Programmed cell death is a normal physiological form of cell death that plays a key role both in the maintenance of adult tissues and in embryonic development. In adults, programmed cell death is responsible for balancing cell proliferation and maintaining constant cell numbers in tissues undergoing cell turnover. For example, about 5 × 1011 blood cells are eliminated by programmed cell death daily in humans, balancing their continual production in the bone marrow. In addition, programmed cell death provides a defense mechanism by which damaged and potentially dangerous cells can be eliminated for the good of the organism as a whole. Virus-infected cells frequently undergo programmed cell death, thereby preventing the production of new virus particles and limiting spread of the virus through the host organism. Other types of insults, such as DNA damage, also induce programmed cell death. In the case of DNA damage, programmed cell death may eliminate cells carrying potentially harmful mutations, including cells with mutations that might lead to the development of cancer.
During development, programmed cell death plays a key role by eliminating unwanted cells from a variety of tissues. For example, programmed cell death is responsible for the elimination of larval tissues during amphibian and insect metamorphosis, as well as for the elimination of tissue between the digits during the formation of fingers and toes. Another well-characterized example of programmed cell death is provided by development of the mammalian nervous system. Neurons are produced in excess, and up to 50% of developing neurons are eliminated by programmed cell death. Those that survive are selected for having made the correct connections with their target cells, which secrete growth factors that signal cell survival by blocking the neuronal cell death program. The survival of many other types of cells in animals is similarly dependent on growth factors or contacts with neighboring cells or the extracellular matrix, so programmed cell death is thought to play an important role in regulating the associations between cells in tissues. Regulation of programmed cell death is mediated by the integrated activity of a variety of signaling pathways, some acting to induce cell death and others to promote cell survival.
(A) Diagrammatic representation of the events of apoptosis. (B) Light micrographs of normal and apoptotic human leukemia cells, illustrating chromatin condensation and nuclear fragmentation during apoptosis. (B, courtesy of D. R. Green/La Jolla Institute for Allergy and Immunology.)
). During apoptosis, chromosomal DNA is usually fragmented as a result of cleavage between nucleosomes. The chromatin condenses and the nucleus then breaks up into small pieces. Finally, the cell itself shrinks and breaks up into membrane-enclosed fragments called apoptotic bodies. Such apoptotic cells and cell fragments are readily recognized and phagocytosed by both macrophages and neighboring cells, so cells that die by apoptosis are efficiently removed from tissues. In contrast, cells that die as a result of acute injury swell and lyse, releasing their contents into the extracellular space and causing inflammation.
Studies of programmed cell death during the development of C. elegans have identified three genes that play key roles in regulating and executing apoptosis. During normal nematode development, 131 somatic cells out of a total of 1090 are eliminated by programmed cell death. Two genes, ced-3 and ced-4, are required for apoptosis to occur; if either of these genes is inactivated, the normal programmed cell deaths do not take place. A third gene, ced-9, functions as a negative regulator of apoptosis. If ced-9 is inactivated by mutation, the cells that would normally survive fail to do so. Instead, they also undergo apoptosis, leading to death of the developing animal. Conversely, if ced-9 is expressed at an abnormally high level, the normal programmed cell deaths fail to occur.
Many cell death signals induce apoptosis via a conserved pathway of regulators, adaptors, and caspases. In C. elegans, the negative regulator Ced-9 inhibits apoptosis by binding to the adaptor Ced-4. In the absence of inhibition by Ced-9, Ced-4 binds two molecules of the caspase Ced-3, resulting in autocleavage and caspase activation. In mammals, regulators of the Bcl-2 family (Ced-9 homologs) act at the mitochondria to control release of cytochrome c, which is required for the binding of caspase-9 to the adaptor Apaf-1 (the Ced-4 homolog). Release of cytochrome c from mitochondria thus signals the activation of caspase-9, which then activates downstream caspases to induce apoptosis.
