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Degradation of chromatin into internucleosomal fragments, a prevailing hallmark of apoptosis in most cells and tissues, has been tightly associated with a Ca^2+ and Mg^2+-dependent endonuclease activity. Several candidate enzymes have been identified as important players in this process. Several decades ago, murine and bovine Ca^2+ and Mg^2+-dependent endonucleases were observed to be inhibited by poly(ADP-ribosyl)ation in a reaction mediated by PARP-1. PARP-1 is one of the earliest nuclear enzymes to be targeted for degradation by caspases during apoptosis. Such cleavage is believed to prevent energy depletion in response to DNA damage generated as a result of an activation of apoptotic endonucleases. We have recently identified, cloned, and characterized DNAS1L3 as the human homolog of the unidentified bovine poly(ADP-ribosyl)ation-regulated endonuclease. In this review, we will describe the efforts of our and other laboratories in the elucidation of a role for this endonuclease during apoptosis. We will discuss its dependence on Ca^2+ and Mg^2+, its inhibition by poly (ADP-ribosyl)ation, and its requirement for PARP-1 cleavage, and subsequent inactivation of PARP-1, for optimal activity during apoptosis.

Poly-ADP-ribosylation has turned out to be a major NAD-consuming process in most eukaryotic cells. Although PARP1 exhibits by far the highest capacity to synthesise poly-ADP-ribose, it is active only in situations that are accompanied by DNA damage. It would appear therefore that, under normal physiological conditions, biosynthesis of NAD should not be of critical importance as it is well-known that there is a mulitude of redox reactions using NAD as cofactor, but they are not accompanied by a net loss of the pyridine nucleotide. However, as will be discussed in this chapter, it has now become clear that besides poly-ADP-ribosylation, there are several important regulatory pathways using NAD as substrate. Since they all involve the cleavage of the glycosidic bond between nicotinamide and the ADP-ribose moiety, continuous biosynthesis of NAD is vital to all cells. Accordingly, over the past few years substantial progress has been made in the molecular characterisation of NAD biosynthetic enzymes. In this chapter the newly recognised NAD-mediated regulatory pathways and the advances in the understanding of NAD biosynthesis will be covered. It will also be highlighted that the relationship between PARP1 and NAD synthesis deserves particular attention under conditions of cellular stress involving DNA damage.

The following review discusses the mechanisms through which cell death may be regulated by poly(ADP-ribose) polymerase-1(PARP-1), a nuclear enzyme that catalyzes the synthesis of long, branching (ADP-ribose)_n polymers from NAD^+. Cell death may be caused by nongenotoxic or genotoxic stimuli. Whereas the former mainly occurs in the form of apoptosis, genotoxic exposure can cause both apoptotic and necrotic cell death, depending on the intensity of DNA damage. Although PARP-1 has been shown to play a role in some models of apoptosis triggered by nongenotoxic stimuli, most studies have found PARP-1 to be dispensable for this form of cell death. However, in many models of necrosis caused by genotoxic damage, PARP-1 appears to be a key player causing necrotic cell death by depleting NAD^+ (the substrate of PARP) and ATP (used in futile attempt to resynthesize NAD^+). The author and his colleagues were among the first to show that upon reaching a genotoxic threshold, PARP-1 ovaractivation switches the “default” apoptotic death to necrosis, likely via cellular energetic depletion. PARP activation has also been shown by us, and later by others, to trigger mitochondrial alterations such as mitochondrial membrane depolarization and secondary superoxide production during necrosis or AIF-mediated, caspase-independent apoptosis. It appears that a finely tuned network involving multilevel mitochondrial-nuclear cross talk between PARP-1, p53, the mitochondrial electron transport chain, energy carriers, as well as nuclear and mitochondrial death mediators regulates genotoxic stress-induced cell death.

