The role of Bcl-2 and its pro-survival relatives in tumourigenesis and cancer therapy

Tumour development requires a combination of defects that allow nascent neoplastic cells to become self-sufficient for cell proliferation and insensitive to signals that normally restrain cell growth. Among the latter, evasion of programmed cell death (apoptosis) has proven to be critical for the development and sustained growth of many, perhaps all, cancers. Apoptotic cell death is regulated by complex interactions between pro-survival members and two subgroups of pro-apoptotic members of the B-cell lymphoma-2 (Bcl-2) protein family. In this invited review article, we reminisce on the discovery of Bcl-2, the first regulator of cell death identified, we discuss the mechanisms that control apoptotic cell death, focussing on how defects in this process promote the development and sustained growth of tumours and also affect their responses to anticancer therapeutics and, finally, we describe how current knowledge of the regulatory networks of apoptosis is exploited to develop novel approaches for cancer therapy.     

Prognostic impact of the expression/phosphorylation of the BH3-only proteins of the BCL-2 family in glioblastoma multiforme

Apoptosis has a crucial role in anti-cancer treatment. The proteins of the BCL-2 family are core members of the apoptotic program. Thus, we postulated that alterations in the expression of BCL-2 protein family, and in particular in that of the Bcl-2 homology domain 3 (BH3)-only proteins (which can neutralized anti-apoptotic proteins or activate pro-apoptotic proteins) could account for differences in the overall survival (OS) of patients. To test this hypothesis, we analyzed the expression of 15 members of the BCL-2 protein family (Bax, Bak, Bok, Bcl-2, Bcl-xl, Bcl-w, Mcl-1, Bad, Bid, Bim, Bik, Bmf, Hrk, Noxa and Puma) in glioblastoma multiforme (GBM) tumors, the most frequent brain tumor in adults. We found that none of the individual expression of these proteins is associated with a significant variation in OS of the patients. However, when all BH3 proteins were pooled to determine a BH3^score, this score was significantly correlated with OS of GBM patients. We also noted that patients with a have high level of phospho-Bad and phospho-Bim displayed a lower OS. Thus, BH3 scoring/profiling could be used as an independent prognostic factor in GBM when globally analyzed.     

Pro-apoptotic Bax is the major and Bak an auxiliary effector in cytokine deprivation-induced mast cell apoptosis

The process of apoptosis in immune cells like mast cells is essential to regain homeostasis after an inflammatory response. The intrinsic pathway of apoptosis is ultimately controlled by the pro-apoptotic Bcl-2 family members Bax and Bak, which upon activation oligomerize to cause increased permeabilization of the mitochondria outer membrane leading to cell death. We examined the role of Bax and Bak in cytokine deprivation-induced apoptosis in mast cells using connective tissue-like mast cells and mucosal-like mast cells derived from bax^−/−, bak^−/− and bax^−/−bak^−/− mice. Although both Bax and Bak were expressed at readily detectable protein levels, we found a major role for Bax in mediating mast cell apoptosis induced by cytokine deprivation. We analyzed cell viability by propidium iodide exclusion and flow cytometry after deprivation of vital cytokines for each mast cell population. Upon cytokine withdrawal, bak^−/− mast cells died at a similar rate as wild type, whereas bax^−/− and bax^−/−bak^−/− mast cells were partially or completely resistant to apoptosis, respectively. The total resistance seen in bax^−/−bak^−/− mast cells is comparable with mast cells deficient of both pro-apoptotic Bim and Puma or mast cells overexpressing anti-apoptotic Bcl-2. These results show that Bax has a predominant and Bak a minor role in cytokine deprivation-induced apoptosis in both connective tissue-like and mucosal-like mast cells.     

