CELLULAR AGING: CELL DEATH

Cell death during aging is an important issue, and it is important to understand what cell death is, and what it is not, as there are several phenomena that use similar terminologies. Perhaps the best known of these is cellular senescence.

Cellular senescence

First identified in the late 1960s by Leonard Hayflick and his collaborators, the term cellular senescence refers to the fact that normal, nonmalignant cells of vertebrates do not divide indefinitely in culture, but in time terminally differentiate and enter a prolonged postmitotic phase, eventually dying in the culture dish. Because the number of divisions that the cells can undergo is inversely related to the age at which they were explanted, cellular senescence has been associated with aging—though there is little evidence that, except in limited situations, individuals at the ends of their lives have "run out of cells." The mechanism for cell senescence is not completely understood. One explanation for the limitation in cell replication is based on the importance of the end-piece, or telomere, of a chromosome. The manner in which DNA is replicated results in the loss of a small portion of DNA (an Okazaki fragment) from the end of the chromosome with each replication. This problem is addressed by the addition to the end of the DNA, during early embryonic life, of a stretch of meaningless, noncoding DNA called a telomere. Thus, at each division a piece of this expendable DNA is shed. Under culture conditions, or in conditions of excessive proliferation, such as chronic challenge to the immune system, the cells eventually use up their telomeres and cease proliferation. Cancerous cells reacquire the embryonic ability to reconstruct the telomere, and thus become immortal.

Cell death: programmed, apoptosis, and necrosis

Cell senescence is distinct from what is properly called cell death. Cells can, of course, encounter violent situations in which proteins precipitate, membranes are ruptured, or their access to energy sources is destroyed. In these situations, cells typically lose the ability to maintain their volumes against osmotic forces, and they swell and rupture (technically, lyse), spilling their contents and provoking an inflammatory response. This process is called either necrosis or oncosis, and is seen in acute situations such as infarct (a region of tissue suddenly deprived of blood flow, as when a clot lodges in a small artery or arteriole), severe chemical toxicity, and extreme thermal damage.

Necrotic cells, generated in an uncontrolled manner, create many problems for an organism because of inflammation and because of the leakage of potentially dangerous chemicals or enzymes. Also, cells lysing from infection may spew out viruses or other pathogens. As a protective mechanism, therefore, organisms can preempt such deaths by invoking a much more biological and controlled response, known as apoptosis, or programmed cell death. These forms of death are a sort of cell suicide, in which cells self-destruct in a controlled and contained manner. All cells carry within themselves the capacity to self-destruct, but are normally restrained from doing so. If this restraint is removed when a cell is challenged, it will default to the self-destruct mode and, assuming that the challenge is not so severe that the cell becomes necrotic, it will undergo this physiological form of death.

The term programmed cell death derived originally from developmental and embryonic observations, and it emphasizes the idea that specific genes regulate the death of cells. Many of these genes have now been recognized and are described below. In developmental situations, death frequently, if surprisingly, requires the synthesis of new proteins, perhaps including those involved in killing the cell. The morphology is more often than not apoptosis. However, apoptosis does not necessarily require protein synthesis and is usually not programmed (meaning that the sequence of death is coded in the genes) except in the generic sense that it was preprogrammed into the cell and simply required release or activation.

The apoptotic cell is recognized by several characteristics. It is a rounded, blebbing cell, with limited permeability of its cell membrane. In an active process, it has moved a component of the inner cell membrane, phosphatidyl serine (PS), to the external surface. The exposed PS will serve as a signal to the phagocytes that will consume it. The chromatin (the complex of protein, DNA, and RNA that can be strained, rendering the chromosome—Greek for colored body—visible) coalesces in the nucleus and frequently marginates, or condenses, along one side of the nuclear membrane. The DNA then fragments into pieces that are multiples of 180 base pairs (nucleosomal fragments), detectable by electrophoresis or by TUNEL (Terminal deoxyUridine Nucleotide End Labeling) in situ. There are, however, many gray areas, and some cells may display intermediate patterns.

