Glucocorticoids inhibit the apoptotic actions of UV-C but not Fas ligand in hepatoma cells: direct evidence for a critical role of Bcl-xL

Our laboratory has shown that glucocorticoids can inhibit apoptosis in rat hepatoma cells; however, the mechanisms are incompletely understood. To address this issue we sought to determine if glucocorticoid inhibition is effective when death is induced by stimuli that more selectively activate either the intrinsic (UV-C) or extrinsic (FasL) apoptotic pathways. Using flow cytometric analysis, we show that pretreatment of HTC cells with dexamethasone (Dex) inhibits UV-C- but not FasL-induced apoptosis. This inhibition requires Dex pretreatment and can be abrogated by the glucocorticoid antagonist RU486 indicating glucocorticoid receptor-mediated action. Dex increases anti-apoptotic Bcl-x(L) at both mRNA and protein levels. The Bcl-x(L) protein level remains elevated even after apoptosis induction with either UV-C or FasL although only UV-C-induced cell death is inhibited. Repression of Bcl-x(L) protein with siRNA abrogates the anti-apoptotic effect of glucocorticoids. Together these data provide direct evidence that Bcl-x(L) mediates glucocorticoid inhibition of UV-C induced apoptosis.     

Glucocorticoids uncover a critical role for ASH2L on BCL-X expression regulation in leukemia cells

Targeting the apoptosis machinery is a promising therapeutic approach in myeloid malignancies. BCL2L1 is a well-known glucocorticoid-responsive gene and a key apoptosis regulator that, when over-expressed, can contribute to tumor development, progression and therapeutic resistance. Moreover, synthetic glucocorticoids, like dexamethasone, are frequently used in the treatment of hematopoietic diseases due to its pro-apoptotic properties. We report here that the trithorax protein ASH2L, considered one of the core subunits of H3K4-specific MLL/SET methyltransferase complexes, contributes to anti-apoptotic BCL-X_L over-expression and cell survival in patient-derived myeloid leukemia cells. We find that the unliganded glucocorticoid receptor (uGR) and ASH2L interact in a common protein complex through a chromatin looping determined by uGR and ASH2L binding to BCL2L1 specific +58 HRE and promoter region, respectively. Upon addition of dexamethasone, GR and ASH2L recruitment is reduced, BCL-X_L expression diminishes and apoptosis is induced consequently. Overall, our findings indicate that uGR and ASH2L may act as key regulatory players of BCL- X_L upregulation in AML cells.     

Shear stress-dependent expression of apoptosis-regulating genes in endothelial cells

Laminar shear stress exerts potent anti-apoptotic effects. Therefore, we analyzed the influence of laminar shear stress on the expression of apoptosis-regulating genes in human umbilical vein endothelial cells (HUVEC). Application of high levels of laminar shear stress (15 and 30 dyn/cm(2)) decreased the susceptibility of HUVEC to undergo apoptosis, whereas low shear stress (1 dyn/cm(2)) had no effect. These diminished signs of apoptosis were accompanied by a decreased mRNA expression of apoptosis-inducing Fas receptor. Furthermore, mRNA and protein expression of anti-apoptotic, soluble Fas isoform FasExo6Del and anti-apoptotic Bcl-x(L) were induced. Surprisingly, high shear stress also elevated mRNA and protein expression of pro-apoptotic Bak. The shear stress-induced up-regulation of Bcl-x(L) and Bak mRNA can be abrogated by inhibition of the endothelial NO synthase. We propose that altered expression of Bcl-x(L) and the Fas system is involved in the protective effect of laminar shear stress against apoptosis in human endothelial cells.     

