[Overexpression of hSav1 promotes Mst1-induced apoptosis in HeLa cells].
OBJECTIVE
To elucidate the effect of hSav1 expression on Mst1-mediated apoptosis in HeLa cells.


METHODS
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.


RESULTS
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).


CONCLUSIONS
Our findings indicate that hSav1 is a newly identified protein that interacts with Mst1 and augments Mst1-mediated apoptosis.
Li
Zhao-Ming
ZM
Center of Gastroenterologic Surgery/Cancer Reseach Institute, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Liu
Wei-Cheng
WC
Dong
Shuo
S
Luo
Xue-Lai
XL
Li
Xiao-Lan
XL
Tao
De-Ding
DD
Gong
Jian-Ping
JP
Hu
Jun-Bo
JB
D017209:Apoptosis; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D006367:HeLa Cells; D017228:Hepatocyte Growth Factor / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D010957:Plasmids; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D018797:Cell Cycle Proteins; D011518:Proto-Oncogene Proteins; C415864:SAV1 protein, human; C082332:macrophage stimulating protein; D017228:Hepatocyte Growth Factor