Programmed Cell Death

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CHAPTER 46 Programmed Cell Death

The Necessity for Cell Death in Multicellular Organisms

The ability to undergo programmed cell death (Box 46-1) is a built-in latent capacity in virtually all cells of multicellular organisms. Cell death is important for embryonic development, maintenance of tissue homeostasis, establishment of immune self-tolerance, killing by immune effector cells, and regulation of cell viability by hormones and growth factors. It has been proposed that most metazoan cells will die if they fail to receive survival signals from other cells. Abnormalities of the cell death program contribute to a number of diseases, including cancer, Alzheimer’s disease, and acquired immune deficiency syndrome (AIDS).

BOX 46-1 Key Terms

Programmed Cell Death: An active cellular process that culminates in cell death. This may occur in response to developmental or environmental cues or as a response to physiological damage detected by the cell’s internal surveillance networks.

Necrosis (Accidental Cell Death): Cell death that results from irreversible injury to the cell. Cell membranes swell and become permeable. Lytic enzymes destroy the cellular contents, which then leak out into the intercellular space, leading to the mounting of an inflammatory response.

Apoptosis: One type of programmed cell death that initially was characterized by a particular pattern of morphologic changes but now is defined by the action of molecular pathways involving cell surface receptors or mitochondria and resulting in the activation of specialized proteases. The name comes from the ancient Greek, referring to shedding of the petals from flowers or leaves from trees. Apoptosis is observed in all metazoans, including both plants and animals.

Apoptotic death occurs in two phases. During the latent phase, the cell looks morphologically normal but is actively making preparations for death. The execution phase is characterized by a series of dramatic structural and biochemical changes that culminate in the fragmentation of the cell into membrane-enclosed apoptotic bodies. Activities that cause cells to undergo apoptosis are said to be pro-apoptotic. Activities that protect cells from apoptosis are said to be anti-apoptotic.

Programmed Cell Death versus Accidental Cell Death: Apoptosis versus Necrosis

Although cells die in many ways, it is useful to focus on the two poles of this spectrum: apoptosis and necrosis. Apoptosis is the most commonly described pathway for programmed cell death, which is cellular suicide resulting from activation of a dedicated intracellular program (Fig. 46-1). Often, these cells appear completely healthy prior to committing suicide. At the other end of the spectrum is necrosis, also called accidental cell death, which occurs when cells receive a structural or chemical insult that kills them outright (Fig. 46-2). Examples of such insults include extremes of temperature and physical trauma. The cell itself can also initiate necrosis in response to certain stimuli, particularly when induction of apoptosis is inhibited. In contrast to the orderly biochemical pathways of apoptosis, which involve the action of enzyme cascades and the consumption of ATP, necrosis typically involves a collapse of normal cell physiology as a result of ATP depletion.

Figure 46-1 apoptosis—active cellular suicide—typically affects single cells. Neighboring cells remain healthy. Apoptotic cell death usually does not lead to an inflammatory response.

Figure 46-2 necrosis is a result of injury to cells. Typically, groups of cells are affected. In most cases, necrotic cell death leads to an inflammatory response (red “angry” macrophages).

Necrosis corresponds to what most of us naively imagine cell death would be like. Owing to lack of cellular homeostasis, water rushes into the dying cell, causing it to swell greatly so that the plasma and organelle membranes burst. As a result, the cell undergoes a generalized process of autodigestion and dissolution, culminating in the spilling of the cytoplasmic contents out into the surroundings (Fig. 46-2). This, in turn, produces local inflammation as phagocytic cells are activated, flock to the site, and ingest the debris (see Chapter 22). Because agents that damage cells act over areas that are large in comparison to the size of a single cell, necrosis often involves large groups of neighboring cells.

In contrast to necrosis, apoptotic cells shrink rather than swelling, as part of a reproducible pattern of structural alterations of both the nucleus and cytoplasm (Fig. 46-1). Apoptosis is a two-stage process. On receipt of the pro-apoptotic signal that triggers the pathway to death, cells enter a latent phase of apoptosis (Fig. 46-3). Although committed to a pathway that leads to their inevitable demise at some later time, cells in the latent phase look as healthy as their neighbors. The duration of the latent phase of apoptosis is extremely variable, ranging from a few hours to several days. The reason for this variability is not known.

