Chapter 7: Mechanisms of Cell Death
Mechanisms of Cell Death: Introduction
Cell death has historically been subdivided into genetically controlled (or programmed) and unregulated mechanisms. Apoptosis has been recognized as a fundamental type of programmed cell death that is activated and repressed by specific genes and pathways. In contrast, necrosis has traditionally been considered an unregulated process and the result of cell death by acute physical trauma or overwhelming stress that is incompatible with cell survival. More recently, however, this strict classification of cell death mechanisms has been revisited, as mechanisms considered “programmed” were in certain instances shown to modulate necrosis and result in a regulated nonapoptotic cell death displaying necrotic morphology (necroptosis). It is also becoming apparent that disabling programmed cell death reveals novel survival mechanisms such as the catabolic autophagy pathway used by cancer cells to tolerate stress and starvation. Thus, cancer cells that acquire defects in programmed cell death are not merely “undead” but rather mobilize a novel physiologic state that actively enables survival. We review here the key aspects of the different cell death mechanisms and their regulation, and how they impact cancer development, progression, and treatment response.
Apoptosis
Apoptosis (or type I programmed cell death) is a genetic pathway for rapid and efficient killing of unnecessary or damaged cells that was initially described by Vogt (1842), and then Kerr et al.1 and Wyllie et al.2 They detailed a novel morphologic process for cell death that included swiftly executed cell shrinkage, blebbing of the plasma membrane, chromatin condensation, and intranucleosomal DNA fragmentation, after which cell corpses are engulfed by neighboring cells and professional phagocytes and degraded. Apoptosis (commonly pronounced ap-a-tow′-sis), a term coined from the Greek apo or from, and ptosis or falling, to make the analogy of leaves falling off a tree. Although underappreciated at the time, once the genes that controlled apoptosis were identified in model organisms and humans, and it was shown that perturbation of this program disturbed development and provoked disease, the importance of apoptosis was generally realized.
Cell death by apoptosis is involved in sculpting tissues in normal development. These developmental cell deaths span the removal of the interdigital webs and tadpole tails, to selection for and against specific B- and T-cell populations essential for controlling the immune response. Proper regulation of apoptosis is critical in that excessive apoptosis is associated with degenerative conditions, while deficient apoptosis promotes autoimmunity and cancer. Furthermore, apoptosis is required for eliminating damaged or pathogen-infected cells as a mechanism for limiting disease, especially cancer. In turn, tumors and pathogens have also evolved elegant mechanisms for disabling apoptosis to facilitate their persistence, often promoting disease progression. In human cancers, multiple mechanisms to disable apoptosis include loss of function of the apoptosis-promoting p53 tumor suppressor and gain of function of the apoptosis-inhibitory and oncogenic B-cell chronic lymphocytic leukemia/lymphoma 2 (BCL2). It became apparent then that cancer progression was aided not only by increasing the rate of cell multiplication through activation of the c-myc oncogene, for example, but also by decreasing the rate of cell elimination through apoptosis, exemplified by gain of BCL2 expression (Fig. 7.1). Indeed, activation of oncogenes such as c-myc or E1A,3–5 or loss of tumor suppressor genes such as Rb,6 can promote apoptosis, providing an explanation for the necessity for inactivation of the apoptotic pathway in many tumors. This may create a physiological state of cancer cells being “primed for death” where the necessity to up-regulate antiapoptotic mechanisms such as BCL2 to oppose oncogene activation poises cancer cells to reactivation of apoptosis providing a therapeutic window for cancer therapy.7
Figure 7.1. Tumor progression through cooperation of proliferative and antiapoptotic functions.
In normal cells in epithelial tissues (green cells) initiating mutational events such as deregulation of c-myc expression deregulate cell growth control and promote abnormal cell proliferation (yellow cells) while triggering a proapoptotic tumor suppression (red apoptotic cells) mechanism that can restrict tumor expansion. Subsequent acquisition of mutations that disables the apoptotic response, exemplified by Bcl-2 overexpression, prevents this effective means of culling emerging tumor cells, thereby favoring tumor expansion. Similar oncogenic events occur in lymphoid tissues.
The effectiveness of many existing anticancer drugs involves or is facilitated by triggering the apoptotic response. Thus, a detailed understanding of the components, molecular signaling events, and control points in the apoptotic pathway has enabled rational approaches to chemotherapy aimed at restoring the capacity for apoptosis to tumor cells. Identification of the molecular means by which tumors inactivate apoptosis has led to cancer therapies directly targeting the apoptotic pathway. These drugs are now being used in the clinic to specifically reactivate apoptosis in tumor cells in which it is disabled to achieve tumor regression.
