title: 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.
abstract: 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.
authors.affiliation: Department of Hospital Pharmacy, School of Medicine, Kobe University, Chuo-ku, Japan.
authors.forename: F
authors.initials: F
authors.lastname: Komada
authors.forename: K
authors.initials: K
authors.lastname: Nishiguchi
authors.forename: Y
authors.initials: Y
authors.lastname: Tanigawara
authors.forename: M
authors.initials: M
authors.lastname: Ishida
authors.forename: X Y
authors.initials: XY
authors.lastname: Wu
authors.forename: S
authors.initials: S
authors.lastname: Iwakawa
authors.forename: R
authors.initials: R
authors.lastname: Sasada
authors.forename: K
authors.initials: K
authors.lastname: Okumura
mesh_terms: 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
chemical_list: D017077:Culture Media, Conditioned; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D013481:Superoxides; D013482:Superoxide Dismutase