Bcl-2 is a better ABT-737 target than Bcl-xL or Bcl-w and only Noxa overcomes resistance mediated by Mcl-1, Bfl-1, or Bcl-B

The novel anticancer drug ABT-737 is a Bcl-2 Homology 3 (BH3)-mimetic that induces apoptosis by inhibiting pro-survival Bcl-2 proteins. ABT-737 binds with equal affinity to Bcl-2, Bcl-xL and Bcl-w in vitro and is expected to overrule apoptosis resistance mediated by these Bcl-2 proteins in equal measure. We have profiled ABT-737 specificity for all six pro-survival Bcl-2 proteins, in p53 wild-type or p53-mutant human T-leukemic cells. Bcl-B was untargeted, like Bfl-1 and Mcl-1, in accord with their low affinity for ABT-737 in vitro. However, Bcl-2 proved a better ABT-737 target than Bcl-xL and Bcl-w. This was reflected in differential apoptosis-sensitivity to ABT-737 alone, or combined with etoposide. ABT-737 was not equally effective in displacing BH3-only proteins or Bax from Bcl-2, as compared with Bcl-xL or Bcl-w, offering an explanation for the differential ABT-737 sensitivity of tumor cells overexpressing these proteins. Inducible expression demonstrated that BH3-only proteins Noxa, but not Bim, Puma or truncated Bid could overrule ABT-737 resistance conferred by Bcl-B, Bfl-1 or Mcl-1. These data identify Bcl-B, Bfl-1 and Mcl-1, but also Bcl-xL and Bcl-w as potential mediators of ABT-737 resistance and indicate that target proteins can be differentially sensitive to BH3-mimetics, depending on the pro-apoptotic Bcl-2 proteins they are complexed with.     

Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function

Apoptosis is initiated when Bcl-2 and its prosurvival relatives are engaged by proapoptotic BH3-only proteins via interaction of its BH3 domain with a groove on the Bcl-2-like proteins. These interactions have been considered promiscuous, but our analysis of the affinity of eight BH3 peptides for five Bcl-2-like proteins has revealed that the interactions vary over 10,000-fold in affinity, and accordingly, only certain protein pairs associate inside cells. Bim and Puma potently engaged all the prosurvival proteins comparably. Bad, however, bound tightly to Bcl-2, Bcl-xL, and Bcl-w but only weakly to A1 and not to Mcl-1. Strikingly, Noxa bound only Mcl-1 and A1. In accord with their complementary binding, Bad and Noxa cooperated to induce potent killing. The results suggest that apoptosis relies on selective interactions between particular subsets of these proteins and that it should be feasible to discover BH3-mimetic drugs that inactivate specific prosurvival targets.     

Beyond Cell Death – Antiapoptotic Bcl-2 Proteins Regulate Migration and Invasion of Colorectal Cancer Cells In Vitro

Migration and invasion of malignant cells are prerequisites for cancer progression and metastasis. The Bcl-2 family of proteins consists of about 25 members and has been extensively studied in the context of apoptosis. Despite the fact that small molecules targeting Bcl-2 proteins have already entered clinical trials, very few studies investigated a role of antiapoptotic Bcl-2 proteins beside cell death in the context of metastasis. The aim of this study was to dissect a potential role of the antiapoptotic Bcl-2 proteins Mcl-1, Bcl-2 and Bcl-x_L on migration and invasion of colorectal cancer cells independent of their cell death control function. We used migration and invasion assays as well as three dimensional cell cultures to analyze colorectal cancer cell lines (HT29 and SW480) after siRNA mediated knockdown or overexpression of Mcl-1, Bcl-2 or Bcl-x_L. We observed neither spontaneous cell death induction nor impaired proliferation of cells lacking Mcl-1, Bcl-2 or Bcl-x_L. In contrast, knockdown of Mcl-1 led to increased proliferation. Strikingly, we demonstrate a profound impairment of both, migration and invasion, of colorectal cancer cells after Mcl-1, Bcl-2 or Bcl-x_L knockdown. This phenotype was completely revised in cells overexpressing Mcl-1, Bcl-2 or Bcl-x_L. The most pronounced effect among the investigated proteins was observed for Bcl-2. The data presented indicate a pivotal role of Mcl-1, Bcl-2 and Bcl-x_L for migration and invasion of colorectal cancer cells independent of their known antiapoptotic effects. Thus, our study illustrates novel antitumoral mechanisms of Bcl-2 protein targeting.     

