Prolongation of murine hybridoma cell survival in stationary batch culture by Bcl-xL expression

While the ectopic expression of the anti-apoptoticprotein Bcl-2 has been shown to significantly increaseboth cell viability and antibody production in batchculture, some cell lines are refractory to thesemanipulations. For example, the NS/O and theP3x63Ag8.653 murine myelomas, which express highendogenous levels of the Bcl-2 homologue Bcl-xL, areboth resistant to the anti-apoptotic effect of Bcl-2.This indicates that, in these cells, Bcl-2 and Bcl-xLmay be functionally redundant. In order to define therole which Bcl-xL plays in hybridoma cultures, we usedthe Sp2/0-Ag14 cell line. This murine hybridomaexpresses low levels of Bcl-xL and is highly sensitiveto apoptosis induction by cycloheximide (CHX) and byamino acid depletion. Bcl-xL-transfected Sp2/0-Ag14cells were more resistant than the wild type and theplasmid-containing cells to apoptosis induced by CHXand by glutamine depletion. Moreover, when compared tothe vector-transfected control, Bcl-xL-Sp2/0 cellsexhibited a substantial increase in viability instationary batch culture. Interestingly, Sp2/0-Ag14cells overexpressing Bcl-xL showed a growth behaviourthat was similar to the parent myeloma cell lineP3x63Ag8.653. Our results suggest that Bcl-xLexpression levels are sufficient to account for therelative robustness of some hybridoma cell lines instationary batch cultures.     

Bcl-xL expression interferes with the effects of L-glutamine supplementation on hybridoma cultures

While feeding protocols and ectopic expression of anti-apoptotic genes have been used to improve the viability of hybridoma cell lines, the effect of the expression levels of survival genes on the behavior of hybridomas following nutrient supplementation is unknown. In this study, we compared the behavior of the Sp2/0-Ag14 hybridoma (Bcl-xL(low)) and the P3x63-Ag8.653 myeloma (Bcl-xL(high)) following culture supplementation with the amino acid L-glutamine (L-Gln). Our data revealed that L-Gln addition substantially increased Sp2/0-Ag14 cell viability and total cell density, concomitant with a decrease in the rate of cell death. This effect was not seen when other amino acids or D-glucose (D-Glc) replaced L-Gln. The improvement in the culture behavior of Sp2/0-Ag14 cells was attributed to a reduction in the rate of accumulation of apoptotic cells. On the other hand, L-Gln supplementation had only a limited effect on the growth of the P3x63-Ag8.653 cells. Interestingly, Sp2/0-Ag14 cells over-expressing Bcl-xL showed a culture behavior upon L-Gln complementation that was similar to the P3x63-Ag8.653 myeloma. These results suggest that the anti-apoptotic gene expression profile of hybridoma cells can markedly impact on the beneficial effects afforded by nutrient supplementation.     

Extension of Sp2/0 hybridoma cell viability through interleukin-6 supplementation

Sp2/0 hybridoma cells die principally by apoptosis in batch culture. We have found that cultures of the Sp2/0 hybridoma exhibit increased viability in response to interleukin 6 (IL-6) supplementation relative to control cultures during serum shiftdown experiments. When shifted from a medium containing 10% fetal bovine serum (FBS) to a medium with 1% FBS, IL-6 supplemented cultures displayed viabilities and viable cell densities similar to control cultures containing 10% FBS. The degree of the survival response induced varied in accordance with the severity of the shiftdown, as cells resuspended in a high serum medium showed little observable enhancement in viability. The extension in culture viability was not accompanied by an observable decrease in growth relative to control cultures, indicating that the effect was not a consequence of growth inhibition. These results suggest the existence of serum components with behavior functionally similar to IL-6, with respect to enhancing cell survival, and that under certain experimental conditions IL-6 serves as a survival factor. In contrast to the extended viability displayed by cultures supplemented with IL-6, Sp2/0 cultures transfected with IL-6 cDNA expression vectors displayed a growth inhibitory response relative to control cultures. This inhibitory response was characterized by an extended lag phase following inoculation, and a decrease in batch culture cell yield. The depression in cell yield varied with serum concentration, with the largest depression occurring at high serum concentrations. We conclude that interactions between components in serum, presumably growth factors, and cytokines play an important role in altering the behavior of industrially relevant cell lines in culture. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 439-446, 1997.     

