The Long-term Biological Responses In NL20 Human Bronchial Epithelial Cells Induced By A Single Dose Of Fe Ion Exposure

Objective:One of the major concerns is the significant risk of carcinogenesis in astronauts after exposure to space radiation such as galactic cosmic rays?GCR?consisting of energetic protons and high-energy charged?HZE?nuclei.Lung cancer has been suggested to be the largest potential cancer risk from space travel.It has been well known that high-LET heavy ions such as Fe ions are more efficient than low-LET X-rays at inducing lung cancer.DNA is the most important target of ionizing radiation.Generally,the genetic mutations and genomic instability caused by unrepaired or incorrectly repaired DNA damage may contribute to the carcinogenesis of ionizing radiation.Reactive oxygen species?ROS?is often involved in carcinogenesis.Cancer cells have higher intracellular ROS levels than their counterpart normal cells.Besides,activation of proto-oncogenes and inactivation of tumor suppressor genes also play an important role in carcinogenesis.Reprimo?RPRM?is a novel downstream effector of p53,which can induce cell cycle arrest at the G2/M checkpoint.And this putative tumor suppressor gene has been found frequently silenced via methylation of its promoter region in several malignances.The aim of this study was to investigate the long-term biological responses in NL20 human bronchial epithelial cells induced by low dose Fe ion radiation,and compare with X-ray exposure.At first,this thesis investigated the difference in DNA damage and repair in NL20 cells after exposed to?particles or X rays.Then,Ku555933,a specific ATM inhibitor,was demonstrated to be able to inhibit DNA damage repair after?particle or X ray exposure.Next,we investigated the long-term biological changes in NL20 cells induced by a single low dose of Fe ions or X-rays.Moreover,the effects of Ku55933 treatment prior to Fe ion exposure on the long-term responses of Fe ions were also studied.Finally,this thesis investigated the effects of Reprimo on the radiosensitivity of H460 cells.These studies paved the way for the further study on the mechanisms underlying lung cancer carcinogenesis induced by low dose of high LET space radiation.Methods:In the first part of this thesis,immunofluorescence of 53BP1 foci?DNA damage surrogate?was used as a biomarker of DNA damage.We used immunofluorescence staining of 53BP1 foci to investigate the kinetics of DNA damage repair after low dose of?particle or X ray exposure.We used the crystal violet assay to determine the optimal concentration of Ku55933 at which can inhibit ATM activation effectively.Cells were pretreated with 10?M Ku55933 for 1 h prior to ionizing exposure.NL20cells were replenished with fresh medium before radiation.We also used immunofluorescence of 53BP1 foci to further investigate the inhibitory effect of Ku55933 on DNA damage repair after low dose of?particle or X ray exposure.In the second part,NL20 cells were irradiated with 10 cGy Fe ions.For Ku55933 pretreatment,NL20 cells were pretreated with Ku55933 for 1 h prior to radiation,then replenished with fresh medium immediately before radiation.Cells were passaged as usual after radiation.And different end points were measured after certain passages.Using micronucleus formation assay to evaluate chromosome aberration.Using cell proliferation assay to detect multiplication rate.Using flow cytometry to determine cell cycle distribution.Using cellular ROS Detection Assay Kit to detect the intracellular ROS levels of cells.Using Western blotting to evaluate the expression of SOD1 and SOD2.Using soft-agar colony forming assay to assess cell transformation.In the third part of this thesis,Using micronucleus formation assay,colony forming assay and flow cytometry to determine cell cycle distribution to observe the changes in radiosensitivity of the progeny of irradiated NL20 cells.And Reprimo expression of the progeny of irradiated NL20 cells was evaluated by Western Blotting.In the fourth part of this thesis,we also explored the association of Reprimo with radiosensitivity by using micronucleus formation assay,colony forming assay and flow cytometry to assess the radiosensitivity of H460 cells when Overexpressing Reprimo.Results:1.At first,for a single dose of irradiation?0.9 Gy of X rays or 0.3 Gy of?particles?,X-irradiation exposure caused more positive cells with 53BP1 foci and more53BP1 foci per nucleus than?particles did at 