Dual targeting Bcl-2 and Bcl-xL augments osteosarcoma response to doxorubicin.

Chemotherapy resistance is the major cause of treatment failure in osteosarcoma, the most common primary bone malignancy, and sensitizing therapeutic strategy is required to improve the clinical outcome. In this study, we discovered that navitoclax, a selective inhibitor of Bcl-2/Bcl-xL, effectively combats chemoresistance in osteosarcoma. Our research revealed that Bcl-2, but not Bcl-xL, is upregulated in osteosarcoma cells that are resistant to doxorubicin. However, venetoclax, a specific inhibitor of Bcl-2, did not exhibit activity against doxorubicin-resistant cells. Further analysis showed that depleting either Bcl-2 or Bcl-xL alone was insufficient to overcome doxorubicin resistance. Only by depleting both Bcl-2 and Bcl-xL significantly reduce the viability of doxorubicin-resistant cells. Similarly, navitoclax not only decreased the viability of doxorubicin-resistant cells but also acted synergistically with doxorubicin in cells sensitive to the drug. To confirm the ability of navitoclax to overcome doxorubicin resistance, we conducted experiments using multiple mouse models of osteosarcoma, both doxorubicin-sensitive and doxorubicin-resistant. The results provided confirmation that navitoclax is effective in overcoming doxorubicin resistance. Our findings demonstrate that simultaneous inhibition of Bcl-2 and Bcl-xL could serve as a novel strategy to sensitize chemoresistant osteosarcoma cells. Moreover, our study presents preclinical evidence supporting the potential of a navitoclax and doxorubicin combination therapy for the treatment of osteosarcoma, paving the way for future clinical investigations.

The potential for citrate to reinforce epigenetic therapy by promoting apoptosis.

Epigenetic drugs induce ATP depletion, promoting a glycolysis-to-oxidative phosphorylation (OXPHOS) shift which sometimes favors tumor growth by promoting necroptosis over apoptosis. To restore effective apoptosis in tumors, we propose that the administration of citrate could inhibit ATP production, activate caspase-8 (a key necroptosis inhibitor), and downregulate key anti-apoptotic proteins (Bcl-xL and MCL1).

Marine Polyether Phycotoxin Palytoxin Induces Apoptotic Cell Death via Mcl-1 and Bcl-2 Downregulation.

Palytoxin is considered one of the most potent biotoxins. As palytoxin-induced cancer cell death mechanisms remain to be elucidated, we investigated this effect on various leukemia and solid tumor cell lines at low picomolar concentrations. As palytoxin did not affect the viability of peripheral blood mononuclear cells (PBMC) from healthy donors and did not create systemic toxicity in zebrafish, we confirmed excellent differential toxicity. Cell death was characterized by a multi-parametric approach involving the detection of nuclear condensation and caspase activation assays. zVAD-sensitive apoptotic cell death was concomitant with a dose-dependent downregulation of antiapoptotic Bcl-2 family proteins Mcl-1 and Bcl-xL. Proteasome inhibitor MG-132 prevented the proteolysis of Mcl-1, whereas the three major proteasomal enzymatic activities were upregulated by palytoxin. Palytoxin-induced dephosphorylation of Bcl-2 further exacerbated the proapoptotic effect of Mcl-1 and Bcl-xL degradation in a range of leukemia cell lines. As okadaic acid rescued cell death triggered by palytoxin, protein phosphatase (PP)2A was involved in Bcl-2 dephosphorylation and induction of apoptosis by palytoxin. At a translational level, palytoxin abrogated the colony formation capacity of leukemia cell types. Moreover, palytoxin abrogated tumor formation in a zebrafish xenograft assay at concentrations between 10 and 30 pM. Altogether, we provide evidence of the role of palytoxin as a very potent and promising anti-leukemic agent, acting at low picomolar concentrations in cellulo and in vivo.

Identification of bicyclic compounds that act as dual inhibitors of Bcl-2 and Mcl-1.

