Role of poly(ADP-ribose) synthetase in inflammation and ischaemia-reperfusion

Oxidative and nitrosative stress can trigger DNA strand breakage, which then activates the nuclear enzyme poly(ADP-ribose) synthetase (PARS). This enzyme has also been termed poly(ADP-ribose) polymerase (PARP) or poly(ADP-ribose) transferase (pADPRT). Rapid activation of the enzyme depletes 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. In this article, Csaba Szabó and Valina Dawson overview the impact of pharmacological inhibition or genetic inactivation of PARS on the course of oxidant-induced cell death in vitro, and in inflammation and reperfusion injury 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. The pharmacological inhibition of poly(ADP-ribose) synthetase is a novel approach for the experimental therapy of various forms of inflammation and shock, stroke, myocardial and intestinal ischaemia-reperfusion, and diabetes mellitus.     

Inhibition of poly (ADP-ribose) synthetase attenuates neutrophil recruitment and exerts antiinflammatory effects

A cytotoxic cycle triggered by DNA single-strand breakage and poly (ADP-ribose) synthetase activation has been shown to contribute to the cellular injury during various forms of oxidant stress in vitro. The aim of this study was to investigate the role of poly (ADP-ribose) synthetase (PARS) in the process of neutrophil recruitment and in development of local and systemic inflammation. In pharmacological studies, PARS was inhibited by 3-aminobenzamide (10-20 mg/kg) in rats and mice. In other sets of studies, inflammatory responses in PARS-/- mice were compared with the responses in corresponding wild-type controls. Inhibition of PARS reduced neutrophil recruitment and reduced the extent of edema in zymosan- and carrageenan-triggered models of local inflammation. Moreover, inhibition of PARS prevented neutrophil recruitment, and reduced organ injury in rodent models of inflammation and multiple organ failure elicited by intraperitoneal injection of zymosan. Inhibition of PARS also reduced the extent of neutrophil emigration across murine mesenteric postcapillary venules. This reduction was due to an increased rate of adherent neutrophil detachment from the endothelium, promoting their reentry into the circulation. Taken together, our results demonstrate that PARS inhibition reduces local and systemic inflammation. Part of the antiinflammatory effects of PARS inhibition is due to reduced neutrophil recruitment, which may be related to maintained endothelial integrity.     

Protection by inhibition of poly (ADP-ribose) synthetase against oxidant injury in cardiac myoblasts In vitro

Peroxynitrite and hydroxyl radical are reactive oxidants produced during myocardial reperfusion injury. In various cell types, including macrophages and smooth muscle cells, peroxynitrite and hydrogen peroxide cause DNA single strand breakage, which triggers the activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS), resulting in cytotoxicity. Using 3-aminobenzamide and nicotinamide, inhibitors of PARS, we investigated the role of PARS in the pathogenesis of myocardial oxidant injury in H9c2 cardiac myoblasts in vitro. Peroxynitrite (100-1000 microM), hydrogen peroxide (0.3-10 microM) and the NO donor compounds S-nitroso-N-accetyl-DL-penicillamine (SNAP) and diethyltriamine NONOate all caused a dose-dependent reduction of the mitochondrial respiration of the cells, as measured by the mitochondrial-dependent conversion of MTT to formazan. Peroxynitrite and hydrogen peroxide, but not the NO donors caused activation of cellular PARS activity. The suppression of mitochondrial respiration by peroxynitrite and hydrogen peroxide, but not by the NO donors, was ameliorated by pharmacological inhibition of PARS. The protection by the PARS inhibitors diminished at extremely high concentrations of the oxidants. Hypoxia (1 h) followed by reoxygenation (1-24 h) also resulted in a significant activation of PARS, and caused a suppression of mitochondrial respiration, which was prevented by inhibition of PARS. Similar to the results obtained with the pharmacological inhibitors of PARS, a fibroblast cell line which derives from the PARS knockout mouse was protected against the suppression of mitochondrial respiration in response to peroxynitrite and reoxygenation, but not to NO donors, when compared to the result of cells derived from wild-type animals. Based on our data, we suggest that activation of PARS plays a role in the myocardial oxidant injury.     

