Bcl-XL is qualitatively different from and ten times more effective than Bcl-2 when expressed in a breast cancer cell line
- Aline A Fiebig†1,
- Weijia Zhu†1,
- Catherine Hollerbach1,
- Brian Leber2 and
- David W Andrews1Email author
© Fiebig et al; licensee BioMed Central Ltd. 2006
Received: 09 February 2006
Accepted: 23 August 2006
Published: 23 August 2006
Bcl-2 and Bcl-XL are anti-apoptotic paralogues that inhibit apoptosis elicited by a wide variety of stimuli, and play critical roles in cancer development and resistance to treatment. Many clinical studies have indicated that expression of these anti-apoptotic proteins in tumours is associated with poor prognosis. It has therefore been assumed that in cells the essential difference between Bcl-2 and Bcl-XL involves regulation of expression and that they are otherwise functionally similar. To examine this issue, we have compared the function of the proteins and of mutants of Bcl-2 and Bcl-XL specifically targeted to different subcellular sites.
We generated clones of the human breast cancer line MCF-7 stably expressing known amounts of Bcl-2, or Bcl-XL as determined by quantitative immunoblotting. Clones expressing equivalent amounts of wild-type and mutants of Bcl-2 and Bcl-XL with subcellular localization restricted to the cytoplasm, endoplasmic reticulum or outer mitochondrial membrane were studied in both MCF-7 and Rat-1 fibroblasts. In MCF-7 cells we measured the functional activities of these proteins in preventing apoptosis induced by four different agents (doxorubicin, ceramide, thapsigargin, TNF-α). Etoposide and low serum were used to compare the effect of Bcl-2, Bcl-XL and mutants located at the endoplasmic reticulum on induction of apoptosis in fibroblasts.
We noted both qualitative and quantitative differences in the functional activity of these two anti-apoptotic proteins in cells: Bcl-2 localized to the endoplasmic reticulum inhibits apoptosis induced by ceramide and thapsigargin but not by doxorubicin or TNFα, while Bcl-XL at the endoplasmic reticulum is active against all four drugs. In fibroblasts Bcl-2 localized to the ER did not prevent cell death due to etoposide whereas Bcl-XL in the same location did. Finally in MCF-7 cells, Bcl-XL is approximately ten times more active than Bcl-2 in repressing apoptosis induced by doxorubicin. This difference can be manifest as a large difference in clonal survival.
When examined in the same cellular context, Bcl-2 and Bcl-XL differ substantially in the potency with which they inhibit apoptosis, mediated in part by differences in the inhibition of specific subcellular pathways.
Apoptosis is a critical process that is dysregulated in tumourigenesis . Bcl-2 was the prototypic anti-apoptotic protein, and Bcl-XL was the first protein discovered with similar function . Since then the Bcl-2 family has expanded to include more than 6 anti-apoptotic and many pro-apoptotic members . Bcl-2 and Bcl-XL display 43 % amino acid identity, share regions of sequence similarity [4, 5] as well as a C-terminal hydrophobic region required for membrane localization , and represent the most recent additions to the Bcl-2 family . Bcl-2 and Bcl-XL appear to function in the same apoptotic pathway , and both confer resistance to multiple chemotherapy agents when tested in experimental systems. Over-expression of either protein is usually associated with poor prognosis in many human cancers (reviewed in 8). However, in some cancer types multiple anti-apoptotic proteins are expressed , and have opposite effects on prognosis [10–12] indicating that there may be subtle, but clinically and biologically relevant functional differences between family members. Experiments in mice with deletion of individual anti-apoptotic genes indicate that the phenotypes are not identical . However, it is generally accepted that this is due to expression in different tissues or in the same tissue but at different times rather than being a consequence of differences in the potency or mechanism of action of the different anti-apoptotic proteins.
The mechanisms of action of Bcl-2 and Bcl-XL are complex, with many postulated interactions with other proteins, and the role of any single interaction in the final phenotype at the cellular level remains ill-defined. Bcl-2 is located at the mitochondrion, endoplasmic reticulum (ER) and the nuclear envelope [14, 15]. Bcl-XL resides in the nuclear envelope, extra-nuclear membranes, including the mitochondrion but also cytosol [16, 17]. Bcl-2 is targeted to membranes by a carboxyl-terminal tail-anchor , and by replacing the tail-anchor with heterologous sequences specific for insertion into either ER or mitochondria, we have created fusion proteins targeted to individual organelles . These targeted mutants defined distinct but overlapping Bcl-2 regulated apoptosis pathways at individual organelles [18–22]. Here we have created similar mutants of Bcl-XL to compare organelle specific inhibition of apoptosis by Bcl-2 and Bcl-XL.
The human breast cancer cell line MCF-7 transfected with plasmids expressing either Bcl-2 or Bcl-XL is an excellent system in which to examine the differences between these two proteins as the cells do not express detectable Bcl-XL, and endogenous Bcl-2 can be drastically reduced by growth in estrogen depleted medium . Therefore, the background due to endogenous anti-apoptotic proteins is minimal, and the effects of exogenously expressed proteins can be monitored. As adherent cells, they can be studied by immunofluorescence microscopy to determine the location of organelle-targeted mutants. MCF-7 cells lack caspase-3 due to a genomic deletion , potentially decreasing feedback activation of other caspases, simplifying the analysis of individual apoptotic pathways . Finally, most human breast cancers originate in epithelial cells that express Bcl-2 or Bcl-XL [26–28]. Clinical studies have demonstrated that increased levels of Bcl-XL in breast carcinoma are associated with a poor outcome . Conversely and paradoxically, Bcl-2 expression confers a better prognosis than lack of expression [10, 11]. However, the association of Bcl-2 with expression of the estrogen receptor (a strong independent predictor of good outcome) has confounded these studies.