Strategies to extend longevity of hybridomas in culture and promote yield of monoclonal antibodies

Summary Cell viability was improved by supplemental feeding of amino acids and vitamins in batch culture of hybridoma cells. Cells could be maintained over a 10 day period following exponential growth at a constant viable cell concentration of 2.1×10⁶ cells/ml. Concentrations of monoclonal antibody (MAb) reached 140 mg/l, a value of nearly four times that found in typical batch culture. Lactate formation appeared to occur only during active exponential growth and not during the stationary phase.     

Fragmented DNA and apoptotic bodies document the programmed way of cell death in hybridoma cultures

Markers of apoptosis were followed in batch hybridoma cultures carried out in protein-free medium. Samples were collected on day 0, representing early exponential phase (viability 91%), and on day 8, corresponding to late stationary phase (viability 8%). The apoptotic index reflecting the relative number of bodies insoluble in 6 M guanidinium hydrochloride in the culture of day 8 (30%) exceeded markedly the index in the culture of day 0 (2.5%). A gel chromatography on Sepharose 2B was developed for quantitative evaluation of fragmented cellular DNA. This analysis, including a correction for nonspecific fragmentation, showed that on day 8 more than 30% of cellular DNA was fragmented, whereas on day 0 it was less than 5%. Control necrotic cells prepared by rapid killing in 1% sodium azide displayed a low apoptotic index (2.4%) and low DNA fragmentation. Electrophoretic patterns in agarose gel showed a typical “ladder” of fragments in the DNA sample of day 8. The demonstration of fragmented cellular DNA and of the high incidence of apoptotic bodies at late stationary phase adds substantial weight to the view that in hybridoma cultures apoptosis represents the prevalent mode of cell death.     

The production of monoclonal antibody in growth-arrested hybridomas cultivated in suspension and immobilized modes.

The effects of the microenvironment and the nature of the limiting nutrient on culture viability and overall MAb productivity were explored using a hybridoma cell line which characteristically produces MAb in the stationary phase. A direct comparison was made of the changes in the metabolic profiles of suspension and PEG-alginate immobilized (0.8 mm beads) batch cultures upon entry into the stationary phase. The shifts in glucose, glutamine, and amino acid metabolism upon entry into the stationary phase were similar for both microenvironments. While the utilization of most nutrients in the stationary phase decreased to below 20% of that in the growth phase, antibody production was not dramatically affected. The immobilized culture did exhibit a 1.5-fold increase in the specific antibody rate over the suspension culture in both the growth and stationary phases. The role of limiting nutrient on MAb production and cell viability was assessed by artificially depleting a specific nutrient to 1% of its control concentration. An exponentially growing population of HB121 cells exposed to these various depletions responded with dramatically different viability profiles and MAb production kinetics. All depletions resulted in growth-arrested cultures and nongrowth-associated MAb production. Depletions in energy sources (glucose, glutamine) or essential amino acids (isoleucine) resulted in either poor viability or low antibody productivity. A phosphate or serum depletion maintained antibody production over at least a six day period with each resulting in a 3-fold higher antibody production rate than in growing batch cultures. These results were translated to a high-density perfusion culture of immobilized cells in the growth-arrested state with continued MAb expression for 20 days at a specific rate equal to that observed in the phosphate- and serum-depleted batch cultures.     