Bcl-x(S) can form homodimers and heterodimers and its apoptotic activity requires localization of Bcl-x(S) to the mitochondria and its BH3 and loop domains

Proteins of the Bcl-2 family regulate apoptosis, some antagonizing cell death and others, such as Bcl-x(S), promoting it. We previously showed that expression of Bcl-x(S) in PC12 cells is a useful system for studying the mechanism of Bcl-x(S)-induced apoptosis. To further investigate this apoptotic effect and its prevention by anti-apoptotic agents, we assessed the role of distinct Bcl-x(S) domains, via the study of their mutations, on the ability of Bcl-x(S) to induce apoptosis and to localize to the mitochondria, as well as the ability of these domains to counteract the effects of anti-apoptotic agents on Bcl-x(S). Deletion of the transmembrane domain (DeltaTM) prevented the localization of Bcl-x(S) DeltaTM to the mitochondria and the ability of this mutant to induce apoptosis. Deletion of the amino acids GD 94-95 from the BH3 domain, or deletion of the loop region, impaired the ability of these mutants to induce apoptosis but not their localization to the mitochondria. Deletion of the BH4 domain or destruction of the caspase cleavage site in the loop region (by replacing amino acid D61 with A61) did not affect either the localization of these mutants to the mitochondria or their ability to induce cell death. It thus appears that Bcl-x(S)-induced apoptosis in PC12 cells is mediated by localization of Bcl-x(S) to the mitochondria by a process that requires the transmembrane domain. Furthermore, once localized to the mitochondria Bcl-x(S) requires the BH3 domain, and to a lesser extent the loop domain, for its subsequent activity. The anti-apoptotic agents Bcl-2 and Bcl-x(L), the caspase inhibitor Z-VAD-FMK, and nerve growth factor (NGF) did not prevent Bcl-x(S) localization to the mitochondria, and did not require the BH4 or the loop domains of Bcl-x(S) for their survival effect. Bcl-x(S) is capable of forming homodimers with itself and heterodimers with Bcl-x(L) or Bcl-2. Accordingly co-expression of Bcl-x(S) DeltaTM with Bcl-x(S), Bcl-2, or Bcl-x(L) leads to a change in the subcellular distribution of Bcl-x(S) DeltaTM, from a diffuse distribution throughout the cell to a more defined distribution. Moreover co-immunoprecipitation experiments directly demonstrated that Bcl-x(S) can associate with GFP-Bcl-x(S), Bcl-x(L), or Bcl-2. These results suggest that such Bcl-x(S) interactions may be important for the mechanism of action of this protein.     

Modification of alternative splicing of Bcl-x pre-mRNA in prostate and breast cancer cells. analysis of apoptosis and cell death

There is ample evidence that deregulation of apoptosis results in the development, progression, and/or maintenance of cancer. Since many apoptotic regulatory genes (e.g. bcl-x) code for alternatively spliced protein variants with opposing functions, the manipulation of alternative splicing presents a unique way of regulating the apoptotic response. Here we have targeted oligonucleotides antisense to the 5'-splice site of bcl-x(L), an anti-apoptotic gene that is overexpressed in various cancers, and shifted the splicing pattern of Bcl-x pre-mRNA from Bcl-x(L) to Bcl-x(S), a pro-apoptotic splice variant. This approach induced significant apoptosis in PC-3 prostate cancer cells. In contrast, the same oligonucleotide treatment elicited a much weaker apoptotic response in MCF-7 breast cancer cells. Moreover, although the shift in Bcl-x pre-mRNA splicing inhibited colony formation in both cell lines, this effect was much less pronounced in MCF-7 cells. These differences in responses to oligonucleotide treatment were analyzed in the context of expression of Bcl-x(L), Bcl-x(S), and Bcl-2 proteins. The results indicate that despite the presence of Bcl-x pre-mRNA in a number of cell types, the effects of modification of its splicing by antisense oligonucleotides vary depending on the expression profile of the treated cells.     

Akt and Bcl-xL promote growth factor-independent survival through distinct effects on mitochondrial physiology

A comparison of Akt- and Bcl-x(L)-dependent cell survival was undertaken using interleukin-3-dependent FL5.12 cells. Expression of constitutively active Akt allows cells to survive for prolonged periods following growth factor withdrawal. This survival correlates with the expression level of activated Akt and is comparable in magnitude to the protection provided by the anti-apoptotic gene Bcl-x(L). Although both genes prevent cell death, Akt-protected cells can be distinguished from Bcl-x(L)-protected cells on the basis of increased glucose transporter expression, glycolytic activity, mitochondrial potential, and cell size. In addition, Akt-expressing cells require high levels of extracellular nutrients to support cell survival. In contrast, Bcl-x(L)-expressing cells deprived of interleukin-3 survive in a more vegetative state, in which the cells are smaller, have lower mitochondrial potential, reduced glycolytic activity, and are less dependent on extracellular nutrients. Thus, Akt and Bcl-x(L) suppress mitochondrion-initiated apoptosis by distinct mechanisms. Akt-mediated survival is dependent on promoting glycolysis and maintaining a physiologic mitochondrial potential. In contrast, Bcl-x(L) maintains mitochondrial integrity in the face of a reduced mitochondrial membrane potential, which develops as a result of the low glycolytic rate in growth factor-deprived cells.     

