The mitochondrial localization of tumor suppressor PTEN promotes apoptosis in A431 cells

Abstract:

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 CoxVⅡ-PTEN-EYFPN1 as a template, CoxⅦ-PTEN was cloned into the shuttle vector pAdTrack-CMV with the restriction endonuclease sites XhoⅠand XbaⅠ. The shuttle plasmid was linearize with PmeⅠand 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 DH5α for propagation. To package the adenoviruses, recombinant plasmid candidate was linearize using PacⅠ 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 10⁷pfu/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.