Hanson, C. Jane; Bootman, Martin D.; Distelhorst, Clark W.; Maraldi, Tullia and Roderick, H. Llewelyn
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|DOI (Digital Object Identifier) Link:||http://dx.doi.org/doi:10.1016/j.ceca.2007.11.014|
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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.
|Item Type:||Journal Article|
|Copyright Holders:||2008 Elsevier Inc|
|Keywords:||calcium; apoptosis; Bcl-2; mitochondria|
|Academic Unit/Department:||Science > Life, Health and Chemical Sciences|
|Depositing User:||Martin Bootman|
|Date Deposited:||24 Oct 2012 13:42|
|Last Modified:||24 Oct 2012 20:47|
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