0.5,1,2 h after radiation.With X-irradiation,the percentage of positive cells with 53BP1 foci and the mean foci number per nucleus decreased 8 h after radiation,and went back to the control level 24h after radiation.The increase and decrease of the percentage of positive cells and the mean foci number per nucleus after?particles were less significant than after X-irradiation.Moreover,similar results were obtained when cells were fractionatedly irradiated.Crystal violet assay was used to determine the optimal concentration of Ku55933,and no statistically significant proliferation inhibition was observed at the concentrations of 2,5,10 or 20?M for 48 h.Previous study has demonstrated that Ku55933 at 10?M can inhibit ATM activation effectively.Thus 10?M was chosen for the optimal concentration for Ku55933.Further study also showed that Ku55933pretreatment prior to?particles or X-irradiation caused longer duration of 53BP1 foci when compared with relative irradiated samples without Ku55933 pretreatment..2.NL20 cells were irradiated with 10 cGy Fe ions.For Ku55933 pretreatment,NL20 cells were pretreated with Ku55933 for 1 h prior to radiation.Cells were passaged as usual after radiation.And different end points were measured after certain passages.The progeny of NL20 cells irradiated with 0.1 Gy of Fe ions showed slightly increased micronucleus formation,significantly decreased proliferation,disturbed cell cycle distribution,obviously elevated intracellular ROS levels accompanied by reduced SOD1 and SOD2 expression,but the progeny of NL20 cells irradiated with 0.9 Gy of X-rays did not show any significant changes.More importantly,Fe ion exposure caused much greater soft-agar colony formation than X-rays in the progeny of irradiated NL20cells.Ku55933 reversed all the changes in the progeny of irradiated cells except the soft agar colony formation.3.The soft-agar colony formation in the 41^st progeny of NL20 cells irradiated with0.1 Gy of Fe ions was greater than the progeny of unexposed group.Some single clones were picked from the soft agar where the progeny of Fe-irradiated NL20 cells or non-irradiated were growing,and were grown as usual for 3-5 passages.These progeny of transformed NL20 cells caused by 0.1 Gy of Fe ions showed greater increased micronucleus formation after 1 Gy X rays comparing with their unexposed counterpart.Moreover,these progeny of transformed NL20 cells caused by 0.1 Gy of Fe ions induced more G2/M arrest than their unexposed counterpart after irradiated with 4 Gy of X-rays.Additionally,the expression of Reprimo was reduced in these progeny of transformed NL20 cells caused by 0.1 Gy of Fe ions when compared with their unexposed counterpart.4.Fourty-eight hours after 1 Gy X-ray exposure,H460 cells with overexpressed Reprimo showed greater micronucleus formation comparing with the negative control.Moreover,H460 cells with overexpressed Reprimo showed much lower survival fraction than the negative control after X-irradiation.In addition,6 and 24 h after 4 Gy X-ray exposure,overexpression of Reprimo in H460 cells caused more G2/M arrest.Conclusions:1.The DNA damage induced by?particles was more complex and was more difficult to repair than that induced by X-rays.Ku55933 pretreatment inhibited DNA damage repair and made DNA damage last longer.2.Compared with the relative control,the progeny of irradiated NL20 cells did not show any changes in both micronucleus formation and radiosensitivity.However,the progeny of NL20 cells irradiated with Fe ions displayed decreased proliferation,disturbed cell cycle distribution manifest as less G1 and more G2/M,elevated intracellular ROS levels and reduced SOD1 and SOD2 expression,most importantly,the progeny of irradiated cells showed greater colony formation in soft agar,suggesting cell transformation.Moreover,ATM inhibition by Ku55933 reversed all the changes in the progeny of irradiated cells except the soft agar colony formation.These data indicated that even a single low dose of Fe ions could induce long-term biological responses such as cell transformation etc.3.These progeny of transformed NL20 cells caused by 0.1 Gy of Fe ions showed higher radiation sensitivity.And the expression of reprimo was reduced in these progeny.These data suggested that radiosensitivity may be related to Reprimo.4.Reprimo overexpression increased radiation sensitivity in H460 cells,suggesting that Reprimo was associated with the radiosensitivity of H460 cells.