Depletion of Bcl-2 by an antisense oligonucleotide induces apoptosis accompanied by oxidation and externalization of phosphatidylserine in NCI-H226 lung carcinoma cells.
Oxidant-induced apoptosis involves oxidation of many different and essential molecules including phospholipids. As a result of this non-specific oxidation, any signaling role of a particular phospholipid-class of molecules is difficult to elucidate. To determine whether preferential oxidation of phosphatidylserine (PS) is an early event in apoptotic signaling related to PS externalization and is independent of direct oxidant exposure, we chose a genetic-based induction of apoptosis. Apoptosis was induced in the lung cancer cell line NCI-H226 by decreasing the amount of Bcl-2 protein expression by preventing the translation of bcl-2 mRNA using an antisense bcl-2 oligonucleotide. Peroxidation of phospholipids was assayed using a fluorescent technique based on metabolic integration of an oxidation-sensitive and fluorescent fatty acid, cis-parinaric acid (PnA), into cellular phospholipids and subsequent HPLC separation of cis-PnA-labeled phospholipids. We found a decrease in Bcl-2 was associated with a selective oxidation of PS in a sub-population of the cells with externalized PS. No significant difference in oxidation of cis-PnA-labeled phospholipids was observed in cells treated with medium alone or a nonsense oligonucleotide. Treatment with either nonsensc or antisense bcl-2 oligonucleotides was not associated with changes in the pattern of individual phospholipid classes as determined by HPTLC. These metabolic and topographical changes in PS arrangement in plasma membrane appear to be early responses to antisense bcl-2 exposure that trigger a PS-dependent apoptotic signaling pathway. This observed externalization of PS may facilitate the 'labeling' of apoptotic cells for recognition by macrophage scavenger receptors and subsequent phagocytic clearance.
Koty
Patrick P
PP
Department of Environmental and Occupational Health, University of Pittsburgh-Graduate School of Public Health, PA, USA.
Tyurina
Yulia Y
YY
Tyurin
Vladimir A
VA
Li
Shang-Xi
SX
Kagan
Valerian E
VE
D017209:Apoptosis / Q000187:drug effects; D002855:Chromatography, Thin Layer; D053938:DNA Fragmentation / Q000187:drug effects; D006801:Humans; D015227:Lipid Peroxidation / Q000187:drug effects; D008168:Lung / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects; D010718:Phosphatidylserines / Q000378:metabolism*; D010743:Phospholipids / Q000032:analysis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D016376:Oligonucleotides, Antisense; D010718:Phosphatidylserines; D010743:Phospholipids; D019253:Proto-Oncogene Proteins c-bcl-2