title: [Gene therapy using a dominant negative form of the protein phosphatase 2A catalytic subunit a driven by a hepatoma tissue-specific promoter achieves effective growth inhibition of hepatoma cells].
abstract: OBJECTIVE
  To generate a gene delivery plasmid carrying the dominant negative form of the protein phosphatase 2A catalytic subunit a (DN-PP2Aca) driven by a hepatocellular carcinoma (HCC) tissue-specific promoter and investigate its ability to inhibit growth of cultured hepatoma cells.
  
  
  METHODS
  The gene delivery plasmid was constructed by PCR-amplifying DN-PP2Aca from wild-type PP2Aca using site-directed mutagenesis and then ligating the sequence-verified amplicon downstream of an alpha-fetoprotein enhancer and phosphoglycerate kinase promoter (AFpg) in the luciferase reporter vector pGL3-Basic. Following transfection into two AFP+ hepatoma cell lines (HepG2 and HepG3) and two AFP- hepatoma cell lines (SK-HEP-1 and L02), the transcriptional activity of the AFpg-driven DN-PP2Aca plasmid was tested using luciferase reporter gene assay and western blotting. The effect on cell growth was tested using MTT assay. Between group differences were assessed by t-test.
  
  
  RESULTS
  The AFpg-driven DN-PP2Aca plasmid showed high transcriptional activity and protein expression in both HepG2 and Hep3B cells. At 72 h after transfection, the proliferation capacities were repressed by 42.65%+/-3.99% (P = 0.0002) and 39.87%+/-3.91% (P = 0.0002) in AFP+ HepG2 and Hep3B cells, respectively (vs. untransfected). In contrast, the plasmid was transcriptionally inactive in and had no effect on proliferation of AFP- cells.
  
  
  CONCLUSIONS
  The AFpg-driven DN-PP2Aca plasmid exhibits selective cytotoxicity against AFP+ hepatoma cells, and may represent a useful gene therapy strategy to treat HCC.
authors.affiliation: Department of Oncology, the First Affiliated Hospital of Soochow University, Suzhou 215006, China.
authors.forename: Dao-ming
authors.initials: DM
authors.lastname: Li
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.forename: Min
authors.initials: M
authors.lastname: Tao
authors.forename: Kai
authors.initials: K
authors.lastname: Chen
authors.forename: Fei-ran
authors.initials: FR
authors.lastname: Gong
authors.forename: Ze-kuan
authors.initials: ZK
authors.lastname: Xu
authors.forename: Zheng
authors.initials: Z
authors.lastname: Chen
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D015316:Genetic Therapy; D005822:Genetic Vectors; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D011401:Promoter Regions, Genetic; D054648:Protein Phosphatase 2 / Q000235:genetics*; D000509:alpha-Fetoproteins / Q000235:genetics
chemical_list: D000509:alpha-Fetoproteins; D054648:Protein Phosphatase 2