[Establishment of Chinese hamster ovary cell lines with reduced expression of glutathione reductase after antisense-oriented gene transfection and assessment of the sensitivity to oxidant injury].
Glutathione reductase (GR) protects tissues from oxidant injury by catalysing the reduction of glutathione disulfide (GSSG) to glutathione (GSH). In order to study the effect of GR in protecting cells from oxidant injury, we generated Chinese hamster ovary (CHO) cell lines stably transformed after antisense-oriented gene transfection. The coding region of the human GR was cloned using revere transcription PCR method and selected by transient expression study in mammalian cells. A clone HGR135 showed overexpression of GR in CHO cells and was proved to have no base substitution. This clone, then, was ligated into MEP4 expression vector in an antisense orientation to the human metallothionein promoter and transfected to CHO cells with polybrene. Among 12 cell lines isolated, G17 showed to have the least GR activity (48% of the control), while another four were mildly GR deficient. Southern hybridization of genomic DNA digests and transformation experiment on E. coli revealed that the promoter-antisense coding region component was integrated. Northern hybridization detected reduced amount of GR transcript but no antisense message. Baseline cellular GSH concentrations were lower in G17 than in control (25.7 +/- 2.5 vs. 36.1 +/- 1.9 nmole/mg protein, P < 0.05), while cellular GSSG concentrations were higher (0.61 +/- 0.19 vs. 0.39 +/- 0.09 nmole/mg protein, P < 0.05). After four hours of treatment of G17 and control cells with increasing doses (1 to 10 mM) of t-butylhydroperoxide (t-BuOOH), cellular GSH concentrations in G17 decreased with an elevation of GSSG concentration at 1 mM followed by no further increase at higher t-BuOOH concentration, while GSSG concentrations increased in the control cells without reduction of GSH concentrations at 1-5 mM t-BuOOH treatment. The concentrations of GSH were lower in G17 than in controls at all doses of t-BuOOH. Four hours of exposure to 10 mM t-BuOOH resulted in greater LDH release in G17 than in control (57.3 +/- 4.7 vs. 32.1 +/- 6.5%, P < 0.05). Similarly, G17 cells released more of their LDH to the media than did CHO cells in response to exposure to 95% O2 for 72 hours (19.3 +/- 5.9 vs. 11.9 +/- 5.4%, P < 0.05). The partial GR deficiency in G17 cells impairs their ability to recycle GSSG and this deficiency offers the best explanation for the increased sensitivity of these cells to injury by t-BuOOH or hyperoxia.
Tonoki
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Department of Pediatrics, Hokkaido University School of Medicine, Sapporo, Japan.
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells / Q000201:enzymology; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D005822:Genetic Vectors; D005980:Glutathione Reductase / Q000235:genetics* / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D016372:RNA, Antisense; D017382:Reactive Oxygen Species / Q000633:toxicity*; D014162:Transfection
D016372:RNA, Antisense; D017382:Reactive Oxygen Species; D005980:Glutathione Reductase