There are many entry points into apoptosis, and there are several variants, depending on tissue type and history. Three of these variants may be summarized as follows:

Caspase-dependent apoptosis. A chief effector of apoptosis is caspase-3, a highly specific protease. Proteases are enzymes that digest proteins. They are divided into several categories, with one classification referring to an amino acid in the enzyme that is essential for its activity. Caspases contain an essential cysteine, contributing the "c" in the name of the enzyme. Also, most proteases recognize a specific amino acid sequence in the substrate and cut the substrate protein at that site. Caspases identify a sequence of four amino acids terminating in aspartic acid. Thus the name caspase is derived from Cysteine ASPartyl protease. Very few proteins have the appropriate sequences, but those that do, and are thus destroyed by caspase-3, include cytoskeletal components that maintain the shape of the cell, enzymes necessary for synthesizing messenger RNA, and enzymes needed for repair of DNA damage. Caspase-3 exists in cells as an inactive proenzyme, with its activity blocked by extra amino acids added during its synthesis. This extra sequence begins with a site recognized by other caspases. Thus, other caspases remove this sequence and activate the enzyme. The active enzyme can also activate other pro-caspase-3 molecules (autoactivation). This process is well described by Earnshaw, Martins, and Kaufmann (1999).

Fas-dependent apoptosis. In many situations, particularly in the immune system, the number of cells is tightly regulated. Cell number has to be increased rapidly to fight an infection and reduced again after the infection subsides. Failure to precisely control numbers may result in autoimmune reactions, in which the body makes antibodies to its own proteins, generating life-threatening inflammations, or to the loss of too many cells, leading to increased susceptibility to infection or an inability to conquer an infection. Thus, mechanisms for cell death are very elaborate in the immune system, though the control mechanisms are used by other cells as well. Many of these cells carry on their surface one member of a family of closely related proteins. One of the most common proteins is called Fas, after an activity first recognized by immunologists. Fas can bind the protein Fas Ligand, which itself may either circulate in the blood or be attached to another cell. Fas bound to Fas Ligand can also attach to one or two similarly linked Fas molecules, forming dimers (two molecules linked) or trimers (three molecules). All of the Fas molecules stretch across the cell membrane to the intracellular side. The dimer or trimer forms interact with other proteins on the inside of the cell in a complex reaction that ultimately results in the freeing of pro-caspase-8 from an inactive bound form. This caspase-8 becomes activated and activates caspase-3, leading to apoptosis. Other receptors in the Fas Ligand family include those binding tumor-necrosis factor, and all members of the family contain similar amino acid sequences and structures, including a region important for the activation of caspases called the death domain.

Fas-independent apoptosis. Apoptosis may also be activated by mechanisms independent of the Fas-FasL pathway. For instance, any of a number of mechanisms may damage mitochondria, leading to the depolarization of the mitochondria, opening of a charge-dependent pore (the mitochondrial membrane permeability transition pore), and leakage of cytochrome c and other mitochondrial components into the cytoplasm. The cytochrome c displaces an inhibitor from pro-caspase-9, allowing its activation, whereupon it activates caspase-3.

Caspase-independent cell death. Some cell deaths, most typically those of large, cytoplasm-rich cells or postmitotic cells, do not rely heavily on caspases. They therefore display a somewhat different morphology from that described below and exhibit rather an autophagic morphology. In autophagy—literally, self-eating—the bulk of the cytoplasm is destroyed in large lysosomal vesicles (autophagosomes) before the morphology becomes more classically apoptotic.

Cell death genes

Many genes are now thought to function primarily in apoptosis. Most consist of families of genes whose different functions reflect the nuances of regulation of cell death. For instance, Fas is a member of a large family of receptor proteins. Caspases, a family including at least eleven enzymes, have evolved from a single caspase in a nematode worm. (Important enzymes are conserved from animal to animal throughout evolution. However, many amino acids may change and animals may keep more than one variant, splitting an original single enzyme into two related enzymes.) Other than the products of the genes mentioned above, there are inhibitors of cell death, such as Bcl-2, which is likewise a member of a family of genes. Since Bcl-2 must form dimers to function, the complexes that it forms with other family members may lead to inhibition or activation of apoptosis. Thus, some family members, such as bax, are proapoptotic. The activity of several genes specifically increases or decreases in apoptosis, but their specific function in apoptosis is not known.

Cell death and aging

"Running out of cells" because of apoptosis does not cause aging. Nevertheless, apoptosis is considered to be an important gerontological issue for many reasons. Diseases that increase with aging include cardiovascular diseases, deterioration of the immune system, neurodegenerative diseases such as senile dementia/Alzheimer's type, and cancer. Apoptosis plays a major role in all of these diseases, as it is ably described by Huber Warner (Warner, Hodes, and Pocinki, 1997; Warner, 1999).

