I. Poly(ADP-Ribose) Metabolism

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Poly(ADP-ribose) polymerase-1 (PARP-1¹; EC 2.4.2.30) [also known as poly(ADP-ribose) synthetase and poly(ADP-ribose) transferase] is a nuclear enzyme present in eukaryotes. PARP-1 is a 116-kDa protein consisting of three main domains: the N-terminal DNA-binding domain containing two zinc fingers, the automodification domain, and the C-terminal catalytic domain (Mazen et al., 1989; de Murcia and Menissier de Murcia, 1994; de Murcia et al., 1994; Schreiber et al., 1995; Szabo, 2000; Smith, 2001) (Fig. 1). The primary structure of the enzyme is highly conserved in eukaryotes (human and mouse enzyme have 92% homology at the level of amino acid sequence) with the catalytic domain showing the highest degree of homology between different species; the catalytic domain contains the so-called PARP signature sequence, a 50-amino acid block showing 100% homology between vertebrates.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Comparison of the domain structures of some PARP enzymes. PARP-1 consists of three main domains: the N-terminal DBD, the automodification domain (AMD), and the C-terminal catalytic domain. Within the DBD, two zinc fingers are responsible for DNA binding and some protein-protein interactions. The DBD also contains an NLS within which the caspase-cleavage site (DEVD) can be found. The automodification domain contains a BRCT motif, which is common in many DNA repair and cell-cycle proteins. PARP-1 participates in various protein-protein interactions through the BRCT motif. The active site of the enzyme found in the C-terminal part is highly conserved in eukaryotes, and this 50-amino acid sequence is also known as "the PARP signature". PARP-2 differs from PARP-1 in the structure of its DBD. Vault PARP (vPARP) has no DBD, and the N terminus is occupied by a BRCT motif. The enzyme has an NLS residing in the C-terminal part. The N terminus of tankyrase-1 and -2 has a histidine-, proline-, and serine-rich (HPS) region or N-terminal domain (NTD), respectively. The remaining parts [ankyrin repeats, sterile alpha motives (SAM), and catalytic domains] are basically the same.

PARP-1 functions as a DNA damage sensor and signaling molecule binding to both single- and double-stranded DNA breaks. Upon binding to damaged DNA mainly through the second zinc-finger domain, PARP-1 forms homodimers and catalyzes the cleavage of NAD⁺ into nicotinamide and ADP-ribose and then uses the latter to synthesize branched nucleic acid-like polymers poly(ADP-ribose) covalently attached to nuclear acceptor proteins (de Murcia et al., 1994; de Murcia and Menissier de Murcia, 1994; Lindahl et al., 1995; Schreiber et al., 1995; Burkle, 2001; Smith, 2001). The size of the branched polymer varies from a few to 200 ADP-ribose units. Because of its high negative charge, the covalently attached ADP-ribose polymer dramatically affects the function of target proteins. In vivo, the most abundantly poly(ADP-ribosylated) protein is PARP-1 itself, and auto-poly(ADP-ribosylation) represents a major regulatory mechanism for PARP-1 resulting in the down-regulation of the enzyme activity. In addition to PARP-1, histones are also considered to be major acceptors of poly(ADP-ribose) (Tanuma et al., 1985; Nagele, 1995). Poly(ADP-ribosylation) confers negative charge to histones, leading to electrostatic repulsion between DNA and histones. This process has been implicated in chromatin remodeling, DNA repair, and transcriptional regulation. Several transcription factors, DNA replication factors, and signaling molecules [NF-B (Oliver et al., 1999), AP-2 (Kannan et al., 1999), Oct-1, YY1 (Oei and Shi, 2001a,b), B-MYB (Cervellera and Sala, 2000), DNA-dependent protein kinase (Ariumi et al., 1999), p53 (Wesierska-Gadek et al., 1996; Kumari et al., 1998; Malanga et al., 1998; Simbulan-Rosenthal et al., 1999b, 2001b; Mendoza-Alvarez and Alvarez-Gonzalez, 2001; Wesierska-Gadek and Schmid, 2001; Tong et al., 2001), topoisomerase I, lamin B, and B23] have been shown to become poly-ADP-ribosylated by PARP-1. The effect of PARP-1 on the function of these proteins is carried out by noncovalent protein-protein interactions and by covalent poly-ADP-ribosylation. Poly-ADP-ribosylation is a dynamic process, indicated by the short (<1 min) in vivo half-life of the polymer (Whitacre et al., 1995). Two enzymespoly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosyl protein lyaseare involved in the catabolism of poly(ADP-ribose), with PARG cleaving ribose-ribose bonds of both linear and branched portions of poly(ADP-ribose) and the lyase removing the protein proximal ADP-ribose monomer (Davidovic et al., 2001).

The regulation of PARP-1 activity is established through different mechanisms. The best characterized mechanism is the down-regulation of enzyme activity through auto-poly-ADP-ribosylation (Kawaichi et al., 1981). Furthermore, nicotinamide, the smaller cleavage product of NAD⁺, also exerts inhibitory effect on PARP-1, allowing negative feedback regulation. Recently, the purines hypoxanthine, inosine, and adenosine also were identified as another class of endogenous PARP inhibitors (Virag and Szabo, 2001). The regulation of PARP activity by purines is possibly relevant under pathophysiological conditions in which intracellular levels of these metabolites reach levels that are high enough to efficiently inhibit PARP. Phosphorylation of PARP by protein kinase C also results in enzyme inhibition (Tanaka et al., 1987; Bauer et al., 1992). The abundance of PARP may also change under certain conditions, suggesting a transcriptional or posttranscriptional regulation (Bergeron et al., 1997; Tramontano et al., 2000; Doucet-Chabeaud et al., 2001). It is not yet clear whether PARP induction significantly alters the poly-ADP-ribosylating capacity of the cells, because even in resting cells, PARP-1 is one of the most abundant nuclear proteins. The interconnection of PARP activation and signal transduction pathways is supported by a report describing DNA strand break-independent PARP activation via the phospholipase C-inositol-1,4,5,-trisphosphate-calcium route (Homburg et al., 2000). The physiological or pathophysiological relevance of this pathway is poorly understood at present.

