The Influence Of CDKs SiRNA Interference On Cell Cycle And Apoptosis

Part OneTHE INFLUENCE OF CDK1 siRNA INTERFERENCE ON CELL CYCLE AND APOPTOSISAbstract BACKGROUND & OBJECTIVE: It is widely accepted fact that the balancebetween cell proliferation and apoptosis is important for tissue homeostasis. What is lesswell known, however, is that the molecular mechanism regulate the balance between cellproliferation and apoptosis. The family of cyclin-dependent kinase complexes (Cdks) arewell known for their role in the cell division cycle,but the precise role of Cdks in apoptosisremains to be defined. In order to investigate the relationship of the master regulators ineukaryotic cell cycle with apoptosis, in the present report, CDK1 siRNA was transfectedinto cells to inhibit the CDK1 gene expression and invesgate its influence on the cell cycleand cell apoptosis. METHODS: The siRNA targeting CDK1 gene was chemicallysynthesized and transfected into Hela cells or SW480 cell by lipofectamine2000. CDK1gene and protein expression level were examined by real-time PCR and westernblot,respectively. In order to demonstrate the change of the cell cycle and apoptosis aftertransfected, we used flow cytometry to detect the cell cycle and used Annexin V/P1technique to study on apoptosis. The morphological change of transfected cell wereexamined under microscopy by Wright-Giemsa stain. RESULTS: CDK1 gene wassuccessfully silenced by siRNA and the CDK1 protein expression level decreasedsignificantly, especially from 48 hour to 60 hour after transfection. The DNA contentanalysis showed that transfection of CDK1 siRNA leaded to cells accumulating in G2/Mphase. No apoptosis happened after transfection of CDK1 siRNA 48h or 60h. More doublenucleus or multi-nucleus cells could be seen under microscopy among the transfected cells.CONCLUSION: The decreased CDK1 expression by gene silence leaded to cell cyclearrest in mitosis, but not induced apoptosis. THE ROLE OF CDK1 siRNA INTERFERENCE IN TAXOL INDUCED APOPTOSISAbstract BACKGROUND & OBJECTIVE: The cell cycle and the cell apoptosis wereintertwined. It was known from our research findings that after receiving apoptosis strikingonly those cells which were during cell cycle progression but not the GO phase cellunderwent apoptosis, we suppose maybe there are some molecular mechanism in cell cycleto regulate the cell apoptosis. This hypothesis prompted us to investigate the relationship ofthe master regulators in eukaryotic cell cycle with apoptosis. In the present report, CDK1siRNA was transfected into cells to interfere the CDK1 expression and invesgate itsinfluence on the taxol induced cell apoptosis, and elucidate the precise role of CDK1 in cellapoptosis. METHODS: The siRNA targeting CDK1 gene was chemically synthesized andtransfected into Hela cells or SW480 cell by lipofectamine2000. To make apoptosis striking,after 48 hours, the transfected cells were treated with 20ug/ml Taxol. Six experimentalgroups have been designed as follows: blank control which has not been treated,negativecontrol in which negative siRNA has been transfected,blank control stimulated byTaxol,negative control stimulated by Taxol,siRNA interfered CDK1,CDK1 silenced bysiRNA and then treated with Taxol. To detect cell cycle changes and apoptosis, we collectedthe cells 12 hours after drug treatment, used Annexin V/PI technique to study apoptosis,and flow cytometry to detect DNA content, thus the change of the cell cycle can be analyzed.The cell lysis were subjected to westernblot analysis for CDK1 and Bcl2 protein expressionchanging. RESULTS: CDK1 protein expression decreased in all siRNA transfected groups significantly. 20ug/ml taxol treatment induced cell apoptosis obviously and cell-cyclearrest in S phase,G2/M phase as early as 12h after addition of drug, while the degree ofits cell cycle arrest and apoptosis were not changed by CDK1 siRNA interference.Furthermore, the anti-apoptotic protein, Bcl-2 protein reduced only when apoptosis-inducing drug was added, but remained constant after transfection. The decrease of Bcl-2protein level related only with taxol treatment regardless of CDK1 siRNA transfection.CONCLUSION: The decreased CDK1 activity by gene silencing did not change theinfluence of Taxol on cell cycle arrest and on apoptosis. It seems likely that Cdk1 mightnot be universally required for apoptosis in the same way that Cdk1 are universally requiredfor the cell cycle. Part ThreeTHE INFLUENCE OF COTRANSFECTION CDK1 AND CDK2siRNA ON CELL CYCLE AND APOPTOSISAbstract BACKGROUND & OBJECTIVE: Investigate of the influence of CDK1 andCDK2 siRNA cotransfection on cell cycle and apoptosis, to explore exact role of cell cyclemaster regulator in apoptosis. METHODS: The siRNA targeting the CDK1 and CDK2genes were synthesized and simultaneously cotransfected into Hela and SW480 cells bylipofectamine2000.48 and 60 hours after the cotransfection, CDK1 and CDK2 or Bcl2protein expressions were examined by westernblot. Cell cycle arrest was analyzed by flowcytometry. We detected apoptosis via the Annexin V/PI method. Study transfected cellmorphological changes under a microscope after Wright-Giemsa Stain. Laser scanningconfocal microscope (LSCM) was used to observe the morphologic changes of theapoptosis cells. RESULTS: CDK1 and CDK2 protein expression was depressed 48 and 60hours after cotransfection. Cotransfection of CDK 1 and CDK2 siRNA led to accumulationof cells in S and G2/M phases and severely triggered apoptosis. An increase of binucleate andmultinucleate cells was observed under the microscope.The morphological change of earlyphase and late phase apoptotic cells could be seen by LSCM,but the anti-apoptotic Bcl2protein level is constant. CONCLUSION: The decreased activities of CDK1 and CDK2 bygene silencing led not only to cell cycle arrest in S phase and G2/M phase, but also inducedapoptosis. It was indirectly tested that CDK2 could compensates for the functionof CDK1 when CDK1 was silenced by siRNA.