To examine different apoptosis pathways in cells expressing Bcl-2, Bcl-XL or one of the targeted mutants, we examined the response of MCF-7 cells to several stimuli that differentially engage organelle specific pathways of apoptosis [29, 30]. To determine how general the qualitative differences between Bcl-2 and Bcl-XL are we also assessed the function of the wild-type proteins and targeted mutants of Bcl-2 and Bcl-XL in a Rat-1 fibroblast cell line used previously to demonstrate differences in the regulation of apoptosis at ER and mitochondria [18, 29, 30].
Doxorubicin was selected to examine the differences between the various cell clones quantitatively, as this drug is used extensively in treating breast cancer patients and it is well characterized as an apoptotic stimulus for MCF-7 cells . Thus, quantitatively assaying doxorubicin-induced apoptosis, both by monitoring the cleavage of PARP and by assessing clonogenic survival in the estrogen receptor positive MCF-7 line represents an excellent cellular context in which to measure and analyze differences between Bcl-2 and Bcl-XL. Unexpectedly, our results demonstrate that Bcl-2 and Bcl-XL differ substantially in the potency with which they inhibit apoptosis, mediated in part by differences in the inhibition of specific subcellular pathways. These data suggest caution in interpreting expression alone as directly proportional to functional activity in clinical samples, and clearly show that Bcl-2 and Bcl-XL are functionally distinct.
Plasmids and recombinant proteins
Plasmids encoding targeted Bcl-2 mutants (Bcl2-acta and Bcl2-cb5) have been previously described . A similar strategy was used for Bcl-XL: the cDNA encoding amino acids 1 – 210 of Bcl-XL was fused either to the sequence encoding the carboxyl-terminus of ActA or the cytochrome b5 hydrophobic tail for mitochondrial or endoplasmic reticulum targeting, respectively. Bcl-XL lacking the carboxyl-terminal sequence was generated by inserting a stop codon after nucleotide 630.
To purify Bcl-XL, DH5α cells were transformed with plasmid encoding a Bcl-XL-intein fusion protein. The fusion protein was expressed and bound to a chitin column; after washing the column, cleavage of the fusion protein was induced with 30 mM DTT. Recombinant glutathione-S-transferase (GST)-Bcl-2ΔTM  was purified on a glutathione column.
MCF-7 cells were analyzed by immunofluorescence as previously described , using rabbit anti-Bcl-XL antisera followed by either a monoclonal antibody to the ER protein calreticulin, or to an inner mitochondrial membrane protein (2G2, ExAlpha Biologicals). Where indicated, FITC was coupled directly to the primary antibody. FITC and rhodamine donkey anti-rabbit and rhodamine donkey anti-mouse were used as secondary antibodies. Cells were analyzed using a Zeiss confocal microscope and associated software (Carl Zeiss LSM510). To assess apoptosis in rat fibroblasts the cells were stained with Hoescht 33342 and Annexin V coupled to Alexa Fluor594 according to specifications of the manufacturer (Molecular Probes) and viewed by epifluorescence microscopy.
Stable transfectants of MCF-7 and Rat-1-MycERTAM (referred to here as Rat-1) cell lines generated using Geneporter (Gene Therapy Systems Inc) were analyzed for expression by quantitative immunoblotting. Apoptosis was induced in Rat-1 cells using etoposide or serum starvation as described previously [29, 30]. To reduce expression of endogenous Bcl-2 in the MCF-7 cells the cells were incubated in phenol red minus αMEM with 10 % charcoal filtered FBS for 6 days prior to each experiment. MCF-7 cells were washed with PBS and incubated in the same medium containing: doxorubicin (at the specified concentrations, for 24 hours), thapsigargin (400 nM, for 60 hours), ceramide (70 μM, for 20 hours), or TNFα and cycloheximide (29 ng/ml and 10 ng/ml, respectively for 30 hours). Floating and adherent cells were harvested and pelleted by centrifugation, the cell pellet washed twice in PBS and resuspended in SDS lysis buffer (10 mM Tris-HCl, pH 7.5; 10 mM NaCl; 1 % SDS and protease inhibitors). An aliquot was removed for protein concentration determination by BCA protein assay (Pierce), and the rest of the cell lysate was diluted 1:1 with hot 4 % SDS, 0.1 M Tris-HCl pH 8.9, 2 mM EDTA, 0.1 % bromophenol blue, 20 % glycerol and 0.25 M DTT, and stored at -80°C.
Cell lysates were processed as previously described . The primary antibodies, source and dilutions used for immunoblotting were as follows: Bcl-2 (sheep polyclonal generated using GST-Bcl-2Δt; our laboratory; 1:10,000), Bcl-XL (rabbit polyclonal generated against Bcl-XL; our laboratory; 1:10,000), PARP (C-2-10, Biomol; 1:20,000), caspases 7, 8 and 9 (ExAlpha Biologicals; 1:4,000). Actin blots to ensure equivalent loading were developed with clone C4 (ICN) diluted at 1:80,000. HRP linked secondary antibodies (Jackson Laboratories) donkey anti-rabbit and donkey anti-mouse were used at dilutions of 1:10,000 and 1:80,000, respectively. To measure expression, the amount of exogenously expressed protein/microgram total protein was determined by quantitative immunoblotting of lysates from each MCF-7 clone. Quantification was performed using a standard curve of defined amounts of recombinant Bcl-2 or Bcl-XL from 3–6 blots, using amounts of protein that were in the linear detection range of enhanced chemiluminescence. The immunoblots were analyzed using the Kodak Image Station system (440CF). A linear regression analysis was performed on the net intensities of the standards and blots in which the lines of best fit with R2 values of ≥ 0.95 were analyzed. To identify appropriate clones of MCF-7 cells for further study, more than thirty were screened. The concentration of Bcl-2 or Bcl-XL in each MCF-7 clone used for further experiments was assayed at least three times and showed low inter-assay variability (<10%).
