Improvement of nonviral p53 gene transfer in human carcinoma cells using glucosylated polyethylenimine derivatives.
Fiche publication
Date publication
mars 2001
Journal
Cancer gene therapy
Auteurs
Membres identifiés du Cancéropôle Est :
Pr DOLIVET Gilles, Pr MERLIN Jean-Louis
Tous les auteurs :
Merlin JL, Dolivet G, Dubessy C, Festor E, Parache RM, Verneuil L, Erbacher P, Behr JP, Guillemin F
Lien Pubmed
Résumé
Polyethylenimine (PEI) derivatives are potent polycationic nonviral vectors for gene transfer. The gene transfer efficiency of glucosylated and galactosylated PEI derivatives was optimized using green fluorescent protein gene as reporter gene in FaDu and PANC3 human carcinoma cell lines. Glucosylated or galactosylated PEI derivatives were found to be slightly less cytotoxic than unsubstituted PEI. Gene transfer efficiency was found to be related to DNA/cell number ratio and optimal gene transfer efficiency was achieved at 4 microg DNA/10(5) cells. PEI-DNA complexes were found to enter cells rapidly and were detected into cytoplasmic vesicles 2 hours post-transfection. Green fluorescent protein gene expression was detected 4-6 hours after transfection and reached maximal value 24 hours post-transfection. The results achieved demonstrated that glucosylated PEI yield higher and longer gene transfer efficiency than unsubstituted PEI. Using glucosylated PEI allowed to achieve significant gene transfer in more than 10% of the total cell population for more than 4 days. These data were then applied to p53 gene transfer in PANC3 cells bearing p53 gene deletion and consequently unable to initiate apoptosis. Using glucosylated PEI, p53 gene transfer was successfully achieved with subsequent recovery of p53 mRNA expression and transient P53 protein expression. P53 protein functionality was further demonstrated because transfected cells underwent apoptosis.
Mots clés
Apoptosis, genetics, Blotting, Western, Breast Neoplasms, genetics, Carcinoma, genetics, Endocytosis, physiology, Female, Formazans, analysis, Gene Expression, Gene Transfer Techniques, Genes, p53, physiology, Genetic Vectors, Glycosylation, Green Fluorescent Proteins, Humans, Indicators and Reagents, Luciferases, analysis, Luminescent Proteins, analysis, Microscopy, Fluorescence, Pancreatic Neoplasms, genetics, Pharyngeal Neoplasms, genetics, Polyethyleneimine, analogs & derivatives, Reverse Transcriptase Polymerase Chain Reaction, Tetrazolium Salts, metabolism, Transfection, Tumor Cells, Cultured
Référence
Cancer Gene Ther.. 2001 Mar;8(3):203-10