Model Organisms Provide Mechanistic Insight into Apoptosis Regulation
Key to elevating the field of programmed cell death from a descriptive to a mechanism-based process was the discovery of genes in the nematode Ceanorhabditis elegans that control cell death, the cell death defective or ced genes.8 Genetic analysis revealed that ced-4 and ced-3 promote cell death, as worms with defective mutations in these genes possessed extra cells. In contrast, the ced-9 gene inhibited the death-promoting function of ced-4 and ced-3, thereby maintaining cell viability.9ced-9 in turn was inhibited by egl-1, thereby promoting cell death. This creates a linear genetic pathway controlled upstream by cell-specific death specification regulators, and downstream by cell corpse engulfment and degradation mechanisms (Fig. 7.2).10 These findings helped propel work in mammalian systems when it became apparent that Ced-9 was homologous to BCL2,11 Ced-3 was homologous to interleukin1-β–converting enzyme, a cysteine protease that would later be classified as a member of the caspase family of aspartic acid proteases,12 Egl-1 was a BH3-only protein homologue,10 and that the proapoptotic factor apoptotic protease-activating factor (APAF-1)-1 identified in mammals was homologous to Ced-4.13
Figure 7.2. Analogous pathways regulate programmed cell death/apoptosis in metazoans.
Regulation of programmed cell death in the nematode Ceanorhabditis elegans (top) and regulation of apoptosis in mammals (bottom). Shaded regions highlight corresponding homologous genes and protein families. In C. elegans, numerous cell death specification genes can up-regulate the transcription of the BH3-only protein Egl-1, which interacts with the antiapoptotic Bcl-2 homologue Ced-9 inhibiting is interaction with Ced-4. Ced-4, the Apaf-1 homologue, in turn, activates the caspase Ced-3, leading to cell death. A variety of engulfment gene products are then responsible for apoptotic corpse elimination and nucleases degrade the genome. In mammals, many survival, damage, and stress events impinge on the numerous members of the BH3-only class of proapoptotic proteins to either activate them to promote apoptosis or suppress their activation to enable cell survival. BH3-only proteins interact with and antagonize the numerous Bcl-2-related multidomain antiapoptotic proteins that serve to sequester proapoptotic Bax and Bak and may also contribute directly to Bax/Bak activation. Bax or Bak is essential for signaling apoptosis by permeabilizing the outer mitochondrial membrane to allow the release of cytochrome c and second mitochondrial-derived activator of caspase (SMAC). Cytochrome c acts as a cofactor for Apaf-1-mediated caspase activation in the apoptosome, and the SMAC amino-terminal four amino acids bind and antagonize the inhibitors of apoptosis proteins that interact with and suppress caspases, leading to their activation, widespread substrate cleavage, and cell death. Many engulfment gene products are responsible for corpse elimination and caspase-activated nucleases in the apoptotic cell itself, and additional nucleases within the engulfing cell are responsible for degradation of the genome.
A similar cell death pathway in the fruit fly Drosophila melanogaster identified Reaper, Hid and Grim as inhibitors of the inhibitors of apoptosis proteins (IAPs) that negatively regulate caspase activation. This eventually led to the identification of their mammalian counterpart second mitochondrial-derived activator of caspase (SMAC), also known as direct IAP-binding protein with low pI (DIABLO).14 These and other studies established the paradigm whereby proapoptotic BH3-only proteins inhibit antiapoptotic Bcl-2 proteins that prevent APAF-1-mediated caspase activation suppressed by IAPs, and the caspase-mediated proteolytic cellular destruction leads rapidly to cell death. It would later be realized that in mammals BH3-only proteins could also act as direct activators of the proapoptotic machinery (see later discussion).
Discovery of Bcl-2 and its Role as an Apoptosis Inhibitor in B-cell Lymphoma
To identify mechanisms of oncogenesis, the bcl-2 gene was cloned from the site of frequent chromosome translocation t(14;18):(q32;q21) in human follicular lymphoma.15–17 This chromosome rearrangement places bcl-2 under the transcriptional control of the immunoglobulin heavy chain locus causing abnormally high levels of bcl-2expression. Distinct from other oncogenes at the time, instead of promoting cell proliferation, bcl-2 promoted B-cell tumorigenesis by the novel concept of providing a survival advantage to cells stimulated to proliferate by c-myc.18 Indeed, engineering high Bcl-2 expression in the lymphoid compartment in mutant mice promotes follicular hyperplasia that progresses to lymphoma upon c-myc translocation, and bcl-2 synergizes with c-myc to produce lymphoid tumors, paralleling events in human follicular lymphoma.19,20 Bcl-2 localizes to mitochondria where it has broad activity in promoting cell survival through suppression of apoptosis,21 provoked by numerous events, including oncogene activation (c-myc, E1A), tumor suppressor activation (p53), growth factor and cytokine limitation, and cellular damage. It also became clear that inactivation of the retinoblastoma tumor suppressor (Rb) pathway promotes a p53-mediated apoptotic response, suggesting that apoptosis was part of a tumor suppression mechanism that responded to deregulation of cell growth.6,22,23 Indeed, apoptotic defects acquired by a variety of means are a common event in human tumorigenesis.