Distinct mechanisms of cardiomyocyte apoptosis induced by doxorubicin and hypoxia converge on mitochondria and are inhibited by Bcl-xL

Hypoxia and doxorubicin can cause cardiotoxicity and loss of myocardial function. These effects are due, in part, to an induction of apoptosis. Herein we identify the apoptotic pathways activated in H9c2 cells in response to hypoxia (O(2)/N(2)/CO(2), 0.5:94.5:5) and doxorubicin (0.5 muM). Although the apoptosis induced was accompanied by induction of Fas and Fas ligand, the death receptor pathway was not critical for caspase activation by either stimulus. Hypoxia induced the expression of endoplasmic reticulum (ER) stress mediators and processed ER-resident pro-caspase-12 whereas doxorubicin did not induce an ER stress response. Most importantly, both stimuli converged on mitochondria to promote apoptosis. Accumulation of cytochrome c in the cytosol coincided with the processing of pro-caspase-9 and -3. Increasing the expression of the anti-apoptotic protein Bcl-x(L), either by dexamethasone or adenovirus-mediated transduction, protected H9c2 cells from doxorubicin- and hypoxia-induced apoptosis. Bcl-x(L) attenuated mitochondrial cytochrome crelease and reduced downstream pro-caspase processing and apoptosis. These data demonstrate that two distinct cardiomyocyte-damaging stimuli converge on mitochondria thus presenting this organelle as a potentially important therapeutic target for anti-apoptotic strategies for cardiovascular diseases.     

A comparison of the properties of a Bcl-xL variant to the wild-type anti-apoptosis inhibitor in mammalian cell cultures

The overexpression of bcl-2 and its homologues is a widely used strategy to inhibit apoptosis in mammalian cell culture systems. In this study, we have evaluated the Bcl-2 homologue, Bcl-x(L) and compared its effectiveness to a Bcl-x(L) mutant lacking most of the non-conserved unstructured loop domain, Bcl-x(L)Delta (deletion of amino acids 26 through 83). The cell line, Chinese hamster ovary (CHO), was genetically modified to express constitutively Bcl-x(L) or the Bcl-x(L) variant and subjected to model apoptotic insults including Sindbis virus (SV) infection, gradual serum withdrawal, and serum deprivation. When cells were engineered to overexpress Bcl-x(L)Delta, cell death due to the SV was inhibited, and Bcl-x(L)Delta provided comparable protection to the wild-type Bcl-x(L) even though expression levels were much lower for the mutant. Furthermore, the cells expressing Bcl-x(L)Delta continued to proliferate following infection while CHO-bcl-x(L) ceased proliferation immediately following infection. As a result, total production of a heterologous protein encoded on the SV was highest in cell lines expressing Bcl-x(L)Delta. Cells expressing the variant Bcl-x(L) also continued to proliferate and showed increased viable cell numbers following gradual serum withdrawal. In contrast, wild-type Bcl-x(L) expressing CHO cells were found to arrest growth but maintain viability following serum withdrawal. Interestingly, CHO cells expressing Bcl-x(L)Delta were also able to recover and return to rapid growth rates much faster than either the wild-type CHO-bcl-x(L) or CHO following the replenishment of fresh complete medium containing 10% FBS. Confocal imaging of yellow fluorescent protein (YFP) fused to the N terminus of Bcl-x(L) and Bcl-x(L)Delta indicated dense aggregates of the Bcl-x(L)Delta while the wild-type protein was distributed throughout the cell in a manner resembling transmembrane localization. As an alternative to complete removal of the loop domain, Bcl-x(L) variants were created in which aspartate residues containing potential caspase recognition sites within the loop domain of Bcl-x(L) were removed. Cell populations expressing various Bcl-x(L)-Asp mutants were exposed to an apoptotic spent medium stimulus, and the cells expressing these Bcl-x(L) variants provided increased viabilities as compared to cells containing wild-type Bcl-x(L) protein. These studies indicate that modification of anti-apoptotic genes can affect multiple cellular properties including response to apoptotic stimuli and cell growth. This knowledge can be valuable in the design of improved apoptosis inhibitors for biotechnology applications.     

