title: Increased oxidant-induced apoptosis in cultured nondividing human retinal pigment epithelial cells.
abstract: OBJECTIVE
  To determine whether long-term cultured nondividing human retinal pigment epithelial (hRPE) cells are sensitive to oxidant-induced apoptosis and whether the Fas pathway is involved in the process.
  
  
  METHODS
  Confluent hRPE cells were maintained for 2 to 3 months in the basal medium (DMEM containing 2% fetal bovine serum) with one medium change per week. DNA synthesis was measured by incorporation of bromodeoxyuridine (BrdU) and the cell cycle was analyzed by flow cytometry. Intracellular glutathione (GSH) and glutathione disulfide (GSSG) were measured by HPLC. Apoptosis was triggered with the oxidant tert-butylhydroperoxide (tBH), recombinant soluble Fas ligand (sFasL), or agonistic anti-Fas antibody (CH-11). Cell viability was assessed by tetrazolium salt (WST-1) assay, and apoptosis was determined by measuring DNA cleavage or phosphatidylserine exposure. FasL and Fas proteins were detected by flow cytometry and Western blot. FasL and Fas transcripts were analyzed by RT-PCR.
  
  
  RESULTS
  After incubation in basal medium for more than 2 months, hRPE cells were largely nondividing and accumulated autofluorescent granules identified by electron microscopy to be lysosomes. Compared with proliferating hRPE cells, the nondividing cells had lower intracellular GSH, GSSG, and GSH/GSSG and a more oxidized redox potential (E(h)). Downregulation of Fas but upregulation of FasL was observed in these cells. The nondividing hRPE cells appeared more susceptible to tBH-induced apoptosis. Similar to proliferating hRPE cells, the apoptosis induced by tBH was preceded by induction of FasL, and antioxidants inhibited both FasL increase and apoptosis. Apoptosis was also inhibited with the antagonistic anti-Fas antibody ZB4. However, the nondividing hRPE cells had decreased sensitivity to apoptosis triggered by sFasL or CH-11.
  
  
  CONCLUSIONS
  Long-term hRPE culture created cells that were nondividing and accumulated autofluorescent granules. The increased sensitivity to tBH-induced apoptosis in these cells was associated with intracellular oxidation and upregulation of FasL. These results suggest that an increase in FasL may contribute to the vulnerability of nondividing hRPE cells to oxidant-induced apoptosis.
authors.affiliation: Department of Ophthalmology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
authors.forename: Shunai
authors.initials: S
authors.lastname: Jiang
authors.forename: Siobhan E
authors.initials: SE
authors.lastname: Moriarty
authors.forename: Hans
authors.initials: H
authors.lastname: Grossniklaus
authors.forename: Kasey C
authors.initials: KC
authors.lastname: Nelson
authors.forename: Dean P
authors.initials: DP
authors.lastname: Jones
authors.forename: Paul
authors.initials: P
authors.lastname: Sternberg
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002455:Cell Division; D002470:Cell Survival; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000096:biosynthesis; D053222:Fas Ligand Protein; D005434:Flow Cytometry; D005978:Glutathione / Q000378:metabolism; D019803:Glutathione Disulfide / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation; D019014:fas Receptor / Q000235:genetics / Q000378:metabolism; D020122:tert-Butylhydroperoxide / Q000494:pharmacology*
chemical_list: C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D019014:fas Receptor; D004247:DNA; D020122:tert-Butylhydroperoxide; D005978:Glutathione; D019803:Glutathione Disulfide