Transcriptional regulation of the rat poly(ADP-ribose) polymerase gene by Sp1

BACKGROUND: Exogenous surfactant therapy of lung donors improves the preservation of normal canine grafts. The current study was designed to determine whether exogenous surfactant can mitigate the damage in lung grafts induced by mechanical ventilation before procurement. METHODS AND RESULTS: Five donor dogs were subjected to 8 hours of mechanical ventilation (tidal volume 45 ml/kg). This produced a significant decrease in oxygen tension (p = 0.007) and significant increases in bronchoscopic lavage fluid neutrophil count (p = 0.05), protein concentration (p = 0.002), and the ratio of poorly functioning small surfactant aggregates to superiorly functioning large aggregates (p = 0.02). Five other animals given instilled bovine lipid extract surfactant and undergoing mechanical ventilation in the same manner demonstrated no significant change in oxygen tension values, lavage fluid protein concentration, or the ratio of small to large aggregates. All 10 lung grafts were then stored for 17 hours at 4 degrees C. Left lungs were transplanted and reperfused for 6 hours. After 6 hours of reperfusion the ratio of oxygen tension to inspired oxygen fraction was 307 +/- 63 mm Hg in lung grafts administered surfactant versus 73 +/- 14 mm Hg in untreated grafts (p = 0.007). Furthermore, peak inspired pressure was significantly (p < 0.05) lower in treated animals from 90 to 360 minutes of reperfusion. Analysis of lavage fluid of transplanted grafts after reperfusion revealed small to large aggregate ratios of 0.17 +/- 0.04 and 0.77 +/- 0.17 in treated versus untreated grafts, respectively (p = 0.009). CONCLUSIONS: Instillation of surfactant before mechanical ventilation reduced protein leak, maintained a low surfactant small to large aggregate ratio, and prevented a decrease of oxygen tension in donor animals. After transplantation, surfactant-treated grafts had superior oxygen tension values and a higher proportion of superiorly functioning surfactant aggregate forms in the air space than untreated grafts. Exogenous surfactant therapy can protect lung grafts from ventilation-induced injury and may offer a promising means to expand the donor pool.     

Random mutagenesis of the poly(ADP-ribose) polymerase catalytic domain reveals amino acids involved in polymer branching

Poly(ADP-ribose) polymerase (PARP) is a multifunctional nuclear zinc finger protein which participates in the immediate response of mammalian cells exposed to DNA damaging agents. Given the complexity of the poly(ADP-ribosylation) reaction, we developed a large-scale screening procedure in Escherichia coli to identify randomly amino acids involved in the various aspects of this mechanism. Random mutations were generated by the polymerase chain reaction in a cDNA sequence covering most of the catalytic domain. Out of 26 individual mutations that diversely inactivated the full-length PARP, 22 were found at conserved positions in the primary structure, and 24 were located in the core domain formed by two beta-sheets containing the active site. Most of the PARP mutants were altered in poly(ADP-ribose) elongation and/or branching. The spatial proximity of some residues involved in chain elongation (E988) and branching (Y986) suggests a proximity or a superposition of these two catalytic sites. Other residues affected in branching were located at the surface of the molecule (R847, E923, G972), indicating that protein-protein contacts are necessary for optimal polymer branching. This screening procedure provides a simple and efficient method to explore further the structure-function relationship of the enzyme.     

Tankyrase, a poly(ADP-ribose) polymerase at human telomeres

Tankyrase, a protein with homology to ankyrins and to the catalytic domain of poly(adenosine diphosphate-ribose) polymerase (PARP), was identified and localized to human telomeres. Tankyrase binds to the telomeric protein TRF1 (telomeric repeat binding factor-1), a negative regulator of telomere length maintenance. Like ankyrins, tankyrase contains 24 ankyrin repeats in a domain responsible for its interaction with TRF1. Recombinant tankyrase was found to have PARP activity in vitro, with both TRF1 and tankyrase functioning as acceptors for adenosine diphosphate (ADP)-ribosylation. ADP-ribosylation of TRF1 diminished its ability to bind to telomeric DNA in vitro, suggesting that telomere function in human cells is regulated by poly(ADP-ribosyl)ation.     