Measurement of cell death
During apoptosis PARP is cleaved into 24 and 89 kDa fragments by caspase-7 in MCF-7 cells  and can be degraded by lysosomal proteases to 62 and 55 kDa or 74 and 42 kDa fragments . The C-2-10 monoclonal antibody recognizes full length and 89 kDa caspase cleaved PARP, but does not recognize the cleavage products of other proteases. Using this and other commercially available antibodies it is only possible to visualize the full length protein, 89 and 24 kDa fragments. Therefore, to assess PARP cleavage by caspase 7 as well as degradation we measured the decrease in the amount of the full length PARP. In each experiment the amount of actin in the samples was also recorded. If the actin blots indicated uneven loading, transfer or development of the blots then the corresponding PARP data was not used. Doxorubicin induced degradation of actin was detected only at drug concentrations higher than 100 μM. Clonogenic survival after doxorubicin exposure was performed as previously described .
Although generally regarded as mechanistically similar, the effectiveness of Bcl-2 and Bcl-XL has not been compared in a single defined cellular context. Such a comparison is difficult because both proteins inhibit multiple different pathways for apoptosis within a single cell line. However, for a defined set of stimuli it is possible to compare Bcl-XL and Bcl-2 in the same context by measuring a common event that occurs at or beyond a point of convergence that is common to multiple cell death pathways. Thus, the first step in comparing Bcl-2 and Bcl-XL is identification of suitable downstream events that can be assayed.
Bcl-2 and Bcl-XL inhibit apoptosis induced by a variety of agents in MCF-7 cells
In vector transfected control cells all four agents induced apoptosis, as assessed by the cleavage of PARP (Figure 1), demonstrating that the effects on PARP occur downstream of a point of convergence. In contrast, only TNFα led to cleavage of caspase 8 (Figure 1). Furthermore, the absence of caspase 8 cleavage with TG, ceramide and doxorubicin establishes that caspase 8 cleavage cannot be used to compare induction of apoptosis by TNFα with the other agents.
MCF-7 cell lines expressing Bcl-XL and Bcl-2 were protected against apoptosis as judged by PARP cleavage induced by TNFα, TG, ceramide and doxorubicin compared to vector transfected (neo), control cells (Figure 1). However, neither Bcl-2 nor Bcl-XL prevented TNFα induced cleavage of caspase 8, indicating that both proteins act downstream of activated caspase 8. Thus both Bcl-2 and Bcl-XL inhibit downstream events elicited by four agents that initiate cell death via different pathways. To compare drugs that induce DNA damage (doxorubicin) and those that act at the ER and do not cause direct changes at the nucleus (TG) methods such as TUNEL staining or chromatin condensation are not optimal. For similar reasons, assessing cleavage of other caspases proved impractical for comparing both a variety of drugs and cells expressing Bcl-2 and Bcl-XL (data not shown).
The data in Figure 1 suggest that for MCF-7 cells, assaying PARP cleavage can be used to determine whether organelle targeted mutants of Bcl-2 and Bcl-XL show the same pattern of protection against the mechanistically distinct apoptosis pathways activated by these four different drugs. While not useful for all of the drugs studied here, we found that chromatin condensation (assessed by Hoescht staining) and externalization of phosphatidylserine (assessed by Annexin V labelling) were useful to compare some subsets of apoptotic agonists particularly in Rat-1 cells (see below).
Expression and subcellular localization of Bcl-2 and Bcl-XL mutants in MCF-7 cells
Bcl-2 and Bcl-XL inhibit multiple different pathways of apoptosis. To examine the inhibition of multiple spatially localized apoptosis pathways, we substituted the tail-anchor from Bcl-XL with one specific for ER (cb5) or mitochondria (acta), as done previously with Bcl-2 . Chimaeras of Bcl-2 or Bcl-XL with the cb5 or acta tail anchors are referred to as Bcl2-cb5 or BclX-cb5, and Bcl2-acta or BclX-acta, respectively. Chimaeras of Bcl-2 and Bcl-XL without a tail-anchor are referred to as Bcl2-Δt and BclX-Δt, respectively. MCF-7 clones are referred to by the proteins expressed. Quantitative western blotting was used to select MCF-7 clones in which expression of the chimaeras was equivalent to that of the wild-type proteins.
In MCF-7 cells Bcl-2 and Bcl-XL regulate organelle specific pathways of apoptosis differently
When apoptosis was elicited by ceramide or TG, both Bcl2-cb5 and BclX-cb5 were as, or more, effective than the respective wild type proteins and mitochondrial targeted mutants (Figure 3). Ceramide treatment that resulted in cleavage of all of the PARP in control cells was not inhibited very well by any of the proteins, so for this agonist a concentration was used that resulted in cleavage of about half the PARP in the control cells (neo). For TG treated control cells, all the PARP was cleaved and/or degraded, and the amount of PARP cleavage in Bcl-2, BclX-acta and BclX-Δt cells indicates that this dose and time of TG treatment is close to the maximum inhibited by these proteins. As Bcl-2 expression is approximately four times higher than Bcl-XL and protection from apoptosis is similar but incomplete, Bcl-XL is at least four times as efficient as Bcl-2 at inhibiting cell death due to TG.