The potential of flow cytometric analysis for the characterization of hybridoma cells in suspension cultures

Flow cytometric (FC) analysis was applied to determine changes at cellular level during the cultivation of hybridoma cell line MN12 in a suspension batch culture. The relative cell size, cytoplasmic and membrane IgG content and the viability were monitored. Besides, the specificity of the cytoplasmic and membrane IgG was ascertained by means of a synthetic peptide containing the antigenic epitope recognized by the antibody. Cell size was found to increase during the exponential growth phase. The viability as determined by FC follows a similar pattern with the viability data obtained by the conventional trypan blue exclusion test. The relative cytoplasmic and membrane IgG contents were high during the exponential growth and low during stationary phase. Measurement of cell cycle distribution and the antibody content in the culture fluid, indicated that the major part of the cytoplasmic IgG is secreted by cells in the G1-phase. It is concluded that flow cytometry is a useful tool to characterize hybridoma cell lines in a suspension batch culture.     

Identification of the major phosphorylation site in Bcl-xL induced by microtubule inhibitors and analysis of its functional significance

Vinblastine and other microtubule inhibitors used as antimitotic cancer drugs characteristically promote the phosphorylation of the key anti-apoptotic protein, Bcl-xL. However, putative sites of phosphorylation have been inferred based on potential recognition by JNK, and no direct biochemical analysis has been performed. In this study we used protein purification and mass spectrometry to identify Ser-62 as a single major site in vivo. Site-directed mutagenesis confirmed Ser-62 to be the site of Bcl-xL phosphorylation induced by several microtubule inhibitors tested. Vinblastine-treated cells overexpressing a Ser-62 --> Ala mutant showed highly significantly reduced apoptosis compared with cells expressing wild-type Bcl-xL. Co-immunoprecipitation revealed that phosphorylation caused wild-type Bcl-xL to release bound Bax, whereas phospho-defective Bcl-xL retained the ability to bind Bax. In contrast, phospho-mimic (Ser-62 --> Asp) Bcl-xL exhibited a reduced capacity to bind Bax. Functional tests were performed by transiently co-transfecting Bax in the context of different Bcl-xL mutants. Co-expression of wild-type or phospho-defective Bcl-xL counteracted the adverse effects of Bax expression on cell viability, whereas phospho-mimic Bcl-xL failed to provide the same level of protection against Bax. These studies suggest that Bcl-xL phosphorylation induced by microtubule inhibitors plays a key pro-apoptotic role at least in part by disabling the ability of Bcl-xL to bind Bax.     

Apoptosis-resistant NS/0 E1B-19K myelomas exhibit increased viability and chimeric antibody productivity under cell cycle modulating conditions

Lymphoid cells expressing sufficient levels of Bcl-2 or E1B-19K are known to resist to induction of apoptosis in glutamine-free or nutrient-limited batch cultures. However, despite the increased viability and prolonged stationary phase achieved in batch culture, product yields are not necessarily improved. Here we have found that expression of E1B-19K in NS/0 myeloma cells cultivated in the presence of certain cell cycle modulators could result in a significant increase in MAb productivity as compared to untransfected control cells. The use of E1B-19K significantly enhanced cell survival in the presence of osmolytes (sorbitol, NaCl), DNA synthesis inhibitors (hydroxyurea, excess thymidine), and the cell culture additive OptiMAb™. E1B-19K myelomas cultivated in the presence of NaCl or OptiMAb™ accumulated in the G1 phase, while those arrested with excess thymidine were blocked in all phases. Interestingly, control NS/0 cells treated with these agents were found to die in a cell-cycle specific manner. Thus, while all G1 and most S phase cells quickly underwent apoptosis, G2/M cells remained alive and maintained MAb secretion for more than 10 days if supplied with adequate nutrients. For both control and E1B-19K cells, incubation with sorbitol or hydroxyurea was detrimental for MAb secretion, while addition of NaCl, excess thymidine and OptiMAb™ resulted in an increased specific MAb productivity as compared to the batch culture. However, this increase resulted in an improvement of final MAb yields only in the case of OptiMAb™. The extension of viability conferred by E1B-19K allowed to further improve the final MAb yield obtained using OptiMAb™ with a 3.3-fold increase for E1B-19K cells as compared to 1.8-fold for control NS/0 cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Phospho-Bcl-xL(Ser62) influences spindle assembly and chromosome segregation during mitosis