Elevated expression of anti-apoptotic proteins, such as Bcl-2 and Mcl-1 contributes to poor prognosis and resistance to current treatment modalities in multiple cancers. Here, we report the design, synthesis and characterization of benzimidazole chalcone and flavonoid scaffold-derived bicyclic compounds targeting both Bcl-2 and Mcl-1 by optimizing the structural differences in the binding sites of both these proteins. Initial docking screen of Bcl-2 and Mcl-1 with pro-apoptotic protein Bim revealed possible hits with optimal binding energies. All the optimized bicyclic compounds were screened for their in vitro cytotoxic activity against two oral cancer cell lines (AW8507 and AW13516) which express high levels of Bcl-2 and Mcl-1. Compound 4d from the benzimidazole chalcone series and compound 6d from the flavonoid series exhibited significant cytotoxic activity (IC50 7.12 µM and 17.18 µM, respectively) against AW13516 cell line. Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) analysis further demonstrated that compound 4d and compound 6d could effectively inhibit the Bcl-2 and Mcl-1 proteins by displacing their BH3 binding partners. Both compounds exhibited potent activation of canonical pathway of apoptosis evident from appearance of cleaved Caspase-3 and PARP. Further, treatment of oral cancer cells with the inhibitors induced dissociation of the BH3 only protein Bim from Mcl-1 and Bak from Bcl-2 but failed to release Bax from Bcl-xL thereby confirming the nature of compounds as BH3-mimetics selectively targeting Bcl-2 and Mcl-1. Our study thus identifies bicyclic compounds as promising candidates for anti-apoptotic Bcl-2/Mcl-1 dual inhibitors with a potential for further development.

The inhibitory role of stigmasterol on tumor growth by inducing apoptosis in Balb/c mouse with spontaneous breast tumor (SMMT).

BACKGROUND: Breast cancer is one of the most common types of cancer in women worldwide. Anti-apoptotic activity of cancer cells is considered the main reason for drug resistance in BC which reduces the 5-year survival rate of patients and is still considered the main obstacle for cancer therapy. Stigmasterol (SS) is natural phytosterols compound in the plant which has been proved to play an important role to lower cholesterol and inducing anti-inflammatory, and anticancer properties. METHODS: In this, study, we aimed to evaluate the effect of SS on the expression of anti-apoptotic genes (Bcl-2 and BCL-XL), and also evaluate its effects on cell apoptosis and cell viability using MCF-7 cell line as well as evaluating its effect on tumor growth of spontaneous breast tumor (SMMT) in vivo. RESULT: SS significantly decreased the expression of Bcl-2 and BCL-XL genes (*P < 0.05), induced apoptosis, and reduced cell proliferation in MCF-7 cell lines. Our in vivo study also indicated that SS could inhibit tumor size after treatment with (0, 10, 20 µM) compared to the normal control. CONCLUSION: SS can be suggested as a potential agent in BC cancer treatment or as an adjuvant based on its ability to decrease the expression of Bcl-2 and BCL-XL genes and induce apoptosis.

Brain-Targeted Codelivery of Bcl-2/Bcl-xl and Mcl-1 Inhibitors by Biomimetic Nanoparticles for Orthotopic Glioblastoma Therapy.

Glioblastoma (GBM) is among the most treatment-resistant solid tumors and often recurrs after resection. One of the mechanisms through which GBM escapes various treatment modalities is the overexpression of anti-apoptotic Bcl-2 family proteins (e.g., Bcl-2, Bcl-xl, and Mcl-1) in tumor cells. Small-molecule inhibitors such as ABT-263 (ABT), which can promote mitochondrial-mediated cell apoptosis by selectively inhibiting the function of Bcl-2 and Bcl-xl, have been proven to be promising anticancer agents in clinical trials. However, the therapeutic prospects of ABT for GBM treatment are hampered by its limited blood-brain barrier (BBB) penetration, dose-dependent thrombocytopenia, and the drug resistance driven by Mcl-1, which is overexpressed in GBM cells and further upregulated upon treatment with ABT. Herein, we reported that the Mcl-1-specific inhibitor A-1210477 (A12) can act synergistically with ABT to induce potent cell apoptosis in U87 MG cells, drug-resistant U251 cells, and patient-derived GBM cancer stem cells. We further designed a biomimetic nanomedicine, based on the apolipoprotein E (ApoE) peptide-decorated red blood cell membrane and pH-sensitive dextran nanoparticles, for the brain-targeted delivery of ABT and A12. The synergistic anti-GBM effect was retained after encapsulation in the nanomedicine. Additionally, the obtained nanomedicine possessed good biocompatibility, exhibited efficient BBB penetration, and could effectively suppress tumor growth and prolong the survival time of mice bearing orthotopic GBM xenografts without inducing detectable adverse effects.

Stayin' alive: BCL-2 proteins in the hematopoietic system.