唯BH3域蛋白与神经细胞凋亡

细胞凋亡在神经细胞的生理性和病理性死亡中起着重要作用。唯BH3域蛋白是Bcl-2家族中的一类仅含有BH3同源结构域的促凋亡分子,它们通过抑制Bcl-2抗凋亡成员的活性或激活Bax/Bak样促凋亡成员的活性来调节细胞凋亡。最近研究表明,唯BH3域蛋白在凋亡的启动及凋亡通路的沟通中发挥着极其重要的作用。     

Apoptosis commitment--translating survival signals into decisions on mitochondria

Most defective and unwanted cells die by apoptosis, cells without damaging the surrounding tissue. Once a an exquisitely controlled genetic programme for removing such cell has committed to apoptosis, the process is remarkably efficient, and is completed within a few minutes of initiation. This point of no retum for an apoptotic cell is commonly held to be the point at which the outer mitochondrial membrane is permeabilised, a process regulated by the Bcl-2 family of proteins. How these proteins regulate this decision point is central to diseases such as cancer where apoptotic control is lost. In this review, we will discuss apoptotic signalling and how a cell makes the irreversible decision to die. We will focus on one set of survival signals, those derived by cell adhesion to the extracellular matrix （ECM）, and use these to highlight the complexities of apoptotic signalling. In particular, we will illustrate how multiple signalling pathways converge to determine critical cell fate decisions.     

Selective involvement of BH3-only proteins and differential targets of Noxa in diverse apoptotic pathways

The BH3-only proteins of the Bcl-2 family are known to mediate mitochondrial dysfunction during apoptosis. However, the identity of the critical BH3-only proteins and the mechanism of their action following treatment by diverse apoptotic stimuli remain to be fully resolved. We therefore used RNAi to screen the entire Bcl-2 family for their involvement in three major apoptotic pathways in HeLa cells. We found that Bcl-xL and Mcl-1 are major inhibitors of apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL), endoplasmic reticulum (ER) stress, and proteasome inhibition. Among the 10 BH3-only proteins, Bid and Noxa were found to be critically involved in TRAIL-induced apoptosis, in which Noxa participates by constitutively binding to Mcl-1. Bim and Noxa were found to be necessary for ER stress-induced apoptosis, in which Noxa assisted Bim function by sequestering Mcl-1 and binding to Bcl-xL. As a critical BH3-only protein, Noxa was strongly upregulated and became associated with both Mcl-1 and Bcl-xL during apoptosis induced by proteasome inhibition. In addition, we found that Noxa became 'Mcl-1 free' following treatment by ER stress and proteasome inhibition, but not after TRAIL treatment. These results defined the critical Bcl-2 network during apoptosis and suggested that Noxa participated in triggering mitochondrial dysfunction in multiple apoptotic pathways through distinct mechanisms.     

Apolipoprotein L2 contains a BH3-like domain but it does not behave as a BH3-only protein

Apolipoproteins of the L family are lipid-binding proteins whose function is largely unknown. Apolipoprotein L1 and apolipoprotein L6 have been recently described as novel pro-death BH3-only proteins that are also capable of regulating autophagy. In an in-silico screening to discover novel putative BH3-only proteins, we identified yet another member of the apolipoprotein L family, apolipoprotein L2 (ApoL2), as a BH3 motif-containing protein. ApoL2 has been suggested to behave as a BH3-only protein and mediate cell death induced by interferon-gamma or viral infection. As previously described, we observed that ApoL2 protein was induced by interferon-gamma. However, knocking down its expression in HeLa cells did not regulate cell death induced by interferon-gamma. Overexpression of ApoL2 did not induce cell death on its own. ApoL2 did not sensitize or protect cells from overexpression of the BH3-only proteins Bmf or Noxa. Furthermore, siRNA against ApoL2 did not alter sensitivity to a variety of death stimuli. We could, however, detect a weak interaction between ApoL2 and Bcl-2 by immunoprecipitation of the former, suggesting a role of ApoL2 in a Bcl-2-regulated process like autophagy. However, in contrast to what has been described about its homologs ApoL1 and ApoL6, ApoL2 did not regulate autophagy. Thus, the role, if any, of ApoL2 in cell death remains to be clarified.     

Neuronal Apoptosis: BH3-Only Proteins the Real Killers?