The biological role of poly(ADP-ribose) is complex and involves nine main functions. First, PARP-1 has been implicated in DNA repair and maintenance of genomic integrity (de Murcia and Menissier de Murcia, 1994; de Murcia et al., 1994,1997; Schreiber et al., 1995; Chatterjee et al., 1999b; Shall and de Murcia, 2000). This "guardian angel" function is indicated by delayed DNA base-excision repair and by a high frequency of sister chromatid exchange in PARP-1-deficient cells exposed to ionizing radiation or treated with alkylating agents (de Murcia et al., 1997). High levels of ionizing radiation and alkylating agents elicit higher lethality in PARP-1-deficient mice when compared with wild-type ones (de Murcia et al., 1997).

Second, PARP-1 also regulates the expression of various proteins at the transcriptional level. Of special importance is the regulation by PARP-1 of the production of inflammatory mediators such as the inducible nitric-oxide synthase (iNOS) (Hauschildt et al., 1992; Le Page et al., 1998; Szabo et al., 1998c; Oliver et al., 1999), intercellular adhesion molecule 1 (ICAM-1) (Zingarelli et al., 1998; Szabo et al., 2001b), and major histocompatibility complex class II (Otsuka et al., 1991). NF-B is a key transcription factor in the regulation of this set of proteins, and PARP has been shown to act as a coactivator in the NF-B-mediated transcription (Oliver et al., 1999). There is currently no consensus in the literature regarding whether the modulation of NF-B-mediated transcription by PARP is dependent on the catalytic activity of the enzyme or, alternatively, on its physical presence (Hassa and Hottiger, 1999; Kameoka et al., 2000; Chang and Alvarez-Gonzalez, 2001; Hassa et al., 2001). Poly(ADP-ribosylation) of histones may also contribute to the transcription-promoting effect of PARP-1, because poly(ADP-ribose) confers negative charge to histones, leading to electrostatic repulsion between histones and DNA. Thus, poly(ADP-ribosylation) can loosen the chromatin structure and can thereby make genes more accessible for the transcriptional machinery. Nuclear receptor-mediated transcription is regulated by PARP-1 in a different manner: PARP-1 seems to suppress nuclear receptor-mediated transcription (Miyamoto et al., 1999).

Third, PARP-1 regulates replication and differentiation. The involvement of PARP-1 in the regulation of replication is supported by observations that poly(ADP-ribose) metabolism is accelerated in the nuclei of proliferating cells (Tanuma et al., 1978; Kanai et al., 1981; Leduc et al., 1988; Bakondi et al., 2002a). Furthermore, PARP-1 is part of the multiprotein replication complex (MRC) (Simbulan-Rosenthal et al., 1996), indicated by copurification of PARP-1 with key components of MRC (Simbulan-Rosenthal et al., 1996; Dantzer et al., 1998). Moreover, several replication factors and centromere proteins have been shown to serve as substrates for PARP (Simbulan-Rosenthal et al., 1996; Saxena et al., 2002). Another mechanism by which PARP may regulate nuclear processes is poly(ADP-ribosylation) of histones facilitating the assembly and deposition of histone complexes on DNA during replication (Boulikas, 1990).

Fourth, poly(ADP-ribosylation) has been implicated in the regulation of telomerase activity. The overexpression of tankyrase-1, a recently discovered PARP enzyme, in telomerase-positive human cells resulted in a gradual and progressive elongation of telomeres (Smith and de Lange, 2000). Besides tankyrase-1, PARP-1 has also been implicated in the maintenance of telomere length. Genetic ablation of PARP-1 has been shown to result in telomere shortening (d'Adda di Fagagna et al., 1999) but others found no difference in telomere length of PARP-proficient and -deficient cells (Samper et al., 2001).

Fifth, PARP-1 activation has been proposed to represent a cell-elimination pathway (Berger et al., 1983, 1986; Schraufstatter et al., 1986b; Sims and Benjamin, 1987; Schreiber et al., 1995; Kleczkowska and Althaus, 1996) through which severely damaged cells are removed from tissues. PARP-1-mediated cell death occurs in the form of necrosis (Schreiber et al., 1995; Virag et al., 1998a,b), which is probably the least desirable form of cell death. During necrotic cell death, the cellular content is released into the tissue, exposing neighboring cells to potentially harmful attacks by proteases and other released factors. This scenario (Fig. 2) is best exemplified by cells that have been exposed to DNA-damaging stimuli. Mild genotoxic noxa cause PARP activation that facilitates DNA repair and cell survival. Severe DNA damage, however, causes overactivation of PAR,P resulting in the depletion of NAD⁺ and ATP and consequently in necrotic cell death (Fig. 2).

View larger version (70K): [in this window] [in a new window]   Fig. 2.   DNA-damage-induced PARP activation: a kiss of life or a kiss of death? DNA-damaging stimuli cause PARP activation. Activated PARP cleaves NAD+ into nicotinamide and ADP-ribose and polymerizes the latter on nuclear-acceptor proteins. Poly(ADP-ribosylation) facilitates DNA repair and thus permits cell survival. Severe DNA damage, however, leads to overactivation of PARP, resulting in NAD+ and ATP depletion and necrotic cell death.

Sixth, poly(ADP-ribose) polymer has been identified recently as an emergency source of energy used by the base-excision machinery to synthesize ATP (Maruta et al., 1997; Oei and Ziegler, 2000).

Seventh, similarly to ubiquitination, poly(ADP-ribose) may also serve as a signal for protein degradation in oxidatively injured cells (Ciftci et al., 2001; Ullrich and Grune, 2001; Ullrich et al., 2001a). Hydrogen-peroxide treatment of K562 cells caused a PARP-1-dependent up-regulation of 20S proteosome activity. During this process, the proteosome becomes poly(ADP-ribosylated), resulting in the enhanced degradation of poly(ADP-ribosylated) histones (Ullrich and Grune, 2001). Immunoprecipitation experiments demonstrated a protein-protein interaction of the functionally active PARP with the proteasome in correlation with the proteasome activity (Ullrich et al., 2001a).

Eighth, in addition to PARP-catalyzed covalent poly(ADP-ribosylation), poly(ADP-ribose) polymers can noncovalently bind to specific (ADP-ribose)_n binding motifs in proteins, such as histones, XRCC1, p53, and DNA polymerase , and thereby modify their function (Althaus et al., 1993; Pleschke et al., 2000). Such (ADP-ribose) polymers can be formed during the catabolism of poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase (Davidovic et al., 2001).

