All cells eventually die either by necrosis or apoptosis. Necrosis is a passive, pathological process induced by cellular injury or accidental means and often involves the simultaneous death of cells in groups (Figure 23.1). Necrotic cells have ruptured cell membranes allowing the cytoplasm and organelles to spill into the surrounding tissue fluids, often inducing an inflammatory response.
In contrast, apoptosis is an active, normal, physiological process that removes individual cells without damaging neighboring cells or inducing inflammation. Cells undergoing apoptosis have a characteristic “blebbed” appearance of their membranes. Apoptosis is as fundamental to cellular and tissue physiology as cell division and differentiation. Disturbance in pathways that regulate apoptosis may result in cancers, autoimmune diseases, and neurodegenerative disorders.
FIGURE 23.1.Cell death by necrosis and apoptosis.
Necrosis is a passive, pathological process induced by acute injury or disease. A group of cells in a localized region of a tissue generally undergo necrosis at the same time after experiencing an insult. Cells that die by necrosis increase in volume and lyse (burst), releasing their intracellular contents. Mitochondria and other intracellular components are released, often inducing a potentially damaging inflammatory response. The necrotic process is completed within several days.
Necrosis and serum enzymes
Because intracellular contents, including enzymes, are released from necrotic cells, measurement of enzymes in serum samples prepared from a patient’s blood is often done to aid in diagnosis and to help to determine a prognosis. For example, most cells contain lactate dehydrogenase (LDH), an enzyme that all cells use to generate ATP from glucose. When cells from any tissue die by necrosis, LDH will appear in the blood. In fact, LDH is often used as a general marker of necrotic cell death.
Cells deprived of survival factors activate an intracellular suicide program and die by a process of programmed cell death called apoptosis (pronounced a?-po?p-to?’si?s apo to?’ sis). The requirement of a cell to receive signals for survival helps to ensure that cells continue to live only when and where they are needed.
Cells undergoing apoptosis shrink in size but do not lyse. Their plasma membrane remains intact but portions of the membrane eventually bud off, or bleb, and lose their asymmetry and ability to attach to neighboring cells in a tissue. The membrane phospholipid phosphatidylserine, which is normally present on the inner membrane leaflet oriented toward the cytosol, inverts and becomes exposed on the cell’s surface. In an active, ATP-requiring process, mitochondria of apoptotic cells release cytochrome c but remain within the membrane blebs (Figure 23.2). Chromatin of apoptotic cells segments and condenses.
FIGURE 23.2.Cellular changes during apoptosis.
Apoptotic cells are engulfed by phagocytic cells, macrophages and dendritic cells, which bind to the phosphatidylserine on the membrane surface (Figure 23.3). A macrophage internalizes and then degrades an apoptotic cell, reducing the risk of inflammation from the cell death. Phagocytic cells also release cytokines including interleukin-10 (IL-10) and transforming growth factor-? (TGF-?) that inhibit inflammation. Therefore, there is no extensive damage done to neighboring cells in a tissue when a nearby resident cell undergoes apoptosis. Apoptosis is completed within a few hours.
FIGURE 23.3.Apoptotic cell removal via phagocytosis.
While necrosis is a traumatic process resulting in widespread cell death, tissue damage, and inflammation, apoptosis has the advantage of eliminating individual cells whose survival would be harmful to the organism or whose elimination is critical for normal development or function.
1. Function of apoptosis: Elimination of damaged cells is an important function of apoptosis. When a cell is damaged beyond repair, infected with a virus, or experiencing starvation or the effects of ionizing radiation or toxins, actions of the tumor suppressor protein p53 (a product of the p53 gene) halt the cell cycle and stimulate apoptosis (Figure 23.4). The normal (wild type) p53 binds to a p53-responsive element within the gene promoter of the proapoptotic protein Bax, triggering programmed cell death. Removal of individual cells by apoptosis saves nutrients needed by other cells and can also halt the spread of a viral infection to other cells. But, mutant forms of p53 can neither halt the cell cycle nor initiate apoptosis. Therefore, abnormal cells expressing mutant p53 can continue to divide and fail to undergo apoptosis, despite the fact that their survival damages the organism.
FIGURE 23.4. Apoptosis in response to DNA damage; role of p53 in apoptosis.
2. Development: Apoptosis is used during development of the embryo. Extensive cell division and differentiation during this period often result in an excess number of cells that must be removed in order for normal development to proceed and for normal function to occur. In a developing vertebrate nervous system, more than half of the nerve cells generated undergo programmed cell death soon after they are formed.