Noxa/Bcl-2 protein interactions contribute to bortezomib resistance in human lymphoid cells

Previous studies have suggested that the BH3 domain of the proapoptotic Bcl-2 family member Noxa only interacts with the anti-apoptotic proteins Mcl-1 and A1 but not Bcl-2. In view of the similarity of the BH3 binding domains of these anti-apoptotic proteins as well as recent evidence that studies of isolated BH3 domains can potentially underestimate the binding between full-length Bcl-2 family members, we examined the interaction of full-length human Noxa with anti-apoptotic human Bcl-2 family members. Surface plasmon resonance using bacterially expressed proteins demonstrated that Noxa binds with mean dissociation constants (K(D)) of 3.4 nm for Mcl-1, 70 nm for Bcl-x(L), and 250 nm for wild type human Bcl-2, demonstrating selectivity but not absolute specificity of Noxa for Mcl-1. Further analysis showed that the Noxa/Bcl-2 interaction reflected binding between the Noxa BH3 domain and the Bcl-2 BH3 binding groove. Analysis of proteins expressed in vivo demonstrated that Noxa and Bcl-2 can be pulled down together from a variety of cells. Moreover, when compared with wild type Bcl-2, certain lymphoma-derived Bcl-2 mutants bound Noxa up to 20-fold more tightly in vitro, pulled down more Noxa from cells, and protected cells against killing by transfected Noxa to a greater extent. When killing by bortezomib (an agent whose cytotoxicity in Jurkat T-cell leukemia cells is dependent on Noxa) was examined, apoptosis was enhanced by the Bcl-2/Bcl-x(L) antagonist ABT-737 or by Bcl-2 down-regulation and diminished by Bcl-2 overexpression. Collectively, these observations not only establish the ability of Noxa and Bcl-2 to interact but also identify Bcl-2 overexpression as a potential mechanism of bortezomib resistance.     

A1/Bfl-1 in leukocyte development and cell death

The function of the anti-apoptotic Bcl-2 family member Bcl2a1/Bfl-1/A1 is poorly understood due to the lack of appropriate loss-of-function mouse models and redundant effects with other Bcl-2 pro-survival proteins upon overexpression. Expression analysis of A1 suggests predominant roles in leukocyte development, their survival upon viral or bacterial infection, as well as during allergic reactions. In addition, A1 has been implicated in autoimmunity and the pathology and therapy resistance of hematological as well as solid tumors that may aberrantly express this protein. In this review, we aim to summarize current knowledge on A1 biology, focusing on its role in the immune system and compare it to that of other pro-survival Bcl-2 proteins.     

Structural biology of the Bcl-2 family and its mimicry by viral proteins

Intrinsic apoptosis in mammals is regulated by protein–protein interactions among the B-cell lymphoma-2 (Bcl-2) family. The sequences, structures and binding specificity between pro-survival Bcl-2 proteins and their pro-apoptotic Bcl-2 homology 3 motif only (BH3-only) protein antagonists are now well understood. In contrast, our understanding of the mode of action of Bax and Bak, the two necessary proteins for apoptosis is incomplete. Bax and Bak are isostructural with pro-survival Bcl-2 proteins and also interact with BH3-only proteins, albeit weakly. Two sites have been identified; the in-groove interaction analogous to the pro-survival BH3-only interaction and a site on the opposite molecular face. Interaction of Bax or Bak with activator BH3-only proteins and mitochondrial membranes triggers a series of ill-defined conformational changes initiating their oligomerization and mitochondrial outer membrane permeabilization. Many actions of the mammalian pro-survival Bcl-2 family are mimicked by viruses. By expressing proteins mimicking mammalian pro-survival Bcl-2 family proteins, viruses neutralize death-inducing members of the Bcl-2 family and evade host cell apoptosis during replication. Remarkably, structural elements are preserved in viral Bcl-2 proteins even though there is in many cases little discernible sequence conservation with their mammalian counterparts. Some viral Bcl-2 proteins are dimeric, but they have distinct structures to those observed for mammalian Bcl-2 proteins. Furthermore, viral Bcl-2 proteins modulate innate immune responses regulated by NF-κB through an interface separate from the canonical BH3-binding groove. Our increasing structural understanding of the viral Bcl-2 proteins is leading to new insights in the cellular Bcl-2 network by exploring potential alternate functional modes in the cellular context. We compare the cellular and viral Bcl-2 proteins and discuss how alterations in their structure, sequence and binding specificity lead to differences in behavior, and together with the intrinsic structural plasticity in the Bcl-2 fold enable exquisite control over critical cellular signaling pathways.     