In the ninth and final function, poly(ADP-ribosylation) may also be involved in the regulation of cytoskeletal organization. A recent study reported morphological alterations in Drosophila overexpressing PARP-1 (Uchida et al., 2001). The overexpression of PARP-1 disrupted the organization of cytoskeletal F-actin, resulting in aberrant cell and tissue morphology. Furthermore, heat-induced PARP expression disrupts the organization of cytoskeletal F-actin in embryos and tissue polarity in adult flies. Whether these morphological alterations are indeed related to PARP-1 function or, alternatively, whether PARP-1 overexpression interferes with the function of cytoplasmic PARP enzymes remains to be seen.

Until recently, PARP activity was believed to result from the function of a single enzyme. After the observation that PARP-1-deficient cells have some residual PARP activity (Shieh et al., 1998), intensive research began to identify enzymes responsible for this activity. In the last 2 years, several other enzymes possessing poly(ADP-ribosylation) activity have been described (Smith, 2001) with the founding member of the PARP enzyme family now designated as PARP-1. Although research on the biological role of these novel PARP enzymes is in the embryonic stage, interesting differences in domain structure (Fig. 1), subcellular localization, tissue distribution, and ability to bind to DNA have already been established.

PARP-2 is a 62-kDa protein of unknown function (Ame et al., 1999). Human and mouse PARP-2 genes were mapped to 14q11.2 and 14C1, respectively. These loci are different from PARP-1 loci. The automodification domain is missing from PARP-2, and the DNA binding domain (DBD) is very different from that of PARP-1 (Ame et al., 1999). Somewhat surprisingly, even though the putative DBD of PARP-2 is devoid of any known DNA binding motifs, DNase I-treated DNA induced PARP-2 activation. DNA binding of PARP is facilitated by the high ratio of basic amino acids in the PARP-2 DBD. Moreover, PARP-2 is capable of auto-poly(ADP-ribosylation); however, it could not poly(ADP-ribosylate) histones, which are prototypical PARP-1 substrates. The enzyme localizes to nuclei and becomes activated in cells upon methylmethanesulfonate-induced DNA damage. PARP-2 has been shown recently to be cleaved by cysteinyl aspartate-specific protease (caspase)-8 (Benchoua et al., 2002).

Vault PARP has been found in vaults (Kickhoefer et al., 1999). Vaults are barrel-shaped ribonucleoprotein particles of arched morphology reminiscent of the vaulted ceilings of cathedrals (Kickhoefer et al., 1996). Their biological role is unknown at present. Vaults were proposed to be part of the nuclear pore complex and have also been implicated in multidrug resistance (Kickhoefer et al., 1996). Vault PARP has been found to associate with and poly(ADP-ribosylate) the major vault protein. The functional significance of this cytoplasmic PARP is as elusive as the biological role of vaults.

Tankyrase-1. The chromosomal end-replication problem has been fascinating researchers for some years. The observation that tumor cells but not untransformed cells can prevent the shortening of their chromosomes by using a ribonucleoprotein enzyme named telomerase opened a new target area for anticancer drug development. Therefore, it is not surprising that among the novel PARPs, tankyrase, a telomere-associated enzyme (Smith et al., 1998), has attracted much attention. Tankyrase-1, a protein containing 24 ankyrin repeats, binds to and poly(ADP-ribosylates) telomere repeat-binding factor 1 (TRF1), a negative regulator of telomerase (Smith et al., 1998). Tankyrase-1 was also found to auto-poly(ADP-ribosylate) itself. Poly(ADP-ribosylation) probably releases TRF from telomeres and inhibits TRF function because overexpression of tankyrase-1 increased telomere length. Like vault PARP, tankyrase does not require DNA for activity. Overexpression of tankyrase-1 in the nucleus diminished the level of unmodified TRF1 in immunoblots and led to reduced immunofluorescence of TRF1 at interphase telomeres (Smith and de Lange, 2000). Long-term overexpression of tankyrase-1 in telomerase-positive human cells resulted in a gradual and progressive elongation of telomeres (Smith and de Lange, 2000). A PARP-deficient form of tankyrase-1 failed to affect TRF1 and did not alter telomere length dynamics, which is consistent with ADP-ribosylation of TRF1 being the main cause of altered telomere homeostasis. Recently, a new tankyrase-1 binding protein has been identified (Seimiya and Smith, 2002). TAB182, a 182-kDa protein has been shown to coimmunoprecipitate with tankyrase-1 from human cells and to serve as an acceptor of poly(ADP-ribosyl)ation by tankyrase-1 in vitro. Like TRF1, TAB182 binds to the ankyrin domain of tankyrase-1 (Seimiya and Smith, 2002).

Poly(ADP-ribosylation) is a dynamic process because poly(ADP-ribose) polymer is rapidly degraded by poly(ADP-ribose) glycohydrolase and ADP ribosyl protein lyase. The half-life of the polymer is estimated to be less than 1 min, indicating a concerted activation of poly(ADP-ribose)-synthesizing and -degrading enzymes. Since its discovery by Miwa and Sugimura in 1971 (Miwa and Sugimura, 1971; Miwa et al., 1974), PARG has not been investigated nearly as intensively as PARP-1. This is mostly because of difficulties in obtaining pure PARG enzyme. The difficulty lies in the low cellular abundance of the enzyme and its sensitivity to proteolytic degradation during purification (Davidovic et al., 2001). PARG is capable of hydrolyzing both terminal ADP-ribose units from poly(ADP-ribose) polymers via exoglycosidic activity and of removing larger oligo(ADP-ribose) fragments via endoglycosidic cleavage (Brochu et al., 1994; Davidovic et al., 2001). Because the K_m value of PARG is much lower for larger (ADP-ribose)_n polymers than for smaller ones (Hatekayama et al., 1986), the enzyme probably removes and catabolizes bigger fragments first. PARG then switches to exoglycosidic mode and removes ADP-ribose units one by one. The proximal ADP-ribose moiety is removed from the acceptor proteins by ADP-ribosyl protein lyase (Oka et al., 1984). The high specific activity of PARG compensates for the low abundance of the enzyme. Nearly 90% inhibition of PARG activity is required (by heat shock) for cellular poly(ADP-ribose) accumulation (Jonsson et al., 1988a,b). The rat and bovine PARG cDNA-s cloned by Sugimura's (Shimokawa et al., 1999) and Jacobson's groups (Lin et al., 1997), respectively, encode 109- to 111-kDa proteins sharing no homology with other proteins other than a protein sequence from Caenorhabditis elegans that is likely to be the PARG enzyme of this organism. Expressed PARG formed stable dimers through leucin zipper-like dimerization domains even under SDS-polyacrylamide gel electrophoresis conditions. PARG contains both a nuclear localization signal (NLS) and a nuclear export signal, providing support for the idea that PARG may shuttle between the nucleus and the cytoplasm (Shimokawa et al., 1999). A PARG shuttle may serve regulatory functions and may also allow PARG to participate in the digestion of poly(ADP-ribose) synthesized by cytoplasmic PARP enzymes.

