The most significant studies of apoptosis in man have been conducted on cells in culture or on lymphocytes. There is comparatively little information on apoptosis in epithelial cells. Apoptotic cells are characterized by nuclear and cytoplasmic changes. The nuclear changes are a condensation of the nuclear chromatin, first as crescentic caps at the periphery of the nucleus, followed by further fragmentation and break-up of the nucleus (Fig. 6-1 top). The fragmentation of the chromatin into small granules of approximately equal sizes is known as karyorrhexis (from Greek, karyon = nucleus, rhexis = breakage), which has now been recognized as a manifestation of apoptosis (Fig. 6-2). The cytoplasm of many apoptotic cells may show shrinkage and membrane blisters. It appears, however, that the cytoplasm may remain relatively intact in squamous cells. As the next stage of cell disintegration, fragments of nuclear material with fragments of adjacent cytoplasm (that may contain various organelles) are packaged into membrane-enclosed vesicles (apoptotic bodies). These packages of cellular debris are phagocytized by macrophages, without causing an inflammatory reaction in the surrounding tissues. One important consequence of apoptosis is that the cell DNA is chopped up into fragments of variable sizes composed of multiples of 185 base pairs. When sorted out by electrophoresis, they form a “DNA ladder” of fragments of diminishing sizes. Because the breaks occur at specific points of nucleotide sequences, they can be recognized by specific probes identifying the break points in the DNA chain. The probes, either labeled with a fluorescent compound or peroxidase, allow the recognition of cells undergoing apoptosis, either by fluorescent microscopy, flow cytometry, or by microscopic observation (Li and Darzynkiewicz, 1999; Bedner et al, 1999). A so-called TUNNEL reaction is a method of documenting apoptosis in cytologic or histologic samples (Gavrieli et al, 1992; Li and Darzynkiewicz, 1999; Sasano et al, 1998).

In paraphrasing a statement by Thornberry and Lazebnik (1998), apoptosis is reminiscent of a well-planned and executed military operation in which the target cell is isolated from its neighbors, its cytoplasm and nucleus are effectively destroyed, and the remains (apoptotic bodies) are destined for burial at sea, leaving no traces behind. Much of the original information on the sequence of events in apoptosis was obtained by studying the embryonal development events in the small worm (nematode), Caenorhabditis elegans. These studies have documented that apoptosis occurs naturally during the developmental stages of the worm to eliminate unwanted cells. It is caused by a cascade of events, culminating in the activation of proteolytic enzymes that effectively destroy the targeted cell. A somewhat similar, but not identical, sequence of events was proposed for mammalian cells.

Apoptosis in mammalian cells is triggered by numerous injurious factors, some known, such as viruses, certain drugs, radioactivity, and some still unknown (summary in Thompson, 1995; Hetts, 1998; review in Nature, 2000). For example, the loss of T4 cells by the human immunodeficiency virus in acquired immunodeficiency syndrome (AIDS) is caused by apoptosis. However, the pathway to apoptosis is extremely complicated because normal cells contain genes that prevent it and genes that promote it. This equilibrium has to be disrupted for the cells to enter the cycle of death.

In brief, it is assumed today that “death signals,” received by the cytoplasm of the cell and mediated by a complex sequence of molecules, lead to activation of proteolytic enzymes, known as caspases that destroy the cytoplasmic proteins, including intermediate filaments, and attack the nuclear lamins, causing the collapse of the nuclear DNA structure. However, the intermediate steps of this sequence of events are enormously complicated. An injury to the molecule p53 (guardian of the genome; see Chap. 3) appears to be an important event (Bennett et al, 1998). Recent studies have documented that genes located on the mitochondrial membrane play a critical role in apoptosis (Brenner and Kroemer, 2000; Finkel, 2001). These genes belong to two families: Bcl, a protooncogene, which protects the cells from apoptosis, and Bax, which favors apoptosis (Zhang et al, 2000). If the proapoptotic molecule prevails, there is damage to the mitochondrial membrane with release of cytochrome C into the cytoplasm. Cytochrome C acts to transform a ubiquitous protein molecule known as zymogen into caspases.

Numerous articles on the subject of apoptosis have appeared in recent years, each addressing a small fragment of the complex puzzle. The key recent articles are cited or listed in the bibliography. The significance of apoptosis goes much beyond a simple morphologic and molecular biologic summary. It is generally thought that the mechanisms of apoptosis, besides playing a key role during embryonal development, may play a key role in cancer and in important degenerative processes such as Alzheimer’s disease. In cancer, suppression of apoptosis may be one of the causes of cell proliferation, so characteristic of this group of disorders. This may explain the role of oncogenes, such as Bcl or Myc, as protecting the cells from apoptosis. It is considered that changes or mutations in molecules controlling DNA damage in replication (such as p53) or molecules governing events in the cell cycle (such as Rb) play a role in these events. It has been proposed that, in degenerative disorders of the brain, such as Alzheimer’s disease, apoptosis may destroy essential centers of memory and control of body functions.

Cells undergoing nonapoptotic forms of necrosis may show extensive cytoplasmic vacuolization (Fig. 6-3). The nuclear changes include homogeneous, dense chromatin known as nuclear homogenization or pyknosis (from Greek, pyknos = thick), nuclear enlargement, and break-down of nuclear DNA, which however, does not form the DNA ladder, characteristic of apoptosis. Necrosis may result in destruction of the cell membranes, resulting in disintegration of the cell and formation of cell debris leading to an inflammatory process. The nuclear material may form fragments or streaks, often recognizable because they stain blue with the common nuclear dye, hematoxylin. Similar events may occur by physical injury to fragile cells if they are inappropriately handled during the technical preparation of
smears or other diagnostic material. In some cancers, the presence of nuclear necrosis is widespread and may be of diagnostic value (see oat cell carcinoma in Chap. 20).

It is often quite impossible to determine morphologically whether a cell died as a consequence of apoptosis or necrosis. There is little doubt that there may be many pathways leading to cell death. What is significant, however, is the role played by necrotic cells as a trigger of inflammatory events, whereas cells dying of apoptosis, as a rule, do not cause any inflammatory reaction.

Cell necrosis may be caused by many of the types of cell injury listed in the opening page of this chapter. Thus, there is a significant overlap between the two modes of cell death. It is not known today why the differences in the mode of dying occurs if the trigger of cell death is the same. It is generally believed that cell necrosis may begin in a manner similar to apoptosis, that is, by activation of a cell membrane molecule or a “death signal,” which is followed by mitochondrial swelling, but this differs from the events in apoptosis because it does not lead to caspase activation (Green and Reed, 1998). Obviously, much is still unknown about cell necrosis, its mechanisms, and consequences.