Apoptosis PARP-1 activity

In aminoglycoside-treated animals, the cells can be led to canonical apop-totic death through activation of caspases. Caspase-9 forms an apoptosome complex with cytochrome c and APAF-1 and leads to apoptosis through activation of caspase-3. Aminoglycosides activate caspases in auditory structures conversely, inhibition of caspase activity successfully blocks neomycin-induced vestibulotoxicity. In contrast, apoptotic markers were essentially absent in a mouse model of chronic kanamycin ototoxicity where death of auditory sensory cells ensued via cathepsins. The activation of cathepsin D was accompanied by the nuclear translocation of endonuclease G, necrotic cleavage of PARP, and activation of p,-calpain, all facets of necrotic cell death. 

ROS play a critical role in initiation of apoptosis through changes in mitochondrial permeability, andpoly(ADP-ribose) polymerase (PARP) activation. These processes provide additional mechanisms for oxidative damage in acute neural trauma and neurodegenerative diseases (Warner et al., 2004). PARP activation is accompanied by the depletion of nicotinamide adenine dinucleotide, NAD. Depletion of NAD leads to depletion of ATP, which in turn promotes neuronal cell death (Zhang etal., 1994 Ishikawaetal., 1999). 

Figure 7.4 Activation of PARP-1 by DNA breaks. PARP-1 is composed of three domains, DNA binding, automodification and catalytic (NAD+ binding) domains (1). In cells, PARP-1 localizes to nucleoli and actively transcribed regions of chromatin by interacting with RNA. When PARP-1 binds to DNA breaks, PARP-1 initiates the poly(ADP-ribosyl)ation reaction by using NAD+ as its substrate (2). PARP-1 itself is the main target of the poly(ADP-ribosyl)ation reaction. ADP-ribose polymers are formed on the automodification domain of PARP-1 (automodification). As a consequence of automodification, PARP-1 dissociates from DNA breaks (3). When cells are committed to apoptosis, PARP-1 is specifically cleaved by an apoptosisspecilic protease, caspase-3, resulting in the formation of a 24kDa N-terminal and 89 kDa C-terminal fragments (4). (see Color Plate 7)...

PARP is a nuclear enzyme involved in DNA repair that is activated in response to DNA damage (27). Early during apoptosis, PARP is cleaved by caspases, primarily by caspase-3 (25). The specific cleavage of this protein that results in distinct 89-kDa and 24-kDa fragments (usually detected electrophoretically) is considered one of the hallmarks of apoptosis. Antibodies that recognize the cleaved PARP products were recently developed and they can be used as immunocytochemical markers of apoptotic cells. The antibody to p89 PARP has been adapted to label apoptotic cells for detection by cytometry (28). The protocol below combines the detection of PARP cleavage and cellular DNA content measurement, which allows one not only to identify and score apoptotic cell populations, but also to correlate apoptosis with the cell cycle position or DNA ploidy. 

For example, leakage of cell content fix>m necrotic cells tri ers inflammation, whereas rapid elimination of apoptotic cells by macrophages protects surrounding cells from secondary damage. Many authors conceive that apoptosis is an aaive process that is finely r ulated while necrosis is a passive and uncontrollable process. However, in view of the active role of poly(ADP-ribose) polymerase (PARP) activation in DNA damage-induced necrosis, this concept needs to be revised. 

While the role of the over activation of PARP-1 in DNA damage-induced necrotic cell death is now widely accepted, its role in apoptosis appears to hugely depend on the experimental conditions such as the cell type and nature of the stimulus. Nonetheless, Valina Dawson s group has shown that PARP activation is required for activation of the mitochondrial route of apoptosis, charaaerized by mitochondrial depolarization, release of AIF ftx>m the mitochondria to the cytosol and caspase-independent, AIF-mediated apoptosis. Notably, these findings are in line with our previous observation that PARP activation mediates mitochondrial alterations during peroxynitrite or hydrogen peroxide-induced necrotic thymocyte death. At the... 

Table 2. PARP activation switches apoptosis to necrosis in DNA damaged cells ... 

Mice overexpressing the DNA-binding domain (DBD) of PARP. (dominant negative PARP inhibition) and PARP-V mice In thymocytes from PARP-DBD overexpressing mice, blockage of PARP activity leads to a drastic increase of p53 expression and activity after DNA damage and correlates with an accelerated onset of Bax expression and apoptosis. Simiiar findings in thymocytes obtained from PARP mice. 32... 

Activation of caspase-3 dependant mitochondrial apoptosis pathway activation of Endo G PARP cleavage Apoptotic DNA fragmentation... 

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