title: Radioresistant Sf9 insect cells undergo an atypical form of Bax-dependent apoptosis at very high doses of γ-radiation.
abstract: OBJECTIVE
  To investigate the underlying mechanisms of cell-death at extremely high doses of radiation in radioresistant Spodoptera frugiperda-9 (Sf9) insect cells.
  
  
  METHODS
  Morphology, cell proliferation and DNA-fragmentation analysis was performed at 500-2000 Gy. Changes in intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), cardiolipin oxidation and Annexin-V externalization were studied using flow-cytometry. Cytochrome-c release was measured using immunofluorescence microscopy. Inhibitors of apoptosis, i.e., Bongkrekic acid (BKA), Caspase-9 inhibitor (C9i), 5-(4-fluorosulfonylbenzoyl) adenosine hydrochloride (FSBA) and Cyclosporin-A (CsA) were used to dissect apoptotic mechanism at many classical steps. Caspase-3 activity was measured using a caspase-activity assay kit.
  
  
  RESULTS
  A dose-dependent induction of typical apoptosis was observed at extremely high doses, marked by extensive apoptotic body formation. However, certain atypical responses such as cellular hypertrophy and the lack of phosphatidylserine-externalization were observed during the initial hours after radiation. Loss of mitochondrial membrane potential observed at 48 h following a 2000 Gy dose was accompanied by an increase in ROS that caused significant cardiolipin oxidation leading to cytochrome-c release, caspase activation and internucleosomal DNA fragmentation. Inhibitors of B-cell lymphoma-2 (Bcl-2)-associated X protein (Bax)-mediated cytochrome-c release, apoptosome formation and caspase-9 effectively prevented radiation-induced apoptosis, strongly suggesting the role of Bax-dependent cell death mechanism.
  
  
  CONCLUSIONS
  Our study demonstrates that the Sf9 insect cells display good homology with human cells in the mitochondria-dependent events during radiation-induced apoptosis, although doses eliciting similar responses were 50-200 times higher than human cells. Factors upstream to mitochondrial damage remain pertinent for a thorough understanding of this extreme radioresistance displayed by lepidopteran cells.
authors.affiliation: Natural Radiation Response Mechanisms Group, Division of Radiation Biosciences, Institute of Nuclear Medicine and Allied Sciences , Delhi.
authors.forename: Sudhir
authors.initials: S
authors.lastname: Chandna
authors.forename: Shubhankar
authors.initials: S
authors.lastname: Suman
authors.forename: Mauli
authors.initials: M
authors.lastname: Chandna
authors.forename: Akshay
authors.initials: A
authors.lastname: Pandey
authors.forename: Vijaypal
authors.initials: V
authors.lastname: Singh
authors.forename: Ashish
authors.initials: A
authors.lastname: Kumar
authors.forename: Bilikere Srinivasarao
authors.initials: BS
authors.lastname: Dwarakanath
authors.forename: Rakesh Kumar
authors.initials: RK
authors.lastname: Seth
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000737:chemistry; D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000528:radiation effects*; D001865:Bongkrekic Acid / Q000737:chemistry; D002308:Cardiolipins / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D049109:Cell Proliferation / Q000528:radiation effects; D016572:Cyclosporine / Q000737:chemistry; D053938:DNA Fragmentation / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D005720:Gamma Rays; D008564:Membrane Potentials; D051336:Mitochondrial Membranes / Q000378:metabolism; D010100:Oxygen / Q000737:chemistry; D011836:Radiation Tolerance; D017382:Reactive Oxygen Species / Q000378:metabolism; D061987:Sf9 Cells; D018411:Spodoptera; D051028:bcl-2-Associated X Protein / Q000378:metabolism*
chemical_list: D017304:Annexin A5; D002308:Cardiolipins; D017382:Reactive Oxygen Species; D051028:bcl-2-Associated X Protein; D001865:Bongkrekic Acid; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D016572:Cyclosporine; D053148:Caspase 3; D053453:Caspase 9; D000241:Adenosine; D010100:Oxygen