*6.3.1. Nuclear DNA damage*

76 Apoptosis and Medicine

independent pathways.

intrinsic apoptotic pathway.

independent proteins can be activated:

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

preventing their function and favouring caspase activation.

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

**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

**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,

**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 caspase-

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