Optimizing vector application for gene transfer into human hepatoblastoma cells.
Gene targeting is currently of distinct interest as an innovative additive treatment option in various malignancies. Its role in pediatric liver tumors has not yet been evaluated thoroughly. For the first time the authors systematically analyzed both lipid-based transfection as well as transduction with adenovirus vectors (Ad) and Sendai virus vectors (SeVV) in order to optimize gene transfer into hepatoblastoma (HB) cells. Two HB cell lines were infected with Ad or SeVV coding for green fluorescent protein (Ad-GFP, SeVV-GFP); transduction efficiencies and apoptosis were assessed using flow cytometry. Furthermore, lipofection of HB cell lines with plasmid-constructs comprising liver-specific promoters was performed using Lipofectamine 2000 and FuGENE 6; lipofection efficiency was monitored by flow cytometry, microscopy, and luciferase activity. The Ad-GFP showed higher transduction rates (61-86%) than the SeVV-GFP (4-24%) depending on the HB cell line used. Infections with first generation SeVV vectors (SeVV-GFP) led to increased target cell apoptosis (7-43%) compared to Ad-GFP (4-16%). The Lipofectamine 2000 revealed a higher transfection efficiency than the FuGENE 6 for both HB cell lines tested. The liver-specific promoters were found to be differently active in the HB cell lines. This study delineates recombinant adenovirus vectors as a promising tool for gene transduction in the HB cells. Furthermore, enhanced activity of the liver-specific promoters in HUH6 cells compared to HepT1 cells supports the observation of varying biological behavior in histologically differing HB tissues.
Warmann
Steven W
SW
Department of Pediatric Surgery, University of Tübingen, Hoppe-Seyler-Str.3, 72076, Tübingen, Germany. Steven.Warmann@med.uni-tuebingen.de
Armeanu
Sorin
S
Heitmann
Heike
H
Ruck
Peter
P
Seitz
Guido
G
Wessels
Johannes T
JT
Lemken
Marie-Luise
ML
Lauer
Ulrich M
UM
Fuchs
Jörg
J
Bitzer
Michael
M
D000256:Adenoviridae; D045744:Cell Line, Tumor; D004337:Drug Carriers; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D018197:Hepatoblastoma / Q000628:therapy*; D006801:Humans; D008055:Lipids; D008113:Liver Neoplasms / Q000628:therapy*; D011401:Promoter Regions, Genetic; D029082:Sendai virus; D014162:Transfection / Q000379:methods*
D004337:Drug Carriers; C411955:FuGene; D008055:Lipids; C086724:Lipofectamine; D049452:Green Fluorescent Proteins