title: The importance of Bcl-xL in the survival of human RPE cells.
abstract: OBJECTIVE
  In normal eyes and in diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR), retinal pigment epithelial (RPE) cell survival is critically important. Bcl-x(L) has been shown to be among the most highly expressed survival factors in cultured human RPE cells. In the current study the effect of Bcl-x(L) blockade on human RPE cell survival was determined under normal conditions and after induced oxidative stress.
  
  
  METHODS
  Cultured human RPE cells from three different donors were transfected with modified, 2'-O-methoxyethoxy Bcl-x(L)-mismatched control antisense oligonucleotides (ASOs), Bcl-x(L)-specific ASOs, and Bcl-x(L) splice switching oligonucleotides (SSOs), which shift the splicing pattern of Bcl-x pre-mRNA from Bcl-x(L) into Bcl-x(S), a proapoptotic factor. RNA and protein were harvested at various time points after transfection. Bcl-x(L) and Bcl-x(S) mRNA transcript levels were analyzed using gene-specific primers with reverse transcription-polymerase chain reaction. Bcl-x(L) protein levels were analyzed using Western blot. Cell viability was measured by WST-1 and lactate dehydrogenase (LDH) assays. The mode of cell death was determined with a cell death ELISA and an M30 assay. To study the effects of oxidative stress, the cells were stimulated after transfection with various concentrations of H(2)O(2.) Cell viability was analyzed by WST-1 (Roche, Indianapolis, IN) and LDH assays.
  
  
  RESULTS
  After Bcl-x(L)-specific ASO and SSO transfections, Bcl-x(L) mRNA and protein levels were significantly reduced. Bcl-x(S) levels were increased after transfection with SSO. By day 8 after plating, the cells transfected with Bcl-x(L)-specific ASO had significantly decreased viability, which was further reduced by day 10. The SSO had an even more potent effect. Cell viability was reduced on day 4 after plating and by day 10, less than 10% of the cells were viable. Apoptotic cell death occurred as early as day 4 after plating. H(2)O(2), used as a model oxidant, further enhanced cell death induced by Bcl-x(L)-specific ASO and SSO.
  
  
  CONCLUSIONS
  Bcl-x(L) plays an important role in human RPE cell survival under normal conditions and when cells are exposed to oxidative stress. Treatment strategies that enhance Bcl-x(L) expression and/or prevent conversion of Bcl-x(L) to Bcl-x(S) may be useful in preventing RPE cell death in AMD. Treatments that reduce Bcl-x(L) and enhance Bcl-x(S) may be useful in inhibiting unwanted RPE cell proliferation in PVR.
authors.affiliation: Department of Ophthalmology, Duke University, Durham, North Carolina.
authors.forename: Nanfei
authors.initials: N
authors.lastname: Zhang
authors.forename: James J
authors.initials: JJ
authors.lastname: Peairs
authors.forename: Ping
authors.initials: P
authors.lastname: Yang
authors.forename: Jillian
authors.initials: J
authors.lastname: Tyrrell
authors.forename: Jennifer
authors.initials: J
authors.lastname: Roberts
authors.forename: Ryszard
authors.initials: R
authors.lastname: Kole
authors.forename: Glenn J
authors.initials: GJ
authors.lastname: Jaffe
mesh_terms: D000328:Adult; D017209:Apoptosis / Q000502:physiology*; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D002648:Child; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008875:Middle Aged; D016376:Oligonucleotides, Antisense; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D014162:Transfection; D051020:bcl-X Protein / Q000235:genetics* / Q000378:metabolism*
chemical_list: C494811:BCL2L1 protein, human; D016376:Oligonucleotides, Antisense; D016877:Oxidants; D012333:RNA, Messenger; D051020:bcl-X Protein; D006861:Hydrogen Peroxide