[The mitochondrial localization of tumor suppressor PTEN promotes apoptosis in A431 cells].
To study the function of mitochondrial PTEN in mediation of cellular apoptosis, the adenoviral recombinant of Mito-PTEN, which contains CoxVII (subunit VII of Cytochrome C Oxidase) gene in N-terminus, were generated. Using CoxV II-PTEN-EYFPN1 as a template, Cox VII-PTEN was cloned into the shuttle vector pAdTrack-CMV with the restriction endonuclease sites Xho I and Xba I . The shuttle plasmid was linearize with Pme I and co-transformed with adenoviral backbone vector pAdeasy-1 into E. coli BJ5183. Following selection and identification, the positive recombinant plasmids were transformed into E. coli Dalpha for propagation. To package the adenoviruses, recombinant plasmid candidate was linearize using Pac I and transfected into HEK-293A cells with Lipofectamine 2000. Through freeze-thaw-vortex cycles, recombinant viral particles were collected and harvested, and utilized to infect 293A cells for further amplification. The method of TCID50 was employed to determine virus titers. With green fluorescent protein (GFP) as marker, the efficiency of transfection and infection was monitored by fluorescence microscopy, and the apoptosis of A431 cells after infection of Mito-PTEN-Ad was analyzed by flow cytometry. Adenoviral recombinant of Mito-PTEN was packaged successfully with the TCID50 as 10 pfu/mL and the expressed protein was detected by western blot. In addition, it has been demonstrated that Mito-PTEN promoted apoptosis of A431 cells. Take together, the successful generation of adenoviral recombinant of Mito-PTEN, which could induce apoptosis in A431 cells, sets up a basis for further functional studies of mitochondrial PTEN and provides us a potential tool for cancer treatment in future.
Yue
Xiao-Jing
XJ
Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. yuexiaojing_mic@yahoo.com.cn
Song
Wei-Dong
WD
Jiang
Xue-Jun
XJ
D000256:Adenoviridae / Q000235:genetics; D017209:Apoptosis; D045744:Cell Line, Tumor; D003576:Electron Transport Complex IV / Q000235:genetics; D006801:Humans; D008928:Mitochondria / Q000502:physiology*; D051059:PTEN Phosphohydrolase / Q000502:physiology*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D025521:Tumor Suppressor Proteins / Q000502:physiology*
D025521:Tumor Suppressor Proteins; D003576:Electron Transport Complex IV; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human