Selective apoptosis “sculpts” the developing tissues. For example, apoptotic death of cells between developing digits must occur for formation of individual fingers and toes (Figure 23.5A). Incomplete apoptosis can result in abnormal structures (Figure 23.5B). Development of a healthy and mature adaptive immune system also requires apoptosis. Negative selection in the thymus, the process by which autoreactive T cells are eliminated from the repertoire of cells, likewise occurs via apoptosis (see also LIR Immunology, p.114).
FIGURE 23.5.Sculpting by apoptosis.
Sonic hedgehog and apoptosis
During embryonic development of vertebrates, a gradient of the signaling molecule Sonic hedgehog (Shh) is released fromthe notochord to direct cells to form patterns in the neural tube.Neural tube cells express Patched 1 (Ptc1), the receptor for Shh.When Shh binds to this receptor, the target cell survives. In theabsence of Shh, cells expressing Ptc1 undergo apoptosis.
3. Homeostasis: In normal, healthy adults, the number of cells is kept relatively constant owing to a balance between cell division and cell death (Figure 23.6). Billions of cells die each hour in the bone marrow and in the epithelia of healthy individuals. When cells are damaged or nonfunctioning, they must be replaced, but generation of new cells must be compensated by cell death to maintain a stable baseline population. Such homeostasis is required to maintain cell number and normal function. If the equilibrium is disturbed, then abnormal growth and tumors or abnormal cell loss can result. A complex system of controls tightly regulates homeostasis. One signaling mechanism that operates in this regard is the Shh-signaling pathway that normally sends an antiapoptotic signal to allow for cell survival. Failure to receive the signal results in apoptosis. However, when the hedgehog system is impaired, the antiapoptotic signal can be sent inappropriately, allowing damaged cells to escape death, with the potential for development of malignancy.
FIGURE 23.6.Homeostatic balance is maintained by a balance of cell growth and cell loss.
Initiation of apoptosis
Specific details of apoptotic mechanisms are cell-type and stimulus dependent; however, research suggests that there are common steps in this process.
1. Apoptosome: An internal cell death program will be initiated if irreparable damage has been sustained to cellular components or to DNA (Figure 23.7). Bax, a proapoptotic protein, is induced and inserted into the mitochondrial membrane to form a channel to allow cytochrome c to exit mitochondria. Cytochrome c in the cytoplasm triggers the formation of the apoptosome, a large protein complex that also requires ATP for its formation. The apoptosome is characteristic of apoptosis triggered by internal signals. To form this complex, cytoplasmic cytochrome c activates the Apaf-1 adaptor protein that in turn activates caspase 9 of the caspase proteolytic cascade that will cleave and destroy cellular proteins and DNA in order to cause cell death by apoptosis.
FIGURE 23.7.Cellular apoptosis via formation of the apoptosome.
2. Death receptors: Death receptors belonging to the tumor necrosis factor receptor (TNFR) gene superfamily can initiate apoptosis from external signals. Individual members of this family recognize specific ligands but not all members of the TNF family initiate cell death. Those that do initiate cell death possess a homologous cytoplasmic sequence termed the “death domain (DD).” Adaptor molecules such as FADD (Fas-associated death domain) and TRADD (TNFR-associated protein) contain such DDs. They interact with the death receptors to transmit the apoptotic signal to the death machinery, via activation of caspase 8 or 10 (Figure 23.8).
FIGURE 23.8.Death-receptor initiation of apoptosis.
The Fas death receptor is a member of the TNFR superfamily that will initiate apoptotic cell death when engaged by the Fas ligand (FasL, also known as CD178). T-cytotoxic cells express FasL that interacts with the Fas death receptor on host cells infected with virus in order to stimulate their apoptotic death. TNFR1 is also involved in death signaling but its death-inducing capability is weak compared to that of Fas (CD95).
Fas ligand–induced apoptosis
Membrane-anchored FasL trimer on the surface of an adjacent cell causes trimerization of the Fas receptor (Figure 23.9). This results in the clustering of the receptors’ DDs, which then recruit the cytosolic adaptor protein FADD by binding to FADD’s death domains. FADD not only contains a DD but also a death effector domain (DED) that binds to an analogous domain repeated in tandem within procaspase 8, the inactive or zymogen form of caspase 8. The complex of Fas receptor (trimer), FADD, and caspase 8 is called the death-inducing signaling complex (DISC). Upon recruitment by FADD, procaspase 8 is able to activate itself. Caspase 8 then activates downstream caspases and commits the cell to apoptosis. Apoptosis triggered by FasL-Fas (CD178:CD95) plays a fundamental role in the regulation of the immune system.
FIGURE 23.9.Apoptosis as a result of FasL binding to the Fas receptor.