The striking difference in activity of Bcl2-cb5 and BclX-cb5 was also evident when TNFα induced apoptosis was assessed qualitatively by staining cells with Hoescht dye (to visualize chromatin condensation) and Annexin V (to visualize externalization of phosphatidylserine at the plasma membrane) (Figure 3B). Staining cannot be used to assess cells treated with doxorubicin as the drug is highly fluorescent. Moreover in MCF-7 cells treated with TNFα, quantification is difficult because the period and extent of chromatin condensation is small and it is followed by extensive chromatin degradation resulting in loss of staining. Nevertheless, it is qualitatively obvious from the images that BclX-cb5 inhibits apoptosis induced by TNFα while Bcl2-cb5 does not.
Thus, targeting Bcl-2 and Bcl-XL to endoplasmic reticulum has drastically different effects on function, depending on the agent used to elicit apoptosis. In contrast, mitochondrial localized Bcl2-acta and BclX-acta were very effective against all four agents, demonstrating that the mitochondria are a point of convergence in the apoptosis pathways induced by a wide variety of drugs (Figure 3A).
Surprisingly, BclX-Δt was considerably more effective than vector control in preventing apoptosis due to all three agents (Figure 3A). This suggests that at least a part of the protective ability of Bcl-XL does not require membrane binding mediated by a tail-anchor. A significant proportion of BclX-Δt measured by quantitative immunoblotting was located in the nucleus by immunofluorescence microscopy (Figure 2, panels D and H) where the relevant binding partners that regulate apoptosis are probably not accessible. Therefore, our data may underestimate the intrinsic activity of BclX-Δt in prevention of apoptosis. We were not able to systematically examine cells expressing Bcl2-Δt to determine the requirement for membrane insertion of this protein because immunoblotting of cells expressing Bcl2-Δt indicated more than half of the molecules were modified as detected by decreased mobility on SDS-PAGE (data not shown). Because this modification has an unknown effect on function, Bcl2-Δt was not examined further.
In rat fibroblasts Bcl-2 and Bcl-XL regulate organelle specific pathways of apoptosis differently
Bcl-XL is ten times more effective than Bcl-2 at preventing apoptosis in MCF-7 cells
Expression of Bcl-2, Bcl-XL and mutant proteins in MCF-7 cells.
Number of Independent Experiments
Expression of Bcl-2 or Bcl-XL proteins (ng/μg total protein) no drug
Expression of Bcl-2 or Bcl-XL proteins (ng/μg total protein) at the EC50 dose of doxorubicin
EC 50 doxorubicin (μM)
Inhibition of PARP degradation.
1.9 ± 0
4.5 ± 1.0
2.5 ± 0.1
22.8 ± 3.3
3.6 ± 0.7
1.6 ± 0.1
30.6 ± 1.0
1.2 ± 0.3
0.2 ± 0.02
13.2 ± 1.7
1.5 ± 0.4
1.3 ± 0.02
2.3 ± 0.1
0.8 ± 0.2
0.7 ± 0.05
85.4 ± 0.6
0.6 ± 0.1
0.5 ± 0.01
8.7 ± 1.7
0.3 ± 0.1
0.2 ± 0.02
97.8 ± 1.5
0.6 ± 0.1
0.6 ± 0.1
32.3 ± 1.8
To accurately measure Bcl-2 and Bcl-XL in the cells we used quantitative immunoblotting. Standard curves on the same blots as the cell lysates demonstrated that detection of protein concentration was linear throughout the range needed for detection of the proteins. It was also essential to screen cells for equivalent expression after growth in estrogen depleted medium as a number of the clones exhibited striking changes in the amount of protein expressed in response to estrogen (data not shown). This is presumably a result of the site of integration of the exogenous DNA in the genome of the MCF-7 cells. Changes in the amount of the anti-apoptotic proteins during apoptosis were measured for all of the proteins, and with the exception of Bcl2-acta after doxorubicin treatment, paralleled each other and did not correlate with functional effects (see below). Furthermore, we extensively tested two separate clones that expressed closely matched levels of Bcl-2 (3.6 and 4.5 ng Bcl-2/μg protein) and performed partial comparisons for independent duplicates of the other cell lines. In all cases we obtained similar results, indicating that observed differences between the cell lines used for quantitative comparisons below were not due to clonal variation, but were due to the expression of the exogenous proteins (data not shown). Thus, we conclude that these clones can be used to compare quantitatively the effects of Bcl-2, Bcl-XL and the targeted mutants.
The assays chosen to measure apoptosis were cleavage of PARP and clonal survival. As shown above, PARP cleavage occurs downstream of convergence of the different apoptosis pathways and there is no feedback activation of caspase 8 after treatment of MCF7 cells with drugs (Figure 1). This lack of feedback amplification, likely due to lack of caspase 3, simplifies the interpretation of PARP cleavage data making it more useful for quantitative comparisons. Finally, PARP cleavage assayed by quantitative immunoblotting appears to have a larger dynamic range than either Hoescht staining or Annexin V labelling (data not shown).