At present there is a poor understanding of the events that lead up to neuronal apoptosis that occurs in neurodegenerative diseases and following acute ischemic episodes. Apoptosis is critical for the elimination of unwanted neurons within the developing nervous system. The Bcl-2 family of proteins contains pro- and anti-apoptotic proteins that regulate the mitochondrial pathway of apoptosis. There is increasing interest in a subfamily of the Bcl-2 family, the BH3-only proteins, and their pro-apoptotic effects within neurons. Recently ischemic and seizure-induced neuronal injury has been shown to result in the activation of the BH3-only protein, Bid. This protein is cleaved and the truncated protein (tBid) translocates to the mitochondria. The translocation of tBid to the mitochondria is associated with the activation of outer mitochondrial membrane proteins Bax/Bak and the release of cytochrome C from the mitochondria. ER stress also has been implicated as a factor for the induction of apoptosis in ischemic neuronal injury. The induction of ER stress in hippocampal neurons has been shown to activate expression of bb3/PUMA, a member of the BH3-only gene family. Activation of PUMA is associated with the activation and clustering of the pro-apoptotic Bcl-2 family member Bax and the loss of cytochrome C from the mitochondria.     

Neither loss of Bik alone, nor combined loss of Bik and Noxa, accelerate murine lymphoma development or render lymphoma cells resistant to DNA damaging drugs

The pro-apoptotic BH3-only protein, BIK, is widely expressed and although many critical functions in developmental or stress-induced death have been ascribed to this protein, mice lacking Bik display no overt abnormalities. It has been postulated that Bik can serve as a tumour suppressor, on the basis that its deficiency and loss of apoptotic function have been reported in many human cancers, including lymphoid malignancies. Evasion of apoptosis is a major factor contributing to c-Myc-induced tumour development, but despite this, we found that Bik deficiency did not accelerate Eμ-Myc-induced lymphomagenesis. Co-operation between BIK and NOXA, another BH3-only protein, has been previously described, and was attributed to their complementary binding specificities to distinct subsets of pro-survival BCL-2 family proteins. Nevertheless, combined deficiency of Bik and Noxa did not alter the onset of Eμ-Myc transgene induced lymphoma development. Moreover, although p53-mediated induction of Bik has been reported, neither Eμ-Myc/Bik^−/− nor Eμ-Myc/Bik^−/−Noxa^−/− lymphomas were more resistant than control Eμ-Myc lymphomas to killing by DNA damaging drugs, either in vitro or in vivo. These results suggest that Bik, even in combination with Noxa, is not a potent suppressor of c-Myc-driven tumourigenesis or critical for chemotherapeutic drug-induced killing of Myc-driven tumours.     

How the Bcl-2 family of proteins interact to regulate apoptosis

Commitment of cells to apoptosis is governed largely by protein-protein interactions between members of the Bcl-2 protein family. Its three sub-families have distinct roles： the BH3-only proteins trigger apoptosis by binding via their BH3 domain to pro-survival relatives, while the pro-apoptotic Bax and Bak have an essential downstream role involving disruption of organellar membranes and induction of caspase activation. The BH3-only proteins act as damage sensors, held inert until their activation by stress signals. Once activated, they were thought to bind promiscuously to pro-survival protein targets but unexpected selectivity has recently emerged from analysis of their interactions. Some BH3-only proteins also bind to Bax and Bak. Whether Bax and Bak are activated directly by these BH3-only proteins, or indirectly as a consequence of BH3-only proteins neutralizing their pro-survival targets is the subject of intense debate. Regardless of this, a detailed understanding of the interactions between family members, which are often selective, has notable implications for designing anti-cancer drugs to target the Bcl-2 family.     