Selective induction of cell death in melanoma cell lines through targeting of Mcl-1 and A1

Melanoma is an often fatal form of skin cancer which is remarkably resistant against radio- and chemotherapy. Even new strategies that target RAS/RAF signaling and display unprecedented efficacy are characterized by resistance mechanisms. The targeting of survival pathways would be an attractive alternative strategy, if tumor-specific cell death can be achieved. Bcl-2 proteins play a central role in regulating survival of tumor cells. In this study, we systematically investigated the relevance of antiapoptotic Bcl-2 proteins, i.e., Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and A1, in melanoma cell lines and non-malignant cells using RNAi. We found that melanoma cells required the presence of specific antiapoptotic Bcl-2 proteins: Inhibition of Mcl-1 and A1 strongly induced cell death in some melanoma cell lines, whereas non-malignant cells, i.e., primary human fibroblasts or keratinocytes were not affected. This specific sensitivity of melanoma cells was further enhanced by the combined inhibition of Mcl-1 and A1 and resulted in 60% to 80% cell death in all melanoma cell lines tested. This treatment was successfully combined with chemotherapy, which killed a substantial proportion of cells that survived Mcl-1 and A1 inhibition. Together, these results identify antiapoptotic proteins on which specifically melanoma cells rely on and, thus, provide a basis for the development of new Bcl-2 protein-targeting therapies.     

Isolation of Bcl-2 binding proteins that exhibit homology with BAG-1 and suppressor of death domains protein

The Bcl-2 oncoprotein is a potent inhibitor of apoptosis and is overexpressed in a variety of different malignancies. Bcl-2 function is regulated through heterodimerization with other members of the Bcl-2 protein family. In addition, several proteins that are not members of the Bcl-2 family can bind to Bcl-2, including BAG-1 protein. In this study, we screened for proteins that bind to Bcl-2, and isolated two additional members of the BAG-1 protein family, BAG-3 and BAG-4. The BAG-4 protein that we cloned also corresponds to the recently isolated suppressor of death domains (SODD) protein, a molecule that binds and inhibits signaling by tumor necrosis factor receptor 1 (TNFR1). Both BAG-3 and BAG-4/SODD were found to physically associate with Bcl-2, and both proteins are well conserved from human to mouse. A region of homology, comprising 68 amino acids, is present in the carboxyl termini of BAG-3 and BAG-4/SODD, and this region corresponds with sequences termed BAG domains that are found in other members of the BAG-1 protein family. In BAG-3 and BAG-4/SODD, the BAG domains appear to constitute the Bcl-2 binding regions of these molecules. BAG-3 and BAG-4/SODD, like BAG-1, were also shown to bind to Hsp70 inside the cell. Moreover, BAG-3 overexpression modestly inhibited apoptosis resulting from cytokine deprivation of IL-3-dependent 32D cells. Together, our findings demonstrate that other members of the BAG-1 protein family, namely BAG-3 and BAG-4/SODD, bind to Bcl-2 and provide a potential link between pathways regulated by Bcl-2 and pathways regulated by Hsp70, as well as TNFR1.     

Vaccinia virus proteins A52 and B14 Share a Bcl-2-like fold but have evolved to inhibit NF-kappaB rather than apoptosis

