| DNA-dependent protein kinase catalytic subunit(DNA-PKcs) is a about 460 k D protein kinase which belongs to the phosphatidylinositol-3-kinase-like kinase family and a DNA-activated serine / threonine protein kinase and heterodimers KU70 / KU80 regu Latory subunit composition together make up the trimeric DNA-dependent protein kinase(DNA-PK). The activity of DNA-PKcs serine / threonine protein kinase phosphorylates not only a variety of proteins involved in DNA damage repair and proteins involved in cell cycle regulation, but also regulates its kinase activity by phosphorylating itself. DNA-PKcs is a multifunctional protein and its major and most thororgh function studied by researchers is involed in DNA double strand breaks repair by nonhomologous end-joining pathway. In addition, DNA-PKcs was also involved in ionizing radiation or chemotherapy-induced abnormal cell division, apoptosis, G1 phase arrest of the cell cycle, or G2 / M arrest and autophagy. In recent years, with the deepening study of DNA-PKcs, we found that DNA-PKcs was abnormally high expressed in tumor tissues. It was reported that inhibition of DNA-PKcs was a valid approach to increase the tumor cell sensitivity to ionizing radiation, anticancer agents Top II poisons and cisplatin.Studies have shown that DNA-PKcs was involved in regulation of autophagy in glioma cells induced by ionizing radiation, but its research in regulating autophagy induced by radiation is little.Autophagy is in eukaryotic cells very critical and conserved process by metaolism and recycle of material and helps to the degradation of long-lived intracellular proteins, cytosolic components, damaged organelles and certain pathogens such that they are able to cycle reuse for maintaining cellular homeostasis, which is essential function about autophagy.In complex enviroment such as lack of essential nutrients, ionizing radiation, chemicals, endogenous reactive oxygen species and replication stress, organism cells make a variety of responses to deal with the adverse environment. Autophagy plays an important role through the consumption of their own material to the cells get a temporary supply in order to promote cell survival. However, autophagy in cancer treatment process is still a very controversial topic, because autophagy not only can stimulate autophagy cell survival, but also induce cell death. Studies have shown that when cells encounter a physiological change, or a certain kind of special treatment such as lethal drug dose, autophagy can promote cell death. Study confirmed autophagy dysfunction are closely related to initiation and development of many diseases.3-methyladenine(3-MA) has been widely used as autophagy inhibitors based on their inhibitory effect on class III PI3 K activity, which is known to be essential for induction of autophagy.Objectives 1. In DNA-PKcs knockdown He La cells line, we investigated how DNA-PKcs regulates autophagy and its possible molecular mechanism by observing cell radiosensitivity and the expression of autophaic proteins after irradiation; 2. Ionizing radiation can induce He La cells G2/M phase arrest, we explored that 3-methyladenine plays a role in ionizing radiation-induced cell cycle arrest.Methods We used constructed sh RNA plasmid targeting the DNA-PKcs, and then backaged the lentivirus of DNA-PKcs sh RNA through lipofectamin 2000 transfection method and infected He La cells, then established a He La cell lines stably knockdowed the expression of DNA-PKcs. Colonies form assay showing resistance to puromycin were isolated and analyzed by western blot; He La-sh NC and He La-sh DNA-PKcs were treated by irradiation alone or combination with autophagic inhibitor 3-methyladenine, then cell proliferation and cell radiosensitivity were detected at different time points(0,12, 24, 48,72h) after 6Gy x-ray treatment by CCK-8 assay; The survival abilities of He La-sh DNA-PKcs cells and He La-sh NC cells treated with different dose were studied by colony formation assay; He La cells were treated by irradiation alone or combination with autophagic inhibitor 3-methyladenine, then the cell cycle and the level of phosphorylated histone H3 at Ser10 in He La at 0, 2, 4, 6, 8, 12 h after 6Gy irradiation were also measured by flow cytometry;He La cells were treated by DNA-PKcs inhibitor NU7026 and autophagy agonist rapamycin,then the protein level of multi-functional ubiquitin-binding protein p62, microtubule light chain protein LC3, mammalian target of rapamycin protein m TOR were deceted by western blot and He La cells were treated by irradiation alone or combination with autophagic inhibitor 3-methyladenine,then the protein level of cycle checkpoint protein Chk2, Cdk1 and Cdc25 C were detected by western blot; We constructed GFP-LC3 expression vector and autophagy was also detected by monitoring the autophagic marker GFP-LC3 puncta per cell under immunofluorescent microscope.Results 1. We found that the expression level of DNA-PKcs in Hela-sh NC was higher than He La-sh DNA-PKcs by western blot and indicated that DNA-PKcs stable knockdown cell line was successfully constructed; 2. CCK-8 assay showed that depression of DNA-PKcs significantly decreased the growth activity of He La cells and increased the cellular sensitivity to ionizing radiation, autophagic inhibitor 3-methyladenine were added in cells on the basis of the irradiation treatment, both control cells or DNA-PKcs knockdown cells, the cell radiosensitivity was further enhanced; 3. Western blot results showed that the protein level of multi-functional ubiquitin-binding protein p62 was decreaed, microtubule light chain protein LC3 was increaced and the phosphorylation of mammalian target of rapamycin protein m TOR at 2481 site was decreaced in DNA-PKcs knockdown cells; 4. He La cells was treated by DNA-PKcs inhibitor NU7026 and the expression of autophagic protein LC3 was increaced then autophagy agonist rapamycin was added and autophagy was further enhanced; 5. Immunofluorescenc showed that autophagic protein expression of GFP-LC3 in DNA-PKcs-depleted cells was increaed, 3MA inhibited autophagy and rapamycin increaced autophagic protein expression of GFP-LC3; 6. The cell cycles of He La cells at different times point after 6Gy treatment were arrested at G2 / M phase by flow cytometry, which are consistent with the pre-test result, the G2 / M phase arrest were destroyed by added 3MA, which indicated 3MA involved may regulation of cycle arrest induced by ionizing radiation; 7. Hela cells were treated by irradiation alone or combination with autophagic inhibitor 3-methyladenine,flow cytometry suggested that the expression level of phosphorylation of histone H3 at Ser10 were further increaced by adding 3MA,which showed that the 3-methyl adenine can promote cells from the G2 phase into the M phase; 8. The expression of ATM(S1981),Chk2(T68), Cdk1(T15) and Cdc25C(S216) were decreced after treatement of 3MA.Conclusion 1. Depression of DNA-PKcs expression prompts the induction of autophagy by ionizing radiation and cellular radiosensitivity; 2. m TOR signaling may involve in the regulation of DNA-PKcs on autophagy processing; 3. 3-methyladenine may regulate cell cycle arrest induced by ionizing radiation by ATM / Chk2 / Cdc25 c / Cdk1 signaling pathway. |
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