First printed in R&D Systems' 2000 Catalog.
A healthy organism is an exquisitely integrated collection of differentiated cells, which maintain a balance between life and death. Some cells are irreplaceable, some cells complete their functions and are then sacrificed, and some cells live a finite lifetime to be replaced by yet another generation. A failure of cells to fulfill their destiny has catastrophic consequences on an organism. Apoptosis1 (also called programmed cell death) is the last phase of a cell's destiny. Apoptosis is the controlled disassembly of a cell. When apoptosis occurs on schedule, neighboring cells and, more importantly, the organism itself, are not adversely affected. Apoptosis gone awry, however, has dire effects.2 When apoptosis occurs in irreplaceable cells, as in some neurodegenerative disorders, functions critical to the organism are lost. When cells fail to undergo apoptosis after serving their purpose, as in some autoimmune disorders, escaped cells adversely affect the organism. When cells become renegade and resist apoptosis, as in cancer, the outlaw cells create a dire situation for the organism. Mistiming of, or errors in, apoptosis can have devastating consequences on development. Apoptotic fidelity is, therefore, critical to the well-being of an organism.
|Figure 1. Release of cytochrome c from the mitochondria can trigger a series of events leading to the activation of effector caspases. For example, pro-caspase-9 is activated when complexed with dATP, APAF-1, and extra-mitochondrial cytochrome c. Following activation, caspase-9 can initiate apoptosis by cleaving additional caspases.|
Apoptosis and necrosis are two major processes by which cells die. Apoptosis is the ordered disassembly of the cell from within.3 Disassembly creates changes in the phospho-lipid content of the plasma membrane outer leaflet. Phosphatidylserine (PS) is exposed on the outer leaflet and phagocytic cells recognizing this change, may engulf the apoptotic cell or cell-derived, membrane-limited apoptotic bodies.4 Necrosis normally results from a severe cellular insult. Both internal organelle and plasma membrane integrity are lost, resulting in spilling of cytosolic and organellar contents into the surrounding environment. Immune cells are attracted to the area and begin producing cytokines that generate an inflammatory response. Thus, cell death in the absence of an inflammatory response may be the best way to distinguish apoptosis from necrosis. Other techniques that have been used to distinguish apoptosis from necrosis in cultured cells and in tissue sections include detecting PS at the cell surface with annexin V binding, DNA laddering, and staining cleaved DNA fragments that contain characteristic ends. At the extremes, apoptosis and necrosis clearly involve different molecular mechanisms. It is not clear if there is cellular death involving both the molecular mechanisms of apoptosis and those of necrosis. Cell death induced by free radicals, however, may have characteristics of apoptosis and necrosis.5
Cytosolic Aspartate-Specific Proteases, called CASPases, are responsible for the deliberate disassembly of a cell into apoptotic bodies. Caspases are present as inactive pro-enzymes, most of which are activated by proteolytic cleavage. Caspase-8, caspase-9, and caspase-3 are situated at pivotal junctions in apoptotic pathways. Caspase-8 initiates disassembly in response to extracellular apoptosis-inducing ligands and is activated in a complex associated with the receptors' cytoplasmic death domains.6 Caspase-9 activates disassembly in response to agents or insults that trigger release of cytochrome c from the mitochondria7,8 and is activated when complexed with dATP, APAF-1, and extra-mitochondrial cytochrome c.9 Caspase-3 appears to amplify caspase-8 and caspase-9 signals into full-fledged commitment to disassembly.9,10 Both caspase-8 and caspase-9 can activate caspase-3 by proteolytic cleavage and caspase-3 may then cleave vital cellular proteins or activate additional caspases by proteolytic cleavage. Many other caspases have been described.11,12
|Figure 2. Removal of a cytokine required for cellular viability leads to the following sequential events: i) loss of kinase (AKT) activity; ii) dephosphorylation of Bad (or some other regulator of Bcl-2/Bcl-x activity); iii) Bad binding to Bcl-x or Bcl-2; iv) loss of normal mitochondrial physiology; v) release of cytochrome c; vi) binding of cytochrome c by Apaf-1 with concomitant activation of caspase-9; vii) amplification by the caspase cascade; viii) cleavage of vital cellular proteins; ix) fission of cells into apoptotic bodies; and finally, x) disappearance of any traces of the cell when the apoptotic bodies are engulfed by either neighboring or phagocytic cells.|
There are two central pathways that lead to apoptosis: i) positive induction by ligand binding to a plasma membrane receptor and ii) negative induction by loss of a suppressor activity. Each leads to activation of cysteine proteases with homology to IL-1 beta converting enzyme (ICE) (i.e., caspases).3 Positive-induction involves ligands related to TNF. Ligands are typically trimeric and bind to cell surface receptors causing aggregation (trimerization) of cell surface receptors.6 The intracellular portion of these receptors contains an 80 amino acid death domain (DD) that through homophilic interactions recruits adaptor proteins to form a signaling complex on the cytosolic surface of the receptor.13 The bringing together of three receptors, thereby orienting the intracellular DDs, appears to be the critical feature for signaling by these receptors. The adaptor complex then recruits caspase-8, caspase-8 is activated, and the cascade of caspase-mediated disassembly proceeds.
