Deciphering the rules of programmed cell death to improve therapy of cancer and other diseases

Apoptosis, the major form of programmed cell death in metazoan organisms, plays critical roles in normal development, tissue homeostasis and immunity, and its disturbed regulation contributes to many pathological states, including cancer, autoimmunity, infection and degenerative disorders. In vertebrates, it can be triggered either by engagement of 'death receptors' of the tumour necrosis factor receptor family on the cell surface or by diverse intracellular signals that act upon the Bcl-2 protein family, which controls the integrity of the mitochondrial outer membrane through the complex interactions of family members. Both pathways lead to cellular demolition by dedicated proteases termed caspases. This review discusses the groundbreaking experiments from many laboratories that have clarified cell death regulation and galvanised efforts to translate this knowledge into novel therapeutic strategies for the treatment of malignant and perhaps certain autoimmune and infectious diseases.     

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。     

The role of apoptosis in the development and function of T lymphocytes

Apoptosis plays an essential role in T cell biology. Thymocytes expressing nonfunctional or autoreactive TCRs are eliminated by apoptosis during development. Apoptosis also leads to the deletion of expanded effector T cells during immune responses. The dysregulation of apoptosis in the immune system results in autoimmunity, tumorogenesis and immunodeficiency. Two major pathways lead to apoptosis： the intrinsic cell death pathway controlled by Bcl-2 family members and the extrinsic cell death pathway controlled by death receptor signaling. These two pathways work together to regulate T lymphocyte development and function.     

Bcl-2-regulated cell death signalling in the prevention of autoimmunity

Cell death mediated through the intrinsic, Bcl-2-regulated mitochondrial apoptosis signalling pathway is critical for lymphocyte development and the establishment of central and maintenance of peripheral tolerance. Defects in Bcl-2-regulated cell death signalling have been reported to cause or correlate with autoimmunity in mice and men. This review focuses on the role of Bcl-2 family proteins implicated in the development of autoimmune disorders and their potential as targets for therapeutic intervention.     

Bax and other pro-apoptotic Bcl-2 family "killer-proteins" and their victim the mitochondrion

Two major intracellular apoptosis signaling cascades have been characterized, the mitochondrial pathway and the death receptor pathway. The mitochondrial pathway is regulated by members of the Bcl-2 protein family. The members of this family can be subdivided into anti- and pro-apoptotic proteins. The pro-apoptotic members are further divided into two groups, the multidomain and the 'BH3 domain only' proteins. When cells are exposed to apoptotic stimulation, pro-apoptotic proteins are activated through post-translational modifications or changes in their conformation. The main site of action of the multidomain proteins are the mitochondria, where these proteins induce permeabilization of the outer membrane resulting in the release of proteins, including cytochrome c, from the intermembrane space. In the cytosol cytochrome c activates caspase cascades ultimately leading to cell death. Mounting evidence indicates that apoptosis is involved in a wide range of pathological conditions. Recent studies suggest that the mitochondrial signaling pathway is involved in several diseases. Although, so far, with the exception of C. elegans, most studies on apoptosis have been performed in mammalian systems, recently homologues to the Bcl-2 family members, including pro-apoptotic members, have been identified in Drosophila and zebrafish. Here the structure and function of the various pro-apoptotic Bcl-2 family members, their effects on mitochondria, and their involvement in diseases are discussed.     

Building blocks of the apoptotic pore: how Bax and Bak are activated and oligomerize during apoptosis

The central role of the Bcl-2 family in regulating apoptotic cell death was first identified in the 1980s. Since then, significant in-roads have been made in identifying the multiple members of this family, characterizing their form and function and understanding how their interactions determine whether a cell lives or dies. In this review we focus on the recent progress made in characterizing the proapoptotic Bcl-2 family members, Bax and Bak. This progress has resolved longstanding controversies, but has also challenged established theories in the apoptosis field. We will discuss different models of how these two proteins become activated and different ‘modes'' by which they are inhibited by other Bcl-2 family members. We will also discuss novel conformation changes leading to Bak and Bax oligomerization and speculate how these oligomers might permeabilize the mitochondrial outer membrane.     