Figure 46-3 the two phases of apoptosis. Note that the latent phase can be subdivided into two stages: a condemned stage, during which the cell is proceeding on a pathway toward death but can still be rescued if it is exposed to anti-apoptotic activities, and a committed stage, beyond which rescue is impossible.

Ultimately, the cells enter the execution phase of apoptosis, lasting about an hour, during which they undergo dramatic morphologic and physiological changes. These include (1) loss of microvilli and intercellular junctions (Fig. 46-4); (2) shrinkage of the cytoplasm; (3) dramatic changes in cytoplasmic motility with activation of violent blebbing (Fig. 46-5); (4) loss of plasma membrane asymmetry, with the distribution of phosphatidylserine being randomized so that it appears in the outer membrane leaflet; (5) hypercondensation of the chromatin and its collapse against the nuclear periphery; and (6) the “explosive” fragmentation of the cell into membrane-enclosed apoptotic bodies that contain remnants of the nucleus, mitochondria, and other organelles. The plasma membrane retains its integrity throughout the entire process. All of these changes are instigated by the action of a specific set of death-inducing proteases, discussed at length later.

Figure 46-4 scanning electron micrograph of intact and apoptotic mouse sarcoma cells. Intact cells are covered with microvilli, whereas apoptotic cells have numerous smooth blebs. These cells were stimulated to undergo apoptosis as a result of interference with RNA metabolism.

(From Wyllie AH, Kerr JFR, Currie AR: Cell death: The significance of apoptosis. Int Rev Cytol 68:251–305, 1980.)

Figure 46-5 apoptosis of a transformed pig kidney cell following exposure to etoposide, a drug used in cancer chemotherapy. The dramatic cytoplasmic blebbing results in the disassembly of the cell into membrane-enclosed vesicles.

(Courtesy of L. M. Martins and K. Samejima, Wellcome Trust Institute for Cell Biology, University of Edinburgh, Scotland.)

In tissues, apoptotic bodies are rapidly phagocytosed by surrounding cells that recognize the phosphatidylserine and other markers exposed on their surface (Fig. 46-6). Apoptosis can thus be considered to be the disassembly of the cell into “bite-sized” vesicles. Because these vesicles remain membrane bound, the cellular contents are not released into the environment. It is important to note that surface markers on apoptotic bodies cause cells that ingest them to secrete anti-inflammatory cytokines. As a result, apoptotic death does not lead to an inflammatory response.

Figure 46-6 phagocytosis of apoptotic cells. a–c, Phagocytosis that occurs when cells express “eat me” signals results in the production of anti-inflammatory cytokines. D, Electron micrograph of a phagocytosed apoptotic body containing a nuclear fragment. The nucleus of the epithelial cell that engulfed this apoptotic body is shown at left. In this case, apoptosis occurred during allo-graft rejection in a pig.

(A, Based on Lauber K, Blumenthal SG, Waibel M, Wesselborg S: Clearance of apoptotic cells: Getting rid of the corpses. Mol Cell 12:277–287, 2004. B, From Wyllie AH, Kerr JFR, Currie AR: Cell death: The significance of apoptosis. Int Rev Cytol 68:251–305, 1980.)

Nonapoptotic Programmed Cell Death

The terms apoptosis and programmed cell death are sometimes viewed as synonymous. However, in a number of well-documented systems, cells undergo programmed cell death without the dramatic structural changes that classically define apoptosis. Thus, all apoptosis is programmed cell death, but the converse is not necessarily true.

When the adult tobacco hawkmoth emerges from its cocoon, its intersegmental muscles undergo programmed cell death that differs in several ways from apoptosis as described earlier. The chromatin does not condense; DNA is not digested; and cytoplasm does not “boil.” Instead, a polyubiquitin gene is induced and plays an important role in intracellular protein degradation (see Chapter 23). Thus, although these muscle cells unquestionably undergo programmed cell death, they apparently do not use the apoptosis pathway.