). Ced-3 is the prototype of a family of more than a dozen proteases, known as caspases because they have cysteine (C) residues at their active sites and cleave after aspartic acid (Asp) residues in their substrate proteins. The caspases are the ultimate effectors or executioners of programmed cell death, bringing about the events of apoptosis by cleaving more than 40 different cell target proteins. Key targets of the caspases include an inhibitor of a DNase which, when activated, is responsible for fragmentation of nuclear DNA. In addition, caspases cleave nuclear lamins, leading to fragmentation of the nucleus, and cytoskeletal proteins, leading to disruption of the cytoskeleton, membrane blebbing, and cell fragmentation.
The caspases are synthesized as inactive precursors that are converted to the active form by proteolytic cleavage, catalyzed by other caspases. The initial activation of a caspase therefore starts off a chain reaction leading to activation of additional caspases and death of the cell. Regulation of caspase activation is thus key to determining cell survival. Ced-4 and its mammalian homolog (Apaf-1) bind to caspases and promote their activation by forming a complex in which two caspases can cleave and activate one another. In contrast, Ced-9 inhibits caspase activation. Mammals encode a whole family of proteins (called the Bcl-2 family) that are related to Ced-9. Some members of the Bcl-2 family, including Bcl-2 itself, function analogously to Ced-9 as inhibitors of caspase activation and programmed cell death. Other members of the Bcl-2 family, however, promote cell death.
In C. elegans, Ced-9 directly regulates caspase activation by binding to Ced-4 and preventing it from activating Ced-3. In mammalian cells, however, members of the Bcl-2 family regulate caspase activation by a less direct mechanism. In particular, Bcl-2 family members appear to act principally at mitochondria, which play a central role in controlling programmed cell death in mammalian cells. One of the key caspases in mammalian cells (caspase-9) is activated, like Ced-3 in C. elegans, by forming a complex with the Ced-4 homolog Apaf-1. However, in contrast to C. elegans, formation of a complex with Apaf-1 is not sufficient for activation of caspase-9. Instead, activation of caspase-9 requires formation of a complex consisting of caspase-9, Apaf-1, and cytochrome c. Under normal conditions of cell survival, cytochrome c is localized to the mitochondrial intermembrane space (see Figure 10.8) while Apaf-1 and caspase-9 are found in the cytosol, so caspase-9 remains inactive. However, many stimuli that trigger cell death, including DNA damage and growth factor deprivation, lead to damage of the mitochondria and release of cytochrome c to the cytosol. In the cytosol, cytochrome c can form a complex with caspase-9 and Apaf-1, leading to caspase-9 activation and cell death. Members of the Bcl-2 family act in the mitochondrial membrane to regulate mitochondrial integrity and cytochrome c release. Bcl-2 family members that inhibit apoptosis (such as Bcl-2 itself) prevent cytochrome c release, whereas Bcl-2 family members that promote cell death act by inducing mitochondrial damage, cytochrome c release, and caspase activation. As discussed below, both caspases and members of the Bcl-2 family are critical targets of the signaling pathways that control survival of mammalian cells.
Some secreted polypeptides signal programmed cell death by activating receptors that directly induce apoptosis of the target cell. These cell death signals are polypeptides belonging to the tumor necrosis factor (TNF) family. They bind to members of the TNF receptor family, which can signal apoptosis in a variety of cell types. One of the best characterized members of this family is the cell surface receptor called Fas, which plays important roles in controlling cell death in the immune system. For example, apoptosis induced by activation of Fas is responsible for killing target cells of the immune system, such as cancer cells or virus-infected cells, as well as for eliminating excess lymphocytes at the end of an immune response.
Binding of ligand to the Fas receptor induces apoptosis by direct activation of caspase-8. Fas ligand consists of three polypeptide chains, so its binding induces receptor trimerization. Caspase-8 bound to the receptor via adaptor molecules is then activated by autocleavage, leading to activation of downstream caspases and cell death.
