| Progression of the cell cycle depended on the activity of a series of protein complexes composed of cyclins and cyclin-dependent kinases(CDKs). The activity of cyclin-CDK complexes was regulated by a group of CDK inhibitors (CKIs). CKIs were classified in two families: CIP/KIP (p27kip1, p21Cip1, p57Kip2) and!NK4(p15, p16, p18, p19). Transition of cell cycle from Glto S phase was promoted by cyclin D-CDK4/CDK6 and cyclin E/CDK2. p27Kip1 and p21 inhibited the activity of these complexes directly by binding to them.DNA damage checkpoint played a pivotal role in appropriate decisions leading to cell survival or cell death. Rapid responses occured when cells were exposed to genotoxic stresses: one is initiating DNA damage checkpoint that leads to arrest in Gl, in order to provide the cell with sufficient time to repair damaged DNA, or leads to arrest in G2 , in order to avoid defective chromosome happening mitosis . On the other hand , if damage to the genome is excessive, the cells initiating apoptosis, when damages cannot be repaired.p27Kip1, a broader range inhibitor of CDKs, inhibited the activity of cyclin D-CDK4/CDK6 and cyclin E/CDK2 by binding to these complexes in Gl phase. In different conditions, the mechanisms of p27Kip1 invovled in regulating cell cycle progression was different. For example, p27Kip1 mainly bound cyclin E/CDK2 in cells treated with TGF- 3 . on the other hand, the activity and function of p27 Kip1 was affected by many physiological agents such as cAMP, IL-2, rampamycin. The expression of p27Kip1 increased after -irradiationin in the fibroblastoma cells, leading to arrest in Gl phase, but its expression markedly decreased after y -irradiation in the NBCC cells, and leaded to arrest in G2/M phase. Several studies have reported that p27 Kip1 involved in a much wider range of cellar processes, such as differentiation , apoptosis and migration, the expression level of p27Kip1 is key for its function in DNAdamage responses.The preliminary results as follows:- Different doses IR induced Gl/S cell cycle checkpoint in HeLa cellscyclin E/CDK2 kinase activity in Hela cells decreased at Ih , then increased at 3h, reached maximum at 12h exposed to2 Gy-irradiation; But its activity upregulation started at Ih, and reached maximum at 12 h exposed to lOGy irradiation .The protein level of cyclin E increased lightly after 2Gy, but it increased from 1 to 24 h after lOGy , level of cyclin E associated with cdk2 increased in response to lOGy, at the same time ,there was no significant changes in the level of cdk2 , while cdk2 levels remained constant in HeLa cells. Our results indicated that the kinetics of cyclin E/CDK2 kinase activity reflected the increase in cyclin E expression levels .The protein level of p27 Klpl increased at Ih , decreased from 6 to 24h after 2Gy, it decreased from 3 to 24 h after lOGy, and the level of p27Kipl associated with Cdk2 decreased, then lost its inhibition function to cyclin-dependent kinase.The protein level of p21 increased lightly from 1 to3 h, then decreased at 6-9 h, reached maximum at 24 h after 2Gy, it increased from 3 to 24 h exposed tolOGy, and the protein level of p21 associated with cdk2 increased, which did not inhibit the cyclin E/CDK2 kinase activity, because of upregulation of cyclin E and deregulation of p27Kipl after irradiation.In contrast to p21, p27Kipl may be a key regulator of cyclin E/CDK2 activity in DNA damage responses .HI The ubiquitin-proteasome pathway invovled in regulation of p27Kiplexpression in response to IRThe ubiquitin-proteasome pathway regulated selective and time-controlled elimination of p27 Kipl. We have known that IR may activate ubiquitin-proteasom pathyway, our experiments focus on whether ubiquitin -proteasome pathway involves in the regulation of p27Klpl during IR responses. Northern blot analysis showed that the mRNA level of p27 K'pl had no changes during IR responses; the level of ubiquitinatedp27Klpl increased following 10Gy-irradiation; MG-132 (proteasome inhibitor) inhibited...
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