Classes of Cells That Undergo Programmed Cell Death

At least six distinct classes of cells undergo programmed cell death (examples are given in Fig. 46-7).

Figure 46-7 a, Types of cells that undergo programmed cell death. B–D, Programmed cell death in the embryonic mouse paw. At day 12.5 of development, the digits are fully connected by webbing. By day 13.5, the webbing has started to die, and by day 14.5, all of the webbing cells are gone. E, Nuclei of cells undergoing programmed cell death take up acridine orange, whereas cells of the surrounding healthy tissue do not.

(Micrographs courtesy of William Wood and Paul Martin, Department of Anatomy and Developmental Biology, University College of London, England.)

Developmentally Defective Cells

During molecular maturation of T-lymphocyte antigen receptors (see Figs. 27-8 and 28-8), immature T cells in the thymus (known as thymocytes) rearrange the genes encoding the receptor a and b chains. Many newly created receptors bind to foreign antigens, but others interact with self-antigens. Cells with receptors recognizing self-antigens are potentially harmful and are eliminated through apoptosis in a process known as negative selection (Fig. 46-8). The drug cyclosporin A, which inhibits apoptosis in thymocytes, can cause autoimmune disease.

Figure 46-8 examples of signals that promote differentiation or programmed cell death of immature thymocytes in the thymus and mature t cells in the periphery. Thymocytes that make functional T-cell receptors and do not recognize self-antigens mature, provided that they receive survival signals, such as interleukin-7. Thymocytes undergo apoptosis if they produce defective T-cell receptor, recognize self-antigens, suffer DNA damage, or receive a death stimulus (glucocorticoid hormone). More than 95% of immature thymocytes die without leaving the thymus.

(Based on Strasser A: The role of BH3-only proteins in the immune system. Nat Rev Immunol 5:189–200, 2005.)

To function properly, the T-cell receptor must recognize major histocompatibility complex (MHC) glycoproteins on other cells during antigen presentation (see Fig. 27-8). T lymphocytes whose T-cell receptors cannot interact with the spectrum of MHC glycoproteins expressed in a given individual are ineffective in the immune response. These cells die by apoptosis in a process known as positive selection (Fig. 46-8). Overall, defects in T-cell receptor assembly are extremely common, and up to 95% of immature T cells die by apoptosis without leaving the thymus.

Similar positive and negative selection steps occur during the maturation of B lymphocytes (see Fig. 28-8), which is accomplished by a combination of gene rearrangements and facilitated mutagenesis. B lymphocytes expressing antibodies directed against self-antigens or producing antibodies whose affinity for antigen is below a critical threshold are eliminated through apoptosis.

Excess Cells

The use of programmed cell death for quality control during development is not limited to the immune system but is also extremely important during brain development. Embryonic ganglia often have many more neurons than are required to enervate their target muscles. Production of excess cells is part of a Darwinian strategy to ensure that a sufficient number of axons reach their targets. Excess neurons that fail to make appropriate connections have no function and are eliminated by programmed cell death. Up to 80% of neurons in certain developing ganglia die in this way. Because of the importance of apoptosis during its development, the brain is often seriously affected in mice that are engineered to lack components of the apoptotic pathway.

Cells That Serve No Function

The elimination of obsolete cells whose function has been completed is most evident in organisms, such as insects and amphibians, that undergo metamorphosis during development. For example, programmed cell death initiated by a burst of thyroid hormone is responsible for resorption of the tadpole tail.

Mammals also use programmed cell death to eliminate obsolete tissues during development. For example, in humans, the digits of hands and feet are connected by a tissue webbing during embryogenesis. Cells in this webbing serve no purpose in the adult and are eliminated by programmed cell death (Fig. 46-7).

During craniofacial development, the hard palate develops from two lateral precursors, each covered in a protective layer of epithelial cells. As the two halves grow together at the midline of the nasopharynx, they remain separated by this covering of epithelium until, in response to a developmental cue, the epithelial cells at the midline undergo programmed cell death. Then the two halves of the palate can fuse. Failure of the epithelial cells to die at the appropriate time can interfere with the fusion of the bone, causing cleft palate.