Functional analysis of a series of phosphorylation mutants reveals that Bcl-xL(Ser62Ala) influences cell entry into anaphase and mitotic exit in taxol-exposed cells compared with cells expressing wild-type Bcl-xL or a series of other phosphorylation mutants, an effect that appears to be independent of its anti-apoptotic activity. During normal mitosis progression, Bcl-xL(Ser62) is strongly phosphorylated by PLK1 and MAPK14/SAPKp38α at the prometaphase, metaphase, and the anaphase boundaries, while it is de-phosphorylated at telophase and cytokinesis. Phospho-Bcl-xL(Ser62) localizes in centrosomes with γ-tubulin and in the mitotic cytosol with some spindle-assembly checkpoint signaling components, including PLK1, BubR1, and Mad2. In taxol- and nocodazole-exposed cells, phospho-Bcl-xL(Ser62) also binds to Cdc20- Mad2-, BubR1-, and Bub3-bound complexes, while Bcl-xL(Ser62Ala) does not. Silencing Bcl-xL expression and expressing the phosphorylation mutant Bcl-xL(Ser62Ala) lead to an increased number of cells harboring mitotic spindle defects including multipolar spindle, chromosome lagging and bridging, aneuploidy with micro-, bi-, or multi-nucleated cells, and cells that fail to resolve undergo mitosis within 6 h. Together, the data indicate that during mitosis, Bcl-xL(Ser62) phosphorylation impacts on spindle assembly and chromosome segregation, influencing chromosome stability. Observations of mitotic cells harboring aneuploidy with micro-, bi-, or multi-nucleated cells, and cells that fail to resolve undergo mitosis within 6 h were also made with cells expressing the phosphorylation mutant Bcl-xL(Ser49Ala) and dual mutant Bcl-xL(Ser49/62Ala).     

抗调亡基因bcl-xl过表达提高工程细胞IVCC及目的蛋白表达的研究

用无血清培养基或化学成分明确的培养基生产治疗用重组蛋白已成为趋势。然而,在此条件下凝血因子、糖蛋白激素等微量糖蛋白的表达十分困难,其主要原因之一是在细胞培养过程中工程细胞大量凋亡造成的细胞密度低和生存期短。通过将早期抗凋亡基因导入工程细胞并进行过表达可改善工程细胞的活细胞密度积分（integral viable cell concentration,IVCC）,提高表达量。该研究将bcl-xl基因导入工程细胞,筛选其高表达细胞株,并验证工程细胞的抗凋亡能力,获得了稳定表达抗凋亡蛋白和目的蛋白的工程细胞株。与母细胞相比,稳定表达Bcl－xL的工程细胞的IVCC提高了50％,最终目的蛋白表达增加超过200％,显示抗凋亡基因bcl-xl的过表达可改善工程细胞在无血清悬浮培养过程中的细胞凋亡,提高表达量,为表达人凝血因子、糖蛋白激素等微量糖蛋白奠定了基础。     

Co-expression of bcl-2 and bag-1, apoptosis suppressing genes, prolonged viable culture period of hybridoma and enhanced antibody production

