1.3 Introduction to apoptosis
1.3.1 Biological significance and morphological / biochemical features
The term ‘programmed cell death’ was originally introduced in 1964, proposing that cell death during development is not of accidental nature but follows a sequence of controlled steps, leading to locally and temporally defined self-destruction of cells (Lockshin and Williams, 1964). Subsequently, the term ‘apoptosis’, a word of Greek origin meaning ‘falling off or dropping off’ in analogy to autumn leaves falling off trees, was used to describe the morphological and biochemical processes, which lead to controlled cellular self-destruction. It was first introduced by Kerr, Wyllie and Currie (Kerr et al., 1972). In general, the process of
apoptosis is of great importance in the development of multi-cellular organisms, as well as in the regulation and maintenance of cell populations in different tissues upon both physiological and pathological conditions. Although apoptosis is by far the most frequent way of programmed cell death, non-apoptotic types of programmed cell death have already been described (Leist and Jaattela, 2001a; Leist and Jaattela, 2001b). The biological significance of apoptosis is highlighted by the following examples: during early development an excess of different cell types is produced, most of which eventually undergo programmed cell death, thereby contributing to the final formation of organs and tissues (Meier et al., 2000). Another example is given by the formation of human limbs. Here separate digits evolve by apoptosis of interdigital mesenchymal tissue (Zuzarte-Luis and Hurle, 2002). Two more examples can be found in brain development, in which one half of all neurons initially created, is disposed of by the organism during the differentiation of neurons and adult brain formation (Hutchins and Barger, 1998) and not to forget the formation of reproductive organs (Meier et al., 2000).
As all cells of an adult organism undergo physiological cell death during its lifetime, this must be balanced with cell proliferation rates in order to maintain homeostasis in terms of constant cell numbers. With reference to the human immune system, apoptosis is of major importance when it comes down to regulation and function. The majority of developing lymphocytes either die during genetic rearrangement events or in formation of the antigen receptor during negative selection or in the periphery. By these means, the pool of highly efficient, non-self-reactive immune cells is strictly controlled. Moreover, lymphocyte numbers are kept relatively constant (Fadeel et al., 1999a; Fadeel et al., 1999b; Rathmell and Thompson, 2002). A last few most important points in terms of biological function of apoptosis are the elimination of damaged, dangerous cells, e.g. cells containing severely damaged DNA that is beyond repair; inappropriate mitogen signalling that stays in conflict with the homeostasis of the cell and might either result in cell cycle arrest or even in apoptosis; elimination of infected cells by microorganisms (Vaux et al., 1988; Vaux and Korsmeyer, 1999).
Taking together all these regulatory functions of apoptosis it becomes clear, what disastrous or even lethal effect a dysfunction or dysregulation has on the organism. Defects in apoptosis are tightly connected with a variety of pathological conditions. While, e.g. by mutation of genes which code for proteins involved in initiation or execution of the signalling cascade (Mullauer et al., 2001), repressed apoptosis might lead to cancer, autoimmune diseases and spreading of viral infections, excessive apoptosis can result in AIDS and iscaemic diseases as well as neurodegenerative disorders, such as Alzheimers’ disease, Parkinson’s disease, Huntington’s disease and amyotrophic lateral sclerosis (Reed, 2002).
Cells undergoing apoptosis always show some characteristic morphological and biochemical features (Cohen, 1993b). Two of them are actually seen as biological hallmarks of apoptosis, namely chromatin condensation and DNA fragmentation. The latter is achieved by activation
of endonucleases like CAD (caspase-activated DNAse) (Reed, 1998; Wyllie et al., 1981;
Wyllie et al., 1984). Moreover, despite cell shrinking and loss of cell-cell contacts, all cell organelles and membranes remain intact. However, apoptotic cells cease to maintain phospholipid asymmetry in their membrane structure. Phosphatidylserine flips to the outer leaflet (Callahan et al., 2000). Even the mitochondrial membrane is subject to apoptosis related changes, including a loss of its electrochemical gradient by formation of pores.
Substances like cytochrome c leak from the mitochrondria into the cytoplasm. In later stages of apoptosis the cell is formed into apoptotic bodies, which are finally phagocytosed by macrophages or adjacent epithelial cells (Cohen, 1993a; Cohen, 1993b; Savill et al., 1989).
This is another crucial step during apoptosis, as it prevents an inactivation of inflammatory processes (Saraste and Pulkki, 2000).
In contrast to apoptosis, we find a second rather uncontrolled process of cell death that is necrosis (Dive et al., 1992). Necrosis occurs, when cells are exposed to an extreme variance of physiological conditions, such as hypothermia and hypoxia, which both cause plasma membrane damage. It is initiated by disturbance of the cell’s ability to keep up its homeostasis. Agents like complement or lytic viruses can cause direct damage to the plasma membrane under physiological conditions. In the following this leads to an uncontrolled influx of water and extracellular ions and finally results in the disruption of organelles and the whole cell. Due to uncontrolled release of cellular contents including lysosomal enzymes into the surrounding extracellular fluid, necrotic cell death is most often associated with extensive tissue damage and initiation of excessive inflammatory responses (Vermes and Haanen, 1994).
