title: In vivo treatment of HCV core-positive HepG2 cells with the transfer             of recombinant caspase-3 using a 2'-5' OAS promoter.
abstract: Hepatitis C virus (HCV) is one of the most common pathogens causing liver-related morbidity and mortality, which affect 170 million individuals worldwide. There is no vaccine available, and current therapy is only partially effective. In a previous study, we constructed a recombinant caspase-3 expression vector under the 2'-5'-oligoadenylate synthetase gene (OAS) promoter (pGL3-OAS-re-caspase-3) and demonstrated that it is an effective gene therapy for HCV core-positive liver cells in vitro. In the present study, the human hepatoma cell line HepG2 was transfected with the pcDNA3.1-HCV-core-EGFP plasmid and selected by G418. Expression of HCV core protein was confirmed by RT-PCR and immunocytochemistry. Both HepG2-expressing HCV core protein and parental HepG2 cells were inoculated subcutaneously into BALB/c mice, respectively. Tumor-bearing mice were treated with an intratumoral injection of pGL3-OAS-re-caspase-3. The mice were sacrificed after 48 h. The correlation between HCV core and caspase-3 expression in tumor tissues was analyzed by immunohistochemical staining and double-label immunofluorescence staining. The subcutaneous hepatoma in vivo mouse models stably expressing HCV core protein and co-expressing HCV core protein and pGL3-OAS-re-caspase-3 were established. Double-label immunofluorescence staining showed that the percentage of co-expression of both HCV core and caspase-3 was 76 ± 6% in the group treated with pGL3-OAS-re-caspase-3. There was a significant increase in the number of apoptotic cells in the group treated with the pGL3-OAS-re-caspase-3 system by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and transmission electron microscopy. The results suggest that the pGL3-OAS-re-caspase-3 construct can effectively induce apoptosis in HCV core-positive hepatocytes in vivo. The results presented strongly suggest that the transfer of pGL3-OAS-re-caspase-3 is an effective and promising gene therapy strategy for HCV infection.
authors.affiliation: Department of Pathology, Xiangya School of Medicine, Central South University, Changsha, Hunan 410013, PR China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Zi
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.forename: Peter S
authors.initials: PS
authors.lastname: Wiegmann
authors.forename: Junming
authors.initials: J
authors.lastname: Luo
authors.forename: Deyun
authors.initials: D
authors.lastname: Feng
mesh_terms: D015088:2',5'-Oligoadenylate Synthetase / Q000235:genetics*; D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000628:therapy; D053148:Caspase 3 / Q000235:genetics* / Q000378:metabolism; D004195:Disease Models, Animal; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000627:therapeutic use; D056945:Hep G2 Cells; D006526:Hepatitis C / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms / Q000628:therapy; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014183:Transplantation, Heterologous; D014758:Viral Core Proteins / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D014758:Viral Core Proteins; C069130:nucleocapsid protein, Hepatitis C virus; D015088:2',5'-Oligoadenylate Synthetase; D053148:Caspase 3