Investigations On GCK Promoted Cell Apoptosis Induced By UV Irradiation

Mitogen-activated protein kinase(MAPK) are sorts of serine / threonine protein kinase which can be actived by many kinds of signal. The MAPK which activated through phosphorylation involve several cell life activities, just like the control gene transcription,induce cell death,regulate cell circle and so on. The effection of inducing cell death, especially to tumor cells by MAPK, had been discussed widely. p38, ERK5, ERK and JNK had been discovered as the four subfamily of MAPK. Now, ERK, JNK, p38MAPK pathway were identified closing to tumor cell apoptosis.JNK is one of very important factor of signal pathway of MAPK. Recent study shows the activation of JNK signal promotes cell apoptosis. JNK activating the transcription factor AP1 through c-Jun and ATF2 phosphorylation, it turns upregulation of FasL and then induce cell death. And another way of JNK induce cell death is trough the phosphorylation of Bc1-2 and Bc1-xL, promotes the releasing of cytochrome C by mitochondria, and then active Caspase cascade to induce cell apoptosis.GCK molecular belongs to Ser/Thr protein kinase. Recent study shows GCK take part in the JNK signal pathway, and exogenous GCK can activate JNK intracellular. Silencing the GCK gene through RNAi can block the JNK pathway activated by LPS and UV. However, it is still unclear about how GCK regulates this signal. Therefore, our study on GCK promoting the cell apoptosis mechanism helped us get more understanding of JNK signal pathway. This study consist of two parts:1.The cloning of human GCK gene and the construction of its transgenic cellsObjective clone human GCK gene and construct the recombinant plasmid vector carrying the target gene which can be expressed stably in mammal cell line HEK293.Methods Human GCK gene was amplified by PCR using the pCMV5-GCK as template and then the PCR product was sequenced. Digested with the restriction endonucleases BglⅡand SalI,the human GCK gene was inserted into pIRES2-EGFP plasmid vector . The recombinant pIRES2-EGFP plasmid vector cotransfected into the HEK293 cell with the Lipfect2000. After 24 hours, HEK293 cell line stably expressing human GCK protein was selected in the presence of G418(800ug/ml).Result The full-length of human GCK gene was cloned and the recombinant pIRES2-EGFP plasmid vector was constructed, The HEK293 cell line expressing human GCK was successfully transfected and selected. Results of RT-PCR and Western-blotting indicated that HEK293 transgenic cells could stably express human GCK protein.Conclusion Cloning of human GCK gene and construction of the recombinant pIRES2-EGFP plasmid vector and HEK293 transgenic cell line stably expressing GCK protein could contribute to further biological function research .2. The study on GCK promoting 293 cell apoptosis induced by UV irradiation Objective Study the mechanism of GCK promoting the UV induced cell apoptosis signal pathway.Methods Culture the GCK-HEK293 transgenic cells,pIRES2-HEK29 transgenic cells,HEK293 cells in 6 well plates with appropriate concentration(2×105/well), after 12h, irradiate cells under UV with different time(2min,4min,5min,6min,8min。). Cells were incubated for another 24h, then collected, staining with PI for 1min, FCM measure the cell mortality. irradiate cells under UV for 5min, after 24h incubate, lysing cells, collected the supernatant for western-bloting for GCK protein.Result 1.After UV irradiation with different time, the mortality of GCK-HEK293 transgenic cells was obviously higher than pIRES2-HEK29 transgenic cells and HEK293 cells; There's no difference between the pIRES2-HEK29 transgenic cells and HEK293 cells.2. UV can promote UV- induced cell death.