Poly(ADP-ribose) synthetase activation mediates mitochondrial injury during oxidant-induced cell death

Reactive oxidant species are important mediators of tissue injury in shock, inflammation, and reperfusion injury. The actions of a number of these oxidants (e.g., hydroxyl radical and peroxynitrite, a reactive oxidant produced by the reaction of nitric oxide and superoxide) are mediated in part by the activation of the nuclear nick sensor enzyme, poly(ADP)-ribose synthetase (PARS), with consequent cellular energy depletion. Here we investigated whether PARS activation contributes to the mitochondrial alterations in cells exposed to oxidants. Authentic peroxynitrite (20 microM), the peroxynitrite-generating compound 3-morpholinosidnonimine, the combination of pyrogallol and S-nitroso-N-acetyl-D,L-penicillamine, as well as hydrogen peroxide induced a time- and dose-dependent decrease in mitochondrial transmembrane potential (delta psi(m)) in thymocytes, as determined by flow cytometry using the mitochondrial potential sensitive dyes DiOC6(3) and JC-1. A time- and dose-dependent increase in secondary reactive oxygen intermediate production and loss of cardiolipin, an indicator of mitochondrial membrane damage, were also observed, as measured by flow cytometry using the fluorescent dyes dihydroethidine and nonyl-acridine orange, respectively. Inhibition of PARS by 3-aminobenzamide or 5-iodo-6-amino-1,2-benzopyrone attenuated peroxynitrite-induced delta psi(m) reduction, secondary reactive oxygen intermediate generation, cardiolipin degradation, and intracellular calcium mobilization. Furthermore, thymocytes from PARS-deficient animals were protected against the peroxynitrite- and hydrogen peroxide-induced functional and ultrastructural mitochondrial alterations. In conclusion, mitochondrial perturbations during oxidant-mediated cytotoxicity are, to a significant degree, related to PARS activation rather than to direct effects of the oxidants on the mitochondria.     

Isolation of cDNA encoding full-length rat (Rattus norvegicus) poly (ADP-ribose) polymerase

Poly(ADP-ribose) polymerase (EC 2.4.2.30) is a nuclear enzyme which binds to DNA breaks and then catalyzes the covalent modification of acceptor proteins with poly(ADP-ribose). Poly(ADP-ribose) polymerase activity contributes to the recovery of proliferating cells from DNA damage and to the maintenance of genomic stability, which may be mediated by effects on chromatin structure, DNA base-excision repair and cell cycle regulation. We established the complete cDNA sequence of rat poly(ADP-ribose) polymerase by RT-PCR and direct sequencing of amplification products and compared it with that of other mammalian species. The amino acid sequence homology is strikingly high. The best conserved regions are the known functional modules of poly(ADP-ribose) polymerase.     

Stimulation of the DNA-dependent protein kinase by poly(ADP-ribose) polymerase

The DNA-dependent protein kinase (DNA-PK) is a heterotrimeric enzyme that binds to double-stranded DNA and is required for the rejoining of double-stranded DNA breaks in mammalian cells. It has been proposed that DNA-PK functions in this DNA repair pathway by binding to the ends of broken DNA molecules and phosphorylating proteins that bind to the damaged DNA ends. Another enzyme that binds to DNA strand breaks and may also function in the cellular response to DNA damage is the poly(ADP-ribose) polymerase (PARP). Here, we show that PARP can be phosphorylated by purified DNA-PK, and the catalytic subunit of DNA-PK is ADP-ribosylated by PARP. The protein kinase activity of DNA-PK can be stimulated by PARP in the presence of NAD+ in a reaction that is blocked by the PARP inhibitor 1, 5-dihydroxyisoquinoline. The stimulation of DNA-PK by PARP-mediated protein ADP-ribosylation occurs independent of the Ku70/80 complex. Taken together, these results show that PARP can modify the activity of DNA-PK in vitro and suggest that these enzymes may function coordinately in vivo in response to DNA damage.     

Transient poly(ADP-ribosyl)ation of nuclear proteins and role of poly(ADP-ribose) polymerase in the early stages of apoptosis