Human bcl-2 and bag-1 DNA were introduced into mouse hybridoma 2E3- O cells and expressed. The expression of bcl-2 in BCMGneo-bcl2 transfectants was confirmed by ELISA and that of bag-1 in pZeo-bag1 was confirmed by western blotting. In batch cultures, the over-expression of bcl-2 prolonged the culture period by 2 days and co-expression of bcl-2 and bag-1 prolonged the culture period by 3 days. The delayed increase in the dead cell number in culture of the bcl-2 and bag-1 cotransfectant indicated the additional antiapoptosis effect of bcl-2 and bag-1 cotransfection in comparison with the bcl-2 only transfection. The bcl-2 transfectants (2E3O-Bcl2) produced antibody twofold per batch culture in comparison with 2E3-O cells transfected with BCMGSneo (2E3O-Mock). Enhancement of this MoAb production was due to the improved survival of the cells and was not due to stimulation of antibody production rate per cell by Bcl-2 expression. And the bcl-2 and bag-1 co-transfectant (2E3O-Bcl2-BAG1) produced antibody approximately fourfold of 2E3O-Mock per batch culture. Enhancement of this MoAb production was due to the improved survival of the cells and was partly due to stimulation of MoAb production rate per cell in the non-growing phase by the cotransfection. The method to engineer hybridoma cells genetically with bcl-2 and bag-1 for increasing viability and productivity would be widely applied for improving antibody productivity of hybridoma cultures. This revised version was published online in August 2006 with corrections to the Cover Date.     

Acetic acid triggers cytochrome c release in yeast heterologously expressing human Bax

Proteins of the Bcl-2 protein family, including pro-apoptotic Bax and anti-apoptotic Bcl-xL, are critical for mitochondrial-mediated apoptosis regulation. Since yeast lacks obvious orthologs of Bcl-2 family members, heterologous expression of these proteins has been used to investigate their molecular and functional aspects. Active Bax is involved in the formation of mitochondrial outer membrane pores, through which cytochrome c (cyt c) is released, triggering a cascade of downstream apoptotic events. However, when in its inactive form, Bax is largely cytosolic or weakly bound to mitochondria. Given the central role of Bax in apoptosis, studies aiming to understand its regulation are of paramount importance towards its exploitation as a therapeutic target. So far, studies taking advantage of heterologous expression of human Bax in yeast to unveil regulation of Bax activation have relied on the use of artificial mutated or mitochondrial tagged Bax for its activation, rather than the wild type Bax (Bax α). Here, we found that cell death could be triggered in yeast cells heterologoulsy expressing Bax α with concentrations of acetic acid that are not lethal to wild type cells. This was associated with Bax mitochondrial translocation and cyt c release, closely resembling the natural Bax function in the cellular context. This regulated cell death process was reverted by co-expression with Bcl-xL, but not with Bcl-xLΔC, and in the absence of Rim11p, the yeast ortholog of mammalian GSK3β. This novel system mimics human Bax α regulation by GSK3β and can therefore be used as a platform to uncover novel Bax regulators and explore its therapeutic modulation.

     

Bladder cancer cell viability inhibition and apoptosis induction by baicalein through targeting the expression of anti-apoptotic genes

The study was aimed to investigate the effect of baicalein, a flavonoid molecule isolated from the plant Oroxylum indicum on bladder cancer cell viability. The results revealed that baicalein treatment of T24 and 253J bladder cancer cells targeted the expression of mRNA and proteins corresponding to the anti-apoptotic genes. RT-PCR assay showed that anti-apoptotic genes were markedly over-expressed in the bladder cancer cells. Exposure of the bladder cancer cells to baicalein at 5 mg/mL doses for 72 h led to reduction in the expression of mRNA levels of antiapoptotic genes. In T24 cells, the levels of BCL2, Bcl-xL, XIAP and surviving was reduced by 65, 69, 58 and 72%, respectively. In T24 and 253J cells exposure to baicalein for 72 h resulted respectively in 39 and 46% reduction in cell viability. Baicalein treatment also induced apoptosis in the bladder cancer cells. In T24 and 253J cells baicalein treatment at 5 mg/mL for 72 h induced apoptosis in 79 and 86% cells respectively. Thus, baicalein mediated reduction in antiapoptotic gene expression inhibits viability and induces apoptosis in bladder cancer cells. Therefore, baicalein is of therapeutic importance for the development of bladder cancer treatment strategy.     

