding number was 105. The doubling generation curve was generated by dividing the cell number with 104 and then transferring the quotient to the logarithm to the base2.
FIG8-B.xls
