In vitro gene targeting in human hepatoblastoma.
Poor treatment results in advanced hepatoblastoma (HB) made alternative treatment approaches desirable. Gene-directed tumor therapy is increasingly investigated in different malignancies. The aim of this study was to analyze possible alternatives of gene transfer into HB cells and to study therapeutic applications based on different strategies. Liposomal transfection of HB cells was assessed using liver-specific promoters, and adenovirus and Sendai virus transductions were performed in vitro. Transfer efficiencies were measured via flow cytometry determining expression of vector-encoded marker gene green fluorescent protein. Gene silencing of the anti-apoptotic bcl-2 gene in HUH6 cells was performed using lipofection of small interfering RNA (siRNA). Additionally, suicide gene therapy was carried out through a yeast-derived cytosine deaminase (YCD)-combined yeast uracil phosphoribosyltransferase (YUPRT)-based adenovirus-mediated gene transfer, leading to a potent intracellular prodrug transformation of 5-fluorocytosine into 5-fluorouracil. Treatment efficiencies were monitored via MTT viability assay. Highest gene transfer rates (86%) were observed using adenovirus transduction. We furthermore observed a significant therapeutic effect of adenovirus-mediated YCD::YUPRT suicide gene transfer. Liposomal-mediated anti-bcl-2 siRNA transfer led to a significant improvement of cisplatin treatment in HUH6 cells. Liver-specific promoters were found to be strongly active in HUH6 cells (mixed HB-derived), but less active in HepT1 cells (embryonal HB-derived). Liposomal transfection and viral transduction are effective approaches to genetically manipulate HB cells in vitro. For the first time, we demonstrate a positive effect of siRNA gene silencing in this malignancy. Additionally, we successfully investigated a model of adenovirus-based suicide gene therapy in HB cell cultures. Our data strongly encourage further studies assessing these alternative treatment approaches.
Warmann
Steven W
SW
Department of Pediatric Surgery, University of Tübingen, Hoppe-Seyler-Street 3, 72076 Tübingen, Germany. Steven.Warmann@med.uni-tuebingen.de
Armeanu
Sorin
S
Frank
Heike
H
Buck
Heike
H
Graepler
Florian
F
Lemken
Marie-Luise
ML
Heitmann
Heike
H
Seitz
Guido
G
Lauer
Ulrich M
UM
Bitzer
Michael
M
Fuchs
Jörg
J
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics; D000256:Adenoviridae; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D018390:Gene Targeting / Q000379:methods*; D018014:Gene Transfer Techniques; D041722:Genes, Transgenic, Suicide; D005822:Genetic Vectors; D018197:Hepatoblastoma / Q000235:genetics / Q000628:therapy*; D006801:Humans; D008081:Liposomes / Q000627:therapeutic use; D008113:Liver Neoplasms / Q000235:genetics / Q000628:therapy*; D009928:Organ Specificity; D011401:Promoter Regions, Genetic; D029082:Sendai virus
D048868:Adaptor Proteins, Signal Transducing; C486655:BMF protein, human; D008081:Liposomes