Study The Relationship And Mechanism Between Unscheduled Cyclin B1 Expression And Apoptosis

Objective:A monoclone cell line was needed for expressing Cyclin B1 induced byTetracycline.Methods:The full sequence of Cyclin B1 was got from fetal liver cDNA library byPCR.After both cutted by BamH1 and X-ball at 37℃overnight,the product of Cyclin B1and empty vector pcDNA4/TO/myc-HisB were ligased at 16℃overnight by T4 DNAligase.The constructed pcDNA4/TO/myc-HisB-Cyclin B1 was sequenced,and the rightone was used to transfect T-REx^TM-293 cells using Lipofectamine^TM2000.The cells werecultured in DMEM containing Blasticidin (5μg/ml)and Zeocin (200μg/ml)for two weeks.Then they were seeded into 96 well plates.So,different clones were got,which amplifyingfrom a single cell.After cyclin B1 induced for different time by Tetracycline,cells wereharvested,and Cyclin B 1 expression was tested by Western blot and flow cytometry.Results:The pcDNA4/TO/myc-HisB-Cyclin B1 vector was successfully constructed,and the sequence was right.The monoclone cells could express Cyclin B 1-myc-his as earlyas 3 hours after Tetracycline added to DMEM,and the highest amount of CyclinB 1-myc-his was expressed 48 hours after Tetracycline added.Conclusions:The monoclone T-REx^TM-293 cell line could express Cyclin B1 inducedby Tetracycline under control. Objective:'Unscheduled expression of Cyclin B1 in G₀/G₁ phase increasing cellapoptosis'was wanted to be confirmed by using a monoclone cell line,which couldexpress Cyclin B1 induced by Tetracycline.Methods:Cells were caused to arrest in G₀/G₁ phase by Thymidine firstly.ThenTetracycline was added to induce Cyclin B1 expression for 48 hours,and thecontrol group was added no Tetracycline.At last,the cell apoptosis was detected byAnnexin V-PI and API methods.The activity of Cdk1 was also confirmed byWestern blot at the same time.Results:Most cells were arrested in G₀/G₁ phase cause of Thymidine.CyclinB1 protein and cell apoptosis increased after Cyclin B1 expression for 48 hoursinduced by Tetracycline.And the activity of Cdk1 (Thr¹⁶¹phosphorylation)was alsoincreased.Conclusions:Unscheduled expression of Cyclin B1 in G₀/G₁ phase couldincrease cell apoptosis. Objective:Study the proteins doing functions in specific apoptosis of G₀/G₁phase cause of Cyclin B1,which can interact with Cyclin B1 or be phosphorylatedby Cyclin B1/Cdk1 complex.Methods:The monoclone cell line could express Cyclin B1 with a Myc and Histag after Tetracycline added.Cells uninduced or induced by Tetracycline for 48hwere both lysed,then 10pg Myc antibody was added to each.IP (immuneprecipitation)was used for detect proteins which might interact with Cyclin B1,thenthey were run in SDS-PAGE.The proteins were confirmed by mass spectrometry,after different bands cutted from SDS-PAGE by coomassie blue staining and silverstaining.Results:The four proteins confirmed by MASS were Cdk1 (Cyclin-dependentkinase 1),Cdk2 (Cyclin-dependent kinase 2),MAD1L1 (Mitotic arrest deficient-likeprotein 1,MAD1-like 1)and heat shock 70kDa protein 8 isoform 1.Conclusions:Cyclin B1 (or Cyclin B1/Cdk1)could interact with Cdk2,MAD1L1and Hsp70,which might had functions in specific apoptosis of G₀/G₁ phase cause ofCyclin B1,besides the activation of Cdk1.