Effect of transfection with superoxide dismutase expression plasmid on superoxide anion induced cytotoxicity in cultured rat lung cells.
Human Cu, Zn-superoxide dismutase (hSOD) cDNA was inserted into a eukaryotic expression plasmid (pRc/CMV) under the control of a cytomegalovirus promoter. The hSOD expression plasmid (pRc/CMV-SOD) was transfected into L2 cells by means of lipofection. The integration of the hSOD gene in genomic DNAs in the cells transfected with pRc/CMV-SOD plasmid was examined by Southern blotting using hSOD cDNA as the probe. However, Southern blots of host cells (without transfection) and CMV cells (pRc/CMV plasmid transfection) indicated no hybridization of hSOD cDNA. Western blots indicated that hSOD was expressed in CMV-SOD cells. The SOD activity in CMV-SOD cells was about twice that in host and CMV cells. Furthermore, this SOD activity in CMV-SOD cells was enhanced for 60 d after the selection of cell clones. After exposure to paraquat and catalase, about 90% of the CMV-SOD cells survived compared with the untreated controls, whereas about 60% of the host cells survived. The production of lipid peroxidation in host cells increased significantly after exposure to both paraquat and catalase, whereas that in CMV-SOD cells did not change. The correlation between the surviving cells and lipid peroxidation was inverse. These results indicated that transfection with the SOD gene was effective against superoxide anion induced cytotoxicity.
Komada
F
F
Department of Hospital Pharmacy, Kobe University, Japan.
Nishiguchi
K
K
Tanigawara
Y
Y
Akamatsu
T
T
Wu
X Y
XY
Iwakawa
S
S
Okumura
K
K
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D015316:Genetic Therapy; D006801:Humans; D008168:Lung / Q000166:cytology; D008969:Molecular Sequence Data; D010269:Paraquat / Q000633:toxicity; D010957:Plasmids; D051381:Rats; D013482:Superoxide Dismutase / Q000235:genetics* / Q000378:metabolism; D013481:Superoxides / Q000633:toxicity*; D014162:Transfection
D013481:Superoxides; D013482:Superoxide Dismutase; D010269:Paraquat