Contrary to the plethora of articles reporting cellular and in vivo effects of PARP inhibition, there are very few articles on the biological role of PARG. Tannin derivatives have been most frequently used to inhibit PARG in vitro. The heterogeneous composition and significant vendor-to-vendor and batch-to-batch variation of tannin represents a major obstacle in PARG pharmacology. Nonetheless, oenothein B, a macrocircular ellagitannin PARG inhibitor, has been shown to suppress mouse mammary tumor virus transcription and to activate tumor-suppressing macrophages (Aoki et al., 1995). Furthermore, gallotannin and nobotanin B have been shown by Swanson's group to protect murine astrocytes from oxidative injury (Ying and Swanson, 2000). Moreover, the same PARG inhibitors and the PARP inhibitor 3-aminobenzamide have been tested in parallel for their cytoprotective effect in hydrogen peroxide, N-methyl-D-aspartate (NMDA), and DNA alkylating agent-induced neuronal and astrocyte cell death model (Ying et al., 2001). Even though inhibition of PARG and PARP had opposing effects on poly(ADP-ribose) formation, both approaches provided remarkable protection to the cells (Ying et al., 2001). We have also observed the cytoprotective effect of gallotannin in oxidatively stressed A549 lung epithelial cells and HaCaT keratinocytes (L. Virag, manuscript in preparation). These findings may open new avenues for pharmacological interventions targeting poly(ADP-ribose) metabolism. However surprising it may sound, it now seems that inhibition of both the poly(ADP-ribose) synthesizing enzyme (PARP) and the catabolizing enzyme (PARG) has similar cytoprotective effect in oxidatively stressed cells. The likely solution for this paradox is that removal of inhibitory poly(ADP-ribose) residues by PARG from the automodification domain of PARP is required for PARP to maintain its active state (Fig. 3.). The inhibition of PARG results in hyper-auto-poly(ADP-ribosylation) of PARP and inhibition of the enzyme. In addition, PARG activity seems necessary for the high poly(ADP-ribose) turnover resulting in NAD⁺ depletion and cell death triggered by DNA-damaging stimuli. These intriguing new data raise several questions: Is PARG inhibition a viable strategy for the treatment of diseases (reperfusion injury, inflammation, shock) in which PARP inhibitors proved useful? How does PARG inhibition and nontransient poly(ADP-ribosylation) affect DNA repair? Are PARP-assisted transcription machineries differentially regulated by PARG and PARP? To address these issues, specific and potent PARG inhibitors as well as efficient molecular biology tools to overexpress or genetically delete PARG are required. Considering the intense effort in this field of research, it is likely that PARG-deficient mice will soon become available and will certainly accelerate PARG research.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   The role of PARG in the reactivation of PARP. PARP activation leads to automodification of PARP, resulting in PARP inhibition. By removing poly(ADP-ribose) from PARP, PARG reactivates PARP and allows for continuous NAD turnover.

The assumption that PARP-1 may be involved in DNA repair was born simultaneously with the identification of the enzyme as a DNA binding protein. Several studies using various pharmacological PARP inhibitors have concluded that PARP-1 plays a role in DNA repair (Burkle, 2001; Ziegler and Oei, 2001). It has been shown, for example, that the PARP inhibitor 3-aminobenzamide retarded the rejoining of DNA strand breaks and enhanced the frequencies of unscheduled DNA synthesis and sister chromatide exchanges in MNNG-treated Chinese hamster ovary and HeLa S3 cells (Park et al., 1983). Furthermore, PARP inhibition rendered cells more sensitive to cytotoxicity induced by DNA-damaging stimuli (Szabo, 2000). Later, using random mutagenesis, Berger's group generated cell lines having low PARP activity and showed that these mutants were hypersensitive to ionizing and UV irradiation, topoisomerase I inhibitors, and a series of different alkylating agents, including alkylsufonates, alkylnitrosoureas, and nitrosoguanidine (Chatterjee et al., 1989, 1990a,b). When molecular biological manipulation has allowed dominant negative inhibition, depletion, or genetic ablation of PARP-1 by overexpression of its DNA binding domain, by expression of antisense PARP-1 RNA, or by homologous recombination, respectively, a contribution of PARP-1 in DNA repair and maintenance of genomic integrity becomes apparent (Molinete et al., 1993; Stevnsner et al., 1994; Schreiber et al., 1995; Shall and de Murcia, 2000; Smulson et al., 2000). Many studies using pharmacological inhibitors of PARP have problems associated with limited selectivity or specificity of many of the compounds used. Thus, from the large number of publications, we restrict our current discussion to what we have learned from the PARP-1-deficient mice and cells derived from them. However, the results obtained from studies using PARP-deficient experimental systems usually do not distinguish between findings related to the physical absence of the enzyme (i.e., "scaffolding" functions) and the lack of PARP's catalytic activity (i.e., the "enzymatic" function).