). TNF and related family members consist of three identical polypeptide chains, and their binding induces receptor trimerization. The cytoplasmic portions of the receptors bind adaptor molecules that in turn bind an upstream caspase called caspase-8. This leads to activation of caspase-8 as a result of self-cleavage, and the activated molecules of caspase-8 can then activate other downstream caspases, thereby initiating a caspase cascade that results in death of the cell.
Caspase-8 not only cleaves other caspases, but it also cleaves a member of the Bcl-2 family called Bid. Bid is one of the Bcl-2 family members that induces rather than protects against apoptosis. Normally, it is retained in inactive form in the cytosol. However, cleavage by caspase-8 allows Bid to translocate to mitochondria where it disrupts the membrane and releases cytochrome c into the cytosol. This leads to activation of caspase-9, further amplifying the caspase cascade initiated by direct activation of caspase-8 at cell death receptors.
Signaling by TNF and related polypeptides is an active process in which stimulation of cell death receptors induces apoptosis. Other signaling pathways act in the opposite direction to promote cell survival by inhibiting apoptosis. These signaling pathways control the fate of a wide variety of cells whose survival is dependent on extracellular growth factors or cell-cell interactions. Indeed, most cells in higher animals are programmed to undergo apoptosis unless cell death is actively suppressed by survival signals from other cells.
As already noted, a well-characterized example of programmed cell death in development is provided by the vertebrate nervous system. About 50% of neurons die by apoptosis, with the survivors having received sufficient amounts of survival signals from their target cells. These survival signals are polypeptide growth factors related to nerve growth factor (NGF), which induces both neuronal survival and differentiation by activating a receptor protein-tyrosine kinase. Other types of cells are similarly dependent upon growth factors or cell contacts that activate signaling molecules (including nonreceptor protein-tyrosine kinases) associated with integrins.
Survival factors such as NGF activate receptor protein-tyrosine kinases, leading to activation of PI 3-kinase and formation of PIP3. PIP3 recruits the protein kinase Akt to the plasma membrane where it is activated as a result of phosphorylation by PDK. Akt then appears to phosphorylate a number of proteins that contribute to cell survival. The targets of Akt that have been implicated in suppression of apoptosis include the Bcl-2 family member Bad, caspase-9, several transcription factors, and the protein kinase GSK-3, which affects cell metabolism and protein synthesis.
). One substrate for Akt is a member of the Bcl-2 family called Bad. Bad is one of the Bcl-2 family members (like Bid) that induces cell death by stimulating the release of cytochrome c from mitochondria. Phosphorylation of Bad by Akt creates a binding site for proteins that sequester Bad in the cytosol, thereby preventing the translocation of Bad to the mitochondrial membrane. Akt may also directly block caspase activation by phosphorylating caspase-9. In addition to these direct effects on components of the cell death machinery, Akt phosphorylates transcription factors that regulate cell survival and another protein kinase (GSK-3) that affects apoptosis as well as regulating cell metabolism and protein synthesis. The PI 3-kinase/ Akt pathway thus regulates cell survival through phosphorylation of a variety of downstream targets, more of which are likely to be identified by future research.
Cell survival is mediated not only by PI 3-kinase/Akt signaling, but also by other signaling pathways including the Ras/Raf/MAP kinase pathway. One mechanism through which this pathway inhibits apoptosis involves the phosphorylation and activation of a protein kinase called RSK by ERK. Like Akt, RSK phosphorylates the Bcl-2 family member Bad, so Bad serves as a site of convergence for the PI 3-kinase/Akt and MAP kinase pathways in signaling cell survival. In addition, ERK and RSK may act by phosphorylating transcription factors that affect the expression of genes that regulate apoptosis. In Drosophila, the MAP kinase pathway suppresses apoptosis by regulating the activity of proteins that directly inhibit caspases, but it is not known if this mechanism is also operative in mammalian cells. Understanding the signals and mechanisms that control cell survival thus remains an active and exciting area of ongoing research, with many questions still to be answered.