Many cancers, such as B-cell lymphoma, are diseases of cell death rather than cell proliferation; that is, the excess of cells arises from the failure of cells to die on schedule rather than from excess production. B-cell lymphoma arises from the inappropriate activation of Bcl-2 (the name of the gene derives from the disease), which protects the cells from death. For many other cancers, conversion to the most dangerous phase involves the loss of a surveillance mechanism, a protein called p53, that can detect abnormal DNA. This change (loss or alteration of p53) occurs in as many as 50 percent of some tumors. P53 can either block mitosis of a cell with abnormal DNA, or, if mitosis has already begun, force the cell into apoptosis. In the absence of p53, the abnormal cell survives. Therefore, mutation or loss of p53 is considered to be an ominous development in the progress of a cancer. For those cancer cells that do not appear to derive directly from abnormalities of apoptosis, there is some hope of eventually targeting signals to cancer cells to force them into apoptosis. (Cancer cells do not typically lose the ability to undergo apoptosis, but they become insensitive to the signals.)

Conversely, many of the problems of the immune system in the elderly stem from a failure to maintain high numbers of specific types of lymphocytes. Throughout life the number of immunocompetent cells is adjusted by a balance of proliferation and specifically induced apoptosis. For instance, loss of cells in AIDS is caused by suicide of cells that are not heavily infected with HIV, but are located near cells that are infected. Biomedical research aims to prevent these losses.

Autoimmune diseases, which also increase with aging, probably result from dysregulation of the controlled and normal down-regulation of the immune system. Indeed, the two best animal models of autoimmune diseases are mice in which Fas and Fas Ligand are mutated and ineffective. In many neurodegenerative diseases, most dramatically Alzheimer's disease, the cells that die have been under chronic stress before they succumb. Dead neurons cannot be replaced, but if the suicide of these stressed cells can be prevented, the disease can be alleviated or prevented. Even in an acute situation, such as an infarct (more frequent among the elderly), only those cells immediately affected by the infarct die by necrosis. Many cells on the periphery are injured, and only later undergo apoptosis. The delayed and apoptotic death of these cells indicates that there is a window in which these cells might be protected, thus lessening damage.

Future expectations

Apoptosis research was quite extensive at the end of the twentieth century, and it focused on several goals:

• Blockage of apoptosis by inhibition of caspases. Several inhibitors of caspases are now known. Although inhibition of caspase may not protect a cell limited for other reasons, it is likely to produce a "zombie" cell, which continues to exist but is incapable of performing a specific function such as conducting an impulse or contracting, and in acute situations such as a heart attack it may buy time.
• Maintenance of challenged systems. For instance, approaches used in addressing the loss of cells in AIDS include supportive therapies and administration of growth factors that are known to suppress apoptosis.
• Targeting of apoptosis. Attempts have been made to control apoptosis by attacking the machinery of apoptosis (such as caspases, particularly in acute emergencies such as stroke), but since most cells possess the machinery for apoptosis, regulation of apoptosis is more likely to focus on identifying the cells to be controlled and arranging a mechanism to specifically target these cells, using inhibitors or ligands to achieve the up- or down-regulation of the apoptosis machinery.

Richard A. Lockshin Zahra Zakeri

See also Cellular Aging: Telomeres; DNA Damage and Repair; Theories of Biological Aging.

BIBLIOGRAPHY

Earnshaw, W. C.; Martins, L. M.; and Kaufman, S. H. "Mammalian Caspases: Structure, Activation, Substrates, and Functions During Apoptosis." Annual Review of Biochemistry 68 (1999): 383–424.

Hayflick, L. "Human Cells and Aging." Scientific American 218, no. 3 (March 1968): 32–37.

Kerr, J. F. R.; Wyllie, A. H.; and Currie, A. R. "Apoptosis: A Basic Biological Phenomenon with Wide-Ranging Implications in Tissue Kinetics." Journal of Cancer 26 (1972): 239–257.

Lowe, S. W. "Cancer Therapy and p53." Current Opinion in Oncology 7, no. 6 (1995): 547–553.

Warner, H. R. "Apoptosis: A Two-Edged Sword in Aging." Annual of the New York Academy of Science 887 (1999): 1–11.

Warner, H. R.; Hodes, R. J.; and Pocinki, K. "What Does Cell Death Have to Do with Aging?" Journal of the American Geriatrics Society 45, no. 9 (1997): 1140–1146.

Zakeri, Z.; Bursch, W.; Tenniswood, M.; and Lockshin, R. A. "Cell Death: Programmed, Apoptosis, Necrosis, or Other?" Cell Death and Differentiation 2 (1995): 83–92.