PARP knockout mice generated in de Murcia's laboratory were highly sensitive to death induced by ionizing radiation or monofunctional alkylating agents (de Murcia et al., 1997). Furthermore, exposure to methylmethanesulfonate or N-methyl-N-nitrosourea of embryonic fibroblasts derived from PARP^/ mice but not from PARP^+/+ mice exhibited a reduced rate of proliferation because of their cell-cycle block in G₂/M (de Murcia et al., 1997; Trucco et al., 1998). PARP-deficient cells also exhibited genomic instability, as evidenced by an increased number of micronuclei (chromatin fragments indicating chromosomal damage) with or without methylmethanesulfonate treatment. Furthermore, PARP^/ fibroblasts exhibited slower rejoining of DNA breaks, as measured with use of the comet (single-cell gel electrophoresis) assay indicating deficient ligation. It has also been investigated whether the short-patch repair system responsible for the replacement of a single mutated nucleotide or the long-patch repair system capable of replacing 7 to 14 nucleotides is more affected by the absence of PARP-1. It was found that lysates from PARP-1-deficient fibroblasts had no long-patch repair activity, and their short-patch repair activity was also reduced by approximately 50%, as compared with PARP-1-proficient cell lysates (Dantzer et al., 2000). From these data, the conclusion can be drawn that PARP-1 contributes to the maintenance of genomic integrity and also enhances base-excision repair in irradiated or alkylating agent-treated cells. The involvement of PARP-1 in genomic surveillance is also indicated by the interaction of PARP-1 with other nick sensors such as DNA ligase III, adaptor factors such as XRCC1 (Masson et al., 1998), and DNA repair effectors such as DNA polymerase  and DNA ligase III, components of the base-excision repair complex. Through the zinc-finger domains or the breast cancer susceptibility protein C terminus (BRCT) motif of the automodification domain, PARP-1 physically associates with these proteins, as indicated by the two-hybrid system or coimmunoprecipitation. Regulation of the activity of these proteins by PARP-1 is carried out both via physical interaction and poly(ADP-ribosylation). The exact nature of the regulatory role of PARP-1 within the base-excision repair complex, however, requires further investigation.

In the last decade, PARP-1 has become widely known among cell biologists as the "death substrate" (Tewari et al., 1995). Indeed, PARP-1 was one of the first identified substrates of caspases, the main executioners of apoptosis (Kaufmann et al., 1993). Therefore, a role for PARP-1 in the regulation of apoptosis has been suggested. Even though there are data in the literature pointing toward a possible role of PARP-1 in apoptosis, more convincing evidence suggests the involvement of PARP-1 in necrosis. Here, we summarize the current knowledge on the role PARP-1 plays in the two main pathways of cell death: apoptosis and necrosis. Most of the studies related to PARP and cell death are likely to pertain to the major PARP isoform, PARP-1. Studies related to the potential role of the other PARP isoforms in cell death are scarce; in one such recent study, nevertheless, it was shown that overexpression of tankyrase-2 is able to induce cell death in fibroblasts (Kaminker et al., 2001).

1. Apoptosis. During apoptosis, caspase-7 and caspase-3 cleave PARP-1 into two fragments: p89 and p24 (Tewari et al., 1995; Germain et al., 1999). These proteases recognize a DEVD motif in the nuclear localization signal of PARP-1 (Lazebnik et al., 1994), and cleavage at this site separates the DNA binding domain from the catalytic domain, resulting in the inactivation of the enzyme. Cleavage fragments contribute to the suppression of PARP activity because p89 and p24 inhibit homoassociation and DNA binding of intact PARP-1, respectively (Kim et al., 2000a,b; D'Amours et al., 2001). The existence of this positive feedback loop in caspase-mediated PARP-1 inactivation suggests that blocking PARP-1 activation is vital for the proper function of the apoptotic machinery. According to this concept PARP cleavage aims at preventing the activation of PARP by the ensuing DNA fragmentation and thereby aims at preserving cellular energy for certain ATP-sensitive steps of apoptosis. Experimental evidence supporting this hypothesis was provided by Herceg and Wang (1999), showing that the expression of a caspase-uncleavable, modified version of PARP in TNF--treated PARP-1 knockout fibroblasts leads to NAD⁺ depletion and necrosis. Inhibition of PARP activity by 3-aminobenzamide blocked both NAD⁺ depletion and cell death. Recent work indicates that PARP-1 cleavage during apoptosis is not simply required to prevent excessive depletion of NAD and ATP, but it is also necessary to release the human Ca²⁺- and Mg²⁺-dependent endonuclease (DNAS1L3) from poly(ADP-ribosyl)ation-mediated inhibition (Boulares et al., 2001). Although caspase-mediated PARP-1 cleavage is well documented, little is known about the possible cleavage of novel PARP enzymes and PARG. Recently PARP-2 has been shown to be cleaved by caspase-8, a caspase which was considered to be an initiator caspase, the proform of which associated with cell-surface death receptors. In ischemia-induced neuronal apoptosis, caspase-8 trans locates into the nucleus and cleaves PARP-2 at a LQMD sequence. PARP-2 cleavage, similar to PARP-1 cleavage, separates the DNA binding end-catalytic domains and inactivates the enzyme (Benchoua et al., 2002). In addition to PARP-1 and PARP-2, PARG also becomes cleaved at a relatively early stage of apoptosis (Affar et al., 2001). However, the biological role of the cleavage of PARP-2 and PARG has not yet been investigated in detail.

