An antisense oligonucleotide to 1-cys peroxiredoxin causes lipid peroxidation and apoptosis in lung epithelial cells.
1-cys peroxiredoxin (1-cysPrx), a member of the peroxiredoxin superfamily, reduces phospholipid hydroperoxides as well as organic peroxides and H(2)O(2). To determine the physiological function(s) of 1-cysPrx, we have used an antisense strategy to suppress endogenous 1-cysPrx in L2 cells, a rat lung epithelial cell line. A 25-base antisense morpholino oligonucleotide was designed to bind a complementary sequence overlapping the translational start site (-18 to +7) in the rat 1-cysPrx mRNA, blocking protein synthesis. Treatment with an antisense oligonucleotide for 48 h resulted in approximately 60% suppression of the 1-cysPrx protein content as measured by immunoblot analysis and an approximately 44% decrease of glutathione peroxidase activity as compared with random oligonucleotide treated and control (vehicle only) cells. Accumulation of phosphatidylcholine hydroperoxide in plasma membranes was demonstrated by high pressure liquid chromatography assay for conjugated dienes (260 pmol/10(6) cells for antisense versus 70 pmol/10(6) cells for random oligonucleotide and control cells) and by fluorescence of diphenyl-1-pyrenylphosphine, a probe for lipid peroxidation. The percentage of cells showing positive staining for annexin V and propidium iodide after antisense treatment was 40% at 28 h and 80% at 48 h. TdT-mediated dUTP nick end labeling assay at 48 h indicated DNA fragmentation in antisense-treated cells that was blocked by prior infection with adenovirus encoding 1-cysPrx or by pretreatment with a vitamin E analogue. The results indicate that 1-cysPrx can function in the intact cell as an antioxidant enzyme to reduce the accumulation of phospholipid hydroperoxides and prevent apoptotic cell death.
Pak
Jhang Ho
JH
Institute for Environmental Medicine, University of Pennsylvania Medical Center, 3620 Hamilton Walk, Philadelphia, PA 19104, USA.
Manevich
Yefim
Y
Kim
Han Suk
HS
Feinstein
Sheldon I
SI
Fisher
Aron B
AB
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017304:Annexin A5 / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D053938:DNA Fragmentation; D004847:Epithelial Cells / Q000473:pathology*; D016650:Fluorescein-5-isothiocyanate / Q000494:pharmacology; D005979:Glutathione Peroxidase / Q000378:metabolism; D015151:Immunoblotting; D020287:In Situ Nick-End Labeling; D015227:Lipid Peroxidation; D008168:Lung / Q000166:cytology*; D008856:Microscopy, Fluorescence; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D009943:Organophosphorus Compounds / Q000378:metabolism; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics*; D054464:Peroxiredoxins; D010713:Phosphatidylcholines / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D011721:Pyrenes / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D013997:Time Factors
D017304:Annexin A5; D000975:Antioxidants; D016376:Oligonucleotides, Antisense; D009943:Organophosphorus Compounds; D010713:Phosphatidylcholines; D010743:Phospholipids; D011721:Pyrenes; D012333:RNA, Messenger; C057590:phosphatidylcholine hydroperoxide; C066322:diphenyl-1-pyrenylphosphine; D010544:Peroxidases; D054464:Peroxiredoxins; D005979:Glutathione Peroxidase; D016650:Fluorescein-5-isothiocyanate; D003545:Cysteine