BH3 mimetics constitute a novel concept of antitumor therapy, inducing apoptosis via inhibition of pro-survival BCL-2 proteins. Programmed cell death is fundamental for physiological hematopoiesis; hence hematological side effects of these compounds are conceivable. Navitoclax and venetoclax have been studied extensively in the clinical setting; our knowledge of the more recently developed BCL-2 protein inhibitors specifically targeting MCL-1 or BCL-XL, however, is restricted mainly to preclinical experiments. To delineate possible adverse effects of novel BH3 mimetics on the human hematopoietic system, this review summarizes current knowledge of the function of specific antiapoptotic BCL-2 proteins in physiological hematopoiesis.

The interplay between apoptosis and cellular senescence: Bcl-2 family proteins as targets for cancer therapy.

Cell death by apoptosis and permanent cell cycle arrest by senescence serve as barriers to the development of cancer. Chemotherapeutic agents not only induce apoptosis, they can also induce senescence known as therapy-induced senescence (TIS). There are, however, controversies whether TIS improves or worsens therapeutic outcome. Unlike apoptosis, which permanently removes cancer cells, senescent cells are metabolically active, and can contribute to tumor progression and relapse. If senescent cells are not cleared by the immune system or if cancer cells escape senescence, they may acquire resistance to apoptotic stimuli and become highly aggressive. Thus, there have been significant efforts in developing senolytics, drugs that target these pro-survival molecules to eliminate senescent cells. The anti-apoptotic Bcl-2 family proteins not only protect against cell death by apoptosis, but they also allow senescent cells to survive. While combining senolytics with chemotherapeutic drugs is an attractive approach, there are also limitations. Moreover, members of the Bcl-2 family have distinct effects on apoptosis and senescence. The purpose of this review article is to discuss recent literatures on how members of the Bcl-2 family orchestrate the interplay between apoptosis and senescence, and the challenges and progress in targeting these Bcl-2 family proteins for cancer therapy.

BCL-XL exerts a protective role against anemia caused by radiation-induced kidney damage.

Studies of gene-targeted mice identified the roles of the different pro-survival BCL-2 proteins during embryogenesis. However, little is known about the role(s) of these proteins in adults in response to cytotoxic stresses, such as treatment with anti-cancer agents. We investigated the role of BCL-XL in adult mice using a strategy where prior bone marrow transplantation allowed for loss of BCL-XL exclusively in non-hematopoietic tissues to prevent anemia caused by BCL-XL deficiency in erythroid cells. Unexpectedly, the combination of total body γ-irradiation (TBI) and genetic loss of Bcl-x caused secondary anemia resulting from chronic renal failure due to apoptosis of renal tubular epithelium with secondary obstructive nephropathy. These findings identify a critical protective role of BCL-XL in the adult kidney and inform on the use of BCL-XL inhibitors in combination with DNA damage-inducing drugs for cancer therapy. Encouragingly, the combination of DNA damage-inducing anti-cancer therapy plus a BCL-XL inhibitor could be tolerated in mice, at least when applied sequentially.

Regulation of tumor necrosis factor-α-induced microvascular endothelial cell hyperpermeability by recombinant B-cell lymphoma-extra large.

BACKGROUND: Tumor necrosis factor-α (TNF-α), a cytotoxic cytokine, induces endothelial cell barrier dysfunction and microvascular hyperpermeability, leading to tissue edema, a hallmark of traumatic injuries. The objective of the present study was to determine whether B-cell lymphoma-extra large (Bcl-xL), an antiapoptotic protein, would regulate and protect against TNF-α-mediated endothelial cell barrier dysfunction and microvascular hyperpermeability. METHODS: Rat lung microvascular endothelial cells were grown as monolayers on Transwell membranes, and fluorescein isothiocyanate-bovine albumin flux (5 mg/mL) across the monolayer was measured fluorometrically to indicate changes in monolayer permeability. The rat lung microvascular endothelial cell adherens junctional integrity and actin cytoskeleton was studied using ß-catenin immunofluorescence and rhodamine phalloidin dye, respectively. Pretreatment of caspase-8 inhibitor (Z-IETD-FMK, 100 µM) for 1 hour and transfection of Bcl-2-homology domain 3-interacting domain death agonist small interfering RNA (10 µM) for 48 hours were performed to study their respective effects on TNF-α-induced (10 ng/mL; 1-hour treatment) monolayer permeability. Recombinant Bcl-xL protein (2.5 µg/ml) was transfected in rat lung microvascular endothelial cells for 1 hour, and its effect on permeability was demonstrated using a permeability assay. Caspase-3 activity was assayed fluorometrically. RESULTS: Z-IETD-FMK pretreatment protected the adherens junctions and decreased TNF-α-induced monolayer hyperpermeability. Bcl-2-homology domain 3-interacting domain death agonist small interfering RNA transfection attenuated the TNF-α-induced increase in monolayer permeability. Recombinant Bcl-xL protein showed protection against TNF-α-induced actin stress fiber formation, an increase in caspase-3 activity, and monolayer hyperpermeability. CONCLUSIONS: Our results have demonstrated the protective effects of recombinant Bcl-xL protein against TNF-α-induced endothelial cell adherens junction damage and microvascular endothelial cell hyperpermeability. These findings support the potential for Bcl-xL-based drug development against microvascular hyperpermeability and tissue edema.