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2. Necrosis. Various stimuli can trigger both apoptotic and necrotic cell death. It is important to note that necrosis is not simply another type of cell death; it represents a more severe form of cell demise compared with apoptosis. Viewing cell death from the point of view of the tissue or organ in which cell death takes place, such a distinction makes sense. In addition to numerous biochemical and morphological differences between apoptosis and necrosis, probably the most distinctive feature of necrosis is the disintegration of the plasma membrane, as opposed to the compaction of apoptotic cells. Leakage of cell content from necrotic cells into the surrounding tissue may contribute to organ injury, whereas apoptotic cells are rapidly cleared from the tissues by macrophages. Using NMDA- or peroxynitrite-treated neurons, Lipton's and Nicotera's groups elegantly demonstrated that apoptosis and necrosis are at two ends of a continuum in which apoptosis is caused by mild stimuli and necrosis is triggered by severe stimuli (Bonfoco et al., 1995; Nicotera et al., 1999). Furthermore, it has also been suggested that both ATP and NAD⁺ are important determinants of the mode of cell death, especially in oxidatively injured cells (Coppola et al., 1995; Klaidman et al., 1996; Leist et al., 1997a, 1999a; Mukherjee et al., 1997; Lelli et al., 1998; Lieberthal et al., 1998; Nicotera et al., 1998; Ran et al., 1999; Crowley et al., 2000). From these observations, it was plausible to hypothesize that PARP as a NAD⁺-catabolizing enzyme may serve as a molecular switch between apoptosis and necrosis. The initial studies on the role of PARP and cell death were performed using pharmacological inhibitors of PARP (most frequently, 3-aminobenzamide and nicotinamide) and have been previously reviewed (Szabo and Dawson, 1998). These agents can have additional actions, such as acting as free-radical scavengers. More recent studies using cells from PARP knockout animals confirmed the role of the PARP pathway in oxidant-mediated cell injury. In the first such study, Heller and coworkers (Heller et al., 1995; Wang et al., 1995) observed that islets of the PARP^/ mice are resistant to NO and oxidant-related injury when compared with the response in islets of the wild-type mice. Similarly, we observed that pulmonary fibroblasts from the PARP^/ mice are protected from peroxynitrite-induced cell injury when compared with the fibroblasts of the corresponding wild-type animals (Szabo et al., 1998c). Furthermore, Eliasson et al. (1997) demonstrated protection by PARP^/ phenotype in brain slices exposed to various oxidants. Thus, the more definitive studies using PARP knockout cells have now fully confirmed the conclusions of the earlier pharmacological studies. With respect to the mode of cell death, the earlier studies provided some clues that it is the necrotic type (e.g., in the Heller study, lactate dehydrogenase release was blocked in PARP-deficient islets), but direct investigations have not been conducted to characterize the mode of cell death. In hydrogen peroxide-treated HT-29 epithelial cells, the inhibition of PARP by 3-aminobenzamide inhibited necrosis but not apoptosis (Watson et al., 1995). One year later, Palomba et al. (1996) found that inhibition by 3-aminobenzamide of hydrogen peroxide-induced necrosis in U937 myeloma cells was associated by increased apoptotic DNA fragmentation and cell blebbing. Later, our group provided evidence for the possible role of PARP-1 in switching default apoptosis to necrosis in oxidatively injured cells. In our experiments, we used thymocytes from PARP-1^+/+ and PARP-1^/ mice and compared their responses to peroxynitrite and hydrogen peroxide (DNA-damaging stimuli) as well as dexamethasone and anti-Fas treatment (non-DNA-damaging agents). While nongenotoxic stimuli (dexamethasone and anti-Fas) triggered equal apoptotic responses, as evidenced by phosphatidylserine exposure, caspase activation, and DNA fragmentation, in wild-type and PARP-1-deficient thymocytes, marked differences could be observed with DNA-damaging oxidative agents. Low concentrations of peroxynitrite and hydrogen peroxide induced apoptosis in both PARP-1^+/+ and PARP-1^/ cells, with 3-aminobenzamide having no effect on the responses. At higher concentrations of the oxidants, necrotic cell death occurred as indicated by propidium iodide uptake, and necrosis was accompanied by decreased output of the apoptotic parameters caspase activation and especially DNA fragmentation. Necrosis and decrease of apoptosis could be prevented by 3-aminobenzamide, indicating that PARP activation was responsible for the apoptosis-to-necrosis switch in severely damaged cells. Furthermore, PARP-1 knockout cells responded to oxidative challenge with a concentration-dependent apoptosis and showed no switch to necrosis. Using pharmacological PARP inhibitors, we found similar biphasic responses in other cell types, including lymphoma cells, pancreatic acinar cells (L. Virág and C. Szabo, unpublished data), and HaCaT keratinocytes (Szabo et al., 2001), and other groups also reported protection by PARP inhibition from necrotic but not from apoptotic cell death (Ha and Snyder, 1999; Filipovic et al., 1999; Palomba et al., 1999; Tentori et al., 2001a). Comparison of PARP^+/+ and PARP^/ fibroblasts provided further support for the existence of PARP-1-mediated apoptosis-to-necrosis switch in oxidatively challenged cells (Ha and Snyder, 1999). Recently, Moroni et al. (2001) published a series of investigations demonstrating that PARP activation may serve as a cell-death switch in vivo in oxygen-glucose deprivation-based models of cerebral ischemia. A role of PARP activation in necrosis is also consistent with the fact that the inhibition or absence of PARP provides the most remarkable protection in disease models such as stroke, myocardial infarction, or mesenteric ischemia-reperfusion injury, which are characterized predominantly by necrotic-type cell death (Miesel et al., 1995; Schreiber et al., 1995). It is noteworthy here that, in addition to the process of NAD⁺ depletion and the induction of mitochondrial dysfunction, part of the PARP overactivation-induced cell necrosis may be related to intracellular acidification: when PARP catabolizes NAD⁺, the "by-product" of the reaction is H⁺, which directly induces intracellular acidification, having direct consequences for cell viability (Affar et al., 2002). It is still a widely held view that necrosis is a futile process that cannot be influenced by pharmacological means (although apoptosis is the sophisticated process which is under the control of a complex cellular machinery and is amenable to pharmacological intervention). The above-listed observationsdemonstrating protection against cell necrosis by inhibition or inactivation of PARP-1prove that the necrotic process, indeed, is amenable to pharmacological interventions. In fact, the massive protection seen in many inflammatory models and in models of reperfusion injury in the absence of functional PARP (see below) may indicate that necrosis and not apoptosis is the probable predominant form of cell death and organ dysfunction in many diseases.