1.3.2 Signalling pathways of apoptosis: molecular mechanisms
Apoptosis can be triggered by various stimuli either from outside the cell i.e. binding of cell surface death receptor ligands such as Fas (Nagata, 1994; Nagata and Golstein, 1995), TNFR1 and DR5 with their ligands FasL, TNF-α and TRAIL (Ashkenazi, 2002) (extrinsic pathway) or from within the cell, via direct DNA damage by cytotoxic drugs or irradiation (Achenbach et al., 2000; Rich et al., 2000).
The extrinsic pathway further distinguishes between type I and type II, depending on the actual cell type. Apoptosis induced via the extrinsic signalling pathway type I is mediated by activation of ‘death receptors’, protein complexes which belong to the tumor necrosis factor receptor (TNFR) gene superfamily (Ashkenazi, 2002; Ashkenazi and Dixit, 1998; Nagata, 1994; Nagata and Golstein, 1995; Vandenabeele et al., 1995a; Vandenabeele et al., 1995b).
After binding of their ligands FasL (synonyms APO-1; CD95), TNF-α or TRAIL, which the receptor recognises by their cysteine rich extracellular subdomain, the receptors trimerise and get activated (Naismith and Sprang, 1998). Subsequently, the cytoplasmic part of the
receptor, termed ‘death domain’ (DD) initiates the following steps in the signalling cascade.
Adapter molecules like FADD (Fas-associated death domain protein) and TRADD (TNF-receptor associated death domain) possess their own death domains, by which they are recruited to the activated DD of the receptor. In this way the death-inducing signalling complex (DISC) is formed (Sartorius et al., 2001). Besides its DD, FADD also possesses a death effector domain (DED), which by interacting with the receptor, DED, recruits procaspase 8 to the DISC. Once bound to the DISC, several procaspase 8 molecules are brought into close proximity. They are assumed to activate each other via autoproteolysis (Denault and Salvesen, 2002), releasing activated caspase 8 molecules. Activated caspase 8 then cleaves and thereby activates several downstream effector caspases, which finally cleave specific substrates consequently causing cell death. One of the most crucial substrates is the endonuclease CAD (caspase-activated DNAase), which is responsible for DNA fragmentation within the nucleus (Scaffidi et al., 1998).
Fig. 1.3.2.1 Receptor-mediated caspase activation via the DISC death-inducing signalling complex (Gewies, 2003 / www.celldeath.de/encyclo/aporev/aporev/htm).
In contrast to the extrinsic type I pathway, the signal generated via the extrinsic type II pathway is not strong enough to start off the above described process on its own. The signal needs to be amplified with the help of the mitochondrial pathway. The pro-apoptotic Bcl-2 family member Bid provides the link in the chain to connect the receptor-mediated caspase activation via the DISC with the mitochondrial pathway. Bid is one of several substrates of the activated caspase 8. Once Bid is cleaved into action, its truncated brother translocates into the mitochondria, where it co-operates with other pro-apoptotic Bcl-2 family proteins like Bax and Bak (Bernardi et al., 1999). Together these proteins initiate the release of cytochrom c and other pro-apoptotic mitochondrial factors into the cytosol (Luo et al., 1998). Of course,
theses processes can be gradually attenuated and counter-acted, when anti-apoptotic proteins interfere, proteins such as Bcl-2, Bcl-XL, Bcl-w, A1 and Mcl-1 (Borner, 2003; Vaux et al., 1988). In this way at certain stages of apoptosis, the fate of a cell can still be tilted towards pro-survival.
When cytochrome c is released into the cytosol, it subsequently binds to the monomeric Apaf-1 in the presence of dATP. This binding causes a conformational change in Apaf-1, leading to an oligomerisation of several Apaf-1 molecules, which finally assemble to form the apoptosome (Salvesen and Renatus, 2002). The apoptosome is a heptameric protein complex with a wheel-like structure. It triggers the activation of initiator pro-caspase 9 (Acehan et al., 2002). Thus activated caspase 9 switches on the caspase cascade, including caspases 3, 6 and 7. Finally, this leads to cleavage of a specific set of substrates, resulting in mediation and amplification of the actual death signal, including all morphological and biochemical features usually observed (Earnshaw et al., 1999; Slee et al., 1999).
Fig. 1.3.2.2 Mitochondria-mediated formation and activation of the apoptosome (Gewies, 2003/
www.celldeath.de/encyclo/aporev/aporev/htm).
Apart from functioning as an amplifier for the extrinsic apoptosis pathway type II, the mitochondrial intrinsic apoptosis pathway also plays a key role in integrating and propagating death signals coming from inside the cell.
Fig. 1.3.2.3 Apoptosis signalling pathways (extrinsic I+II; intrinsic), (Gewies, 2003 www.celldeath.de/encyclo/aporev/aporev/htm).
These stimuli include DNA damage, oxidative stress, mitogen starvation, radiation, in addition to those changes induced by cytotoxic drugs (Kaufmann and Earnshaw, 2000;
Wang, 2001). In biological terms, induction and propagation of pro-apoptotic signals are run in the same way as described for the extrinsic apoptosis pathway.