Effects Of Cyclin D1 Over-expression On Activities Of ClC-3 Chloride Channels

Objective:To construct the eukaryotic expression vectors of human cyclin D1gene and express them in poorly differentiated nasopharyngeal carcinoma cells (CNE-2Z) in order to study the effects of cyclin D1 over-expression on activities of ClC-3 chloride channels.Methods:The full-length cyclin D1 was cloned from CNE-2Z cells by RT-PCR. The cDNA fragments were inserted into pIRES2-EGFP and pEGFP-C2 plasmids and confirmed by restriction enzymes digestion, PCR and sequencing. The recombinant vectors were transfected into CNE-2Z cells via LipofectamineTM2000, and the expression of cyclin D1 in the cells was examined by immunofluorescence and Western blot. To confirm the interactions between cyclin D1 and ClC-3 chloride channels by fluorescence resonance energy transfer (FRET), record the volume-activated chloride currents of cyclin D1 over-expression cells by patch clamp, study the changes of volume-activated chloride currents when dialyzing CDK6 antibody and using the inhibitor of CDK4/6.Result:Agarose gel electrophoresis showed a 918 bp band of the RT-PCR products, which matched the expected size. Restriction enzyme digestion, PCR and sequencing demonstrated successful construction of the recombinant vectors. CNE-2Z cells transfected with the recombinant vectors expressed cyclin D1 protein or cyclin D1-GFP protein as were verified by immuno fluorescence and Western blot. The FRET phenomenon showed the interactions between cyclin D1 and ClC-3, the distance of which was less 10 nm. The volume-activated chloride currents of cyclin D1 over-expression cells increased more than control cells; the volume-activated chloride currents decreased when dialyzing CDK6 antibody; chloride currents could be triggered in ISO solution containing 1 nM FCH (Fascaplysin chloride hydrate, CDK4/6 inhibitor), which couldn't be activated more in HYPO solution containing 1 nM FCH; along with increasing of FCH, the activated chloride currents decreased and could be activated again in in HYPO solution containing FCH. Conclusion:We have cloned cyclin D1 gene and constructed its eukaryotic expression vectors that can be expressed in nasopharyngeal carcinoma cells. Cyclin D1 could affect on chloride channel ClC-3 directly via CDK4/6 phosphorylating channels, the effects of CDK4 turn off chloride channels and the effects of CDK6 turn on chloride channels.