Neuronal injury resulting from glutamate receptor-mediated excitotoxicity has been implicated in a wide spectrum of neurological disorders. Following dramatic results in the preclinical setting, anti-excitotoxic neuroprotective agents have been used in clinical trials for stroke and head injury, but the results have generally been unsuccessful. Hence, alternative targets in the excitotoxic cascade appear to be required. Poly(ADP-ribosyl)ation has been linked to the pathogenesis of numerous disorders of the CNS, including excitotoxicity and ischemic injury. A presumed cascade of glutamate receptor activation leading to excessive free radical formation, DNA damage and then overactivation of PARP-1 is based on studies with drugs that block these various steps. Along this classical view, experiments in our laboratory have shown that the intracellular depletion of ATP and NAD induced by PARP-1 overactivation leads to necrotic cell death in ischemic and excitotoxic models and that PARP-1 inhibitors are protective against necrotic but not apoptotic neuronal death. Therefore, it appears reasonable to propose PARP-1 inhibitors as useful therapeutic agents in pathological brain conditions where necrosis predominates.

Poly (adenosine 5′-diphosphate ribose) polymerase-1 (PARP-1) is an abundant chromatin-bound enzyme which is present in the nuclei of most cells. The physiological role of PARP-1 involves its activation by single strand breaks in DNA after which it transfers ADP-ribose moieties from nicotinamide adenine dinucleotide (NAD^+) to various nuclear proteins including histones and even to PARP-1 itself (automodification) forming extended chains of ADP-ribose. This reaction leads to the generation of nicotinamide, which, via negative feedback, inhibits PARP-1 activity. However, continuous or excessive activation of PARP-1 (and perhaps other, less well characterized members of the family of poly[ADP-ribose] polymerases, collectively termed “PARP”) during ischemia-reperfusion (I-R) leads to excessive PARP activation resulting in a substantial depletion in intracellular levels of NAD^+. As NAD^+ functions as an electron carrier in the mitochondrial respiratory chain, its depletion rapidly leads to a fall in intracellular levels of adenosine triphosphate (ATP). Moreover, nicotinamide can be recycled back to NAD^+ in a reaction which consumes ATP and thus continuous/excessive activation of PARP results in a fall in ATP via two different mechanisms ultimately leading to cell death—a pathophysiological process commonly referred to as The PARP Suicide Hypothesis. Oxygen-derived radicals such as superoxide anions and hydroxyl radicals cause strand breaks in DNA, activation of PARP and depletion of NAD^+ and ATP. Peroxynitrite, which is generated when equimolar amounts of nitric oxide react with superoxide anions, also causes strand breaks in DNA, activation of PARP and ultimately cell death. Inhibitors of PARP activity can reduce the renal dysfunction caused by I-R of the kidney in vivo and also reduce cellular injury and death caused by oxidative stress to renal cells in culture. In this chapter we review the current evidence that PARP activation plays an important role of the pathophysiology of renal I-R injury and how PARP inhibitors could provide a new therapeutic strategy for patients suffering I-R injury of the kidney.

Oxidative and nitrosative stress triggers DNA strand breakage, which then activates the nuclear enzyme poly(ADP-ribose) polymerase (PARP). Activation of PARP may dramatically lower the intracellular concentration of its substrate, nicotinamide adenine dinucleotide, thus slowing the rate of glycolysis, electron transport and subsequently ATP formation. This process can result in cell dysfunction and cell death. Here I review the impact of pharmacological inhibition or genetic inactivation of PARP on the course of various forms of shock and inflammation in vivo. A major trigger for DNA damage in pathophysiological conditions is peroxynitrite, a cytotoxic oxidant formed by the reaction between the free radicals nitric oxide and superoxide. Here I review (a) some of the evidence supporting the production of DNA-damaging oxygen- and nitrogen-derived oxidants and free radicals and oxidants in various forms of shock and inflammation, (b) the evidence demonstrating PARP activation in these conditions and (c) the effects of pharmacological inhibition or PARP or genetic deficiency of PARP-1. Pharmacological inhibition of PARP emerges as a novel approach for the experimental therapy of various forms of inflammation and shock.

