Contaminants within bacterial plasmid preparations trigger apoptosis in liposome transfected OVCAR3, but not in SKOV3 or AZ224 human ovarian cancer cells.
Toxicity associated with plasmid/liposome transfection of eucaryote cells has been attributed to the inherent toxicity of cationic lipid formulations and also to bacterial contaminants of plasmid DNA preparations, such as lipopolysaccharides (LPS). Certain plasmid preparations were observed to trigger apoptosis in DNA/liposome transfected OVCAR3 human epithelial ovarian cancer cells. In contrast, AZ224 and SKOV3 cells were unaffected under the same conditions. Agarose gel electrophoresis with recovery of the plasmid DNA removed the toxic component, but not purification by phenol/chloroform extraction or isopicnic CsCl ultracentrifugation. The toxicity of individual preparations correlated with the concentration of bacterial LPS. However, polymixin B could not neutralise the toxicity and neither could the effect be reproduced by the addition of bacterial LPS to non-toxic plasmid preparations. Surprisingly, the conditioned medium of OVCAR3 cells undergoing apoptosis was found to kill non-transfected OVCAR3 cells but not AZ224 or SKOV3 cells. This observation illustrates the possibility that unpredictable contaminants of bacterial plasmid preparations are able to cause cell death in the context of plasmid/liposome transfection in a cell-type specific way. It emphasizes the importance of achieving maximal plasmid DNA purity when performing DNA transfection experiments that focus on cell survival.
Neyns
B
B
Laboratory of Medical Oncology and Department of Medical Oncology, Oncologisch Centrum, Akademisch Ziekenhuis Vrije Universiteit Brussel, Belgium.
De Rijcke
M
M
Vermeij
J
J
Teugels
E
E
Zeinoun
Z
Z
De Grève
J
J
D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D016923:Cell Death / Q000187:drug effects / Q000235:genetics; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004352:Drug Resistance, Microbial / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000737:chemistry / Q000187:drug effects / Q000235:genetics*; D005260:Female; D015870:Gene Expression / Q000502:physiology; D049452:Green Fluorescent Proteins; D006801:Humans; D008070:Lipopolysaccharides / Q000008:administration & dosage / Q000378:metabolism; D008081:Liposomes / Q000008:administration & dosage; D008164:Luminescent Proteins / Q000235:genetics; D009355:Neomycin / Q000494:pharmacology; D010051:Ovarian Neoplasms / Q000235:genetics / Q000473:pathology*; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
D017077:Culture Media, Conditioned; D008070:Lipopolysaccharides; D008081:Liposomes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D009355:Neomycin