Comparison of the dose response curve for Bcl2-cb5 with the control demonstrates a complete lack of activity for Bcl2-cb5 at every concentration of doxorubicin tested (Figure 6A). To compare different cell lines directly, dose response curves were used to obtain an EC50, defined as the drug concentration that caused the disappearance and/or cleavage of half of the pre-treatment PARP (Figure 5 arrows, left panel).
The absolute amount of the anti-apoptotic proteins in each cell line and the apoptosis index were used to derive a measure of functional activity, defined as the EC50 of doxorubicin in μM (Figure 6A) divided by molar amount of anti-apoptotic protein expressed per μg of cell protein. Calculation of functional activity in this way permits normalization of the data for differences in the expression level of the exogenous proteins. However, such normalization is complicated by the fact that the amount of protein expressed is altered by drug treatment. Therefore, to determine the true extent of variation within the data and provide estimates of the relative efficacy of the different proteins, functional activities were calculated using both the amount of exogenous protein expressed prior to drug treatment and the amount of exogenous protein present at the time of assay at the EC50 for doxorubicin for each clone. The results of both calculations were compared by normalizing to Bcl-2 clone #2 which was arbitrarily set to 1). As a control to show the amount of variation for Bcl-2 we examined clone #5 as part of this analysis. Using this method to determine activity, it is clear that on a molar basis Bcl-XL prevented PARP degradation at a concentration of doxorubicin that is at least ten times higher than was inhibited by Bcl-2 (Figure 6C). Part of this increased efficacy may be due to the fact that the Bcl-XL associated with the ER is effective, whereas Bcl-2 is not able to prevent doxorubicin induced apoptosis from the ER. However, even when targeted to mitochondria where both proteins are active against doxorubicin induced apoptosis, Bcl-XL is much more efficacious than Bcl-2 (Figure 6C).
The shape of the dose response curve for Bcl2-acta differs from that of both wt Bcl-2, and BclX-acta (Figure 6A and 6B). Unlike the other mutants, addition of 5–20 μM doxorubicin strongly reduced the amount of Bcl2-acta as determined by quantitative immunoblotting. Because the same effect was seen in several different independently derived cell clones including those expressing more or less Bcl2-acta than the clone used here (data not shown) we presume that Bcl2-acta is more susceptible to a protease that is activated by low doses of doxorubicin. It is unclear whether this proteolytic activity is related to the organelle specific degradation of Bcl-2 that has been reported in other cell types after exposure to cytotoxic agents . We did not observe any pro-apoptotic caspase cleavage products of Bcl-2  or Bcl-XL  that have been described in other systems, possibly due lack of caspase 3 in MCF-7 cells.
Dose response curves were also used to assess the requirement of membrane binding for Bcl-XL activity. In many cell types a substantial fraction of Bcl-XL is cytoplasmic (e.g. 17), or as in these MCF-7 cells, peripherally bound to membranes when cells are not stressed (Zhu, Leber and Andrews, unpublished data). BclX-Δt remains nucleo-cytoplasmic during apoptosis but when assayed with doxorubicin it has higher functional activity than Bcl-2 (Figure 6C). Nevertheless, this activity is much less than that of Bcl-XL. BclXL-cb5 is also membrane bound and is substantially more active than BclXL-Δt. However, we cannot be sure how much of the activity of the latter is under-estimated by mis-targeting to putatively inactive sites in nucleus, as noted above (Figure 3, panels D and H). Thus, a significant amount of the activity of BclX-cb5 may not be due to specific location at the ER and interaction with resident membrane proteins at that organelle, but rather by sequestering and inactivating cytoplasmic pro-apoptotic proteins. A similar mechanism has been proposed for the activity of Bcl2-cb5 in preventing apoptosis by sequestering Bad .
Our results indicate that Bcl-XL and Bcl-2 are not functionally interchangeable. The organelle targeted mutants demonstrate that in both MCF-7 and Rat-1 cells Bcl-2 and Bcl-XL are qualitatively different: BclX-cb5 prevents apoptosis due to TNFα, doxorubicin (Figures 3, 6) and etoposide (Figure 4), whereas Bcl2-cb5 is ineffective. This extends our concept of organelle specific pathways of apoptosis by demonstrating that within a single cell type, a stressful stimulus elicits events at the ER differentially inhibited by Bcl-XL compared to Bcl-2. As apoptosis can be inhibited at either ER or mitochondria, this implies that changes at both organelles are necessary for apoptosis . The most likely reason that when corrected for expression targeted mutants appear more effective than wild-type proteins is that at equal levels of expression there is twice as much protein at a single organelle. We speculate that for this reason the targeted mutants more efficiently inhibit one essential pathway whereas the wild-type proteins partially inhibit two pathways (Figure 6C).
The difference in efficacy we observed for BclX-cb5 and BclX-acta suggests that inhibition of apoptosis by sequestration of cytoplasmic effectors (the presumed mechanism of action for BclX-cb5) may be an activity independent from another function of the Bcl-XL that integrates into mitochondria. It is likely that this latter activity involves inhibiting oligomerization of Bax and Bak .
We also noted a quantitative difference between Bcl-2 and Bcl-XL in preventing doxorubicin induced apoptosis: Bcl-XL is about ten times more efficient than Bcl-2. This marked difference may explain the different prognostic values of Bcl-2 compared to Bcl-XL expression in human breast cancer samples independent of the presence of the estrogen receptor.
