Apoptosis in Animals

Apoptosis is derived from Greek words meaning "falling away" or "discarding" (Kerr et al., 1972). Although there are some variations in the apoptosis processes in animal cells, the sequence of cytological and biochemical events listed in Table 1-3 characterizes this process. It must be noted at the outset that not all cells undergoing what seems to be apoptosis show all these symptoms (Schulze-Osthoff et al., 1994; Leist and Jaattela, 2001), so apoptosis may not be a single pathway, but a braided stream with some ability to flow through different channels. In addition, several different cell death programs may run in parallel and share some components (Ameisen, 2002). Nonetheless, apoptosis is important for the normal development and health of animals (Meier et al., 2000). The role of apoptosis in determining the longevity of an animal is not clear (Lockshin and Zakeri, 1996; Warner et al., 1997).

Once one starts to examine the molecular details of apoptosis and its controls, one soon becomes overwhelmed with the number of protein-protein interactions and the terminology. The literature is an alphabet soup of acronyms, and often, they differ for the same or related genes. It seems inappropriate to try to cover all these details here. Moreover, this picture and the acronyms are changing very rapidly as new information is added. Nonetheless, these are the standard to which plant studies must be compared. Apoptosis seems to consist of three phases: (1) initiation, (2) execution and (3) cleanup of the cell remains (e.g., autophagic elimination).

Table 1-3. The Main Features of Apoptosis in Animal Cells



Cytoplasmic shrinkage, vacuolization and fragmentation Nuclear chromatin coalesces into clumps along the outer margin

Nuclear condensation and fragmentation Membrane blebbing ^ vesicles (apoptotic bodies) which may include nuclear fragments Phagocytosis of cell fragments

Cascade of caspase activation

Endolytic cleavage of many cell proteins, exs., lamins (nuclear membrane structural proteins) and poly ADPribose polymerase, (PARP a DNA repair enzyme) DNA cleavage into discrete nucleosomal fragments Exposure of phosphatidyl serine on the inner surface of the plasma membrane Binding and docking of the phagocyctes

Apoptosis can be initiated by a wide range of factors ranging from internal programming to external factors such as chemicals and environmental stress (Raff, 1992). This triggers a signaling pathway that appears to involve Ca+2 flux and protein phosphorylation. Interaction of ligands with membrane-spanning death receptors recruits certain intracellular proteins to the death domain of the receptor and initiates a caspase cascade.

Caspases are cysteine proteases that cut specifically after an aspartate. There are several different types of caspases, and they are present in the cytosol as inactive proenzymes that are activated after the death signal is triggered. They are recruited into cell-death signaling complexes through protein-protein interactions that lead to self-cleavage, oligomerization and activation. Although the release of cytochrome c from mitochondria may trigger the caspase cascade, there is an alternative pathway not involving the mitochondria (Finkel, 2001; Hunot and Flavell, 2001). Along the way, some antagonistic protein-protein interactions seem to be important in regulating apoptosis, e.g., the BCL-2 family mostly inhibits apoptosis by inhibiting caspase activation and preventing changes in the permeability of the mitochondrial membrane. However, one member of this family, BAX, promotes apoptosis, and this illustrates the complexity of these protein-protein interactions.

Apoptosis is sometimes called death by a thousand cuts due to the prominent role of the caspases, which cleave proteins internally. Nonetheless, several lines of evidence (e.g., caspase inhibitors and genetic knockouts of caspases) suggest that apoptosis can proceed in the absence of those specific caspases (Borner and Monney, 1999; Nicotera et al., 1999; Joza et al., 2001; Kumar and Vaux, 2002). Does this type of death represent a different pathway, perhaps one of the others cited above (Section II) or is it just a different channel in the braid of channels collectively called apoptosis or does it involve some other type of caspase that simply escaped the inhibitory treatments?

Very likely, the endolytic cleavage of the caspase substrates directly accounts for many of the characteristic changes occurring during apoptosis (Cohen, 1997). For example, cutting of the lamins in the nuclear envelope may result in the blebbing of the nucleus, and inactivation of the DNA-repair enzyme PARP [poly(ADP-ribose) polymerase] may contribute to the formation of DNA fragments; however, activation of an endonuclease seems necessary to cleave the DNA (Ameisen, 2002).

The nicking of DNA and cleavage into multiples of nucleosomal fragments are considered hallmarks of the apoptosis process. They are often measured respectively by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) staining of the nuclei to detect 3' breaks in the DNA and gel electrophoresis of the DNA to form a ladderlike pattern (internucleosomal fragments) on the gel. Certainly, these are good markers, but they are not absolute; many exceptions occur (Danon et al., 2000; Joza et al., 2001) and false positives may occur (Wang et al., 1996). Furthermore, if the DNA is cleaved after the nucleosomal proteins are removed or if the DNA fragments are rapidly degraded, the "laddered" DNA fragments will not accumulate. In addition, a TUNEL-positive reaction may also occur in oncosis (Zakeri, 1998). What this adds up to is that there are no absolute criteria for defining/detecting apoptosis (certainly not DNA fragmentation), and it appears to be more of a syndrome. Again, this may merely be a manifestation of the braided channel nature of the process. These considerations are important in determining whether or not apoptosis occurs in plants.

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