Caspase family of proteases
Caspases are a family of proteases (enzymes whose substrates are proteins) that are major effectors of apoptotic cell death. They are members of the cysteine protease class, which is named after a cysteine amino acid residue present within the catalytic site of the enzyme molecule. Caspases are synthesized in inactive zymogen or proenzyme forms and are activated to become functional proteases when needed. This posttranslational modification ensures that the enzymes can be activated rapidly when required.
1. Classification of caspases: Caspases are grouped based on their function (Figure 23.10). Eleven members of the caspase family have been identified in humans. Some are not involved in apoptosis. Caspase 1 is involved in cytokine maturation, caspases 4 and 5 are involved in inflammation, and caspase 14 is important in skin development. The remaining caspases are involved in apoptosis and are grouped into either the initiator or the effector families of apoptotic caspases.
FIGURE 23.10.Classification and roles of caspases.
Initiator caspases include caspases 2, 8, 9, and 10. These possess characteristic regions or domains such as caspase recruitment domains (CARD) in caspases 2 and 9 and DED in caspases 8 and 10 that enable the protease to interact with molecules that regulate their activity. Initiator caspases cleave inactive proenzyme forms of effector caspases, resulting in their activation. Effector caspases include caspases 3, 6, and 7. These “executioner caspases” proteolytically cleave protein substrates with the cell, causing the apoptotic demise of the cell.
2. The caspase cascade: This process is the sequential proteolytic activation of one caspase after another in an orderly fashion during the initiation of apoptosis. Caspase inhibitors regulate the process. The cascade can be activated by various stimuli, including the apoptosome, death receptors, and granzyme B released by cytotoxic T cells. The apoptosome and death receptors activate initiator caspases; the apoptosome activates caspase 9, while death receptors activate caspases 8 and 10. Granzyme B activates caspases 3 and 7, which are effector caspases.
Targets of caspases include nuclear and cytoplasmic proteins. In many instances, the exact role played by the cleavage of caspase substrates is not understood and how the destruction of the protein relates to apoptosis is often unclear. Nuclear lamins, structural fibrous proteins in the nucleus, are targets of caspases. Additionally, DNA fragmentation factor 45/inhibitor of caspase-activated DNAse is cleaved, allowing caspase-activated DNAse to enter the nucleus and fragment DNA, causing the characteristic laddering pattern of DNA in apoptotic cells (Figure 23.11). The DNA is cleaved by an endonuclease into fragments that are multiples of the same size, corresponding to the length of the nucleosome coil, for example, 2, 4, 6, 8, etc. A distinctive 180-bp ladder is seen in the DNA of cells undergoing apoptosis. Poly ADP ribose polymerase is also known to be proteolytically cleaved by caspases during the apoptotic process, as is Bid, a member of the Bcl-2 family.
FIGURE 23.11.DNA fragmentation or laddering in apoptotic cells.
Members of the Bcl-2 Family
In apoptotic cells, the ratio of prosurvival to proapoptotic proteins changes to favor the proapoptotic proteins. Many of these proteins are members of the Bcl-2 protein family. Prosurvival (antiapoptotic) members of the Bcl-2 family include Bcl-2 and Bcl-xL, while prodeath members include Bak and Bax (Figure 23.12). In most cell types, death receptor signaling results in the activation of caspase 8, a protease which catalyzes the cleavage of Bid to tBid (Figure 23.13). Bak and Bax can then translocate from the cytosol to the outer mitochondrial membrane, permeabilize it, and facilitate the release of proapoptotic proteins including cytochrome c and Smac/DIABLO, an antagonist of inhibitors of apoptosis proteins (IAPs).
FIGURE 23.12.Prosurvival and prodeath members of the Bcl-2 family.
FIGURE 23.13.Bcl-2 family members in apoptosis.
Apoptosis in disease
1. Cancer: A leading cause of death, cancer arises when the homeostatic balance between cell division and apoptosis is altered. Decreased apoptosis in cancer cells may result from altered expression of apoptosis regulatory proteins. Overexpression of the antiapoptotic Bcl-2 protein in lymphocytes that also express the myc oncogene can result in lymphoma. When chromosomal translocations move the Bcl-2 gene next to the immunoglobulin heavy chain locus, lymphoma can also result. The Bcl-2 gene has also been implicated in breast, prostate, and lung carcinomas and in melanoma. Resistance to cancer chemotherapy in some tumors may also be caused by the overexpression of Bcl-2 and defective apoptosis.