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.     

Bcl-2 family regulation of neuronal development and neurodegeneration

Neuronal cell death is a key feature of both normal nervous system development and neuropathological conditions. The Bcl-2 family, via its regulation of both caspase-dependent and caspase-independent cell death pathways, is uniquely positioned to critically control neuronal cell survival. Targeted gene disruptions of specific bcl-2 family members and the generation of transgenic mice overexpressing anti- or pro-apoptotic Bcl-2 family members have confirmed the importance of the Bcl-2 family in the nervous system. Data from studies of human brain tissue and experimental animal models of neuropathological conditions support the hypothesis that the Bcl-2 family regulates cell death in the mature nervous system and suggest that pharmacological manipulation of Bcl-2 family action could prove beneficial in the treatment of human neurological conditions such as stroke and neurodegenerative diseases.     

Methods of assaying Bcl-2 and Bax family proteins in yeast

Bcl-2 family proteins play an evolutionarily conserved role in regulating the life and death of the cell. Certain proapoptotic members of the Bcl-2 family, Bax and Bak, have intrinsic cytotoxic activities in that they not only induce or sensitize mammalian cells to undergo apoptosis but also display a lethal phenotype when ectopically expressed in two yeast species Saccharomyces cerevisiae and Schizosaccharomyces pombe. Furthermore, the antiapoptotic Bcl-2 and Bcl-XL proteins can protect yeast against Bax-mediated lethality, suggesting that the death-regulatory functions of these Bcl-2 family proteins are well preserved in yeast. These observations provide the opportunity to study the function of Bcl-2 family proteins in genetically tractable yeast and to apply classical yeast genetics and functional cloning approaches to the dissection of programmed cell death pathway regulated by Bcl-2 family proteins. We describe here methods used in our laboratory to express and to study the functions of Bcl-2 family proteins in both the budding yeast S. cerevisiae and the fission yeast S. pombe.     

The role of Bcl-2 family members in tumorigenesis

The Bcl-2 family consists of about 20 homologues of important pro- and anti-apoptotic regulators of programmed cell death. The established mode of function of the individual members is to either preserve or disturb mitochondrial integrity, thereby inducing or preventing release of apoptogenic factors like Cytochrome c (Cyt c) from mitochondria. Recent findings also indicate further Bcl-2-controlled mitochondria-independent apoptosis pathways. Bcl-2 represents the founding member of the new and growing class of cell death inhibiting oncoproteins. In this review, we try to briefly summarize current models of Bcl-2 family function and to outline the work demonstrating the influence of deregulated Bcl-2 family member expression on tumorigenesis and cancer therapy. Since several Bcl-2 homologues, in addition to influencing apoptotic behaviour, also impinge on cell cycle progression, we discuss possible implications of this additional role for the expression of Bcl-2 family members in tumor cells.     

A conserved domain in Bak, distinct from BH1 and BH2, mediates cell death and protein binding functions.

Regulation of the cell death program involves physical interactions between different members of the Bcl-2 family that either promote or suppress apoptosis. The Bcl-2 homolog, Bak, promotes apoptosis and binds anti-apoptotic family members including Bcl-2 and Bcl-xL. We have identified a domain in Bak that is both necessary and sufficient for cytotoxic activity and binding to Bcl-xL. Sequences similar to this domain were identified in Bax and Bip1, two other proteins that promote apoptosis and interact with Bcl-xL, and were likewise critical for their capacity to kill cells and bind Bcl-xL. Thus, the domain is of central importance in mediating the function of multiple cell death-regulatory proteins that interact with Bcl-2 family members.     