Our experiments demonstrate that small differences in the amount of anti-apoptosis proteins lead to large differences in cell survival. This is especially notable with Bcl-XL, and has important implications for clinical correlative studies where the ability to distinguish between different levels of expression of Bcl-XL and other anti-apoptotic proteins by immunohistochemistry is limited, and in which intercellular variability in the expression of Bcl-XL is within this critical range. While important clinical correlations have been found in many studies, our results suggest that biologically relevant variation may not always be detectable by these assays.
Because anti-apoptotic Bcl-2 family proteins participate in complex pathways with many components, the qualitative and quantitative differences in function for Bcl-2 and Bcl-XL may be mediated by altered binding affinity to other proteins that affect apoptosis (including but not limited to pro-apoptotic Bcl-2 family members, as has been recently reported, 46). Membrane binding is also important for many aspects of the function of these proteins, including the binding of pro-apoptotic Bcl-2 family proteins [47, 48]. Therefore, the mechanisms and consequences of membrane integration of different anti-apoptotic proteins [39, 46, 49–51] is an important component in elucidating the differences between family members. Our results suggest that efficient sequestration of pro-apoptotic proteins depends on both the intrinsic preferences of Bcl-2 and Bcl-XL for particular targets as well as which organelle membrane the proteins are associated with. The most important general implication of our findings however, is that the exact mechanism of action in preventing apoptosis must be determined independently and specifically for each Bcl-2 family protein.
When examined in the same cellular context, Bcl-2 and Bcl-XL differ substantially in the potency with which they inhibit apoptosis, mediated in part by differences in the inhibition of specific subcellular pathways.
This work was supported by grant FRN12517 from the Canadian Institutes of Health Research to DWA and BL. CH was supported by the McMaster University BMT/Leukemia Fund. DWA holds the Canada Research Chair in Membrane Biogenesis.
- Hanahan D, Weinberg RA: The hallmarks of cancer. Cell. 2000, 100: 57-70. 10.1016/S0092-8674(00)81683-9.View ArticlePubMed
- Boise LH, Gonzalez-Garcia M, Postema CE, Ding L, Lindsten T, Turka LA, Mao X, Nunez G, Thompson CB: Bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell. 1993, 74: 597-608. 10.1016/0092-8674(93)90508-N.View ArticlePubMed
- Aravind L, Dixit VM, Koorin EV: Apoptotoic molecular machinery: vastly increased complexity in vertebrates revealed by genome comparisons. Science. 2001, 291: 1279-1284. 10.1126/science.291.5507.1279.View ArticlePubMed
- Muchmore SW, Sattler M, Liang H, Meadows RP, Harlan JE, Yoon HS, Nettesheim D, Chang BS, Thompson CB, Wong SL, Ng SL, Fesik SW: X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death. Nature. 1996, 381: 335-341. 10.1038/381335a0.View ArticlePubMed
- Petros AM, Medek A, Nettesheim DG, Kim DH, Yoon HS, Swift K, Matayoshi ED, Oltersdorf T, Fesik SW: Solution structure of the anti-apoptotic protein bcl-2. Proc Nat Acad Sci USA. 2001, 98: 3012-3017. 10.1073/pnas.041619798.PubMed CentralView ArticlePubMed
- Evans DE, Mansel RE: Molecular evolution and secondary structure conservation in the B-cell lymphoma leukemia (bcl-2) family of proto-oncogene products. J Mol Evol. 1995, 41: 775-783. 10.1007/BF00173157.View ArticlePubMed
- Chao DT, Linette GP, Boise LH, White LS, Thompson CB, Korsmeyer SJ: Bcl-XL and Bcl-2 repress a common pathway of cell death. J Exp Med. 1995, 182: 821-828. 10.1084/jem.182.3.821.View ArticlePubMed
- Cory S, Adams JM: The Bcl-2 family: regulators of the cellular life-or-death switch. Nature Rev Cancer. 2: 647-656. 10.1038/nrc883.
- Kitada S, Anderson J, Akar S, Zapata JM, Takayama S, Krajewski S, Wan HG, Zhan X, Bullrich F, Croce CM, Rai K, Hines J, Reed JC: Expression of apoptosis-regulating proteins in chronic lymphocytic leukemia: correlations with In vitro and In vivo chemoresponses. Blood. 1998, 91: 3379-3389.PubMed
- Lipponen P, Pietilainen T, Kosma VM, Aaltomaa S, Eskelinen M, Syrjanen KJ: Apoptosis suppressing protein bcl-2 is expressed in well-differentiated breast carcinomas with favourable prognosis. Pathol. 1995, 177: 49-55. 10.1002/path.1711770109.
- Gasparini BM, Donglioni C, Palma PL: Expression of Bcl-2 protein predicts efficacy of adjuvant treatments in operable node positive breast cancers. Clin Cancer Res. 1995, 1: 189-198.PubMed
- Olapadu OI, Adeyanju MO, Safa AR, Hagos F, Mick R, Thompson CB: Overexpression of Bcl-x protein in primary breast cancer is associated with high grade and nodal metastases. Cancer J Sci Am. 1997, 3: 230-237.