Lovastatin-induced up-regulation of the BH3-only protein, Bim, and cell death in glioblastoma cells

The mechanism of lovastatin-induced cell death was examined in three established human glioblastoma cell lines; U87, U251, and U138. Changes in potential modifiers of apoptosis, including Bcl-2 family proteins and MAP kinase targets after such lovastatin treatment, were evaluated. Lovastatin (5 microm) treatment causes extensive cell death in two of the cell lines, U87 and U251; but only minimal in a third, U138. Lovastatin-induced death occurs in correlation with significantly increased levels of the BH3-only protein, Bim. The up-regulation of Bim levels was directly associated with an increased incidence of apoptosis. Lovastatin treatment in U87 cells results in activation of targets of three major mitogen-activating protein kinase cascades including Erk1/2, JNK and p38. Changes in levels of Bim, as well as increase phosphorylation of Erk1/2, c-jun, and p38 are all prevented by co-incubation of lovastatin and the isoprenylation metabolite, geranylgeranyl pyrophosphate. Inhibition of the MAP kinase pathways failed to block the increased expression of Bim expression or cell death. Further elucidation of the mechanisms of lovastatin-induced up-regulation of Bim and apoptosis in glioblastoma cells are important in determining a potential role for lovastatin as a chemotherapy agent.     

Debcl, a proapoptotic Bcl-2 homologue, is a component of the Drosophila melanogaster cell death machinery

Bcl-2 family of proteins are key regulators of apoptosis. Both proapoptotic and antiapoptotic members of this family are found in mammalian cells, but no such proteins have been described in insects. Here, we report the identification and characterization of Debcl, the first Bcl-2 homologue in Drosophila melanogaster. Structurally, Debcl is similar to Bax-like proapoptotic Bcl-2 family members. Ectopic expression of Debcl in cultured cells and in transgenic flies causes apoptosis, which is inhibited by coexpression of the baculovirus caspase inhibitor P35, indicating that Debcl is a proapoptotic protein that functions in a caspase-dependent manner. debcl expression correlates with developmental cell death in specific Drosophila tissues. We also show that debcl genetically interacts with diap1 and dark, and that debcl-mediated apoptosis is not affected by gene dosage of rpr, hid, and grim. Biochemically, Debcl can interact with several mammalian and viral prosurvival Bcl-2 family members, but not with the proapoptotic members, suggesting that it may regulate apoptosis by antagonizing prosurvival Bcl-2 proteins. RNA interference studies indicate that Debcl is required for developmental apoptosis in Drosophila embryos. These results suggest that the main components of the mammalian apoptosis machinery are conserved in insects.     

BH3-only proteins trigger cytochrome c release, but how?

The mitochondrial apoptosis pathway has been neatly ordered. Mitochondrial apoptosis is governed by Bcl-2 family proteins, and their respective contributions determine the release of cytochrome c. It is clear that, among the Bcl-2 family, BH3-only proteins are the triggers: activation of BH3-only proteins by apoptotic stimuli initiates the process. BH3-only proteins cause cytochrome c release by activating Bax and/or Bak, and the anti-apoptotic group of Bcl-2-like proteins prevents this. However, it is curiously uncertain how BH3-only proteins activate Bax/Bak. Current models suggest that this is either through direct interaction--although this interaction is not detectable experimentally--or by the neutralisation of Bcl-2-like proteins. Here we discuss the context in which these models are placed and attempt to weigh the evidence.     

Apoptotic blocks and chemotherapy resistance: strategies to identify Bcl-2 protein signatures.

Acquired or innate resistance to chemotherapy is a major drawback of cancer therapeutics, which is frequently seen in epithelial cancers. However, the molecular mechanisms underlying chemotherapy resistance remain poorly understood. The mitochondrial pathway is a critical death pathway common to many different types of chemotherapy. Aberrations in this pathway can result in resistance to chemotherapy. The Bcl-2 family of proteins control commitment to programmed cell death by mitochondrial apoptosis. In this review, we will summarize the strategies in determining the components of apoptotic defects responsible for chemotherapy resistance, mainly focused on Bcl-2 protein network.     

