**6.2. Cell death effectors released from mitochondria**

The release of proteins from the intermembrane space and the loss of membrane-associated mitochondrial functions lead to cell death. Albeit is not clear the mechanism, mitochondrial destabilization provokes release of factors that mediate in caspase-dependent and independent pathways.

Cell Death and Cancer, Novel Therapeutic Strategies 77

DNase I in order to facilitate DNA processing. Endo G nuclease activity has been observed even in the presence of caspase inhibitors. Therefore, similarly to AIF, Endo G participates in caspase-independent cell death mechanisms. The physiological role of Endo G, however,

Besides its function as IAP inhibitor, **Omi/HtrA2** has been implicated in caspaseindependent cell death mechanisms by virtue of its serine protease activity. This effector molecule cleaves proteins such as ped/pea-15 [49], HAX-1 [50], or RIP-1 [51]. However, the

AIF and Endo G activity together with Omi/HtrA2 serine protease activity are considered

Mitochondria play a central role in programmed cell death pathways and integrate different signals coming from other organelles, being MMP, a point of no return, as already

The tumour suppressor p53 mediates DNA damage response, either by stimulating DNA damage response or by inducing apoptosis. As a transcription factor, p53 transactivates Bcl-2 proteins (Bad, Bid, Puma and Noxa) [52], which induce MMP and release of proteins from the intermembrane space [12]. After DNA damage, p53 can also induce the expression of **p**53-**i**nduced protein with a **d**eath **d**omain (PIDD), which activates nuclear caspase-2. PIDD associates with RAIDD, forming a signalling platform known as the PIDDosome [53]. The PIDDosome can activate caspase-2 and the transcription factor NFkB in response to DNA damage [54]. Caspase-2 acts upstream of the mitochondria by inducing Bid cleavage, Bax translocation and cytochrome c release. The role of caspase-2 in apoptosis, however, is controversial. It is now thought that caspase-2 functions as a tumour suppressor gene regulating the cell cycle machinery [55]. p53 may also induce apoptosis by transcriptionindependent mechanisms, for instance, by directly interacting with Bak, Bax, Bcl-2 and Bcl-XL at the outer mitochondrial membrane [56]. The activity of p53 can be promoted by glycogen synthase kinase 3β (GSK3β) binding both in the nucleus and in the mitochondria,

responsible for the recently called **caspase-independent intrinsic apoptosis** [11].

which in turns promotes cytochrome c release and caspase-3 activation [57].

The ER has primordial roles in normal physiological and survival processes. These include intracellular calcium homeostasis, protein secretion and lipid biosynthesis [58]. Apoptosis can be initiated as a consequence of stress in the ER. This stress condition can be caused by calcium homeostasis alteration, glucose deprivation, hypoxia, low redox potential, excessive protein synthesis or defective protein secretion. These insults can cause accumulation of

*6.3.2. Endoplasmic reticulum: Unfolded protein response* 

needs to be established.

mentioned.

*6.3.1. Nuclear DNA damage* 

exact mechanism as to how this occurs is still unknown.

**6.3. Afferent signals from other organelles** 

**Cytochrome c** is a protein localized in the intermembrane mitochondrial space where it participates in the electron transport between complex III and IV of the mitochondrial respiratory chain. As already mentioned, cytochrome c is involved in the apoptosome formation and caspase cascade activation [45]. Cytochrome c release is a crucial step in the intrinsic apoptotic pathway.

**Smac/DIABLO** means **S**econd **m**itochondrial **a**ctivator of **c**aspases or **D**irect **IA**P **B**inding protein with **LO**w pI. This 29 kDa protein is also localized in the intermembrane space and is released to the cytosol upon activation by certain apoptotic stimuli, where it binds IAPs, preventing their function and favouring caspase activation.

**Omi/HtrA2** functions in a similar way to Smac/Diablo. This 36 kDa protein belongs to a highly conserved protein family. In healthy cells, Omi/HtraA2 is located in the intermembrane space. Upon apoptotic triggering (TRAIL, UV radiation, staurosporine, etc.) is released to the cytosol where it binds and inactivates IAPS promoting caspase activation.

In addition, to the caspase-dependent effectors already mentioned, other caspaseindependent proteins can be activated:

**AIF** stands for Apoptosis Inducing Factor. AIF is a 57 kDa flavoprotein localized in the intermembrane space whose aminoacid sequence resembles ferrodoxin. AIF is expressed as a 67 kDa precursor that possesses two mitochondrial localization sequences in its aminoterminal end. Once in the mitochondria, AIF precursor is cleaved giving rise to the mature protein that is believed to have an oxidoreductase function based on its FAD domain, playing an important physiological role in oxidative phosphorylation. AIF translocates to the nucleus in response to apoptotic stimuli where it induces chromatin condensation and large-scale (50 Kbp) DNA fragmentation by an unknown mechanism, leading to apoptosis in a caspase-independent fashion. It has been recently suggested that Steroid receptor coactivator-interactive protein prevents AIF release from the mitochondria [46]. Conversely, calcium-activated calpain promotes AIF release from the mitochondria [47] and poly-ADPribose-plymerase 1 (PARP-1) activity is necessary for AIF translocation to the nucleus [48].

**Endo G** or Endonuclease G also participates in caspase-independent cell demise mechanisms. It belongs to a family of Mg2+-dependent endonucleases. Endo G is a 30 kDa mitochondrial protein that, like AIF, is synthesized as a precursor form and its mitochondrial localization sequence is cleaved when the protein reaches the intermembrane space. Endo G is released from the mitochondria upon certain apoptotic stimuli such as UV radiation or anti-Fas antibodies, and it translocates to the nucleus, where it cleaves chromatin DNA into nucleosomal fragments. Endo G cooperates with exonucleases and DNase I in order to facilitate DNA processing. Endo G nuclease activity has been observed even in the presence of caspase inhibitors. Therefore, similarly to AIF, Endo G participates in caspase-independent cell death mechanisms. The physiological role of Endo G, however, needs to be established.

Besides its function as IAP inhibitor, **Omi/HtrA2** has been implicated in caspaseindependent cell death mechanisms by virtue of its serine protease activity. This effector molecule cleaves proteins such as ped/pea-15 [49], HAX-1 [50], or RIP-1 [51]. However, the exact mechanism as to how this occurs is still unknown.

AIF and Endo G activity together with Omi/HtrA2 serine protease activity are considered responsible for the recently called **caspase-independent intrinsic apoptosis** [11].