Overexpressed Bcl-x(L) prevents bacterial superantigen-induced apoptosis of thymocytes in vitro

bcl-x, a homologous gene of bcl-2, has an anti-apoptotic function and appears to play a critical role in the development of lymphoid systems. To investigate the effect of overexpressed Bcl-x(L) on the development of T lymphocytes, we established two lines of transgenic mice by using Emu-chicken bcl-x(L) (cbcl-x(L)) transgene, where the cBcl-x(L) protein was expressed mainly in lymphoid cells. Although thymocytes and splenocytes from cbcl-x(L) transgenic mice are resistant to apoptosis in vitro, clonal deletion of thymocytes, recognizing endogenous self-superantigens in the thymus, still normally proceeded and no self-reactive T cells were found in the spleen of the transgenic mice. To dissect clonal deletion, we utilized two in vitro models, thymocytes/antigen presenting cells co-culture system and fetal thymus organ culture system. In both, bacterial superantigen staphylococcus aureus enterotoxin B (SEB) induces apoptosis of T cells with Vbeta8+ T cell receptor (TCR) reacting to SEB, which mimics clonal deletion of self-reactive thymocytes in vivo. SEB-induced depletion of Vbeta8+ T cells from thymocytes when taken from the transgenic mice was effectively inhibited. The data might raise the possibility that cell death process involved in clonal deletion in the thymus is a form of apoptosis inhibited by Bcl-x(L).     

The super anti-apoptotic factor Bcl-xFNK constructed by disturbing intramolecular polar interactions in rat Bcl-xL

A powerful artificial anti-apoptotic factor will be useful for medical applications of the future therapies for many diseases by prolonging survival of sick cells. For constructing it, we designed the super anti-apoptotic factor by disturbing three intramolecular polar interactions among alpha-helix structures of Bcl-x(L). The resultant mutant Bcl-x(L), named Bcl-xFNK, was expected to make the pore-forming domain more mobile and flexible than the wild-type. When overexpressed in Jurkat cells, Bcl-xFNK was markedly more potent in prolonging survival following apoptosis-inducing treatment with a kind of cell death cytokines (anti-Fas), a protein kinase inhibitor (staurosporine), cell cycle inhibitors (TN-16, camptothecin, hydroxyurea, and trichostatin A), or oxidative stress (hydrogen peroxide and paraquat) than wild-type Bcl-x(L). Furthermore, the transfectants of bcl-xFNK became more resistant against a calcium ionophore and even a heat treatment than wild-type Bcl-x(L). In addition, Bcl-xFNK showed marked anti-apoptotic activity in Chinese hamster ovary and Jurkat cells deprived of serum. Thus, Bcl-xFNK may be the first mutant generated by site-directed mutagenesis of Bcl-x(L) with a gain-of-function phenotype. Interestingly, Bcl-xFNK was found to allow interleukin-3-dependent FDC-P1 to grow without interleukin-3, but not BaF/3. In Bcl-xFNK transfectants of FDC-P1 and Jurkat, the p42/p44 mitogen-activated protein kinase was activated by 2 to 5 times, but not in those of BaF/3 and Chinese hamster ovary. Bcl-xFNK might gain a new function to activate the mitogen-activated protein kinase in a cell-type specific manner. The findings of this study suggest that the central alpha5-alpha6 pore-forming region of anti-apoptotic factor Bcl-x(L) has a pivotal role in suppressing apoptosis.     