The BH4 domain of Bcl-X(L) rescues astrocyte degeneration in amyotrophic lateral sclerosis by modulating intracellular calcium signals.

Collective evidence indicates that motor neuron degeneration in amyotrophic lateral sclerosis (ALS) is non-cell-autonomous and requires the interaction with the neighboring astrocytes. Recently, we reported that a subpopulation of spinal cord astrocytes degenerates in the microenvironment of motor neurons in the hSOD1(G93A) mouse model of ALS. Mechanistic studies in vitro identified a role for the excitatory amino acid glutamate in the gliodegenerative process via the activation of its inositol 1,4,5-triphosphate (IP(3))-generating metabotropic receptor 5 (mGluR5). Since non-physiological formation of IP(3) can prompt IP(3) receptor (IP(3)R)-mediated Ca(2+) release from the intracellular stores and trigger various forms of cell death, here we investigated the intracellular Ca(2+) signaling that occurs downstream of mGluR5 in hSOD1(G93A)-expressing astrocytes. Contrary to wild-type cells, stimulation of mGluR5 causes aberrant and persistent elevations of intracellular Ca(2+) concentrations ([Ca(2+)](i)) in the absence of spontaneous oscillations. The interaction of IP(3)Rs with the anti-apoptotic protein Bcl-X(L) was previously described to prevent cell death by modulating intracellular Ca(2+) signals. In mutant SOD1-expressing astrocytes, we found that the sole BH4 domain of Bcl-X(L), fused to the protein transduction domain of the HIV-1 TAT protein (TAT-BH4), is sufficient to restore sustained Ca(2+) oscillations and cell death resistance. Furthermore, chronic treatment of hSOD1(G93A) mice with the TAT-BH4 peptide reduces focal degeneration of astrocytes, slightly delays the onset of the disease and improves both motor performance and animal lifespan. Our results point at TAT-BH4 as a novel glioprotective agent with a therapeutic potential for ALS.

Prostacyclin receptor-mediated ATP release from erythrocytes requires the voltage-dependent anion channel.

Erythrocytes have been implicated as controllers of vascular caliber by virtue of their ability to release the vasodilator ATP in response to local physiological and pharmacological stimuli. The regulated release of ATP from erythrocytes requires activation of a signaling pathway involving G proteins (G(i) or G(s)), adenylyl cyclase, protein kinase A, and the cystic fibrosis transmembrane conductance regulator as well as a final conduit through which this highly charged anion exits the cell. Although pannexin 1 has been shown to be the final conduit for ATP release from human erythrocytes in response to reduced oxygen tension, it does not participate in transport of ATP following stimulation of the prostacyclin (IP) receptor in these cells, which suggests that an additional protein must be involved. Using antibodies directed against voltage-dependent anion channel (VDAC)1, we confirm that this protein is present in human erythrocyte membranes. To address the role of VDAC in ATP release, two structurally dissimilar VDAC inhibitors, Bcl-x(L) BH4(4-23) and TRO19622, were used. In response to the IP receptor agonists, iloprost and UT-15C, ATP release was inhibited by both VDAC inhibitors although neither iloprost-induced cAMP accumulation nor total intracellular ATP concentration were altered. Together, these findings support the hypothesis that VDAC is the ATP conduit in the IP receptor-mediated signaling pathway in human erythrocytes. In addition, neither the pannexin inhibitor carbenoxolone nor Bcl-x(L) BH4(4-23) attenuated ATP release in response to incubation of erythrocytes with the ß-adrenergic receptor agonist isoproterenol, suggesting the presence of yet another channel for ATP release from human erythrocytes.

Activation of the permeability transition pore by Bax via inhibition of the mitochondrial BK channel.

