Apoptosis or programmed cell death is a highly regulated, energy-dependent form of cell death with a characteristic morphological appearance that involves cellular shrinkage and chromatin condensation. Apoptosis is responsible for the removal of unwanted or supernumerary cells during development, as well as in adult homeostasis (Jacobson et al. 1997). Apoptosis is also the predominant form of cell death triggered by cytotoxic drugs in tumor cells (Solary et al. 2000).
There are two pathways leading to apoptosis: the intrinsic pathway and the extrinsic (death receptor) pathway (Fig. 1). In the intrinsic pathway, the mitochondrion fulfills a dual function, (a) as an integrator of multiple pro-apoptotic signaling cascades or damage pathways, and (b) as a coordinator of the catabolic reactions culminating in apoptosis. In response to multiple apoptotic signals of different origins (Ferri and Kroemer 2001), the outer mitochondrial membrane becomes permeabilized, resulting in the release of molecules normally confined to the intermembrane space. Such proteins translocate from mitochondria to the cytosol in a reaction that is controlled by Bcl-2 and Bcl-2-related proteins (Kroemer and Reed 2000). Various molecular mechanisms have been proposed to account for the permeabilization of the outer mitochondrial membrane. These include pore formation in the external mitochondrial membrane by proteins such as Bax (alone or in combination with these proteins) and physical disruption of the outer membrane as a result of mitochondrial matrix swelling (resulting from the formation of nonspecific pores in the inner membrane and/or increased net influx of ions and water) (Marzo et al. 1998; Zamzami and Kroemer 2001). Mitochondrial intermembrane molecules include cytochrome c, apoptosis-inducing factor (AIF), endonuclease G (EndoG), Omi/HtrA2, and second mitochondria-derived activator of caspases (Smac), also called DIABLO. Cytochrome c, once in the cytosol, interacts with Apaf-1 (apoptotic protease activation factor-1), thereby triggering the ATP-dependent oligomerization of Apaf-1, while exposing its CARD domain (caspase recruitment domain) (Li et al. 1997; Hu et al. 1999). Oligomerized Apaf-1 then binds to cytosolic procaspase-9 in a homotopic interaction involving the CARD domain of caspase-9, thereby leading to the formation of the so-called apoptosome, the caspase-9 activation complex. Activated caspase-9 triggers the proteolytic maturation of pro-caspase-3, setting on the activation in the cytosol of a caspase cascade that leads to the limited pro-teolytic cleavage of intracellular, structural, and regulatory proteins, leading to membrane blebbing, chromatin condensation, and nuclear DNA fragmentation (Li et al. 1997). In contrast to cytochrome c, AIF and EndoG directly translocate to the nucleus and trigger caspase-independent nuclear changes (Susin et al. 1999; Joza et al. 2001). Smac/DIABLO and Htra2/Omi activate apoptosis by neutralizing the inhibitory activity of the IAPs (inhibitory apoptotic proteins) that associate with and inhibit caspases (Du et al. 2000) (Fig. 1).
The extrinsic pathway involves plasma membrane death receptors. These receptors (TNF-R1, CD95/APO-1/Fas, TRAIL-R1, TRAIL-R2, DR3, DR6, etc.) belong to the superfamily of TNF receptors (Nagata 1997). Death receptors contain an intracytoplasmic domain called the death domain. Upon ligation of TNF, Fas, or related death receptors, a complex protein known as the death-
inducing signaling complex (DISC) form at the cytosolic C-terminus of the receptor. This complex includes adaptor cytosolic proteins such as TRADD (TNF receptor death domain protein) or FADD (Fas-associated death domain protein) that recruit procaspase-8 (and often procaspase-10), thereby provoking their proteolytic autoactivation to generate active caspase-8 (and perhaps caspase-10). Downstream of caspase-8 and -10, two alternative pathways can trigger apoptotic cell death. One involves the direct activation of other cas-pases (in the so-called type 1 cells), while the other requires the intervention of mitochondria (in the so-called type 2 cells) and therefore converges to the above-mentioned mitochondrial apoptotic pathway (Scaffidi et al. 1999). In type 2 cells, caspase-8 cleaves and activates the pro-apoptotic Bcl2 family protein Bid, which then translocates to mitochondria and triggers permeabi-lization of the outer membrane.
One of the best-studied mediators of apoptosis are the caspases (Thornberry and Lazebnik 1998). Caspases are cysteine proteases expressed in virtually all animal cells. They are synthesized as inactive proenzymes (procaspases) and can be classified into two main groups according to the length of their N-terminal prodomain. Procaspases with a short prodomain (procaspase-3, -6 and -7) are effectors of apoptotic cell death by cleaving essential cellular sub strates. Procaspases with a long prodomain (procaspase-8, -9 and -10) are usually the initiators of a caspase cascade. The serine proteases calpains and cathepsins have also been involved in apoptotic pathways, either working in synergy with caspases or by inducing a caspase-independent apoptotic cell death (Jaattela 2002). In addition or alternatively (Ferri et al. 2000; Susin et al. 2000), proteins such as AIF or EndoG may act independently of cas-pases and constitute a direct molecular link between outer mitochondrial membrane permeabilization and nuclear chromatin condensation. Indeed, in several paradigms of cell death, inhibition of caspases will lead to abortive, presumably AIF-dependent nuclear apoptosis (Daugas et al. 2000; Hisatomi et al. 2001; Joza et al. 2001; Loeffler et al. 2001).
The apoptotic process is tightly regulated. An abnormal increase in apoptosis leading to the unwarranted demise of cells is involved in several pathological processes such as myocardial infarction, stroke, neurodegenerative disease, and AIDS (Kroemer and Reed 2000). In contrast, a deficit in apoptosis is involved in cancer development. Proteins of the Bcl2 family, IAPs, and recently heat shock proteins have been demonstrated to control the apoptotic process at different key points. In this chapter, we will discuss the potential apoptosis modulating functions of different Hsps, putting special emphasis on the nature of their molecular partners and their consequent role in tumorigenicity.
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