3. Complex Role of PARP-1 in DNA Damage-Induced Cell Death. After more than 30 years since the discovery of PARP-1, a considerable controversy still exists over the role the enzyme plays in DNA-damage signaling and especially DNA damage-induced cell death. The two sides of the coin are represented by groups claiming that PARP-1 is an indispensable cellular survival factor and by other scientists viewing PARP as a perpetrator of cell death, as described earlier in this article (Fig. 4). This controversy mainly results from differences in experimental approaches with special regard to the use of different DNA-damaging stimuli (alkylating agents, ionizing radiation, or free radicals and oxidants) (Fig. 4) and different cytotoxicity assays measuring either apoptosis or necrosis. This issue now seems to be resolvable in a unifying concept (Fig. 5). According to this concept (Fig. 5), cells exposed to DNA-damaging agents can enter three pathways determined by the intensity of stimulus: 1) PARP-1 activated by mild genotoxic stimuli facilitates DNA repair by signaling cell-cycle arrest and by interacting with DNA repair enzymes such as XRCC1 and DNA-dependent protein kinase. As a result, DNA damage is repaired, and cells survive without the risk of passing on mutated genes. 2) More severe DNA damage induces apoptotic cell death during which caspases, the main executor enzymes of the apoptotic process, inactivate PARP-1 by cleaving it into two fragments (p89 and p24). This pathway allows cells with irreparable DNA damage to become eliminated in a safe way. Cleavage of PARP is believed to aim at preventing the activation of PARP by the ensuing DNA fragmentation and thereby preventing cells from the pathological sequelae of the third route in which cells die by necrosis, a less controlled mechanism posing danger for bystander cells. 3) The third route is induced by extensive DNA breakage that is usually triggered by a massive degree of oxidative or nitrosative stress (hydroxyl radical, peroxynitrite, nitroxyl anion). The overactivation of PARP depletes the cellular stores of its substrate NAD⁺ and consequently ATP. The severely compromised cellular energetic state inhibits the apoptotic cell death process to proceed, because many steps of apoptosis are known to depend on ATP (Kass et al., 1996; Richter et al., 1996; Stefanelli et al., 1997; Ferrari et al., 1998; Feldenberg et al., 1999; Chalmers-Redman et al., 1999; Leist et al., 1999b). PARP activation can quickly take predominance over caspase activation-mediated apoptosis because the very rapid activation of PARP that occurs within minutes after DNA damage, as opposed to the slower kinetics of caspase activation. Pharmacological PARP inhibition or the absence of PARP in PARP-deficient mice preserves cellular ATP and NAD⁺ pools in oxidatively stressed cells and thereby allows them to function normally or, if the apoptotic process has initiated, to use the apoptotic machinery and die by apoptosis instead of by necrosis. We and others have shown that inhibiting or deleting PARPin parallel with decreased necrosisresults in a dramatic increase in the output of apoptotic parameters (caspase activity, DNA fragmentation, phosphatidylserine exposure), providing convincing support for this scenario (Palomba et al., 1996; Virag et al., 1998b).

View larger version (29K): [in this window] [in a new window]   Fig. 4.   The Yin-Yang of PARP activation. The death-promoting and the cytoprotective effects of poly(ADP-ribosylation) represent two seemingly opposing faces (the Yin and Yang) of PARP. Oxidative stress-induced DNA breakage causes a high level of PARP activation, leading to the depletion of NAD+ and ATP and consequently to necrotic cell death. On the other hand, poly(ADP-ribosylation) facilitates DNA repair in cells subjected to treatment with alkylation agents or ionizing radiation.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   The intensity of DNA-damaging stimuli determines the fate of cells: survival, apoptosis, or necrosis. Depending on the intensity of the stimulus, genotoxic agents can trigger three different pathways. In the case of mild DNA damage, poly(ADP-ribosylation) facilitates DNA repair and thus survival (route 1). More severe genotoxic stimuli activate the p53-dependent (or possibly independent) apoptotic pathway (route 2). The most severe DNA damage may cause excessive PARP activation, depleting cellular NAD+/ATP stores. NAD+/ATP depletion blocks apoptosis and results in necrosis (route 3). The inhibition of PARP in cells entering route 1 inhibits repair and thus diverts cells to route 2 (broken arrow). The inhibition of PARP in cells entering route 3 preserves cellular energy stores and thus enables apoptotic machinery to operate (broken arrow).

Involvement of PARP-1 in the regulation of replication is supported by observations that poly(ADP-ribose) metabolism is accelerated in the nuclei of proliferating cells (Tanuma et al., 1978; Kanai et al., 1981; Leduc et al., 1988; Bakondi et al., 2002a). Several lines of evidence suggest that PARP-1 is part of the MRC (Simbulan-Rosenthal et al., 1996). PARP-1 copurifies with DNA polymerase  and , DNA primase, DNA helicase, DNA ligase, topoisomerases I and II, and key components of MRC (Simbulan-Rosenthal et al., 1996; Dantzer et al., 1998; Bauer et al., 2001). Furthermore, several centromere proteins (Saxena et al., 2002) and replication factors such as DNA polymerase , topoisomerase I and II, and proliferating cell nuclear antigen have been shown to be poly(ADP-ribosylated) (Simbulan-Rosenthal et al., 1996). Moreover, poly(ADP-ribosylation) of histones was also proposed to facilitate the assembly and deposition of histone complexes on DNA during replication (Boulikas, 1990). Nonetheless, the exact role of PARP-1 in the regulation of replication is still controversial. Inhibition of PARP by pharmacological or molecular biological means (anti-PARP-1 antisense, knockout cells, dominant-negative PARP inhibition by overexpression of the DNA binding domain of PARP) has been shown to inhibit replication, cell proliferation, and differentiation in various experimental models (D'Amours et al., 1999). However, PARP-1 has also been proposed to be a negative regulator for the initiation of DNA replication (Eki, 1994).

Given that replication and differentiation are closely coupled processes, the above-mentioned experimental data may provide rationale for a differentiation-modifying effect of PARP. Indeed, inhibition of PARP has been shown to interfere with differentiation in various cellular models. Some myeloid leukemia cell lines can be induced to undergo differentiation toward the monocyte/macrophage or neutrophil granulocyte linage. In NB4 acute promyelocytic leukemia and HL-60 acute myelocytic leukemia cells, PARP levels were dramatically modulated during monocyte/macrophage and neutrophilic differentiation (Bhatia et al., 1995). PARP inhibitors (5-methylnicotinamide, 3-methoxybenzamide, and 3-aminobenzamide) were found to inhibit differentiation of human granulocyte-macrophage progenitor cells to the macrophage lineage (Francis et al., 1983). Differentiation to the neutrophil-granulocyte lineage was much less affected (Francis et al., 1983). In other studies, overexpression of PARP arrested NB4 cells and blocked all trans-retinoic acid-induced terminal neutrophilic differentiation (Bhatia et al., 1996). Furthermore, plasmacytic differentiation of Daudi lymphoma cells was impaired in the presence of PARP inhibitors (Exley et al., 1987). Importance of cell type-specific differences is also underlined by observations that benzamide PARP inhibitors induced melanogenesis and differentiation of melanoma cells (Durkacz et al., 1992). Poly(ADP-ribosylation) has also been implicated in erythroid differentiation (Rastl and Swetly, 1978; Morioka et al., 1979; Terada et al., 1979; Sugiura et al., 1984), chicken limb bud mesenchymal cell differentiation (Nishio et al., 1983; Cherney et al., 1985), and trophoblastic cell differentiation during tumorigenesis (Masutani et al., 2001).

