title: Gene transfer in hepatocarcinoma cell lines: in vitro optimization of a virus-free system.
abstract: Many approaches exist for hepatic gene delivery, including viral vectors and non-viral vectors. In this study, we tested a panel of liposomes to transfer pAGO, a plasmid containing one copy of herpes simplex virus (HSVtk) gene, and pYED11, a plasmid containing two copies of the HSVtk gene, into a murine hepatocarcinoma cell line (Hepa 1-6) and a human hepatocarcinoma cell line (Hep-G2). The efficiency of gene delivery and expression was characterized by beta-galactosidase staining, flow cytometric analysis and quantitative lacZ activity. Different combinations of liposomes and DNA and the ratio of the concentration of liposome to DNA were tested. The efficient transfer was shown with DOTAP followed by transfectam and lipofectamine. Under these conditions, we tested the cytotoxicity of ganciclovir (GCV) exposure on Hepa 1-6 and Hep-G2 transfected separately with liposome-pAGO and liposome-pYED11 complexes. This study demonstrates the in vitro efficacy of each liposome tested to transduce the HSVtk gene into hepatocarcinoma cell lines. The transfer of two copies of the HSVtk gene rendered cells 1.5 times more sensitive to GCV than cells transduced by pAGO as compared to controls. This was achieved most efficiently by the DOTAP-pYED11 complex. Thus, pYED11 may be considered as an alternative to pAGO as a gene transfer vector.
authors.affiliation: Laboratoire du Service d'Oncologie Médicale, Hôpital de la Pitié-Salpétrière, Paris France.
authors.forename: A M
authors.initials: AM
authors.lastname: Ghoumari
authors.forename: O
authors.initials: O
authors.lastname: Rixe
authors.forename: S V
authors.initials: SV
authors.lastname: Yarovoi
authors.forename: A
authors.initials: A
authors.lastname: Zerrouqi
authors.forename: R
authors.initials: R
authors.lastname: Mouawad
authors.forename: T
authors.initials: T
authors.lastname: Poynard
authors.forename: P
authors.initials: P
authors.lastname: Opolon
authors.forename: D
authors.initials: D
authors.lastname: Khayat
authors.forename: C
authors.initials: C
authors.lastname: Soubrane
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000633:toxicity*; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D015774:Ganciclovir / Q000633:toxicity*; D006801:Humans; D008081:Liposomes; D008113:Liver Neoplasms; D008114:Liver Neoplasms, Experimental; D051379:Mice; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics*; D013937:Thymidine Kinase / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection / Q000379:methods*; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000096:biosynthesis
chemical_list: D000998:Antiviral Agents; D008081:Liposomes; D011994:Recombinant Proteins; D013937:Thymidine Kinase; D001616:beta-Galactosidase; D015774:Ganciclovir