Mitochondria are crucially involved in the intrinsic pathway of apoptosis. Upon induction of apoptosis, proapoptotic proteins of the Bcl-2 family, in particular Bax and Bak, transfer the death signal to the organelle. The outcome is release of proapoptotic factors, such as cytochrome c, and mitochondrial changes, such as depolarization. Details of the mechanism by which Bax mediates mitochondrial alterations, however, are unknown. Using the single-channel patch-clamp method, we studied mitoplasts (vesicles of inner membrane) from rat astrocyte and liver mitochondria and intact murine glioma mitochondria to determine the action of proapoptotic Bax and antiapoptotic Bcl-xL on the mitochondrial Ca(2+)-activated channel (mtBK) and the permeability transition pore (mtPTP). Bax (1 nM) inhibited the open probability of the mtBK, whereas Bcl-xL or control proteins had no effect. Incubating mitochondria with iberiotoxin, an inhibitor of mtBK, induced the release of cytochrome c. Bcl-xL inhibited the effects of Bax on mtBK. Furthermore, in patch-clamp studies Bcl-xL inhibited the mtPTP itself, whereas Bax had no direct effect on the mtPTP. We conclude that Bax exerts its proapototic effect by inhibiting mitochondrial K(+) channels, whereas Bcl-xL exerts its antiapoptotic effect by inhibiting the effects of Bax on mitochondrial potassium channels and by direct inhibition of the mtPTP.

An anti-apoptotic peptide improves survival in lethal total body irradiation.

Cell penetrating peptides (CPPs) have been used to deliver the anti-apoptotic Bcl-xL-derived BH4 peptide to prevent injury-induced apoptosis both in vitro and in vivo. Here we demonstrate that the nuclear localization sequence (NLS) from the SV40 large T antigen has favorable properties for BH4 domain delivery to lymphocytes compared to sequences based on the HIV-1 TAT sequence. While both TAT-BH4 and NLS-BH4 protected primary human mononuclear cells from radiation-induced apoptotic cell death, TAT-BH4 caused persistent membrane damage and even cell death at the highest concentrations tested (5-10 microM) and correlated with in vivo toxicity as intravenous administration of TAT-BH4 caused rapid death. The NLS-BH4 peptide has significantly attenuated toxicity compared to TAT-BH4 and we established a dosing regimen of NLS-BH4 that conferred a significant survival advantage in a post-exposure treatment model of LD90 total body irradiation.

[Progress in small-molecule inhibitors of Bcl-2 family proteins].

Apoptosis is an essential factor in keeping homeostasis of the organism. Apoptosis is regulated by a series of cytokines. Bcl-2 family proteins are key regulators of apoptosis. The Bcl-2 family includes both anti- and pro-apoptotic proteins with opposing biological functions. Their interaction regulates the transmission of the apoptosis signal. High expression of anti-apoptotic members such as Bcl-2 and Bcl-xL are commonly found in human cancers. In recent years, following the disclosing of the crystal structures of Bcl-2 family proteins, researchers have paid attention to the development of the small molecule inhibitors of Bcl-2 family proteins. This article reviews the progress in this field from the view of drug design.

Bcl-x(L) inhibits Bax-induced alterations in mitochondrial respiration and calcium release.

Apoptosis is a natural cell elimination process involved in a number of physiological and pathological events. This process can be regulated by members of the Bcl-2 family. Bax, a pro-apoptotic member of this family, accelerates cell death, while the pro-survival member, Bcl-x(L), can antagonize the pro-apoptotic function of Bax to promote cell survival. In the present study, we have evaluated the effect of Bcl-x(L) on Bax-induced alterations in mitochondrial respiration and calcium release. We found that in primary cultured astrocytes, recombinant Bcl-x(L) is able to antagonize Bax-induced decrease in mitochondrial respiration and increase in mitochondrial calcium release. In addition, we found that Bcl-x(L) can lower the calcium store in the endoplasmic reticulum, thus limiting potential calcium flux induced by apoptosis. This regulation of calcium flux by Bcl-x(L) may represent an important mechanism by which this protein promotes cell survival.

Bcl-xL antisense oligonucleotide and cisplatin combination therapy extends survival in SCID mice with established mesothelioma xenografts.