Synthetic peptides and non-peptidic molecules as probes of structure and function of Bcl-2 family proteins and modulators of apoptosis

The Bcl-2 family includes a growing number of proteins that play an essential role in regulating apoptosis or programmed cell death. Members of this family display diverse biological functions and can either inhibit or promote cell death signals. Abnormal gene expression of some Bcl-2 family members such as Bcl-2 that inhibits apoptosis is found in a wide variety of human cancers and contributes to the resistance of tumor cells to conventional therapies through interfering with the cell death signals triggered by chemotherapeutic agents. As such, elucidating the structure-function and mechanism of the Bcl-2 family is important for understanding some of the fundamental principles underling the death and survival of cells and of practical value for developing potential therapeutics to control apoptosis in pathological processes. Synthetic peptides derived from homologous or heterogeneous domains in Bcl-2 family proteins that might mediate different biological activities provide simplified and experimentally more tractable models as compared to their full-length counterparts to dissect and analyze the complex functional roles of these proteins. Non-peptidic molecules identified from random screening of natural products or designed by rational structure-based techniques can mimic the effect of synthetic peptides by targeting similar active sites on a Bcl-2 family member protein. In this article, we review recent progress in using these synthetic peptides and non-peptidic mimic molecules to obtain information about the structure and function of Bcl-2 family proteins and discuss their application in modulating and studying intracellular apoptotic signaling.     

Current concepts in Bcl-2 family member regulation of female germ cell development and survival

Since the cloning of the bcl-2 gene in 1985, considerable progress has been made in elucidating the function of Bcl-2 and related proteins in controlling apoptosis. Although much of this work initially relied on the ectopic expression of bcl-2 gene family members in cell lines in vitro, a number of genetically manipulated mice have been generated to better understand the in vivo significance of specific family members to organ development and homeostasis. Of the many tissues that exhibit apoptosis at some point during fetal or postnatal life, the female gonads arguably possess one of the highest and most protracted incidences of apoptosis, associated with development and maturation of the germ line. Moreover, female germ cells (oocytes) are, for as-yet poorly understood reasons, extremely vulnerable to a host of pathological insults, such as anti-cancer therapies, that ultimately cause premature ovarian failure and infertility due to accelerated oocyte death. Accordingly, efforts to understand the occurrence and regulation of apoptosis in the ovary are of considerable importance from both biological and clinical perspectives. This review will highlight what is known of apoptosis in the female gonads, and the role that Bcl-2 family members play in regulating this process.     

Structural biology of the Bcl-2 family of proteins

The proteins of the Bcl-2 family are important regulators of programmed cell death. Structural studies of Bcl-2 family members have provided many important insights into their molecular mechanism of action and how members of this family interact with one another. To date, structural studies have been performed on six Bcl-2 family members encompassing both anti- (Bcl-x(L), Bcl-2, KSHV-Bcl-2, Bcl-w) and pro-apoptotic (Bax, Bid) members. They all show a remarkably similar fold despite an overall divergence in amino acid sequence and function (pro-apoptotic versus anti-apoptotic). The three-dimensional structures of Bcl-2 family members consist of two central, predominantly hydrophobic alpha-helices surrounded by six or seven amphipathic alpha-helices of varying lengths. A long, unstructured loop is present between the first two alpha-helices. The structures of the Bcl-2 proteins show a striking similarity to the overall fold of the pore-forming domains of bacterial toxins. This finding led to experiments which demonstrated that Bcl-x(L), Bcl-2, and Bax all form pores in artificial membranes. A prominent hydrophobic groove is present on the surface of the anti-apoptotic proteins. This groove is the binding site for peptides that mimic the BH3 region of various pro-apoptotic proteins such as Bak and Bad. Structures of Bcl-x(L) in complex with these BH3 peptides showed that they bind as an amphipathic alpha-helix and make extensive hydrophobic contacts with the protein. These data have not only helped to elucidate the interactions important for hetero-dimerization of Bcl-2 family members but have also been used to guide the discovery of small molecules that block Bcl-x(L) and Bcl-2 function. In the recently determined structure of the anti-apoptotic Bcl-w protein, the protein was also found to have a hydrophobic groove on its surface capable of binding BH3-containing proteins and peptides. However, in the native protein an additional carboxy-terminal alpha-helix interacts with the hydrophobic groove. This is reminiscent of how the carboxy-terminal alpha-helix of the pro-apoptotic protein Bax binds into its hydrophobic groove. This interaction may play a regulatory role and for Bax may explain why it is found predominately in the cytoplasm prior to activation. The hydrophobic groove of the pro-apoptotic protein, Bid protein, is neither as long nor as deep as that found in Bcl-x(L), Bcl-2, or Bax. In addition, Bid contains an extra alpha-helix, which is located between alpha1 and alpha2 with respect to Bcl-x(L), Bcl-2, and Bax. Although there are still many unanswered questions regarding the exact mechanism by which the Bcl-2 family of proteins modulates apoptosis, structural studies of these proteins have deepened our understanding of apoptosis on the molecular level.     