Vaccinia virus (VACV), the prototype poxvirus, encodes numerous proteins that modulate the host response to infection. Two such proteins, B14 and A52, act inside infected cells to inhibit activation of NF-kappaB, thereby blocking the production of pro-inflammatory cytokines. We have solved the crystal structures of A52 and B14 at 1.9 A and 2.7 A resolution, respectively. Strikingly, both these proteins adopt a Bcl-2-like fold despite sharing no significant sequence similarity with other viral or cellular Bcl-2-like proteins. Unlike cellular and viral Bcl-2-like proteins described previously, A52 and B14 lack a surface groove for binding BH3 peptides from pro-apoptotic Bcl-2-like proteins and they do not modulate apoptosis. Structure-based phylogenetic analysis of 32 cellular and viral Bcl-2-like protein structures reveals that A52 and B14 are more closely related to each other and to VACV N1 and myxoma virus M11 than they are to other viral or cellular Bcl-2-like proteins. This suggests that a progenitor poxvirus acquired a gene encoding a Bcl-2-like protein and, over the course of evolution, gene duplication events have allowed the virus to exploit this Bcl-2 scaffold for interfering with distinct host signalling pathways.     

Critical role of anti-apoptotic Bcl-2 protein phosphorylation in mitotic death

Microtubule inhibiting agents (MIAs) characteristically induce phosphorylation of the major anti-apoptotic Bcl-2 family members Mcl-1, Bcl-2 and Bcl-xL, and although this leads to Mcl-1 degradation, the role of Bcl-2/Bcl-xL phosphorylation in mitotic death has remained controversial. This is in part due to variation in MIA sensitivity among cancer cell lines, the dependency of cell fate on drug concentration and uncertainty about the modes of cell death occurring, thus making comparisons of published reports difficult. To circumvent problems associated with MIAs, we used siRNA knockdown of the anaphase-promoting complex activator, Cdc20, as a defined molecular system to investigate the role, specifically in mitotic death, of individual anti-apoptotic Bcl-2 proteins and their phosphorylated forms. We show that Cdc20 knockdown in HeLa cells induces mitotic arrest and subsequent mitotic death. Knockdown of Cdc20 in HeLa cells stably overexpressing untagged wild-type Bcl-2, Bcl-xL or Mcl-1 promoted phosphorylation of the overexpressed proteins in parallel with their endogenous counterparts. Overexpression of Bcl-2 or Bcl-xL blocked mitotic death induced by Cdc20 knockdown; phospho-defective mutants were more protective than wild-type proteins, and phospho-mimic Bcl-xL was unable to block mitotic death. Overexpressed Mcl-1 failed to protect from Cdc20 siRNA-mediated death, as the overexpressed protein was susceptible to degradation similar to endogenous Mcl-1. These results provide compelling evidence that phosphorylation of anti-apoptotic Bcl-2 proteins has a critical role in regulation of mitotic death. These findings make an important contribution toward our understanding of the molecular mechanisms of action of MIAs, which is critical for their rational use clinically.     

Amphipathic tail-anchoring peptide and Bcl-2 homology domain-3 (BH3) peptides from Bcl-2 family proteins induce apoptosis through different mechanisms

Bcl-2 homology domain-3 (BH3) peptides are potent cancer therapeutic reagents that target regulators of apoptotic cell death in cancer cells. However, their cytotoxic effects are affected by different expression levels of Bcl-2 family proteins. We recently found that the amphipathic tail-anchoring peptide (ATAP) from Bfl-1, a bifunctional Bcl-2 family member, produced strong pro-apoptotic activity by permeabilizing the mitochondrial outer membrane. Here, we test whether the activity of ATAP requires other cellular factors and whether ATAP has an advantage over the BH3 peptides in targeting cancer cells. Confocal microscopic imaging illustrates specific targeting of ATAP to mitochondria, whereas BH3 peptides show diffuse patterns of cytosolic distribution. Although the pro-apoptotic activities of BH3 peptides are largely inhibited by either overexpression of anti-apoptotic Bcl-2 or Bcl-xL or nullification of pro-apoptotic Bax and Bak in cells, the pro-apoptotic function of ATAP is not affected by these cellular factors. Reconstitution of synthetic ATAP into liposomal membranes results in release of fluorescent molecules of the size of cytochrome c from the liposomes, suggesting that the membrane permeabilizing activity of ATAP does not require additional protein factors. Because ATAP can target to the mitochondrial membrane and its pro-apoptotic activity does not depend on the content of Bcl-2 family proteins, it represents a promising candidate for anti-cancer drugs that can potentially overcome the intrinsic apoptosis-resistant nature of cancer cells.     

