title: Differential protection by wildtype vs. organelle-specific Bcl-2 suggests a combined requirement of both the ER and mitochondria in ceramide-mediated caspase-independent programmed cell death.
abstract: BACKGROUND
  Programmed cell death (PCD) is essential for development and homeostasis of multicellular organisms and can occur by caspase-dependent apoptosis or alternatively, by caspase-independent PCD (ciPCD). Bcl-2, a central regulator of apoptosis, localizes to both mitochondria and the endoplasmic reticulum (ER). Whereas a function of mitochondrial and ER-specific Bcl-2 in apoptosis has been established in multiple studies, corresponding data for ciPCD do not exist.
  
  
  METHODS
  We utilized Bcl-2 constructs specifically localizing to mitochondria (Bcl-2 ActA), the ER (Bcl-2 cb5), both (Bcl-2 WT) or the cytosol/nucleus (Bcl-2 DeltaTM) and determined their protective effect on ceramide-mediated ciPCD in transiently and stably transfected Jurkat cells. Expression of the constructs was verified by immunoblots. Ceramide-mediated ciPCD was induced by treatment with human recombinant tumor necrosis factor and determined by flow cytometric measurement of propidium iodide uptake as well as by optical analysis of cell morphology.
  
  
  RESULTS
  Only wildtype Bcl-2 had the ability to efficiently protect from ceramide-mediated ciPCD, whereas expression of Bcl-2 solely at mitochondria, the ER, or the cytosol/nucleus did not prevent ceramide-mediated ciPCD.
  
  
  CONCLUSIONS
  Our data suggest a combined requirement for both mitochondria and the ER in the induction and the signaling pathways of ciPCD mediated by ceramide.
authors.affiliation: Institut für Immunologie, Christian-Albrechts-Universität Kiel, 24105 Kiel, Germany. dadam@email.uni-kiel.de
authors.forename: Andrea
authors.initials: A
authors.lastname: Deerberg
authors.forename: Justyna
authors.initials: J
authors.lastname: Sosna
authors.forename: Lutz
authors.initials: L
authors.lastname: Thon
authors.forename: Claus
authors.initials: C
authors.lastname: Belka
authors.forename: Dieter
authors.initials: D
authors.lastname: Adam
mesh_terms: D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D002518:Ceramides / Q000633:toxicity; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D019169:Jurkat Cells; D008928:Mitochondria / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection
chemical_list: D002518:Ceramides; D019253:Proto-Oncogene Proteins c-bcl-2; D020169:Caspases