A transient burst of poly(ADP-ribosyl)ation of nuclear proteins occurs early, prior to commitment to death, in human osteosarcoma cells undergoing apoptosis, followed by caspase-3-mediated cleavage of poly(ADP-ribose) polymerase (PARP). The generality of this early burst of poly(ADP-ribosyl)ation has now been investigated with human HL-60 cells, mouse 3T3-L1, and immortalized fibroblasts derived from wild-type mice. The effects of eliminating this early transient modification of nuclear proteins by depletion of PARP protein either by antisense RNA expression or by gene disruption on various morphological and biochemical markers of apoptosis were then examined. Marked caspase-3-like PARP cleavage activity, proteolytic processing of CPP32 to its active form, internucleosomal DNA fragmentation, and nuclear morphological changes associated with apoptosis were induced in control 3T3-L1 cells treated for 24 h with anti-Fas and cycloheximide but not in PARP-depleted 3T3-L1 antisense cells exposed to these inducers. Similar results were obtained with control and PARP-depleted human Jurkat T cells. Whereas immortalized PARP +/+ fibroblasts showed the early burst of poly(ADP-ribosyl)ation and a rapid apoptotic response when exposed to anti-Fas and cycloheximide, PARP -/- fibroblasts exhibited neither the early poly (ADP-ribosyl)ation nor any of the biochemical or morphological changes characteristic of apoptosis when similarly treated. Stable transfection of PARP -/- fibroblasts with wild-type PARP rendered the cells sensitive to Fas-mediated apoptosis. These results suggest that PARP and poly(ADP-ribosyl)ation may trigger key steps in the apoptotic program. Subsequent degradation of PARP by caspase-3-like proteases may prevent depletion of NAD and ATP or release certain nuclear proteins from poly(ADP-ribosyl)ation-induced inhibition, both of which might be required for late stages of apoptosis.     

Selected nuclear matrix proteins are targets for poly(ADP-ribose)-binding

Recent evidence suggests that poly(ADP-ribose) may take part in DNA strand break signalling due to its ability to interact with and affect the function of specific target proteins. Using a poly(ADP-ribose) blot assay, we have found that several nuclear matrix proteins from human and murine cells bind ADP-ribose polymers with high affinity. The binding was observed regardless of the procedure used to isolate nuclear matrices, and it proved resistant to high salt concentrations. In murine lymphoma LY-cell cultures, the spontaneous appearance of radiosensitive LY-S sublines was associated with a loss of poly(ADP-ribose)-binding of several nuclear matrix proteins. Because of the importance of the nuclear matrix in DNA processing reactions, the targeting of matrix proteins could be an important aspect of DNA damage signalling via the poly ADP-ribosylation system.     

Effects of inhibitors of the activity of poly (ADP-ribose) synthetase on the liver injury caused by ischaemia-reperfusion: a comparison with radical scavengers

1. Poly (ADP-ribose) synthetase (PARS) is a nuclear enzyme activated by strand breaks in DNA which are caused by reactive oxygen species (ROS) and peroxynitrite. Excessive activation of PARS may contribute to the hepatocyte injury caused by ROS in vitro and inhibitors of PARS activity reduce the degree of reperfusion injury of the heart, skeletal muscle and brain in vivo. Here we compared the effects of various inhibitors of the activity of PARS with those of deferoxamine (an iron chelator which prevents the generation of hydroxyl radicals) and tiron (an intracellular scavenger of superoxide anion) on the degree of hepatic injury caused by ischaemia and reperfusion of the liver in the anaesthetized rat or rabbit. 2. In the rat, ischaemia (30 or 60 min) and reperfusion (120 min) of the liver resulted in significant increases in the serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) indicating the development of liver injury. Intravenous administration of the PARS inhibitors 3-aminobenzamide (3-AB, 10 mg kg(-1) or 30 mg kg(-1)), 1,5-dihydroxyisoquinoline (ISO, 1 mg kg(-1)) or 4-amino-1,8-naphthalimide (4-AN, 3 mg kg(-1)) before reperfusion did not reduce the degree of liver injury caused by ischaemia-reperfusion. 3. In contrast to the PARS inhibitors, deferoxamine (40 mg kg(-1)) or tiron (300 mg kg(-1)) significantly attenuated the rise in the serum levels of AST and ALT caused by ischaemia-reperfusion of the liver of the rat. 4. In the rabbit, the degree of liver injury caused by ischaemia (60 min) and reperfusion (120 min) was also not affected by 3-AB (10 mg kg(-1)) or ISO (1 mg kg(-1)). 5. These results support the view that the generation of oxygen-derived free radicals mediates the liver injury associated with reperfusion of the ischaemic liver by mechanism(s) which are independent of the activation of PARS.     