Populations of cells that are fully functional may become obsolete as a result of physiological changes in the status of an organism. For example, in male mammals, certain accessory glands of the reproductive system are regulated by the levels of circulating male hormone. If hormone levels fall below a critical threshold, these organs, including the prostate, virtually disappear in a very brief time as their constituent cells undergo massive apoptotic death. Should levels of circulating androgens rise again, the remaining prostatic stem cells proliferate and reconstruct the gland. A similar cycle of growth and involution is seen in the mammary gland of female mammals, which exhibits substantial differences in size and cellular composition in the lactating and nonlactating states. Interference with survival signaling by sex hormones is one important strategy that is commonly used in the treatment of breast and prostate cancer.

Programmed cell death is also used to eliminate certain populations of cells that never served any function to begin with. The Müllerian ducts develop into the female oviduct. In male embryos, progenitors of the Müllerian ducts develop, even though they have no function. Programmed cell death eliminates the constituent cells of these embryonic ducts.

Cells Whose Cell Cycle Is Perturbed

Chapters 40 to 43 describe how biochemical circuits called checkpoints regulate the cell cycle. If DNA is damaged, checkpoint activation blocks cell-cycle progression while repair processes operate. An important downstream effector of checkpoints, the p53 transcription factor, induces the expression of genes encoding proteins that arrest the cell cycle as well as genes encoding proteins that induce cell death. It is generally thought that if the damage cannot be repaired quickly, the pro-death factors win out, and the outcome is apoptosis. Types of DNA damage that commonly trigger cell death are double-strand breaks induced by ionizing radiation and DNA breaks or other damage induced by chemotherapeutic agents.

A second important cell-cycle checkpoint regulates the transition from the G1 phase to the S phase. Passage of the restriction point (see Fig. 41-7) represents the commitment of the cell to undergo another cycle of DNA replication and division. Restriction point control centers on the regulation of the E2F family of transcription factors. However, E2F not only regulates genes that promote cell-cycle progression; it also induces the expression of genes that promote apoptosis. It is now thought that if E2F is activated too strongly, as, for example, where restriction point control has broken down (see Fig. 41-10), its function as a death inducer takes over, and the cells undergo apoptosis. Cells that die in response to inappropriate signals to proliferate include those that are infected by certain viruses or overexpress genes involved in cell proliferation (such as c-myc and c-fos [Fig. 46-14]). This ability to recognize an inappropriate stimulus to proliferate and respond to it by undergoing apoptosis may be an important defense against cancer.

Figure 46-14 Bcl-2 protects cells from oncogene-induced cell death. Chinese hamster ovary cells die when they are induced to express abnormally high levels of the c-myc protein, but simultaneous expression of the Bcl-2 protein rescues them from this effect. These cells contain many copies of the c-myc gene under control of a promoter that is activated when the cells are briefly exposed to high temperature (43°C for 90 minutes). The curves show the percentage of viable cells remaining at various times following the induction of c-myc expression. The Bcl-2 gene was introduced into these cells on a plasmid molecule under the control of a viral promoter, which is always active. The blue line represents the parental cell line lacking either the cloned c-myc or Bcl-2 genes. (Note that about 40% of these cells die following the heat treatment used to induce c-myc expression.) The yellow line shows that the cells that produce high levels of c-myc protein alone rapidly die by apoptosis. The red line shows that cells expressing both the c-myc and Bcl-2 proteins survive the treatment almost as well as the parental cells.

(From Bissonnette RP, Echeverri F, Mahboubi A, et al: Apoptotic cell death induced by c-myc is inhibited by bcl-2. Nature 359:552–556, 1992.)

Virus-Infected Cells

Cells that harbor infectious agents, such as viruses, are harmful to the organism. Cytotoxic T lymphocytes eliminate virus-infected cells by causing them to undergo programmed cell death either by apoptosis or by a second related pathway.

At least part of the loss of mature CD4⁺ T helper cells (see Fig. 28-8) in people who are infected with HIV-1 results from programmed cell death. When exposed to agents that normally stimulate cell proliferation, these cells instead undergo apoptosis. Paradoxically, it ap-pears that many of these dying cells are not themselves infected with HIV.