A possible role of poly(ADP-ribosylation) in the regulation of transcription has been indicated by findings reporting frequent association of PARP with transcriptionally active regions of chromatin (de Murcia et al., 1986; Lindahl et al., 1995). Furthermore, suppression of inducible-protein synthesis by PARP inhibitors has also been reported. For example, Yamada et al. (1990a) found that in pancreatic islet cells, nicotinamide and 3-aminobenzamide attenuated IFN-- and TNF--induced expression of class II but not class I major histocompatibility molecules. Similar results have also been reported in human thyroid cells and human astrocytes (Hiromatsu et al., 1992; Taniguchi et al., 1993; Qu et al., 1994). Moreover, inhibition of PARP by nicotinamide, 3-methoxybenzamide, and 3-aminobenzamide or 5-iodo-6-amino-1,2-benzopyrone (INH₂BP) has been shown to inhibit cytokine-induced iNOS expression in various cell types (Hauschildt et al., 1991, 1992; Pellat-Deceunynck et al., 1994; Szabo et al., 1998). Furthermore, treatment of interleukin-1 -stimulated rabbit synovial fibroblasts with 3-aminobenzamide resulted in reduced collagenase synthesis, indicating the involvement of PARP in the regulation of collagenase production (Ehrlich et al., 1995). Later, the role of PARP as transcriptional regulator was confirmed with PARP-deficient cells. Our group showed defective iNOS expression both at the protein and at the mRNA level in bacterial lipopolysaccharide (LPS)- and IFN- -stimulated PARP-1^/ fibroblasts compared with wild-type cells (Szabo et al., 1998). Furthermore, PARP inhibition or inactivation reduces the expression of ICAM-1, P-selectin, and E-selectin and of mucosal addressin cell adhesion molecule-1 in cytokine-stimulated human umbilical vein endothelial cells (Zingarelli et al., 1998; Oshima et al., 2001; Sharp et al., 2001). Moreover, decreased expression of these adhesion molecules has also been found after reperfusion injury in the hearts of PARP-1-deficient mice compared with their wild-type counterparts (Zingarelli et al., 1998).

The question arises as to how PARP regulates transcription. One component of the transcription-regulating activity of PARP may be the regulation of chromatin structure and function. Poly(ADP-ribosylation) confers negative charge to histones resulting in electrostatic repulsion between DNA and histones. Loosening histone-DNA interactions may render DNA regions more accessible to the transcriptional machinery and thus may enhance transcription. Indeed, it was reported that basal PARP activity regulates histone shuttling and nucleosomal unfolding (Althaus et al., 1994).

Meisterernst et al. (1997) provided further molecular details to our understanding of how PARP regulates transcription. Their work identified PARP-1 as a functional component of the positive cofactor-1 activity. PARP enhanced transcription by acting during preinitiation complex formation, but it did so at a step after the binding of transcription factor IID. This transcriptional activation was independent of DNA damage and required the amino-terminal DNA binding domain but not the carboxyl-terminal catalytic region (Meisterernst et al., 1997). The coactivator function of PARP was suppressed by NAD⁺, probably as a result of auto-ADP-ribosylation. These results supported a model in which the binding of PARP-1 to DNA and members of the transcription complex facilitates transcription, whereas catalytic activity of PARP has a transcription-inhibitory effect.

Another important milestone in establishing PARP as a transcriptional regulator was a report from de Murcia's laboratory (Oliver et al., 1999). Given the known anti-inflammatory and transcription inhibitory effect of PARP inhibition, they hypothesized a possible interaction between PARP-1 and NF-B, a key transcription factor regulating the expression of several elements of inflammation such as cytokines, chemokines, adhesion molecules, and inflammatory mediators (e.g., iNOS, and the inducible form of cyclooxygenase). They showed that PARP-1-deficient cells were defective in NF-B-dependent transcription activation, but not in its nuclear translocation, in response to TNF- (Oliver et al., 1999). Treating mice with LPS resulted in the rapid activation of NF-B in macrophages from PARP-1^+/+ but not from PARP-1^/ mice. PARP-1-deficient mice were extremely resistant to LPS-induced endotoxic shock (Oliver et al., 1999). The molecular basis for this resistance relied on an almost complete abrogation of NF-B-dependent accumulation of TNF- in the serum and a down-regulation of iNOS, leading to decreased NO synthesis.

Recent studies attempted to delineate the relative importance of PARP catalytic activity versus PARP as a structural protein in its stimulatory role on NF-B activation, and they yielded contrasting results. For instance, Hassa et al. (2001) demonstrated that a PARP-1 mutant lacking enzymatic and DNA binding activity interacted comparably with the wild-type PARP-1 with p65 or p50, concluding that the enzymatic activity of the enzyme is not essential for its interaction with NF-B. In contrast, Chang and Alvarez-Gonzalez (2001) concluded that NF-B p50 DNA binding was dependent on the presence of NAD⁺; DNA binding by NF-B p50 was not efficient in the absence of NAD⁺ and was blocked in the presence of 3-aminobenzamide, allowing for the conclusion that NF-B p50 DNA binding is protein-poly(ADP-ribosyl)ation-dependent. It is possible that these interactions are dependent on the cell type, the model system, and the nature of the stimulus used. Using the hydrochloride salt of N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide (designated PJ34), a potent and specific PARP inhibitor, a suppression of NF-B-mediated gene expression was found in immunostimulated macrophages (Jagtap et al., 2002), but no alterations in NF-B activation were seen in endothelial cells stimulated in the presence of high extracellular glucose concentration (Soriano et al., 2001c).

The identification of genes, the expression of which is regulated by PARP, has also been carried out using DNA chip technology. In wild-type and PARP-deficient fibroblasts, 91 of 11,000 genes were found to be differentially expressed (Simbulan-Rosenthal et al., 2000), suggesting a role for PARP-1 as a basal transcriptional regulator. This technology will hopefully be used for the systematic delineation of genes affected by PARP in stimulated cells and also in vivo in various forms of inflammation.

	II. Pharmacological Inhibition of PARP

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The endogenous inhibitor of PARP, nicotinamide, and the compound 3-aminobenzamide have long served as "benchmark" inhibitors of PARP, i.e., experimental agents suitable for laboratory investigations. These compounds inhibit the enzyme with a low potency, have limited cell uptake