Anti-apoptotic defense of bcl-2 gene against hydroperoxide-induced cytotoxicity together with suppressed lipid peroxidation,enhanced ascorbate uptake,and upregulated Bcl-2 protein

Although it is well known that Bcl-2 can prevent apoptosis, the Bcl-2's anti-apoptotic mechanism is not fully understood. Here, we investigate the mechanism of oxidant-induced cell death and to investigate the role of Bcl-2 in the tert-butyl hydroperoxide (t-BuOOH)-induced oxidant injury in Rat-1 fibroblasts and their bcl-2 transfected counterparts, b5 cells. Treatment with t-BuOOH causes mitochondrial disfunction and induced morphological features consistent with apoptosis more markedly in Rat-1 cells than in b5 cells. The hydroperoxide t-BuOOH at concentrations less than 100 nM for as long as 48 h or with higher concentrations (up to 100 microM) for only 3 h induces death in Rat-1 cells, whereas their bcl-2 transfectants were significantly resistant to cytotoxicity by both time and all concentration other than 100 microM. The similar results were obtained also for DNA strand cleavages as detected by TUNEL stain. The bcl-2 transfectants significantly suppressed t-BuOOH-induced increases in both lipid peroxidation and caspase-3 activation 3 and 1 h after t-BuOOH exposure, respectively, but failed to suppress either caspase-1 activation or an enhanced production of the intracellular reactive oxygen species (ROS). Intracellular uptake of [1-(14)C] ascorbic acid (Asc) into the bcl-2 transfectants was superior to that into the non-transfectants always under examined conditions regardless of serum addition to culture medium and cell density. Upregulation of Bcl-2 proteins was rapidly induced after t-BuOOH exposure in the transfectants, but not in non-transfectants, and restored till 24 h to the normal Bcl-2 level. Thus suppressions of both lipid peroxidation and the subsequent cell death events such as caspase-3 activation and DNA cleavage were concerned with the inhibitory effects of Bcl-2 on the t-BuOOH-induced cytotoxicity. And some of these events may correlate with Bcl-2 expression-induced partial enhanced anti-oxidant cellular ability including enrichment of intracellular Asc and oxidative stress-induced upregulation of Bcl-2 protein. On the other hand, ROS production and caspase-1 activation were not related to cytoprotection by Bcl-2.     

A1/Bfl-1 expression is restricted to TCR engagement in T lymphocytes

We analyzed regulation of the prosurvival Bcl-2 homologue A1, following T-cell receptor (TCR) or cytokine receptor engagement. Activation of CD4(+) or CD8(+) T cells by antigenic peptides induced an early but transient IL-2-independent expression of A1 and Bcl-xl mRNA and proteins, whereas expression of Bcl-2 was delayed and required IL-2. Cytokines such as IL-2, IL-4, IL-7 or IL-15 prevented apoptosis of activated T cells that effect being associated with the maintenance of Bcl-2, but not of A1 expression. However, restimulation of activated or posteffector T cells with antigenic peptide strongly upregulated A1 mRNA and maintained A1 protein expression. IL-4, IL-7 or IL-15 also prevented cell death of naive T cells. In those cells, cytokines upregulated Bcl-2, but not A1 expression. Therefore, in naive, activated and posteffector T cells, expression of A1 is dependent on TCR but not on cytokine receptor engagement, indicating that A1 is differently regulated from Bcl-xl and Bcl-2.     

Bax alpha perturbs T cell development and affects cell cycle entry of T cells.

Bax alpha can heterodimerize with Bcl-2 and Bcl-X(L), countering their effects, as well as promoting apoptosis on overexpression. We show that bax alpha transgenic mice have greatly reduced numbers of mature T cells, which results from an impaired positive selection in the thymus. This perturbation in positive selection is accompanied by an increase in the number of cycling thymocytes. Further to this, mature T cells overexpressing Bax alpha have lower levels of p27Kip1 and enter S phase more rapidly in response to interleukin-2 stimulation than do control T cells, while the converse is true of bcl-2 transgenic T cells. These data indicate that apoptotic regulatory proteins can modulate the level of cell cycle-controlling proteins and thereby directly impact on the cell cycle.     

