title: The cellular concentration of Bcl-2 determines its pro- or anti-apoptotic effect.
abstract: Bcl-2 is an oncoprotein that is widely known to promote cell survival by inhibiting apoptosis. We explored the consequences of different expression paradigms on the cellular action of Bcl-2. Using either transient or stable transfection combined with doxycycline-inducible expression, we titrated the cellular concentration of Bcl-2. With each expression paradigm Bcl-2 was correctly targeted to the endoplasmic reticulum and mitochondria. However, with protocols that generated the greatest cellular concentrations of Bcl-2 the structure of these organelles was dramatically altered. The endoplasmic reticulum appeared to be substantially fragmented, whilst mitochondria coalesced into dense perinuclear structures. Under these conditions of high Bcl-2 expression, cells were not protected from pro-apoptotic stimuli. Rather Bcl-2 itself caused a significant amount of spontaneous cell death, and sensitised the cells to apoptotic agents such as staurosporine or ceramide. We observed a direct correlation between Bcl-2 concentration and spontaneous apoptosis. Expression of calbindin, a calcium buffering protein, or an enzyme that inhibited inositol 1,4,5-trisphosphate-mediated calcium release, significantly reduced cell death caused by Bcl-2 expression. We further observed that high levels of Bcl-2 expression caused lipid peroxidation and that the deleterious effects of Bcl-2 could be abrogated by the reactive oxygen species (ROS) scavenger Trolox. When stably expressed at low levels, Bcl-2 did not corrupt organelle structure or trigger spontaneous apoptosis. Rather, it protected cells from pro-apoptotic stimuli. These data reveal that high cellular concentrations of Bcl-2 lead to a calcium- and ROS-dependent induction of death. Selection of the appropriate expression paradigm is therefore crucial when investigating the biological role of Bcl-2.
authors.affiliation: Laboratory of Molecular Signalling, Babraham Institute, Babraham, Cambridge CB2 4AT, UK.
authors.forename: C Jane
authors.initials: CJ
authors.lastname: Hanson
authors.forename: Martin D
authors.initials: MD
authors.lastname: Bootman
authors.forename: Clark W
authors.initials: CW
authors.lastname: Distelhorst
authors.forename: Tullia
authors.initials: T
authors.lastname: Maraldi
authors.forename: H Llewelyn
authors.initials: HL
authors.lastname: Roderick
mesh_terms: D017209:Apoptosis; D020013:Calcium Signaling; D002478:Cells, Cultured; D016257:Fura-2 / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015227:Lipid Peroxidation; D018613:Microscopy, Confocal; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D014162:Transfection
chemical_list: D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D049452:Green Fluorescent Proteins; D016257:Fura-2