Elucidation of the relationship between poly-ADP-ribosylation and carcinogenesis has markedly progressed by the recent development of knockout or transgenic mice models of poly(ADP-ribose) polymerase (Parp)-1, Parp-2, and poly(ADP-ribose) glycohydrolase (Parg). Parp-1 is involved in base excision repair (BER), single- and |double-strand break repair, and chromosomal stability. These multiple functions explain why Parp-1 deficiency enhances carcinogenesis induced by alkylating agents and that in aged animals. Parp-1 is also involved in transcriptional regulation through protein-protein interaction as a coactivator and/or poly-ADP-ribosylation reaction and is possibly involved in epigenetic alteration during carcinogenesis and modulation of tumor phenotypes. Parp-1-dependent cell-death accompanying NAD depletion may be another important issue in carcinogenesis because this process could lead to the selection of Parp-1 deficient cells due to their survival advantage during cancer growth. The relationship of Parp-2, Parp-3, tankyrase and Parg with carcinogenesis is also discussed.

The compelling evidence for the role of poly(ADP-ribose) polymerase(s) (PARP) in the cellular reaction to genotoxic stress was the stimulus to develop inhibitors as therapeutic agents to potentiate DNA-damaging anticancer therapies. The earliest inhibitors, the benzamides, developed in the 1980s provided “proof of principle” evidence that such an approach was feasible but they lacked the potency and specificity for advanced preclinical evaluation. Over the last two decades potent PARP inhibitors have been developed using structure activity relationships (SAR) and crystal structure analysis. These approaches have identified key desirable features for potent inhibitor-enzyme interactions. The resulting PARP inhibitors are up to 1,000 times more potent than the classical benzamides. These novel potent inhibitors have helped define the therapeutic potential of PARP inhibition. They significantly enhance the in vitro cytotoxicity of DNA monofunctional alkylating agents e.g., temozolomide, topoisomerase I poisons and ionising radiation. PARP inhibitors increase the antitumour activity of these three classes of anticancer agents in vivo, in some cases resulting in complete tumour regression. On the basis of these extremely promising preclinical data, clinical trials with a PARP inhibitor, in combination with temozolomide, commenced in June 2003 in the UK. This trial will allow the evaluation of PARP inhibition as a therapeutic manoeuvre in cancer for the first time.

In this Chapter, we review the evidence suggesting that the family of poly(ADP-ribose) polymerases (PARPs) is involved in regulation of the aging process. First, as genotoxic stress, mainly produced by reactive oxygen species, is believed to be the major driving force of cellular aging, the importance of mechanisms that counteract it or revert its consequences is quiet obvious. A pivotal pathway for eliminating oxidative DNA damage, spontaneously formed abasic sites, or DNA single strand breaks is DNA base-excision repair and its activity is facilitated by PARP-1 and PARP-2. In line with these observations, the capacity of mononuclear blood cells to synthesize poly(ADP-ribose), largely reflecting PARP-1 activity, is positively correlated with the life span of the donor species in mammalians. Second, maintenance of telomere length is very important for replicating cells to avoid cellular senescence and replicative crisis. Two important regulators are the poly(ADP-ribose) polymerases tankyrase-1 and tankyrase-2, which inhibit via modification of TRF-1 its negative influence on telomerase activity. Third, the interaction of PARP-1 with proteins important in preventing premature aging and retarding age-related disease like the Werner syndrome protein (WRN) further supports the importance of PARP-1 in this process. Forth, as several PARPs are components of the mitotic apparatus with apparent regulatory function, they counteract genomic instability also on this level of defence, i.e., beyond DNA repair. Fifth, by interacting with important cell cycle regulators such as p53, PARPs and probably their product, poly(ADP-ribose), take an active part in DNA damage surveillance and regulation of cell division. In conclusion, PARP proteins are crucial players in the cellular responses to various kinds of impaired functionality of genomic DNA, as they represent versatile tools to fight all kinds of threats to genomic integrity, thus keeping in check aging-related dysfunction and disease. In doing so, they might be importanthelpers to keep the speed of the aging process low and may also contribute to attaining “healthy aging”, i.e., a state of being old, yet free from major age-related disease or disability.