High-yield expression and purification of soluble forms of the anti-apoptotic Bcl-x(L) and Bcl-2 as TolAIII-fusion proteins

A method is presented to produce large amounts of Bcl-2 and Bcl-x_L, two anti-apoptotic proteins of considerable biomedical interest. Expression constructs were prepared in which the Escherichia coli protein TolAIII, known to promote over expression of soluble product, was added to the N-terminus of Bcl-2 or Bcl-x_L proteins, which had their C-terminal hydrophobic anchors deleted. Here the expression of these TolAIII-fusion constructs, followed by a two-step metal-affinity based purification protocol is described. The method delivers at least 20 and 10 mg of more than 90% pure TolAIII-Bcl-x_LΔC and TolAIII-Bcl-2(2)ΔC proteins, respectively, per liter of E. coli cell culture. The proteins are released by proteolysis with thrombin providing >12 mg of Bcl-x_LΔC or >6 mg of Bcl-2(2)ΔC per liter of E. coli cell culture with a purity of more than 95%. Whereas Bcl-x_LΔC is soluble both before and after TolAIII removal, Triton X-100 can significantly increase the extraction of TolAIII- Bcl-2(2)ΔC from the bacterial cells and its subsequent solubility. Far-UV CD spectroscopy demonstrated that they both have an α-helical structure. Fluorescence spectroscopy was used to quantitatively analyze the binding of the respiratory inhibitor antimycin A to recombinant Bcl-2 and Bcl-x_L proteins as well as the displacement of this ligand from the hydrophobic pocket with BH3 Bad-derived peptide. Purified Bcl-x_LΔC and Bcl-2(2)ΔC both protect isolated mitochondria from Bax-induced release of cytochrome c. The ensemble of data shows that the expressed proteins are correctly folded and functional. Therefore, the TolAIII-fusion system provides a convenient tool for functional characterization and structural studies of anti-apoptotic proteins.     

Inhibition of Mitochondrial Neural Cell Death Pathways by Protein Transduction of Bcl-2 Family Proteins

Bcl-2 and other closely related members of the Bcl-2 family of proteins inhibit the death of neurons and many other cells in response to a wide variety of pathogenic stimuli. Bcl-2 inhibition of apoptosis is mediated by its binding to pro-apoptotic proteins, e.g., Bax and tBid, inhibition of their oligomerization, and thus inhibition of mitochondrial outer membrane pore formation, through which other pro-apoptotic proteins, e.g., cytochrome c, are released to the cytosol. Bcl-2 also exhibits an indirect antioxidant activity caused by a sub-toxic elevation of mitochondrial production of reactive oxygen species and a compensatory increase in expression of antioxidant gene products. While classic approaches to cytoprotection based on Bcl-2 family gene delivery have significant limitations, cellular protein transduction represents a new and exciting approach utilizing peptides and proteins as drugs with intracellular targets. The mechanism by which proteins with transduction domains are taken up by cells and delivered to their targets is controversial but usually involves endocytosis. The effectiveness of transduced proteins may therefore be limited by their release from endosomes into the cytosol.     

Alpha 5/6 helix domains together with N-terminus determine the apoptotic potency of the Bcl-2 family proteins

A critical process in apoptosis is the permeabilization of the mitochondrial outer membrane (MOM). This process is known to be regulated by the multi-domain Bcl-2 family proteins. For example, the pro-apoptotic proteins Bax and Bak are responsible for forming pores at MOM. The anti-apoptotic proteins (including Bcl-2, Mcl-1 and Bcl-xL), on the other hand, can inhibit this pore-forming process. Interestingly, although these two subgroups of proteins perform opposite apoptotic functions, their structures are very similar. This raises two highly interesting questions: (1) Why do these structurally similar proteins play opposite roles in apoptosis? (2) What are the roles of different functional domains of a Bcl-2 family protein in determining its apoptotic property? In this study, we generated a series of deletion mutants and substitution chimera, and used a combination of molecular biology, bio-informatics and living cell imaging techniques to answer these questions. Our major findings are: (1) All of the Bcl-2 family proteins appear to possess an intrinsic pro-apoptotic property. (2) The N-termini of these proteins play an active role in suppressing their pro-apoptotic function. (3) The apoptotic potency is positively correlated with membrane affinity of the alpha 5/6 helix domains. (4) Charge distribution flanking the alpha 5/6 helices is also important for the apoptotic potency. These findings explain why different members of Bcl-2 family proteins with similar domain composition can function oppositely in the apoptotic process.