- Ranger AM, Malynn BA, Korsmeyer SJ: Mouse models of cell death. Nature Gen. 2001, 28: 113-8. 10.1038/88815.View Article
- Krajewski S, Tanaka S, Takayama S, Schibler MJ, Fenton W, Reed JC: Investigation of the subcellular distribution of the bcl-2 oncoprotein: residence in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membranes. Cancer Res. 1993, 53: 4701-4714.PubMed
- Janiak F, Leber B, Andrews DW: Assembly of Bcl-2 into microsomal and outer mitochondrial membranes. J Biol Chem. 1994, 269: 9842-9849.PubMed
- Gonzalez-Garcia M, Perez-Ballestero R, Ding L, Duan L, Boise LH, Thompson CB, Nunez G: bcl-XL is the major bcl-x mRNA form expressed during murine development and its product localizes to mitochondria. Development. 1994, 120: 3033-3042.PubMed
- Hsu YT, Wolter KG, Youle RG: Cytosol-to-membrane redistribution of Bax and Bcl-X(L) during apoptosis. Proc Natl Acad Sci USA. 1997, 94: 3668-3672. 10.1073/pnas.94.8.3668.PubMed CentralView ArticlePubMed
- Zhu W, Cowie A, Wasfy GW, Penn LZ, Leber B, Andrews DW: Bcl-2 mutants with restricted subcellular location reveal spatially distinct pathways for apoptosis in different cell types. EMBO J. 1996, 15: 4130-4141.PubMed CentralPubMed
- Hacki J, Egger L, Monney L, Conous S, Rosse T, Fellay I, Borner C: Apoptotic crosstalk between endoplasmic reticulum and mitochondria controlled by bcl-2. Oncogene. 2000, 19: 2286-2295. 10.1038/sj.onc.1203592.View ArticlePubMed
- Rudner J, Lepple-Wienhues A, Budach W, Berschauer J, Friedrich B, Wesselborg S, Schulze-Osthoff K, Belka C: Wild-type, mitochondrial and ER-restricted Bcl-2 inhibit DNA damage-induced apoptosis but do not affect death receptor-induced apoptosis. J Cell Sci. 2001, 114: 4161-4172.PubMed
- Thomenius MJ, Wang NJ, Reineks EZ, Distelhorst CJ: Bcl-2 on the endoplasmic reticulum regulates Bax activity by binding to BH-3 only proteins. J Biol Chem. 2003, 278: 6243-6250. 10.1074/jbc.M208878200.View ArticlePubMed
- Annis MG, Yethon JA, Leber B, Andrews DW: There is more to life and death than mitochondria:Bcl-2 proteins at the endoplasmic reticulum. Biochim Biophys Acta. 2004, 1644: 115-123. 10.1016/j.bbamcr.2003.07.001.View ArticlePubMed
- Teixeira C, Reed JC, Pratt MA: Estrogen promotes chemotherapeutic drug resistance by a mechanism involving Bcl-2 proto-oncogene expression in human breast cancer cells. Cancer Res. 1995, 55: 3902-3907.PubMed
- Janicke RU, Sprengart ML, Wati MR, Porter AG: Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J Biol Chem. 1998, 273: 9357-9360. 10.1074/jbc.273.16.9357.View ArticlePubMed
- Yang XH, Sladek TL, Liu X, Butler BR, Froelich CJ, Thor AD: Reconstitution of caspase 3 sensitizes MCF-7 breast cancer cells to doxorubicin and etoposide-induced apoptosis. Cancer Res. 2001, 61: 348-354.PubMed
- Hockenberry DM, Zutter M, Hickey W, Nahm M, Korsmeyer SJ: Bcl-2 protein is topographically restricted in tissues characterized by apoptotic cell death. Proc Natl Acad Sci USA. 88: 6961-6965. 10.1073/pnas.88.16.6961.
- Feuerhake F, Sigg W, Hofter EA, Dimpfl T, Welsch U: Immunohistochemical analysis of Bcl-2 and Bax expression in relation to cell turnover and epithelial differentiation markers in the non-lactating human mammary gland. Cell Tissue Res. 299: 47-58.
- Krajewski S, Krajewski M, Shabaik A, Wang HG, Irie S, Fong L, Reed JC: Immunohistochemical analysis of in vivo patterns of Bcl-XL expression. Cancer Res. 54: 5501-5507.
- Lee ST, Hoeflich KP, Wasfy GW, Woodgett JR, Lebe B, Andrews DW, Hedley DW, Penn LZ: Bcl-2 targeted to the endoplasmic reticulum can inhibit apoptosis induced by Myc but not etoposide in Rat-1 fibroblasts. Oncogene. 1999, 18: 3520-8. 10.1038/sj.onc.1202716.View ArticlePubMed
- Annis MG, Zamzami N, Zhu W, Penn LZ, Kroemer G, Leber B, Andrews DW: Endoplasmic reticulum localized Bcl-2 prevents apoptosis when redistribution of cytochrome c is a late event. Oncogene. 2001, 20: 1939-52. 10.1038/sj.onc.1204288.View ArticlePubMed
- Germain M, Affar EB, D'Amours D, Dixit VM, Salvesen GS, Poirier GG: Cleavage of automodified poly(ADP-ribose) polymerase during apoptosis. Evidence for involvement of caspase-7. J Biol Chem. 1999, 274: 28379-84. 10.1074/jbc.274.40.28379.View ArticlePubMed
- Gobeil S, Boucher CC, Nadeau D, Poirier GG: Characterization of the necrotic cleavage of poly(ADP-ribose) polymerase (PARP-1): implication of lysosomal proteases. Cell Death Differ. 2001, 8: 588-94. 10.1038/sj.cdd.4400851.View ArticlePubMed
- Hahn SM, Liebmann MD, Cook J, Fisher J, Goldspiel B, Venzon D, Mitchell JB, Kaufman D: Taxol in combination with doxorubicin or etoposode: possible antagonism In Vitro. Cancer. 1993, 72: 2705-11. 10.1002/1097-0142(19931101)72:9<2705::AID-CNCR2820720930>3.0.CO;2-K.View ArticlePubMed
- Denmeade SR, Isaacs JT: The SERCA pump as a therapeutic target: making a "smart bomb" for prostate cancer. Cancer Biol Ther. 2005, 4: 14-22.View ArticlePubMed
- Ogretmen B, Hannan YA: Biologically active sphingolipids in cancer pathogenesis and treatment. Nat Rev Cancer. 2004, 604-16. 10.1038/nrc1411.
- Minotti G, Menna P, Salvatorellai E, Cairo G, Gianni L: Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol Rev. 2004, 56: 185-229. 10.1124/pr.56.2.6.View ArticlePubMed
- Hsu H, Shu HB, Pan MG, Goeddel DV: TRADD-TRAF2 and TRADD-FADD interactions define two distinct TNF receptor 1 signal transduction pathways. Cell. 1996, 84: 299-308. 10.1016/S0092-8674(00)80984-8.View ArticlePubMed
- Sundararajan R, Cuconati A, Nelson D, White E: Tumor necrosis factor-alpha induces Bax-Bak interaction and apoptosis, which is inhibited by adenovirus E1B 19K. J Biol Chem. 2001, 276: 45120-7. 10.1074/jbc.M106386200.View ArticlePubMed
- Jeong S-Y, Gaume B, Lee Y-J, Hsu Y-T, Ryu S-W, Yoon S-H, Youle RJ: Bcl-XL sequesters its C-terminal membrane anchor in soluble, cytosolic homodimers. EMBO J. 2004, 19: 2146-55. 10.1038/sj.emboj.7600225.View Article
- Gottschalk AR, Boise LH, Thompson CB, Quintans J: Identification of immunosuppressant-induced apoptosis in a murine B-cell line and its prevention by bcl-x but not bcl-2. Proc Natl Acad Sci USA. 1994, 91: 7350-4. 10.1073/pnas.91.15.7350.PubMed CentralView ArticlePubMed
- Johnson DE: Noncaspase proteases in apoptosis. Leukemia. 2000, 14: 1695-1703. 10.1038/sj.leu.2401879.View ArticlePubMed
- Fadeel B, Hassan Z, Hellstrom-Lindberg E, Henter J-I, Orrenius S, Zhitovsky B: Cleavage of Bcl-2 is an early event inchemotherapy-induced apoptosis of human myeloid leukemia cells. Leukemia. 1999, 13: 719-28. 10.1038/sj/leu/2401411.View ArticlePubMed
- Cheng EH, Jirsch DG, Clem RJ, Ravi R, Kastan MB, Bedi A, Ueno K, Hardwick JM: Conversion of Bcl-2 to Bax-like death effector by caspases. Science. 1997, 278: 1966-8. 10.1126/science.278.5345.1966.View ArticlePubMed
- Clem RJ, Cheng EH, Karp CC, Kirsch DG, Kirsch DG, Ueno K, Takahashi A, Kastan MB, Griffen DE, Earnshaw WC, Veliuona MA, Hardwick JM: Modulation of cell death by Bcl-XL through caspase interaction. Proc Natl Acad Sci USA. 1998, 95: 554-9. 10.1073/pnas.95.2.554.PubMed CentralView ArticlePubMed
- Brown M, Attardi LD: The role of apoptosis in cancer development and treatment response. Nature Rev Cancer. 2005, 5: 231-237. 10.1038/nrc1560.
- Willis SN, Chen L, Dewson G, Wei A, Naik E, Fletcher JI, Adams JM, Huang DCS: Proapoptotic Bak is sequestered by Mcl-1 and Bcl-XL, but not by Bcl-2, until displaced by BH3-only proteins. Genes Dev. 2005, 19: 1294-1305. 10.1101/gad.1304105.PubMed CentralView ArticlePubMed
- Kim PK, Annis MG, Dlugosz PJ, Leber B, Andrews DW: During apoptosis Bcl-2 changes membrane topology at both the endoplasmic reticulum and mitochondria. Mol Cell. 2004, 14: 523-9. 10.1016/S1097-2765(04)00263-1.View ArticlePubMed
- Annis MG, Soucie EL, Dlugosz PJ, Cruz-Aguado J, Penn LZ, Leber B, Andrews DW: Bax forms multispanning monomers that oligomerize to permeabilize membranes during apoptosis. EMBO J. 2005, 24: 2096-103. 10.1038/sj.emboj.7600675.PubMed CentralView ArticlePubMed
- Kaufman T, Schlipf S, Sanz J, Neubert K, Stein R, Borner C: Characterization of the signal that directs Bcl-XL, but not Bcl-2, to the mitochondrial outer membrane. J Cell Biol. 2003, 160: 53-64. 10.1083/jcb.200210084.View Article
- Wilson-Annan J, O'Reilly LA, Crawford SA, Hausmann G, Beaumont JG, Parma LP, Chen L, Lackmann M, Lithgow T, Hinds MG, Day CL, Adams JM, Huang DCS: Proapoptototic activity of BH3-only proteins trigger membrane integration of prosurvival Bcl-w and neutralize its activity. J Cell Biol. 1993, 162: 877-87. 10.1083/jcb.200302144.View Article
- Dlugosz P, Billen LP, Annis MG, Zhu W, Zhang Z, Lin J, Leber B, Andrews DW: Bcl-2 changes conformation to inhibit Bax oligomerization. EMBO J. 2006, 25: 2287-96. 10.1038/sj.emboj.7601126.PubMed CentralView ArticlePubMed
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/6/213/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.