Central to the apoptotic cell death pathway are the Bcl-2 family members. These proteins are distributed on ER and mitochondrial membranes, where they have been reported to function in regulating calcium homeostasis. The effect of Bcl-2 overexpression on cellular calcium homeostasis however, remains controversial. To resolve this controversy we determined whether the overexpression paradigm (i.e. transient versusstable) was responsible. To this end, Bcl-2 fused to GFP was initially overexpressed in a transient or stable manner in HEK293 cells and its effects on cellular architecture and apoptosis determined. Data are presented as means ± S.E.M. with n indicating the number of experiments. Statistical significance was determined by a one tailed Student’s t test.

Using confocal microscopy of multiple fields of view, we observed that transient over-expression of Bcl-2 significantly disrupted organelle structure, causing the mitochondria to fragment and collapse around the nucleus (93 ± 3.4 of all cells, n = 3, P < 0.05). Apoptosis was determined by quantitating sub-G1 DNA content of propidium iodide-labelled cell populations by FACS. Transiently transfected cells were sensitised to staurosporine-induced apoptosis (4 µM for 4 h). Furthermore, transient expression of Bcl-2 in the absence of staurosporine treatment induced apoptosis. In contrast, stable expression of Bcl-2 did not perturb the structure and distribution of the mitochondria and ER to the same degree as transient overexpression (33 ± 3.5 %, n = 3 P < 0.05). In addition, only 6 % of cells stably overexpressing Bcl-2 (n = 2) became apoptotic following staurosporine treatment whereas 25 % of non-transfected cells did (n = 3). Since stable transfection is the optimal paradigm for increasing Bcl-2 expression, we investigated its role in calcium homeostasis in the WEHI7.2 T-lymphocyte cell-line which is a well-characterised cellular model for apoptosis. Cytosolic and ER lumenal calcium was monitored by single-cell imaging of Fura2-AM and digitonin permeabilised Fura2FF loaded cells respectively. InsP₃ induced calcium release was stimulated by application of antibody to the CD3 component of the T-cell receptor (1:40 dilution) or a cell permeant InsP₃ ester (25 µM). The cytosolic calcium transient induced by either CD3 ligation or InsP₃-ester application was decreased from 97 ± 13 nM and 350 ± 20 nM in control cells to 38 ± 10 nM and 140 ± 20 nM (n = 3, P < 0.005) in Bcl-2 overexpressing cells respectively. No decrease in ER lumenal calcium content (1, 010 ± 300 nM in controls versus1, 000 ± 250 nM in Bcl-2 expressing cells, n = 3, P = 0.815) or InsP₃R abundance (n = 3) was observed. By co-immunoprecipitation we detected an interaction between Bcl-2 and the InsP₃R (n = 3). In addition, we found that Bcl-2 overexpression significantly decreased the affinity of the InsP₃R for its ligand to 7.5 ± 0.2 nM from 4.8 ± 0.2 nM in control cells (n = 3, P < 0.001).

Our findings suggest that when stably expressed Bcl-2 protects cells form apoptosis by regulating InsP₃ induced calcium release. In addition, transient overexpression of Bcl-2, which severely disrupts organelle structure, may have abnormal consequences for calcium signalling.