Poly (ADP-ribose) synthetase activation mediates pulmonary microvascular and intestinal mucosal dysfunction in endotoxin shock

Endotoxin shock is known to impair critical cellular functions and is associated with the development of multiple organ dysfunction. Recent in vitro and in vivo studies demonstrated that oxidants produced during shock and inflammation trigger the activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS), resulting in intracellular energetic failure and tissue dysfunction. Here we examined the role of PARS activation in the development of barrier dysfunction of the intestine and lung during endotoxemia in rats. Ileal mucosal permeability was assessed by the measurement of the lumen to plasma directional passage of the hydrophil solute sodium fluorescein. Microvascular permeability in the lung was examined by the measurement of the extravasation of Evans blue. Inhibition of PARS was achieved by treating the animals with 3-aminobenzamide 30 min prior and 3 hr after lipopolysaccharide injection (10 mg/kg). Endotoxemia (E. coli bacterial lipopolysaccharide, 5-10 mg/kg) resulted in an increased epithelial permeability in the ileum and a microvascular hyperpermeability and neutrophil accumulation in the lung in 6 hr. The PARS inhibitor 3-aminobenzamide significantly reduced the lipopolysaccharide-induced hyperpermeability in both organs, without affecting neutrophil deposition. Thus, PARS activation plays a role in mediating endothelial and epithelial dysfunction and hyperpermeability during endotoxin shock.     

Induction of hepatic poly(ADP-ribose) polymerase by peroxisome proliferators, non-genotoxic hepatocarcinogens

The binding capacity of FK506 binding protein (FKBP) was examined after 2-h hemispheric ischemia in the gerbil brain in order to clarify the precise mechanism of the neuroprotective effects of FK506. Firstly, the FK506 binding was evaluated in vitro in the normal gerbil brain using 1 nM [3H]dihydro-FK506 as a specific ligand. FK506 binding sites were distributed in a rather homogeneous manner, although the greatest binding was noted in the hippocampus CA1. Secondly, Scatchard analysis demonstrated that the binding sites of FK506 could be composed of two components in each brain region. Thirdly, 18 Mongolian gerbils were divided into two groups: an ischemia group (n = 12) and a sham group (n = 6). The right common carotid artery was ligated to induce hemispheric ischemia for 2 h in the ischemia group. The local cerebral blood flow was measured at the end of the experiment by the [14C]iodoantipyrine method. The ligated animals with levels of local cerebral blood flow in the lateral nuclei of the thalamus of less than 50 ml/100 g/min were utilized as the ischemia group (n=6) for further data analysis. No significant differences in FK506 binding between the ischemia and sham groups were observed in any regions. The above data indicate that the binding capacity of FKBP tends to remain normal during 2-h ischemia, suggesting that FK506 may exert its neuroprotective effects through its binding to FKBP in the brain during the early phase of cerebral ischemia.     

Protection against peroxynitrite-induced fibroblast injury and arthritis development by inhibition of poly(ADP-ribose) synthase

Peroxynitrite, a cytotoxic oxidant formed from nitric oxide (NO) and superoxide, induces DNA strand breakage, which activates the nuclear enzyme poly(ADP-ribose) synthase (PARS; EC 2.4.2.30). The cellular function of PARS was determined in fibroblast lines from PARS knockout animals (PARS-/-) and corresponding wild-type animals (PARS+/+), with the aid of the lipophilic PARS inhibitor 5-iodo-6-amino-1,2-benzopyrone (INH2BP). We investigated the role of PARS in peroxynitrite-induced fibroblast injury in vitro and also in the development of arthritis in vivo. Exposure of embryonic fibroblasts from the PARS+/+ animals to peroxynitrite caused DNA single-stand breakage and PARS activation and caused an acute suppression of mitochondrial respiration. INH2BP protected the PARS+/+ cells against the suppression of mitochondrial respiration in response to peroxynitrite (50-100 microM). Similarly to PARS inhibition with INH2BP, the PARS-/- cells were protected against peroxynitrite-induced injury. The protection against cellular injury by PARS-/- phenotype or INH2BP waned when cells were challenged with higher concentrations of the oxidant. Inhibition of PARS by INH2BP or by PARS-/- phenotype reduced inducible nitric-oxide synthase (iNOS; EC 1.14.13.39) mRNA levels and inhibited production of NO in immunostimulated cells. INH2BP had no peroxynitrite scavenging or hydroxyl radical scavenging effects, and it exerted no additional (nonspecific) effects in the PARS-/- cells. In collagen-induced arthritis, significant staining for nitrotyrosine, a marker of peroxynitrite formation, was found in the inflamed joints. Oral treatment with INH2BP (0.5 g/kg, daily), starting at the onset of arthritis (day 25), delayed the development of the clinical signs at days 26-35 and improved histological status in the knee and paw. Our data demonstrate that deletion of PARS by genetic manipulation or pharmacological inhibition of PARS protects against oxidant-induced cellular injury in vitro and exhibits anti-inflammatory effects in vivo.     