A Critical role for Bim in retinal ganglion cell death

Optic nerve transection results in the death of retinal ganglion cells (RGCs) by apoptosis. Apoptosis is regulated by the Bcl-2 family of proteins, of which the Bcl-2 homology (BH3) -only proteins forms a subset. As BH3-only proteins have been shown to play a significant role in regulating cell death in the central nervous system, we wished to investigate the role of Bcl-2 interacting mediator of cell death (Bim), a prominent member of this protein family in the regulation of cell death in the RGC layer using in vitro retinal explants. In this study, we use an innovative retinal shaving procedure to isolate the cells of the ganglion cell layer to use for western blotting. Members of the BH3-only protein family are down-regulated during retinal development and are not normally expressed in the adult retina. Using this procedure, we demonstrate that Bim is re-expressed and its expression is increased over time following axotomy. Expression of Bad and Bik decreases over the same time course, whereas there is no indication that Bid and Puma are re-expressed. We show that explants from Bim knockout mice are resistant to axotomy-induced death when compared with their wild-type counterparts. Genetic deletion of Bim also prevents caspase 3 cleavage. The activity of Bim can be negatively regulated by phosphorylation. We show that the decrease of Bim phosphorylation correlates with a decrease in expression of survival kinases such as pAkt and pERK over the same time course. These results implicate Bim re-expression as being essential for axotomy-induced death of RGCs and that phosphorylation of Bim negatively regulates its activity in RGCs.     

Protease signalling in cell death: caspases versus cysteine cathepsins

Proteases were, for a long time, mainly considered as protein degrading enzymes. However, in the last decade this view has changed dramatically, and the focus is now on proteases as signalling molecules. One of the best examples is apoptosis, the major mechanism used by eukaryotes to remove superfluous, damaged and potentially dangerous cells, in which a number of proteases have been found to play a central role. Of these the caspases have been considered to be the major players. However, more recently, other proteases have been increasingly suggested as being important in apoptosis, in particular the cysteine cathepsins. In this review the roles of caspases and cysteine cathepsins in apoptosis signalling are compared and discussed.     

Bcl-2 family member Bcl-G is not a proapoptotic protein

The three major subgroups of the Bcl-2 family, including the prosurvival Bcl-2-like proteins, the proapoptotic Bcl-2 homology (BH)3-only proteins and Bax/Bak proteins, regulate the mitochondrial apoptotic pathway. In addition, some outliers within the Bcl-2 family do not fit into these subgroups. One of them, Bcl-G, has a BH2 and a BH3 region, and was proposed to trigger apoptosis. To investigate the physiological role of Bcl-G, we have inactivated the gene in the mouse and generated monoclonal antibodies to determine its expression. Although two isoforms of Bcl-G exist in human, only one is found in mice. mBcl-G is expressed in a range of epithelial as well as in dendritic cells. Loss of Bcl-G did not appear to affect any of these cell types. mBcl-G only binds weakly to prosurvival members of the Bcl-2 family, and in a manner that is independent of its BH3 domain. To understand what the physiological role of Bcl-G might be, we searched for Bcl-G-binding partners through immunoprecipitation/mass spectroscopy and yeast-two-hybrid screening. Although we did not uncover any Bcl-2 family member in these screens, we found that Bcl-G interacts specifically with proteins of the transport particle protein complex. We conclude that Bcl-G most probably does not function in the classical stress-induced apoptosis pathway, but rather has a role in protein trafficking inside the cell.     

Detection of Bcl-2 family member Bcl-G in mouse tissues using new monoclonal antibodies

Bcl-G is an evolutionarily conserved member of the Bcl-2 family of proteins that has been implicated in regulating apoptosis and cancer. We have generated monoclonal antibodies that specifically recognise mouse Bcl-G and have used these reagents to analyse its tissue distribution and subcellular localisation using western blotting, immunohistochemistry and immunofluorescence. We found that Bcl-G predominantly resides in the cytoplasm and is present in a wide range of mouse tissues, including the spleen, thymus, lung, intestine and testis. Immunohistochemical analyses revealed that Bcl-G is expressed highly in mature spermatids in the testis, CD8⁺ conventional dendritic cells (DCs) in hematopoietic tissues and diverse epithelial cell types, including those lining the gastrointestinal and respiratory tracts. The Bcl-G monoclonal antibodies represent new tools for studying this protein, using a variety of techniques, including immunoprecipitation and flow cytometry.