Control of Apoptosis by Bcl-2 Family Members
Bcl-2 is the first member of what is now a large family of related proteins that regulate apoptosis and are conserved among metazoans including worms, flies, and mammals, and also viruses.24–28 Multidomain Bcl-2 family members containing Bcl-2 homology regions 1-4 (BH1-4) are either antiapoptotic (Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and Bfl-1/A1, and virally encoded Bcl-2 homologues such as E1B 19K), or proapoptotic (Bax and Bak). Antiapoptotic proteins can block apoptosis by binding and sequestering Bax and Bak or by indirectly preventing Bax and Bak activation (Fig. 7.3).25,29,30
Figure 7.3. Regulation of apoptosis by the Bcl-2 family of proteins in mammals.
A: Schematic of apoptosis regulation by the Bcl-2 family. Cytotoxic events activate, while survival signaling events suppress the activity of the BH3-only class of Bcl-2 family members (orange). BH3-only proteins are controlled at the transcription level and also by numerous posttranscriptional events that modulate phosphorylation, proteolysis, localization, sequestration, and protein stability. Once activated, BH3-only proteins disrupt functional sequestration of Bak and Bax by the multidomain antiapoptotic Bcl-2-like proteins (blue) and may also directly facilitate Bax/Bak activation. Although Bak is commonly membrane-associated in a complex with Mcl-1 and Bcl-xL in healthy cells, Bax resides in the cytoplasm as an inactive monomer with its carboxy-terminus occluding the BH3-binding hydrophobic cleft.138 Bax activation thereby additionally requires a change in protein conformation and membrane translocation by an unknown mechanism that may be facilitated by tBid binding. Binding specificity among BH3-only proteins for antiapoptotic Bcl-2-like proteins determines which complexes are disrupted, with some BH3-only proteins having broad specificity and others do not. Survival and death signaling events can also modulate apoptosis by targeting the multidomain antiapoptotic proteins either by antagonizing their function to promote apoptosis or induction their function to promote survival. ABT-737 is a rationally designed BAD BH3 mimetic that can bind Bcl-2, Bcl-xL, and Bcl-w but not Mcl-1 that can promote apoptosis where survival does not depend on Mcl-1. Once activated, Bax or Bak oligomerization promotes apoptosis. B: Tumor necrosis factor-α (TNF-α) apoptotic signaling induces mitochondrial membrane translocation and a conformational change exposing the amino-terminus of Bax (visualized here by the Bax-NT antibody) and apoptosis, which is blocked by sequestration of Bax by the antiapoptotic viral Bcl-2 homologue E1B 19K. The human cancer cell line (HeLa cells) with or without E1B 19K expression, were then left untreated or treated with TNF/CHX. The localization of conformationally altered Bax (Bax-NT) and cytochrome c (left and middle panels), or E1B 19K and cytochrome c (right panel), are shown. The proapoptotic stimulus (TNF/CHX) induces Bax activation, mitochondrial translocation, and cytochrome c release from mitochondria that leads to caspase activation and apoptotic cell death, whereas expression of E1B 19K sequesters Bax thereby blocking cytochrome c release from mitochondria, caspase activation, and apoptotic cell death. The yellow and red arrows, respectively, mark cells with partial or complete cytochrome c release from mitochondria upon TNF/CHX treatment.
Bax and Bak are functionally redundant and required for signaling apoptosis through mitochondria, and deficiency in Bax and Bak produces a profound defect in apoptosis. Bax and Bak are considered the core apoptosis machinery controlled directly or indirectly by antiapoptotic Bcl-2-like proteins and proapoptotic BH3-only proteins. Remarkably, mice deficient in both Bax and Bak develop relatively normally, suggesting that other death mechanisms can compensate for loss of apoptosis in development.31 In healthy cells, Bak is bound and sequestered by Mcl-1 and Bcl-xL at cellular membranes, whereas Bax resides in the cytosol in a latent form and requires activation and translocation to membranes, where it is either sequestered by antiapoptotic Bcl-2-like proteins or otherwise induces apoptosis (Fig. 7.3).