Negative induction of apoptosis by loss of a suppressor activity involves the mitochondria.8 Release of cytochrome c from the mitochondria into the cytosol serves as a trigger to activate caspases.7 Permeability of the mitochondrial outer membrane is essential to initiation of apoptosis through this pathway. Proteins belonging to the Bcl-2 family appear to regulate the membrane permeability to ions and possibly to cytochrome c as well.14 Although these proteins can themselves form channels in membranes, the actual molecular mechanisms by which they regulate mitochondrial permeability and the solutes that are released are less clear. The Bcl-2 family is composed of a large group of anti-apoptosis members that when overexpressed prevent apoptosis and a large group of pro-apoptosis members that when overexpressed induce apoptosis. The balance between the anti-apoptotic and pro-apoptotic Bcl-2 family members may be critical to determining if a cell undergoes apoptosis. Thus, the suppressor activity of the anti-apoptotic Bcl-2 family appears to be negated by the pro-apoptotic members.
Many members of the pro-apoptotic Bcl-2 family are present in cells at levels sufficient to induce apoptosis. However, these members do not induce apoptosis because their activity is maintained in a latent form. Bax is present in the cytosol of live cells. After an appropriate signal, Bax undergoes a conformational change and moves to the mitochondrial membrane where it causes release of mitochondrial cytochrome c into the cytosol.15,16 BID is also present in the cytosol of live cells. After cleavage by caspase-8, it moves to the mitochondria where it causes release of cytochrome c17,18 possible by altering the conformation of Bax.19 Similarly, BAK appears to undergo a conformational change that converts it from an inactive to an active state.20 Thus, understanding the molecular mechanisms responsible for regulating the Bcl-2 family activities creates the potential for pharmaceutical intervention to control apoptosis.
The viability of many cells is dependent on a constant or intermittent supply of cytokines or growth factors. In the absence of an apoptosis-suppressing cytokine, cells may undergo apoptosis. Bad is a pro-apoptotic member of the Bcl-2 family21 and is sequestered in the cytosol when cytokines are present. Cytokine binding to its receptor induces the phosphorylation of Bad. Cytokine binding can activate PI3 kinase, which phosphorylates Akt/PKB, which in turn phosphorylates Bad.22 Phosphorylated Bad is sequestered in the cytosol by the 14-3-3 protein.23 Removal of the cytokine turns the kinase pathway off, the phosphorylation state of Bad shifts to the dephosphorylated form, and dephosphorylated Bad causes release of cytochrome c from the mitochondria.
Suppression of the anti-apoptotic members or activation of the pro-apoptotic members of the Bcl-2 family leads to altered mitochondrial membrane permeability resulting in release of cytochrome c into the cytosol.24-28 In the cytosol, or on the surface of the mitochondria, cytochrome c is bound by the protein Apaf-1 (apoptotic protease activating factor27,29), which also binds caspase-930 and dATP. Binding of cytochrome c triggers activation of caspase-9, which then accelerates apoptosis by activating other caspases. Release of cytochrome c from mitochondria has been established by determining the distribution of cytochrome c in subcellular fractions of cells treated or untreated to induce apoptosis.24,25,31,32 Cytochrome c was primarily in the mitochondria-containing fractions obtained from healthy, non-apoptotic cells and in the cytosolic non-mitochondria-containing fractions obtained from apoptotic cells. Using mitochondria-enriched fractions from mouse liver, rat liver, or cultured cells it has been shown that release of cytochrome c from mitochondria is greatly accelerated by addition of Bax,33,34 fragments of BID,35-37 and by cell extracts.24
|Figure 3. The inhibitor of apoptosis proteins, XIAP, cIAP-1, cIAP-2, and Survivin, can prevent proteolytic processing of procaspases-3, -6 and -7 by blocking cytochrome c-induced activation of pro-caspase-9.|
The induction of apoptosis or progression through the process of apoptosis is inhibited by a group of proteins called Inhibitors of Apoptosis (IAPs). These proteins contain a BIR (baculovirus IAP repeat) domain near the amino-terminus.38-41 The BIR domain can bind some caspases.39 Many members of the IAP family of proteins block proteolytic activation of caspase-3 and -7.38,39,42-44 XIAP, cIAP-1 and cIAP-2 appear to block cytochrome c-induced activation of caspase-9, thereby preventing initiation of the caspase cascade.43 Since cIAP-1 and cIAP-2 were first identified as components in the cytosolic death domain-induced complex41 associated with the TNF family of receptors, they may inhibit apoptosis by additional mechanisms.
FLIP/FLAME is highly homologous to caspase-8.45-48 It does not, however, contain the active site required for proteolytic activity. FLIP appears to compete with caspase-8 for binding to the cytosolic receptor complex, thereby preventing the activation of the caspase cascade in response to members of the TNF family of ligands. The exact in vivo influence of the IAP family of proteins on apoptosis is not clear.