Bcl-xL functions as a dominant regulator of apoptotic cell death and is implicated in chemotherapeutic resistance of malignant pleural mesothelioma (MPM). Mesothelioma cell lines demonstrate increasing levels of Bcl-xL as resistant clones are selected in vitro. Moreover, upon introduction of antisense oligonucleotides specific to Bcl-xL mRNA, MPM cells are sensitized to chemotherapeutic agents. Here we describe the therapeutic effects of a novel combination therapy, Bcl-xL antisense oligonucleotide (ASO 15999) and cisplatin, on mesothelioma cell lines in vitro and in vivo; in addition, efficacy of ASO 15999 in decreasing tumor load as well as its effect on survival in an animal model. Finally, we initiated preliminary toxicity studies involved with intraperitoneal (IP) injections of ASO 15999 into mice. This novel combination, with doses of cisplatin four times below established IC(50) levels, significantly decreased viability of MPM cell lines after 48 hr. The growth of established mouse flank human tumor xenografts was reduced with intra-tumor administration of ASO 15999. Local spread and development of IP xenografts was reduced with treatments of ASO alone, and survival of mice afflicted with these xenografts was prolonged after administration of ASO alone and ASO 15999 + cisplatin combination therapy. These findings suggest that ASO 15999 sensitizes MPM cell lines to the toxic effects of cisplatin. ASO 15999 induced reduction of Bcl-xL is effective in slowing the progression of human mesothelioma cell lines both in vitro and in vivo. More notably, the combination of Bcl-xL ASO and cisplatin extends survival in an orthotopic tumor xenograft model.

Persistent mitochondrial dysfunction and oxidative stress hinder neuronal cell recovery from reversible proteasome inhibition.

Oxidative stress, proteasome impairment and mitochondrial dysfunction are implicated as contributors to ageing and neurodegeneration. Using mouse neuronal cells, we showed previously that the reversible proteasome inhibitor, [N-benzyloxycarbonyl-Ile-Glu (O-t-bytul)-Ala-leucinal; (PSI)] induced excessive reactive oxygen species (ROS) that mediated mitochondrial damage and a caspase-independent cell death. Herein, we examined whether this insult persists in neuronal cells recovering from inhibitor removal over time. Recovery from proteasome inhibition showed a time and dose-dependent cell death that was accompanied by ROS overproduction, caspase activation and mitochondrial membrane permeabilization with the subcellular relocalizations of the proapoptotic proteins, Bax, cytochrome c and the apoptosis inducing factor (AIF). Caspase inhibition failed to promote survival indicating that cell death was caspase-independent. Treatments with the antioxidant N-acetyl-cysteine (NAC) were needed to promote survival in cell recovering from mild proteasome inhibition while overexpression of the antiapoptotic protein Bcl-xL together with NAC attenuated cell death during recovery from potent inhibition. Whereas inhibitor removal increased proteasome function, cells recovering from potent proteasome inhibition showed excessive levels of ubiquitinated proteins that required the presence of NAC for their removal. Collectively, these results suggest that the oxidative stress and mitochondrial inhibition induced by proteasome inhibition persists to influence neuronal cell survival when proteasome function is restored.

GADD153 expression does not necessarily correlate with changes in culture behavior of hybridoma cells.

BACKGROUND: The acute sensitivity of some hybridoma cell lines to culture-related stresses severely limits their productivity. Recent developments in the characterization of the stress signals modulating the cellular phenotype revealed that the pro-apoptotic transcription factor Gadd153 could be used as a marker to facilitate the optimization of mammalian cell cultures. In this report, we analyzed the expression of Gadd153 in Sp2/0-Ag14 murine hybridoma cells grown in stationary batch culture and subjected to two different culture optimization paradigms: L-glutamine supplementation and ectopic expression of Bcl-xL, an anti-apoptotic gene. RESULTS: The expression of Gadd153 was found to increase in Sp2/0-Ag14 cells in a manner which coincided with the decline in cell viability. L-glutamine supplementation prolonged Sp2/0-Ag14 cell survival and greatly suppressed Gadd153 expression both at the mRNA and protein level. However, Gadd153 levels remained low after L-glutamine supplementation even as cell viability declined. Bcl-xL overexpression also extended Sp2/0-Ag14 cell viability, initially delayed the induction of Gadd153, but did not prevent the increase in Gadd153 protein levels during the later phase of the culture, when cell viability was declining. Interestingly, L-glutamine supplementation prevented Gadd153 up-regulation in cells ectopically expressing Bcl-xL, but had no effect on cell viability. CONCLUSION: This study highlights important limitations to the use of Gadd153 as an indicator of cell stress in hybridoma cells.