Establishment and characterization of conditions required for tumor colonization by intravenously delivered bacteria

Transient gene expression (TGE) provides a method for quickly delivering protein for research using mammalian cells. While high levels of recombinant proteins have been produced in TGE experiments in HEK 293 cells, TGE efforts in the commercially prominent CHO cell line still suffer from inadequate protein yields. Here, we describe a cell-engineering strategy to improve transient production of proteins using CHO cells. CHO-DG44 cells were engineered to overexpress the anti-apoptotic protein Bcl-x(L) and transiently transfected using polyethylenimine (PEI) in serum-free media. Pools and cell lines stably expressing Bcl-x(L) showed enhanced viable cell density and increased production of a glycosylated, therapeutic fusion protein in shake flask TGE studies. The improved cell lines showed fusion protein production levels ranging from 12.6 to 27.0 mg/L in the supernatant compared to the control cultures which produced 6.3-7.3 mg/L, representing a 70-270% increase in yield after 14 days of fed-batch culture. All Bcl-xL-expressing cell lines also exhibited an increase in specific productivity during the first 8 days of culture. In addition to increased production, Bcl-x(L) cell lines maintained viabilities above 90% and less apoptosis compared to the DG44 host which had viabilities below 60% after 14 days. Product quality was comparable between a Bcl-xL-engineered cell line and the CHO host. The work presented here provides the foundation for using anti-apoptosis engineered CHO cell lines for increased production of therapeutic proteins in TGE applications.     

Anti-apoptotic Bcl-2 Family Proteins Disassemble Ceramide Channels

Early in mitochondria-mediated apoptosis, the mitochondrial outer membrane becomes permeable to proteins that, when released into the cytosol, initiate the execution phase of apoptosis. Proteins in the Bcl-2 family regulate this permeabilization, but the molecular composition of the mitochondrial outer membrane pore is under debate. We reported previously that at physiologically relevant levels, ceramides form stable channels in mitochondrial outer membranes capable of passing the largest proteins known to exit mitochondria during apoptosis (Siskind, L. J., Kolesnick, R. N., and Colombini, M. (2006) Mitochondrion 6, 118-125). Here we show that Bcl-2 proteins are not required for ceramide to form protein-permeable channels in mitochondrial outer membranes. However, both recombinant human Bcl-x(L) and CED-9, the Caenorhabditis elegans Bcl-2 homologue, disassemble ceramide channels in the mitochondrial outer membranes of isolated mitochondria from rat liver and yeast. Importantly, Bcl-x L and CED-9 disassemble ceramide channels in the defined system of solvent-free planar phospholipid membranes. Thus, ceramide channel disassembly likely results from direct interaction with these anti-apoptotic proteins. Mutants of Bcl-x L act on ceramide channels as expected from their ability to be anti-apoptotic. Thus, ceramide channels may be one mechanism for releasing pro-apoptotic proteins from mitochondria during the induction phase of apoptosis.     

Growth factors inactivate the cell death promoter BAD by phosphorylation of its BH3 domain on Ser155

The Bcl-2 family protein BAD promotes apoptosis by binding through its BH3 domain to Bcl-x(L) and related cell death suppressors. When BAD is phosphorylated on either Ser(112) or Ser(136), it forms a complex with 14-3-3 in the cytosol and no longer interacts with Bcl-x(L) at the mitochondria. Here we show that phosphorylation of a distinct site Ser(155), which is at the center of the BAD BH3 domain, directly suppressed the pro-apoptotic function of BAD by eliminating its affinity for Bcl-x(L). Protein kinase A functioned as a BAD Ser(155) kinase both in vitro and in cells. BAD Ser(155) was found to be a major site of phosphorylation induced following stimulation by growth factors and prevented by protein kinase A inhibitors but not by inhibitors of the phosphatidylinositol 3-kinase/Akt pathway. Growth factors inhibited BAD-induced apoptosis in both a Ser(112)/Ser(136)- and a Ser(155)-dependent fashion. Thus, growth factors engage an anti-apoptotic signaling pathway that inactivates BAD by direct modification of its BH3 cell death effector domain.     

