[Liposomal DNA transfection of human sarcoma cells with p53 alterations].
An vital assay allows to optimize liposomal transfection for human tumor cells via FACS. Various cationic lipids were tested to analyse the reporter gene expression (green fluorescent protein, GFP) in different soft tissue sarcoma (STS) cells with known genetic alterations. Furthermore, the cellular uptake of fluorescence-labeled oligodeoxynucleotides (ODN's) was determined. The results obtained with two self-established sarcoma cell lines (LMS6-93, US8-93) were compared with ATCC sarcoma cell lines (Saos-2, A-204, RD) and fibroblast cells. We found maximal 37% cells expressing GFP 24 h post-transfection. All mesenchymal (tumor) cells but not fibroblast cells could be transfected in a cell-specific and lipid-dependent manner. In kinetic studies highest transfection rates were determined between 24 and 48 h, whereas the GFP expression is downregulated after 72 h. Furthermore, we found transfectability is p53 mutation-independent and a relative low toxicity of the new lipids (Lipotaxi and Clonfectin) in comparison to other lipids (Lipofectin, Lipofectamine). By a cell sorting system sarcoma cell lines expressing the reporter gene could be enriched up to 84% of the living cell population. Labeled ODN's were taken up more efficiently (> 90%) when they were mixed with lipids before, but ODN's alone were incorporated into sarcoma cells only in a low percentage (< 10%) and concentration. STS cell cultures showed also a relative high ODN uptake compared with cell lines. We propose the liposomal transfection strategy as an efficient method which can be applied to adherent-growing tumor cells. The method allows simultaneously to study transfection rates, apoptosis and cell cycle alterations in vitro. Furthermore, in future, extension on ex vivo and in vivo transgene expression (xenotransplanted sarcomas) will be evaluated.
Meye
A
A
Institut für Pathologie, Universität Halle-Wittenberg.
Würl
P
P
Löhn
M
M
Lasch
J
J
Bache
M
M
Hinze
R
R
Holzhausen
H J
HJ
Schmidt
H
H
Rath
F W
FW
Taubert
H
H
D015972:Gene Expression Regulation, Neoplastic; D016158:Genes, p53; D049452:Green Fluorescent Proteins; D006801:Humans; D008081:Liposomes; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012509:Sarcoma; D014162:Transfection / Q000379:methods*; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis
D008081:Liposomes; D008164:Luminescent Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D049452:Green Fluorescent Proteins