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.

Dao-ming LI; Wei LI; Min Tao; Kai Chen; Fei-ran Gong; Ze-kuan XU; Zheng Chen

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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.

Author: Dao-ming LI ¹ ; Wei LI ; Min TAO ; Kai CHEN ; Fei-ran GONG ; Ze-kuan XU ; Zheng CHEN
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1. Department of Oncology, the First Affiliated Hospital of Soochow University, Suzhou 215006, China.
Publication Type:Journal Article
MeSH: Carcinoma, Hepatocellular; genetics; metabolism; Enhancer Elements, Genetic; Genetic Therapy; Genetic Vectors; Hep G2 Cells; Humans; Liver Neoplasms; genetics; metabolism; Mutation; Promoter Regions, Genetic; Protein Phosphatase 2; genetics; alpha-Fetoproteins; genetics
From: Chinese Journal of Hepatology 2013;21(6):459-463
CountryChina
Language:Chinese
Abstract:
OBJECTIVETo 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.
METHODSThe 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.
RESULTSThe 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.
CONCLUSIONThe AFpg-driven DN-PP2Aca plasmid exhibits selective cytotoxicity against AFP+ hepatoma cells, and may represent a useful gene therapy strategy to treat HCC.