Discovery of a potent and selective Bcl-2 inhibitor using SAR by NMR

The Bcl-2 family of proteins plays a major role in the regulation of apoptosis, or programmed cell death. Overexpression of the anti-apoptotic members of this family (Bcl-2, Bcl-x_L, and Mcl-1) can render cancer cells resistant to chemotherapeutic agents and therefore these proteins are important targets for the development of new anti-cancer agents. Here we describe the discovery of a potent, highly selective, Bcl-2 inhibitor using SAR by NMR and structure-based drug design which could serve as a starting point for the development of a Bcl-2 selective anti-cancer agent. Such an agent would potentially overcome the Bcl-x_L mediated thrombocytopenia observed with ABT-263.     

Paradox of Bcl‐2 (and p53): why may apoptosis‐regulating proteins be irrelevant to cell death?

Although the Bcl-2 family members and p53 are involved in the regulation of apoptosis, the status of apoptotic machinery (eg caspases) plays a major role in determining the mode and timing of cell death. If the apoptotic machinery is lost, inhibited, or intrinsically inactivated, the "death stars", Bcl-2 and p53, may become irrelevant to cell death. In this light, high levels of Bcl-2 may indicate that downstream apoptotic pathways are still functional. This explains why Bcl-2 overexpression can be a marker of chemosensitivity and favorable prognosis in certain cancers and why retention of wild-type p53 may manifest inactivation of caspases in aggressive cancers.     

Mitochondria as the central control point of apoptosis

Mitochondria play a major role in apoptosis triggered by many stimuli. They integrate death signals through Bcl-2 family members and coordinate caspase activation through the release of cytochrome c as a result of the outer mitochondrial membrane becoming permeable. The mechanisms that lead to this permeability are not yet completely understood. Here, we attempt to summarize our current view of the mechanisms that lead to the efflux of many proteins from mitochondria during apoptosis and the role played by Bcl-2 family proteins in the control of this event.     

The conserved N-terminal BH4 domain of Bcl-2 homologues is essential for inhibition of apoptosis and interaction with CED-4.

Bcl-2 and close homologues such as Bcl-xL promote cell survival, while other relatives such as Bax antagonize this function. Since only the pro-survival family members possess a conserved N-terminal region denoted BH4, we have explored the role of this amphipathic helix for their survival function and for interactions with several agonists of apoptosis, including Bax and CED-4, an essential regulator in the nematode Caenorhabditis elegans. BH4 of Bcl-2 could be replaced by that of Bcl-x without perturbing function but not by a somewhat similar region near the N-terminus of Bax. Bcl-2 cell survival activity was reduced by substitutions in two of ten conserved BH4 residues. Deletion of BH4 rendered Bcl-2 (and Bcl-xL) inactive but did not impair either Bcl-2 homodimerization or ability to bind to Bax or five other pro-apoptotic relatives (Bak, Bad, Bik, Bid or Bim). Hence, association with these death agonists is not sufficient to promote cell survival. Significantly, however, Bcl-xL lacking BH4 lost the ability both to bind CED-4 and antagonize its pro-apoptotic activity. These results favour the hypothesis that the BH4 domain of pro-survival Bcl-2 family members allows them to sequester CED-4 relatives and thereby prevent apoptosis.