Construction of cyclin D1 recombinant plasmids and its expression in human nasopharyngeal carcinoma cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct the eukaryotic expression vectors of human cyclin D1 gene and express them in poorly differentiated nasopharyngeal carcinoma cells (CNE-2Z cells).</p><p><b>METHODS</b>The full-length cyclin D1 was cloned from CNE-2Z cells by RT-PCR. The cDNA fragments were inserted into pIRES2-EGFP plasmids and pEGFP-C2 plasmids and confirmed by restriction enzyme digestion, PCR and sequencing. The recombinant vectors were transfected into CNE-2Z cells via Lipofectamine 2000, and the expression of cyclin D1 in the cells was examined by immunofluorescence and Western blotting.</p><p><b>RESULTS</b>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 immunofluorescence and Western blotting.</p><p><b>CONCLUSION</b>We have cloned cyclin D1 gene and constructed its eukaryotic expression vectors that can be expressed in nasopharyngeal carcinoma cells, which may facilitate the study of the role of cyclin D1 in the development of nasopharyngeal carcinoma.</p>

Ca(2+)-dependent potassium channels play important roles in regulatory volume decrease in human nasopharyngeal carcinoma cells / 生理学报

It has been shown that cell volume regulation mechanisms play important roles in various cell functions. We demonstrated previously that volume-activated chloride channels were involved in cell volume regulation. The present study aimed to clarify the roles of various types of potassium channels in regulatory volume decrease (RVD) induced by hypotonic challenges in human nasopharyngeal carcinoma cells (CNE-2Z cells). The whole-cell patch clamp technique was used to record hypotonic challenge-induced potassium currents. During current recordings, cells were held at 0 mV and stepped to +/-46 and +/-92 mV, repeatedly. The cell volume was computed from cell diameters. The changes of cell volume were monitored and analyzed by the time-lapse imaging technique. The results showed that the exposure to 160 mOsm/L hypotonic solution caused the cells to swell by (144.5+/-4.2)%, activated a potassium current (59.2 pA/pF+/-13.3 pA/pF at 92 mV), and induced RVD. Cell volume was recovered from hypotonic challenge-induced swelling by (48.9+/-4.6)% after 20 min. The potassium current (at 92 mV) and RVD were inhibited by the calcium-dependent potassium channel blocker, clotrimazole (100 mumol/L), by (98.5+/-2.8)% and (89.3+/-4.9)%, respectively. Depletion of extracellular calcium prevented the activation of the hypotonic challenge-induced potassium current and inhibited the process of RVD. The voltage-gated potassium channel blocker, 4-AP (5 mmol/L), partially inhibited the hypotonic challenge-activated potassium currents by (66.6+/-5.3)% (at 92 mV). These results suggest that the Ca(2+)-dependent potassium channel is the main component of volume-activated potassium channels and plays an important role in volume regulation of CNE-2Z cells. The voltage-gated potassium channels may also contribute in part to the formation of the volume-activated potassium current.