Control of Multidomain Bcl-2 Family Proteins by the BH3-only Proteins
Bax and Bak deficiency abrogates the ability of BH3-only proteins to induce apoptosis, placing them upstream and dependent on the core apoptosis machinery.32 BH3-only protein Bcl-2 family members (Bim, Bid, Nbk/Bik, Puma, Bmf, Bad, and Noxa) are proapoptotic and antagonize the survival activity of antiapoptotic Bcl-2-like proteins by binding and displacing Bax and Bak to allow apoptosis (BH3-only proteins as neutralizers of Bcl-2) (Fig. 7.4).30 The different BH3-only proteins respond to specific stimuli to activate apoptosis (Fig. 7.3). For example, Bim induces apoptosis in response to taxanes, Puma and Noxa are transcriptional targets of and mediate apoptosis in response to p53 activation, Bad signals apoptosis on growth factor withdrawal, Nbk/Bik promotes apoptosis in response to inhibition of protein synthesis, and Bid is required for apoptosis signaled by death receptors. All of these signals are transduced from the BH3-only proteins to other members of the Bcl-2 family by protein-protein interactions.
Figure 7.4. Modes of apoptosis activation by BH3-only proteins.
The neutralization mode (top), de-repression mode (middle), and sensitizer mode (bottom). See text for explanation.
The BH3 region of BH3-only proteins binds to a hydrophobic cleft in the multidomain Bcl-2-like antiapoptotic proteins that also supports Bax and Bak binding,33,34 causing their displacement (neutralization mode; Fig. 7.4).30 Differential binding specificities among the BH3 regions of the different BH3-only proteins determine whether they bind one or more Bcl-2-like proteins and displace Bax or Bak or both.35 Noxa binds and antagonizes Mcl-1, whereas Bad binds and antagonizes Bcl-2 and Bcl-xL, necessitating cooperation between Noxa and Bad function for efficient apoptosis. In contrast, Bim, Bid, and Puma have broader binding specificity and antagonize Mcl-1, Bcl-2, and Bcl-xL to release both Bax and Bak to induce apoptosis. Bim, the active form of Bid (truncated Bid or tBid) and possibly Puma can also be direct activators of Bax and Bak. For example, tBid can bind to latent, inactive Bax and promote its conformational change and translocation to the mitochondrial membrane that is required for apoptosis.36 BH3-only proteins can inhibit Bcl-2-like proteins, releasing these direct activators of Bax and Bak to promote apoptosis in the de-repression mode (Fig. 7.4). BH3-only proteins that only interact with Bcl-2-like proteins can release activator BH3-only proteins to promote apoptosis in the sensitizer mode (Fig. 7.4). Thus, apoptosis induction by BH3-only proteins can occur through neutralization, de-repression and sensitizer functions.28
Importantly, it is this BH3 interaction with Bcl-2 that is the molecular basis for the BH3-mimetic class of proapoptotic, Bcl-2–antagonizing anticancer drugs (Fig. 7.5).33,37–39 This detailed understanding of the Bcl-2 family member protein interactions and function is allowing rational, apoptosis-targeted therapy (see later discussion).
Figure 7.5. Three-dimensional structure of Bcl-xL with bound Bad BH3 ligand and ABT-737.
Space-filling model of Bcl-xL illustrating the hydrophobic cleft binding the 25-mer peptide (green helix) of the Bad BH3 (left) or the rationally designed BH3-mimetic ABT-737 (right).
Role of Mitochondrial Membrane Permeabilization in Apoptosis
Once activated, Bax and Bak oligomerize in the mitochondrial outer membrane, rendering it permeable to proapoptotic mitochondrial proteins cytochrome c and SMAC.40–44 How Bcl-2 family members permeabilize membranes is not entirely clear but it is likely related to a change in topology of the proteins in the membrane and formation of a channel or pore. Once released into the cytoplasm, cytochrome c interacts with the WD40 domains of APAF-1 in the apoptosome, a wheel-like particle with sevenfold symmetry that serves as a scaffold for caspase-9 activation.45 SMAC functions to antagonize the caspase inhibitors, the IAP proteins, to facilitate caspase activation. The amino-terminus of SMAC binds to IAPs, neutralizing their caspase-inhibitor function. Subsequent effector caspase activation (e.g., caspase-3), leads to the rapid, orderly dismantling of the cell and cell death without activating the innate immune response.46