Liposome-mediated transfection of fetal lung epithelial cells: DNA degradation and enhanced superoxide toxicity.
Cationic liposomes, 1:1 (mol/mol) 1,2-dioleoyldimethylammonium chloride-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, were used to transfect primary cultures of distal rat fetal lung epithelial cells with pCMV4-based plasmids. A DNA-to-lipid ratio of 1:10 to 1:15 (wt/wt) optimized DNA uptake over a 24-h exposure. At a fixed DNA-to-lipid ratio of 1:15, chloramphenicol acetyltransferase (CAT) reporter gene expression declined at lipid concentrations > 2.5 nmol/cm2 cell surface area, whereas DNA uptake remained concentration dependent. CAT expression peaked 48 h after removal of the liposome-DNA complex, declining thereafter. Reporter gene expression was increased, and supercoiled cDNA degradation was reduced by the addition of 0.2 mM nicotinamide and 10 microM chloroquine. Rat fetal lung epithelial cells transfected with two different expression cassettes had an increased susceptibility to superoxide-mediated cytotoxicity. This could be attributed to a nonspecific delivery of exogenous DNA or some other copurified factor. The DNA-dependent increase in superoxide-mediated cytotoxicity, but not basal levels of cytotoxicity, was inhibited by the addition of 0.2 mM nicotinamide and 10 microM chloroquine.
Tanswell
A K
AK
The Medical Research Council Group in Lung Development and Lung Biology Programme, Hospital for Sick Children Research Institute, University of Toronto, Toronto, Ontario, Canada.
Staub
O
O
Iles
R
R
Belcastro
R
R
Cabacungan
J
J
Sedlackova
L
L
Steer
B
B
Wen
Y
Y
Hu
J
J
O'Brodovich
H
H
D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics*; D003587:Cytomegalovirus; D004247:DNA / Q000378:metabolism*; D004337:Drug Carriers; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D005333:Fetus; D017930:Genes, Reporter; D005822:Genetic Vectors; D008081:Liposomes; D008168:Lung / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D010714:Phosphatidylethanolamines; D010957:Plasmids; D000644:Quaternary Ammonium Compounds; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D013481:Superoxides / Q000633:toxicity; D014162:Transfection / Q000379:methods*
D004337:Drug Carriers; D008081:Liposomes; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; D011994:Recombinant Proteins; D013481:Superoxides; C015831:dimethyldioctadecylammonium; C020888:1,2-dielaidoylphosphatidylethanolamine; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase