Bcl-XL is qualitatively different from and ten times more effective than Bcl-2 when expressed in a breast cancer cell line

Plasmids encoding targeted Bcl-2 mutants (Bcl2-acta and Bcl2-cb5) have been previously described [18]. 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 [13] was purified on a glutathione column.

MCF-7 cells were analyzed by immunofluorescence as previously described [18], 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-Myc^ERTAM (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 [18]. 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%).

During apoptosis PARP is cleaved into 24 and 89 kDa fragments by caspase-7 in MCF-7 cells [31] and can be degraded by lysosomal proteases to 62 and 55 kDa or 74 and 42 kDa fragments [32]. 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 [33].

The drugs used to induce apoptosis are known to promote apoptosis via different pathways: experiments with MCF-7 and Rat-1 fibroblasts suggested that TG and ceramide induce apoptosis at the endoplasmic reticulum upstream of events at mitochondria [29, 30]. The initial target site of thapsigargin (TG) is the ER Ca⁺⁺ pump SERCA2 [34]. C₂ ceramide is a soluble version of a signalling molecule generated at the ER [35]. Mechanisms regulated at the endoplasmic reticulum are not expected to be involved in doxorubicin induced apoptosis [30] as it inhibits topoisomerase II and generates free radicals [36]. TNFα activates caspase 8 to cleave Bid which in turn triggers Bax/Bak to permeabilize mitochondria [37, 38]. To determine an appropriate way to compare apoptosis elicited by these different mechanisms in MCF-7 cells, we examined cells treated with the different agents for cleavage of caspases and PARP as well as using Hoescht and Annexin V staining. Apoptosis was measured at a variety of concentrations; however, in Figure 1 a single concentration and time point are shown for each drug to facilitate visual comparison. The dose and time were selected to maximize the difference between control and Bcl-2/Bcl-XL expressing 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).

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 [18]. 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.

Bcl2-cb5 and Bcl2-acta are localized to ER and mitochondria, respectively in several different cell lines [18‐21, 30]. However, localization of BclX-cb5 and BclX-acta has not been examined previously. We therefore stained MCF-7 cells expressing targeted mutants with polyclonal antisera to either Bcl-XL (Figure 2, panels A-D, I-J) or Bcl-2 (not shown), and examined them by indirect immunofluorescence using confocal microscopy. Cells were co-stained with antibodies recognizing either an inner mitochondrial membrane protein (Figure 2, 2G2, panels E-H) or an ER protein (anti-calreticulin, Figure 2, panels K-L). As expected, both BclX-acta and Bcl2-acta (data not shown) are targeted to mitochondria in MCF-7 cells, as indicated by the extensive co-localization observed with 2G2 (Figure 2, panels A, E), but not with the endoplasmic reticulum chaperone calreticulin (Figure 2, panels I, K). The distribution of wild-type Bcl-XL co-localized with 2G2 indicating that in MCF-7 cells the Bcl-XL expressed exogenously is located primarily at mitochondria (Figure 2, panels C, G). Wild-type Bcl-2 had a similar distribution in MCF-7 cells (data not shown). By contrast, BclX-cb5 (Figure 2, panels B, F) and Bcl2-cb5 (data not shown) do not localize to mitochondria, but instead exhibited a net-like cytoplasmic staining that co-localized with calreticulin (Figure 2 panels J, L). Bcl-XL without a tail-anchor (BclX-Δt) was distributed diffusely throughout the cytoplasm and in the nucleus without accumulating at mitochondria (Figure 2, panels D, H). The significant amount of nuclear staining seen with this construct suggests that the Bcl-XL tail-anchor prevents nuclear import, perhaps because it is essential for the formation of Bcl-XL dimers [39] that unlike the monomer, are too large to diffuse through the nuclear pore. The localization of Bcl2-acta, Bcl2-cb5, BclX-acta, BclX-cb5 and BclX-Δtm detected by immunofluorescence was also confirmed by subcellular fractionation of cell lysates (data not shown).

Using MCF-7 clones expressing Bcl-2, Bcl-XL or targeted mutants, we assessed the differences in inhibition of apoptosis induced by the agents characterized above. The dose and time were selected such that most of the PARP in the vector expressing control cells is cleaved to the characteristic 89 kDa fragment (ΔPARP). This dose was chosen because the closer the treatment is to the limit of inhibition, the more sensitive the assay is to differences between mutants. As expected, in cells expressing Bcl-2 or Bcl2-acta there was much less PARP cleavage. However, Bcl2-cb5 is largely ineffective at preventing doxorubicin induced cleavage of PARP. To our surprise, BclX-cb5 functioned differently than Bcl2-cb5 and prevented PARP cleavage as effectively as Bcl-XL and BclX-acta. Although BclX-Δtm was less effective than Bcl-XL it was much more effective at preventing PARP cleavage than Bcl2-cb5. Similarly, there was little to no inhibition of TNFα induced PARP cleavage by Bcl2-cb5 (Figure 3). In contrast, BclX-cb5 was relatively effective at inhibiting apoptosis induced by TNFα. This suggests that there is a fundamental difference in the function of Bcl-2 and Bcl-XL at the endoplasmic reticulum. Although based on this data alone it is formally possible that Bcl2-cb5 is misfolded and non-functional the fact that Bcl2-cb5 prevents apoptosis induced by either ceramide or TG demonstrated that Bcl2-cb5 is a functional protein (Figure 3).

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.

To determine if the difference between Bcl2-cb5 and BclX-cb5 is specific to MCF-7 cells we expressed Bcl-2, Bcl-XL, Bcl2-cb5 and BclX-cb5 in rat fibroblasts. We have shown previously that Bcl2-cb5 prevents apoptosis induced by agents that target the ER but does not inhibit apoptosis initiated at mitochondria in Rat-1 cells [29, 30]. Based on our earlier results we compared cell death induced by etoposide and low serum. Because we are comparing only these two agents it is also possible to use other assays in addition to PARP cleavage to measure the induction of apoptosis in the Rat-1 cell clones. As expected, control cells transfected with the empty vector (neo) are very sensitive to both etoposide and low serum (Figure 4), as a result PARP is cleaved, the nuclei condense as shown by Hoescht staining, and lipid asymmetry at the plasma membrane is lost resulting in externalization of phosphatidylserine (detected by labelling cells with Annexin V). In Rat-1 cells nuclear condensation is dramatic and quantitative thus, the results can be quantified from micrographs (Figure 4C). Bcl-2, Bcl-XL and BclX-cb5 all inhibited, but did not completely eliminate, nuclear condensation. Bcl2-cb5 was not effective at inhibiting etoposide induced nuclear condensation. Similarly, the fraction of cells that are annexin V positive is similar for the controls and Bcl2-cb5 expressing cells, whereas it is much reduced for cells expressing Bcl-2, Bcl-XL and importantly BclX-cb5 (Figure 4B–C). The most likely explanation for this result is that similar to MCF-7 cells, ER localized Bcl2-cb5 is unable to inhibit etoposide induced apoptosis even though it can be inhibited by BclX-cb5 proteins located at the same organelle. To demonstrate that this is a bona fide difference in activity between the two proteins and not a result of Bcl2-cb5 being inactive in rat fibroblasts, apoptosis was also induced in the cells by growth in low serum. Under these treatment conditions both Bcl2-cb5 and BclX-cb5 prevent apoptosis as well as the wild type proteins (Figure 4). Although all of the Bcl-2 family proteins prevent nuclear condensation as measured by Hoescht staining, none of the proteins prevented shrinkage of the nucleus (Figure 4) that occurs when cells are grown in low serum. While the difference in efficacy of BclX-cb5 and Bcl2-cb5 is obvious for these clones, these cells express unknown quantities of endogenous Bcl-2 and Bcl-XL complicating quantitative assessment.

It is clear from the results above that Bcl-2 and Bcl-XL inhibit many different forms of apoptosis. Previous reports suggest that expression levels of Bcl-2 or Bcl-XL in cells determine the extent of inhibition [40]. While there are some indications in the data above that Bcl-XL is more efficient than Bcl-2 at inhibiting apoptosis, quantitative comparison of Bcl-2 and Bcl-XL requires selection of transfected clones stably expressing known amounts of protein that does not vary with multiple passages, unlike what can be observed with pools of transfectants used in previous studies. As expected for stable clones of cell lines, all the cells expressed the transfected protein after expansion, as assessed by immunofluorescence (data not shown). Because our preliminary experiments demonstrated that Bcl-XL was more effective than Bcl-2 at preventing apoptosis, transfected cell lines grown in estrogen depleted medium (to decrease endogenous Bcl-2) were identified that consistently expressed roughly equipotent amounts of either Bcl-2 or Bcl-XL (range 1.2 to 4.5 ng /μg protein or 0.3 to 0.8 ng /μg protein, respectively and summarized in Table 1).

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).

A further qualitative difference between inhibition of apoptosis by Bcl-2 and Bcl-XL was evident at doses of doxorubicin that were effective at overcoming Bcl-2/Bcl-XL resistance. At these concentrations, cleavage of PARP at the site that generates the 89 kDa product characteristic of cleavage by Caspase 7 was evident in Bcl-2 expressing lines (Figure 5, left panels, arrow). By contrast, cleavage of PARP at this site was minimal in cells expressing Bcl-XL even at high doses (with the exception of cells expressing BclX-Δt, Figure 3). Instead, the total amount of PARP was decreased, indicating the degradation products in these cells were not recognized by the antibody (Figure 5, left panels). As many other proteases, including lysosomal proteases, are active during apoptosis [41], this suggests that Bcl-XL expression almost completely inhibits activation of effector caspases but not other proteases that mediate doxorubicin induced cell death. Since the 89 kDa product was eventually also degraded in Bcl-2 expressing cells, these additional proteases are active in clones expressing either protein. Degradation of PARP is still a specific indicator of programmed cell death and not a result of a general loss of cell protein as shown by blotting for actin (see below).

Loss of full length PARP is the most appropriate measure of cell death since both cleavage and degradation are accounted for (Figure 5, right panel). Using this metric as an apoptosis index we constructed doxorubicin dose response curves for control cells (neo) and cell clones expressing Bcl-2, Bcl2-cb5 or Bcl2-acta (Figure 6A) and for Bcl-XL, BclX-cb5, BclX-acta or BclX-Δt (Figure 6B) from immunoblots. This data confirms that for a wide range of drug concentrations the differences in inhibition of apoptosis noted with a single concentration of doxorubicin (Figure 3A) are preserved.

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 [42]. We did not observe any pro-apoptotic caspase cleavage products of Bcl-2 [43] or Bcl-XL [44] 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 [21].

As an independent and more clinically relevant means of comparing the effects of Bcl-2 and Bcl-XL on apoptosis [45], we measured clonal survival by re-plating efficiency of MCF7 cells exposed to 5μM doxorubicin for 1 hour [33]. This assay allows us to determine if the different effects on PARP cleavage and Annexin V labelling translate to the biologically and clinically relevant outcome of cell survival [37]. Consistent with results observed with measurement of residual PARP after exposure to doxorubicin, Bcl-XL was much more effective than Bcl-2 in promoting cell survival (Figure 7A). Furthermore, when corrected for expression level, the targeted mutants of Bcl-XL exhibited a similar order of efficacy, with BclX-acta > BclX-cb5 > BclX-Δt, although Bcl-XL was more effective than BclX-acta (Figure 7B). To our surprise Bcl2-cb5 promoted clonal survival after exposure of cells to 5 μM Doxorubicin for one hour even though it was unable to prevent cleavage of PARP after exposure to the same amount of drug for 24 hours. Therefore, the replating assay detected residual activity of Bcl2-cb5 not detectable with the PARP cleavage assay. However, the rest of the clonal survival data parallels that observed for cleavage of PARP.