title: Differential mechanisms of conjunctival cell death induction by ultraviolet irradiation and benzalkonium chloride.
abstract: OBJECTIVE
  To determine the molecular mechanisms of conjunctival cell death on exposure to the quaternary ammonium preservative benzalkonium chloride (BAC) and ultraviolet (UV) irradiation.
  
  
  METHODS
  Chang conjunctival cells, either wild-type or stably transfected with various constructs encoding antiapoptotic molecules or transiently transfected with siRNA targeting the beclin-1 gene, were exposed to BAC or UV radiation Cell death was analyzed morphologically with fluorescence and electron microscopy, and molecular mechanisms of death were studied by using immunofluorescence, cell fractionation, caspase substrates, and immunoblot analysis, with or without immunoprecipitation. The main results were controlled in IOBA-NHC cells.
  
  
  RESULTS
  Both agents induced cytochrome c release from the mitochondria, caspase activation, and nuclear chromatin condensation, suggesting caspase-dependent apoptosis. These events are prevented by stable expression of Bcl-2 protein. Both agents also induced a redistribution of Fas in plasma membrane rafts and the Fas-ligand-independent formation of a death-inducing complex leading to caspase-8 activation. Stable expression of either a dominant negative construct of Fas-associated death domain (FADD) or the long or short isoform of FADD-like interleukin-1-beta-converting enzyme inhibitory protein (FLIP) inhibited caspase-8 activation in response to both UV radiation and BAC. However, these proteins, as well as permeant peptides and baculovirus p35 caspase-inhibitors, delayed more efficiently the UV irradiation-induced than the BAC-induced nuclear chromatin condensation. BAC specifically activated a caspase-independent pathway by inducing the mitochondrial release of apoptosis-inducing factor. BAC-treated cells contain autophagosomes/autolysosomes, a characteristic feature of autophagy, and siRNA-mediated downregulation of the beclin-1 gene, whose product is crucial for autophagy, increases BAC toxicity.
  
  
  CONCLUSIONS
  UV irradiation induces typical, caspase-dependent cell death, whereas death induced by BAC associates features of caspase-dependent and -independent apoptosis counteracted by an autophagic process.
authors.affiliation: INSERM (Institut National de la Santé et de la Recherche Médicale) U517, Faculty of Medicine, Dijon, France.
authors.forename: Nelly
authors.initials: N
authors.lastname: Buron
authors.forename: Olivier
authors.initials: O
authors.lastname: Micheau
authors.forename: Séverine
authors.initials: S
authors.lastname: Cathelin
authors.forename: Pierre-Olivier
authors.initials: PO
authors.lastname: Lafontaine
authors.forename: Catherine
authors.initials: C
authors.lastname: Creuzot-Garcher
authors.forename: Eric
authors.initials: E
authors.lastname: Solary
mesh_terms: D017209:Apoptosis / Q000187:drug effects* / Q000528:radiation effects*; D051033:Apoptosis Inducing Factor / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects; D001548:Benzalkonium Compounds / Q000494:pharmacology*; D020169:Caspases / Q000378:metabolism; D018929:Cell Culture Techniques; D003228:Conjunctiva / Q000187:drug effects* / Q000528:radiation effects*; D045304:Cytochromes c / Q000378:metabolism; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000187:drug effects / Q000528:radiation effects; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D015151:Immunoblotting; D008928:Mitochondria / Q000378:metabolism; D011310:Preservatives, Pharmaceutical / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014466:Ultraviolet Rays; D019014:fas Receptor / Q000378:metabolism
chemical_list: D051033:Apoptosis Inducing Factor; D001548:Benzalkonium Compounds; D011310:Preservatives, Pharmaceutical; D019253:Proto-Oncogene Proteins c-bcl-2; D034741:RNA, Small Interfering; D019014:fas Receptor; D045304:Cytochromes c; D020169:Caspases