Apoptosis PARPs

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. 

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. 

Caspase-10 FLICE-2, Mch4 Apoptosis Caspase-7 PARP pro-Caspase-9 Caspase-3... 

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). 

Immunohistochemistry, on the other hand, enables identification of activated caspases or their cleaved products in fixed archival tissue sections. This technique allows identification of cell(s) undergoing caspase activation, as well as analysis of the distribution of cell(s) in the tissue. Specific antibodies to various caspases are now commercially available, the most frequently studied being caspase-3. Studies in various human tissues and cells have shown that immunohistochemical detection of activated caspase-3 is a useful tool for identifying apoptotic cells in archival material, even before all of the morphological features of apoptosis occur [84-86]. Several target proteins cleaved by caspases can also be detected by immunohistochemistry for example PARP [87], actin [88, 89], and lamin B [90]. 

Abundances are either increased or decreased in the nucleus (Table 13.1) for proteins thought to participate in DNA repair (the nucleolin isoforms, high mobility group protein 1), in protein folding (cyclophilin B, also known as proline isomerase), in apoptosis (78K glucose-regulated protein, PARP-1), and cell cycle... 

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