Protective effect of transfection with secretable superoxide dismutase (SOD) (a signal sequence-SOD fusion protein coding cDNA) expression vector on superoxide anion-induced cytotoxicity in vitro.
For ex vivo gene therapy, superoxide dismutase (SOD) must be secreted into the extracellular space and delivered to damaged cells. Recombinant DNA technique can be used to produce a secretory protein that is fused to a non-secretory protein and a signal peptide of another secretory protein gene. We constructed a secretable SOD eukaryotic expression vector which expresses human SOD cDNA by fusing it to the signal peptide DNA sequence of the human interleukin-2 (IL-2) gene. The ILSOD cDNA constructed by PCR-based gene expression was ligated into the multicloning site of the pRc/CMV plasmid (pRc/CMV-ILSOD). Rat lung epithelial like cells (L2 cells) were transfected with pRc/CMV-ILSOD by lipofection. The extracellular SOD activity of ILSOD-L2 cells (transfected cells with pRc/CMV-ILSOD) was 3 times as high as that of host cells. We used the xanthin (X)/xanthin oxidase (XO) system to produce superoxide anions at the extracellular space. We initially investigated the direct cytotoxicity of superoxide anions upon cells. Host and ILSOD-L2 cells were killed by using X/XO, although the sensitivity of the ILSOD-L2 cells to X/XO induced cytotoxicity was significantly decreased compared with that of host cells. The production of lipid peroxidated substances in the host in the presence of X/XO increased to about twice the control (absence of X/XO) level. However, that of ILSOD-L2 cells did not change in the presence of X/XO. Therefore, ILSOD-L2 cells were resistant to X/XO induced lipid peroxidation. These findings indicated that ILSOD gene transfection protected against direct oxidant stress by X/XO. We then investigated the effect of extracellular SOD secreted from ILSOD-L2 cells on extracellular superoxide anion induced cytotoxicity in normal cells. The conditioned media of host cells had no significant effect upon X/XO induced cytotoxicity. However, the conditioned media of ILSOD-L2 cells protected against X/XO induced cytotoxicity. Furthermore, the conditioned medium of ILSOD-L2 cells was more effective than that of host cells against the production of lipid peroxidated substances by normal cells under conditions of oxidative stress. These results indicated that non-secretable protein could be delivered to target cells by means of DNA engineering. This strategy could thus provide an ex vivo means of applying gene therapy using non-secretable proteins.
Komada
F
F
Department of Hospital Pharmacy, School of Medicine, Kobe University, Chuo-ku, Japan.
Nishiguchi
K
K
Tanigawara
Y
Y
Ishida
M
M
Wu
X Y
XY
Iwakawa
S
S
Sasada
R
R
Okumura
K
K
D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D018076:DNA, Complementary / Q000235:genetics; D004847:Epithelial Cells; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D005110:Extracellular Space / Q000201:enzymology; D015870:Gene Expression; D006801:Humans; D007424:Intracellular Fluid / Q000201:enzymology; D015227:Lipid Peroxidation / Q000187:drug effects; D008168:Lung / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010084:Oxidation-Reduction; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D013482:Superoxide Dismutase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D013481:Superoxides / Q000633:toxicity*; D014162:Transfection
D017077:Culture Media, Conditioned; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D013481:Superoxides; D013482:Superoxide Dismutase