Aven and Bcl-xL enhance protection against apoptosis for mammalian cells exposed to various culture conditions

A balance between proliferation and cell death is critical for achieving desirable high cell densities in mammalian cell culture. In this study, we evaluate a recently discovered anti-apoptotic gene, aven, and examine its effectiveness alone and in combination with a member of the Bcl-2 family, bcl-xL. The commercially popular cell line, Chinese hamster ovary (CHO), was genetically modified to constitutively express aven, bcl-xL, and the two genes in combination. Cells were exposed to several model insults that simulate severe bioreactor environments, including serum deprivation, spent medium, and Sindbis virus infection, as well as staurosporine, a known chemical inducer of apoptosis. CHO cells exhibited DNA fragmentation, a hallmark of apoptosis, after exposure to these model insults. After exposure to serum deprivation, 4- and 5-day spent medium, and staurosporine, cells expressing Aven provided limited protection against cell death when compared with the protection afforded by cells expressing Bcl-xL alone. However, the highest survival levels for all insults were achieved when Aven was expressed in combination with Bcl-xL. In fact, Aven appeared to act synergistically to enhance the protective function of Bcl-xL for several insults, because the protective function of the two genes expressed together in one cell line often exceeded the additive protective levels of each anti-apoptosis gene expressed alone. Surprisingly, Aven expression provided a mildly pro-apoptotic response in CHO isolates infected with Sindbis virus. However, CHO cells expressing both Bcl-xL and Aven showed protection against Sindbis virus infection due to the inhibitory properties of the bcl-xL anti-apoptosis gene. This study shows that combinatorial anti-apoptosis cell engineering strategies may be the most effective mechanisms for providing extended protection against cell death in mammalian cell culture.     

Expression of MARCKS Effector Domain Mutants Alters Phospholipase D Activity and Cytoskeletal Morphology of SK-N-MC Neuroblastoma Cells

Stable overexpression of myristoylated alanine-rich C-kinase substrate (MARCKS) is known to enhance phorbol ester stimulation of phospholipase D (PLD) activity and protein kinase Cα (PKCα) levels in SK–N–MC neuroblastoma cells. In contrast, expression of MARCKS mutants (S152A or S156A) lacking key PKC phosphorylation sites within the central basic effector domain (ED) had no significant effect on PLD activity or PKCα levels relative to vector control cells. Like control cells, those expressing wild type MARCKS were elongated and possessed longitudinally oriented stress fibers, although these cells were more prone to detach from the substratum and undergo cell death upon phorbol ester treatment. However, cells expressing MARCKS ED mutants were irregularly shaped and stress fibers were either shorter or less abundant, and cell adhesion and viability were not affected. These results suggest that intact phosphorylation sites within the MARCKS ED are required for PLD activation and influence both membrane-cytoskeletal organization and cell viability.     

Expression of BHRF1 improves survival of murine hybridoma cultures in batch and continuous modes

Cell death by apoptosis limits growth and productivity in most animal cell cultures. It is therefore desirable to define genetic interventions to generate robust cell lines with superior performance in bioreactors, either by increasing specific productivity, life-span of the cultures or both. In this context, forced expression of BHRF1, an Epstein–Barr virus-encoded early protein with structural and functional homology with the anti-apoptotic protein Bcl-2, effectively protected hybridomas in culture and delayed cell death under conditions of glutamine starvation. In the present study, we explored the potential application of BHRF1 expression in hybridomas for long-term apoptosis protection under different biotechnological process designs (batch and continuous) and compared it to strategies based on Bcl-2 overexpression. Our results confirmed that long-term maintenance of the anti-apoptotic effect of BHRF1 can be obtained using bicistronic configurations conferring enhanced protection compared to Bcl-2, even in the absence of selective pressure. Such protective effect of BHRF1 is demonstrated both in batch and continuous culture. Moreover, a further analysis at high cell densities in semi-continuous perfusion cultures indicated that the mechanism of action of BHRF1 involves cell cycle arrest in G0–G1 state and this is translated in lower numbers of dead cells.     

