Cell Death: A One-Way Journey to the Graveyard

Tissue homeostasis is ensured by the correct balance between cell proliferation and death, the latter mainly occurring through a multi-step program, named apoptosis, which ultimately leads to the breakdown of cellular DNA and proteins. Apoptosis is activated under physiological developmental conditions, during metamorphosis and atrophy of tissues and organs, sexual differentiation and cell turnover, and can also be triggered by various external stimuli, including DNA damage, growth factor deprivation and metabolic stress. The main features of apoptosis will be described in detail. Although apoptosis is recognised as the main type of programmed cell death, cells may die by alternative mechanisms, e.g. autophagy and necrosis. Their properties will be discussed in this review. APOPTOSIS: A HISTORICAL OVERVIEW Individual cells may die through a finely orchestrated sequence of biochemical and morphological changes which define the so-called Programmed Cell Death (PCD). This notion was first developed when Lockshin and Williams described the controlled cell death occurring during insect development [1-4], and further extended to different organisms [5-8]. After the discovery of PCD in insects, the beststudied animal model was the nematode Caenorhabditis elegans, which is characterised by the selective elimination of 131 out of 1090 cells during embryo development [9]. Genetic studies in this organism allowed the identification of the four essential gene products governing PCD: CED-3 (Cell Death Abnormal), a cystein-protease involved in protein degradation; CED-4, the upstream activator of CED-3; CED-9, able to bind CED-4, thus abolishing its death function; EGL-1, a CED-9-interacting protein that prevents CED-9/CED-4 interaction, thereby promoting PCD [9-11]. Apoptosis is the most well known form of PCD, defined for the first time by Kerr, Currie and Wyllie as an event reminiscent of the falling of leaves from trees [12]. The greek word apoptosis was used by Hippocrates to describe the removal of bone cells after a fracture and by Galen to define the falling of scabs [13], and was found in a Spanish dictionary of medicine written in 1878 [14]. DISTINCTIVE FEATURES OF APOPTOTIC CELLS Morphological Changes Apoptotic cells can be easily recognised by morphological hallmarks, such as nuclear shrinkage, chromatin condensation and aggregation, changes in the surface of plasma membrane, appearance of blebs and consequent formation of the so-called apoptotic bodies. Apoptosis can trigger cell death without rising of any inflammatory response or autoimmune reaction, because *Address correspondence to this author at the IGM-CNR, Via Abbiategrasso 207, 27100 Pavia, Italy; Tel: +39-0382-546334; Fax: +390382-422286; E-mail: [email protected] apoptotic cells display “eat-me signals”, including phosphatidylserine [15], annexin I [16], a large variety of sugars, such as mannose and galactose [17], and ICAM-3 [18] (reviewed in [19]), which favour their recognition by phagocytes [20-22]. However, as recently reviewed [23], the non-immunogenic nature of apoptosis is presently debated, given that the apoptotic response elicited by anti-cancer therapies could exert an immunostimulatory side effect [24]. Degradation of Proteins Apoptotic cells undergo controlled and organised DNA and protein digestion, carried out by specific endonucleases and proteolytic enzymes, respectively [25]. Among the proteases that promote the orderly destruction of structural and regulatory proteins [25], the best known are caspases. Caspases are cytosolic proteases so-named because of their mechanism of action: they contain a cystein residue in their catalytic site and cleave their substrates after an aspartic acid [26]. To date, 14 distinct mammalian caspases have been identified. Their primary structure consists of an N-terminal pro-domain, a large subunit, and a small subunit linked by a region flanked by two aspartate residues [27]. As illustrated in Fig. (1), mammalian caspases with a long pro-domain are named initiator caspases; those with a short pro-domain are called effector caspases [28-30]. Caspases are present within the cell as inactive zymogens and are converted into active enzymes only in response to apoptotic stimuli. The conversion of initiator caspase-8 [31] and -9 [32] into active proteases occurs through their dimerisation, while the transformation of effector pro-caspases to mature enzymes requires one cleavage to remove the N-terminal pro-domain and one to separate the large from the small subunit. These cleavages are followed by heterodimerisation of the subunits (Fig. 1). Once activated, caspases can in turn activate other caspases. Many targets of caspases are known, some of them involved in the maintenance of cell structure, such as actin, fodrin, tubulin and lamin; others, with an enzymatic activity, control DNA metabolism [33]. A recent survey revealed that at least 400 substrates of caspases exist [34]. The cleavage of structural proteins impairs their function in cytoskeleton dynamics. Analogously, most enzymes, once proteolysed, are generally inactive. The nuclear protein PARP-1 (poly(ADP-ribose) polymerase-1), which is involved in the recognition of DNA 28 The Open Biology Journal, 2008, Volume 1 Giansanti and Scovassi single and double strand breaks, is the most utilised apoptotic marker. Its proteolysis by caspases converts the 113 kDa protein into two inactive fragments of 89 and 24 kDa easily detectable by immunological assays [35,36]. Although caspases held the predominant role in proteolysis, there is growing evidence that other proteolytic enzymes are involved in the execution of apoptosis. A family of serine proteases proved to degrade proteins during apoptosis [37]. Among them, the HtrA2/Omi (high temperature requirement protein A) protease plays a crucial role in mitochondrial homeostasis and is released from mitochondria into the cytosol during apoptosis, thus being able to antagonize anti-apoptotic factors [38]. Cathepsins are proteases normally localised intralysosomally; after a specifical signal that targets lysosomes, cathepsins are released into the cytoplasm, where they trigger apoptosis [39,40]. Another class of proteolytic enzymes involved in apoptosis are calpains (i.e. calciumdependent proteases), which are present in the cytosol as zymogens and activated when Ca is released from the endoplasmic reticulum into the cytosol [41,42].