Overexpression of hSav1 promotes Mst1-induced apoptosis in HeLa cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To elucidate the effect of hSav1 expression on Mst1-mediated apoptosis in HeLa cells.</p><p><b>METHODS</b>Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and cotransfected into HeLa cells. Triple immunofluorescent labeling of hSav1, Mst1 and nucleus was performed to determine their subcellular localization. Plasmids pCMV-HA-hSav1 and/or pcDNA/4TO-Flag-Mst1 were transfected into HeLa cells, and 36 hours later cisplatin (50 micromol/L) as a pro-apoptotic agent was added for 14 hours. Cell apoptosis was analyzed by annexin V/PI assay.</p><p><b>RESULTS</b>Plasmids pCMV-HA-hSav1 and pcDNA/4TO-Flag-Mst1 were constructed and the authenticity of constructs was verified by sequencing. The binding in vitro showed that hSav1 could be detect from the anti-Mst1 immunoprecipitation complex. The immunofluorescent labeling showed that hSav1 and Mst1 had the same localization in cells. Overexpressed protein hSav1 did not induce a significant cell apoptosis. However, co-expression of hSav1 with Mst1 resulted in a significant increase of apoptosis above the level seen with Mst1 alone (24.5% +/- 2.4% vs. 39.3% +/- 4.0%, P < 0.05).</p><p><b>CONCLUSION</b>Our findings indicate that hSav1 is a newly identified protein that interacts with Mst1 and augments Mst1-mediated apoptosis.</p>

Effects of MST1 on cell proliferation and apoptosis of human breast carcinoma cell line MCF-7 / 肿瘤

Objective: To elucidate effects of mammalian sterile 20-like kinase 1 (MST1) gene on cell proliferation and apoptosis of human breast carcinoma cell line MCF-7. Methods: This study constructed plasmid pCMV-FLAG-MST1 and transfected it into human breast carcinoma cell line MCF-7 via mediation by Lipofectamine 2000. We detected the efficiency of transfection by Western blotting. The effect of MST1 on cell proliferation was measured by MTT assay at 12, 24, 36, and 48 h after transfection. The effect of MST1 on cell proliferation was also determined by BrdU incorporation assay at 36 h after transfection. Cisplatin was added into cultured cells at 36 h to induce apoptosis. Forteen hours later cell apoptosis was detected by Annexin V staining. Results: This study successfully constructed plasmid pCMV-FLAG-MST1. The MTT and BrdU incorporation assay showed that the cell proliferation was significantly inhibited after transfection of pCMV-FLAG-MST1. Annexin V staining demonstrated that cell apoptotic rate was relatively higher after overexpression of MST1. Conclusion: In summary, overexpression of MST1 decreases cell proliferation and induced apoptosis of human breast carcinoma cell line MCF-7.