Hepatocyte growth factor induces GATA-4 phosphorylation and cell survival in cardiac muscle cells

Hepatocyte growth factor (HGF) is released in response to myocardial infarction and may play a role in regulating cardiac remodeling. Recently, HGF was found to inhibit the apoptosis of cardiac muscle cells. Because GATA-4 can induce cell survival, the effects of HGF on GATA-4 activity were investigated. Treatment of HL-1 cells or primary adult rat cardiac myocytes with HGF, at concentrations that can be detected in the human serum after myocardial infarction, rapidly enhances GATA-4 DNA-binding activity. The enhanced DNA-binding activity is associated with the phosphorylation of GATA-4. HGF-induced phosphorylation and activation of GATA-4 is abolished by MEK inhibitors or the mutation of the ERK phosphorylation site (S105A), suggesting that HGF activates GATA-4 via MEK-ERK pathway-dependent phosphorylation. HGF enhances the expression of anti-apoptotic Bcl-x(L), and this is blocked by dominant negative mutants of MEK or GATA-4. Forced expression of wild-type GATA-4, but not the GATA-4 mutant (S105A) increases the expression of Bcl-x(L). Furthermore, expression of the GATA-4 mutant (S105A) suppresses HGF-mediated protection of cells against daunorubicin-induced apoptosis. These results demonstrate that HGF protects cardiac muscle cells against apoptosis via a signaling pathway involving MEK/ERK-dependent phosphorylation of GATA-4.     

The ability of Bcl-x(L) and Bcl-2 to prevent apoptosis can be differentially regulated

Although expression of Bcl-2 has been shown to prevent apoptosis under many circumstances, there are several systems in which Bcl-2 fails to promote cell survival. We have previously reported that Bcl-2 and Bcl-x(L) display differential ability to protect WEHI-231 cells from multiple inducers of apoptosis. A possible explanation for this paradox was provided by the discovery of Bax. Bax is a Bcl-2-related protein which can inhibit the ability of Bcl-2 to enhance the survival of growth factor-dependent cell lines in the absence of growth factor. Consistent with the possibility that Bcl-2 function in WEHI-231 cells is inhibited by Bax, WEHI-231 cells were found to express a high level of Bax. To directly test the effects of Bax expression on Bcl-x(L) function, FL5.12 cells were transfected with both genes. Although Bax overexpression can inhibit Bcl-2 from prolonging cell survival upon growth factor withdrawal, Bax overexpression did not inhibit Bcl-x(L) from preventing apoptosis in this cell line. Although Bcl-2 and Bcl-x(L) were both found to be able to form heterodimers with Bax, the majority of Bax in both cases was not complexed to a partner. Our data suggest that Bcl-x(L) does not function by simply preventing the formation of Bax homodimers which promote cell death. Instead Bax appears to display selectivity in its ability to inhibit Bcl-2 but not Bcl-x(L) from prolonging survival. Furthermore, our data suggest that the abilities of Bcl-2 and Bcl-x(L) to promote cell survival are not identical and can be independently regulated within a cell. Regulation of a cell's apoptotic threshold is likely to result from a complex set of interactions among Bcl-2 family members and other, as yet uncharacterised, regulators of apoptosis.     

Resistance to TGF-beta-induced apoptosis in regenerating hepatocytes

Treatment with transforming growth factor beta (TGF-beta) of hepatocytes from two different proliferative conditions, such as fetal development and adult liver regeneration, shows that regenerating cells respond to this cytokine in terms of growth inhibition, but are less sensitive than the fetal ones to the apoptosis induced by this factor. Regenerating TGF-beta treated cells show higher cell viability and lower percentage of apoptotic cells than the fetal treated ones. Furthermore, TGF-beta treated regenerating hepatocytes maintain a well-preserved parenchyma-like organization. Treatment with TGF-beta induces the loss of mitochondrial transmembrane potential in fetal but not in regenerating hepatocytes and activation of caspase-3 is lower in regenerating than in fetal cells. Regenerating hepatocytes show higher intracellular levels of some antiapoptotic proteins, such as Bcl-x(L) and c-IAP-1 and, interestingly, they present higher intracellular glutathione levels, which might provide of mechanisms to avoid potential dangerous effects of the oxidative stress-mediated apoptosis induced by TGF-beta. In fact, treatment with BSO (a glutathione synthesis inhibitor) restores the response of regenerating hepatocytes to TGF-beta in terms of cell death. In conclusion, increased levels of Bcl-x(L) and cIAP-1 and higher intracellular glutathione levels could confer resistance to the apoptosis induced by TGF-beta during liver regeneration.