In situ staining for poly(ADP-ribose) polymerase activity using an NAD analogue

Poly(ADP-ribose) polymerase (PARP) is a highly abundant nuclear enzyme which metabolizes NAD, in response to DNA strand breakage, to produce chains of poly(ADP-ribose) attached to nuclear proteins. PARP activation has been implicated in ischemia/reperfusion injury, but its biological significance is not fully understood. We have modified an existing in situ method for detection of PARP activity by using an NAD analogue in which adenine is modified by an "etheno" (vinyl) bridge. Etheno-NAD serves as a PARP substrate in an initial enzymatic reaction; a specific antibody to ethenoadenosine is then used in an immunohistochemical reaction to detect the production of modified poly(ADP-ribose). The method produces strong and specific labeling of nuclei in which PARP has been activated, i.e., those in which DNA strand breaks have been produced, and the results can be analyzed by microscopy, flow cytometry, or colorimetry. The method is applicable to cultured cells in several formats and to frozen tissue sections. The particular characteristics of the new method may assist in future in situ studies of PARP activation.     

Identification of commonly recognized epitopes on a rare autoantigen: poly(ADP-ribose) polymerase

To analyze mechanisms of autoantibody production, epitope mapping of a rare autoantigen, poly(ADP-ribose) polymerase, was performed. A cDNA fragment (1873 bp long), which was already confirmed to encode the autoepitopes of this protein, was subcloned into a protein expression plasmid pEX. Several deletion mutants were produced by enzymatic treatments of this construct. PCR-amplified cDNA fragments were also individually subcloned into this vector. The recombinant proteins produced in Escherichia coli by these vectors were tested for their respective antigenicities by immunoblotting. It was found that all positive sera tested (seven cases) strongly recognized common epitopes in a restricted region of the molecule. Furthermore, three out of the seven positive sera were found to recognize other parts of the molecule. The data suggest possible mechanisms for the formation of anti-poly(ADP-ribose) polymerase autoantibodies.     

Protease activation during nitric oxide-induced apoptosis: comparison between poly(ADP-ribose) polymerase and U1-70kDa cleavage

Nitric oxide (NO) promotes apoptotic cell death in the mouse macrophage cell line RAW 264.7 and in the human promyelocytic leukaemia cell line U937, which exemplifies p53-dependent and p53-independent executive death pathways. Here, we followed the cleavage of two caspase substrates during NO-intoxication, assaying poly(ADP-ribose) polymerase and U1-70kDa small ribonucleoprotein (U1-70kDa) degradation. By using pharmacological inhibitors, we found that Z-aspartyl-2,6-dichlorobenzoyloxymethylketone (Z-Asp-CH2-DCB; 100 microM), a caspase-like protease inhibitor, completely blocked S-nitrosoglutathione (GSNO)-induced apoptosis in both RAW 264.7 and U937 cells (IC50 = 50 microM for RAW 264.7 macrophages vs. IC50 = 33 microM for U937 cells). Notably, a characterized caspase-3 (Ac-DEVD-CHO) inhibitor left NO-induced DNA fragmentation and the appearance of an apoptotic morphology unaltered, although completely blocking caspase-3 activity. However, Z-Asp-CH2-DCB suppressed protease-mediated U1-70kDa cleavage and DNA fragmentation in parallel. In contrast, poly(ADP-ribose) polymerase cleavage in U937 cells was only delayed by Z-Asp-CH2-DCB, while poly(ADP-ribose) polymerase digestion in RAW 264.7 macrophages proceeded unaltered. We further compared U1-70kDa and poly(ADP-ribose) polymerase cleavage in stably Bcl-2 transfected RAW 264.7 macrophages. Rbcl2-2, a Bcl-2 overexpressing clone, suppressed DNA fragmentation and U1-70kDa digestion in response to GSNO, although allowing delayed but complete poly(ADP-ribose) polymerase degradation. Conclusively, poly(ADP-ribose) polymerase cleavage not causatively coincided with the appearance of other apoptotic parameters. Our results suggest that NO-induced apoptosis demands a Z-Asp-CH2-DCB inhibitable caspase activity, most likely distinct from caspase-3 and caspase-1. NO-mediated executive apoptotic signaling results in U1-70kDa and poly(ADP-ribose) polymerase cleavage. Whereas U1-70kDa digestion closely correlates to the occurrence of apoptotic parameters such as DNA fragmentation or an apoptotic morphology, poly(ADP-ribose) polymerase-breakdown does not.