2. Autoimmune conditions: Rheumatoid arthritis, systemic lupus erythematosus, and type 1 diabetes mellitus are examples of autoimmune conditions that may result in part from defective apoptosis (see LIR Immunology, Chapter 16). A prominent feature of autoimmune diseases is the failure of T cells that recognize self to undergo negative selection via apoptosis. The autoreactive cells then survive and proliferate. Defective apoptosis has sometimes been attributed to abnormal expression of proteins involved in apoptotic signaling. For example, some studies indicate that T cells infiltrating rheumatoid synovium express high levels of Bcl-2 and are resistant to Fas-induced apoptosis. Defective Fas death receptors and increased apoptosis in pancreatic islets may cause destruction of pancreatic ? cells and development of type 1 diabetes mellitus.
Enhanced apoptosis can also impact progression of infection with human immunodeficiency virus (HIV) to the immune-compromised state of AIDS. Inappropriate apoptotic depletion of CD4+ T helper cells causes marked decreases in these T cells in affected individuals. Some HIV proteins inactivate antiapoptotic Bcl-2 and other HIV proteins promote Fas-mediated apoptosis.
3. Neurodegenerative illnesses: Other types of disorders may also result in part from increased apoptosis. Schizophrenia, a chronic neurodegenerative illness, is characterized by delusions, hallucinations, and changes in emotional state. Although the mechanisms underlying these deficits are largely unknown, recent postmortem data implicate a role for altered neuronal apoptosis. Apoptotic regulatory proteins and DNA fragmentation patterns appear to be altered in several cortical regions in individuals with schizophrenia. In individuals with Alzheimer’s disease, localized apoptosis may contribute to early neurite and synapse loss, leading to the initial cognitive decline. Additionally, many individuals infected with the HIV virus develop a syndrome of neurologic deterioration known as HIV-associated dementia (HAD). HAD appears to be associated with active caspase 3 in the affected brain regions, leading to speculation that pharmacologic interventions aimed at the caspase pathway may be beneficial.
Laboratory assessment of apoptosis
Several laboratory assays exist to assess apoptosis. In addition to the methods described below, analysis of expression of proapoptotic proteins, such as Bax, and measurement of caspase activity can also be done.
- DNA laddering: Visualization of DNA laddering is perhaps the oldest technique available to detect that apoptosis has occurred. Since the genomic DNA of apoptotic cells is degraded into approximately 180 base pair fragments, a characteristic laddering appearance is revealed on agarose gel electrophoresis (see Figure 23.11).
- TUNEL: The TUNEL method, which stands for terminal uridine deoxynucleotidyl transferase nick end labeling, detects DNA fragmentation based on the presence of strand breaks or nicks in the DNA. The terminal deoxynucleotidyl transferase enzyme catalyzes the addition of dUTPs that have been labeled for the experiment.
- Annexin 5: The Annexin 5 affinity assay is also useful for the detection of cells early in the apoptotic process. Annexins are a family of proteins that bind to phospholipids in cell membranes. Annexin 5 binds to phosphatidylserine, which, in healthy cells, is present on the inner membrane leaflet. Soon after a cell has initiated steps toward programmed cell death, phosphatidylserine flip-flops to the outer membrane leaflet. A labeled antibody to Annexin 5 can be used to detect cells displaying phosphatidylserine on their outer leaflet, indicating that they have initiated the apoptotic process.
- Flow cytometry: This procedure can be used to measure cell size and granularity of cells within a population, both of which differ in apoptotic and normal cells. Because apoptotic cells shrink in size, the forward angle light scatter will reveal an apoptotic population of less intensity compared with normal cells. Granularity of apoptotic cells is increased compared with that of normal cells, as indicated by side scatter.
- Cell death occurs via one of the two processes: necrosis, a pathological process, or apoptosis, a physiological process.
- Necrotic cells rupture and release their contents, including enzymes, to the extracellular media, often inducing an inflammatory response.
- Apoptotic cells undergo a programmed form of cell death with blebbing membranes that remain intact. Their engulfment by phagocytic cells prevents their death from causing an inflammatory response.
- Apoptosis is important for elimination of damaged cells in development and for tissue homeostasis.
- Apoptosis may be stimulated by an internal process resulting in assembly of an apoptosome or by extracellular signals via death receptors.
- Apoptotic cells show a change in their ratio of proapoptotic and prosurvival protein members of the Bcl-2 protein family to favor the proapoptotic proteins.
- Caspase proteases catalyze cleavage of cellular proteins, culminating in apoptosis.
- Insufficient apoptosis in certain cells can result in cancer and autoimmune conditions, while excess apoptosis can lead to neurodegenerative illnesses and may play a role in the development of AIDS from HIV infection.