Mcl‐1 overexpression leads to higher viabilities and increased production of humanized monoclonal antibody in Chinese hamster ovary cells

Bioreactor stresses, including nutrient deprivation, shear stress, and byproduct accumulation can cause apoptosis, leading to lower recombinant protein yields and increased costs in downstream processing. Although cell engineering strategies utilizing the overexpression of antiapoptotic Bcl‐2 family proteins such as Bcl‐2 and Bcl‐x_L potently inhibit apoptosis, no studies have examined the use of the Bcl‐2 family protein, Mcl‐1, in commercial mammalian cell culture processes. Here, we overexpress both the wild type Mcl‐1 protein and a Mcl‐1 mutant protein that is not degraded by the proteasome in a serum‐free Chinese hamster ovary (CHO) cell line producing a therapeutic antibody. The expression of Mcl‐1 led to increased viabilities in fed‐batch culture, with cell lines expressing the Mcl‐1 mutant maintaining ～90% viability after 14 days when compared with 65% for control cells. In addition to enhanced culture viability, Mcl‐1‐expressing cell lines were isolated that consistently showed increases in antibody production of 20–35% when compared with control cultures. The quality of the antibody product was not affected in the Mcl‐1‐expressing cell lines, and Mcl‐1‐expressing cells exhibited 3‐fold lower caspase‐3 activation when compared with the control cell lines. Altogether, the expression of Mcl‐1 represents a promising alternative cell engineering strategy to delay apoptosis and increase recombinant protein production in CHO cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Bcl-xL acts as an inhibitor of IP3R channels,thereby antagonizing Ca2+-driven apoptosis

Anti-apoptotic Bcl-2-family members not only act at mitochondria but also at the endoplasmic reticulum, where they impact Ca²⁺ dynamics by controlling IP₃ receptor (IP₃R) function. Current models propose distinct roles for Bcl-2 vs. Bcl-xL, with Bcl-2 inhibiting IP₃Rs and preventing pro-apoptotic Ca²⁺ release and Bcl-xL sensitizing IP₃Rs to low [IP₃] and promoting pro-survival Ca²⁺ oscillations. We here demonstrate that Bcl-xL too inhibits IP₃R-mediated Ca²⁺ release by interacting with the same IP₃R regions as Bcl-2. Via in silico superposition, we previously found that the residue K87 of Bcl-xL spatially resembled K17 of Bcl-2, a residue critical for Bcl-2’s IP₃R-inhibitory properties. Mutagenesis of K87 in Bcl-xL impaired its binding to IP₃R and abrogated Bcl-xL’s inhibitory effect on IP₃Rs. Single-channel recordings demonstrate that purified Bcl-xL, but not Bcl-xL^K87D, suppressed IP₃R single-channel openings stimulated by sub-maximal and threshold [IP₃]. Moreover, we demonstrate that Bcl-xL-mediated inhibition of IP₃Rs contributes to its anti-apoptotic properties against Ca²⁺-driven apoptosis. Staurosporine (STS) elicits long-lasting Ca²⁺ elevations in wild-type but not in IP₃R-knockout HeLa cells, sensitizing the former to STS treatment. Overexpression of Bcl-xL in wild-type HeLa cells suppressed STS-induced Ca²⁺ signals and cell death, while Bcl-xL^K87D was much less effective in doing so. In the absence of IP₃Rs, Bcl-xL and Bcl-xL^K87D were equally effective in suppressing STS-induced cell death. Finally, we demonstrate that endogenous Bcl-xL also suppress IP₃R activity in MDA-MB-231 breast cancer cells, whereby Bcl-xL knockdown augmented IP₃R-mediated Ca²⁺ release and increased the sensitivity towards STS, without altering the ER Ca²⁺ content. Hence, this study challenges the current paradigm of divergent functions for Bcl-2 and Bcl-xL in Ca²⁺-signaling modulation and reveals that, similarly to Bcl-2, Bcl-xL inhibits IP₃R-mediated Ca²⁺ release and IP₃R-driven cell death. Our work further underpins that IP₃R inhibition is an integral part of Bcl-xL’s anti-apoptotic function.Subject terms: Cancer, Cell biology, Molecular biology