Basal and hydrogen peroxide-induced poly-ADP ribosylation in J774 macrophages. Untreated ( A,B ) and hydrogen peroxide- treated J774 cells were stained with the bio-NAD method as described in Materials and Methods. A low-intensity nuclear staining could be visualized with the assay in untreated cells, reflecting baseline PARP activity ( A,B ). Mitotic cells displayed increased poly-ADP ribosylating activity (arrows). In response to hydrogen peroxide (500 ␮ M), a markedly increased nuclear staining could be detected in J774 cells ( C ) but not in cells pretreated with the PARP inhibitor PJ34 (5 ␮ M) ( D ). Bars: A,C,D ϭ 8 ␮ m; B ϭ 3 ␮ m. 

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... polymerase (PARP) is a nuclear enzyme that becomes activated in response to DNA damage (de Murcia and Menissier de Murcia 1994). Activated PARP cleaves NAD ϩ to nicotinamide and ADP-ribose and polymerizes the latter on nuclear acceptor proteins such as histones, transcription factors, and PARP itself. Poly-ADP ribosylation contributes to DNA repair and to the maintenance of genomic stability (Wang et al. 1997; Simbulan–Rosenthal et al. 1999; Muiras and Bürkle 2000). During inflammation, ischemia–reperfusion, or shock, free radical/oxi- dant-induced DNA single-strand breakage triggers the over-activation of PARP, leading to depletion of NAD ϩ (Szabó and Dawson 1998; Szabó 2000). In an effort to re-synthesize NAD ϩ , ATP is also consumed, resulting in necrotic type cell death (Schraufstatter et al. 1986; Virág et al. 1998a,b). This PARP-mediated pathway of cell suicide has been implicated in the death of immune-stimulated macrophages as well as in peroxynitrite- or hydrogen peroxide-induced dysfunction or cell death of thymocytes, macrophages, endothelial cells, neuronal cells, and fibroblasts (Zingarelli et al. 1996; Szabó et al. 1998; Virág et al. 1998b; Soriano et al. 2001). Inhibition of PARP activity by pharmacological inhibitors or the absence of functional PARP enzyme in PARP knockout animals provided significant protection in animal models of a wide variety of diseases, including various forms of inflammation, shock, stroke, myocardial ischemia, dia- betes, and diabetic endothelial dysfunction (Szabó and Dawson 1998; Szabó 2000). For measurement of PARP activity, the incorporation of radioactivity from isotope-labeled NAD ϩ into TCA-precipitable proteins is considered the gold standard (Schraufstatter et al. 1986). Alternatively, poly(ADP-ribose), the product of the PARP-catalyzed reaction can be purified from cells and tissues by a te- dious procedure and the polymer can be quantitated by HPLC (Kiehlbauch et al. 1993; Shah et al. 1995). A more convenient approach to assessment of PARP activation is the detection of poly(ADP-ribose) by a monoclonal anti-poly(ADP-ribose) antibody in West- ern blots, dot-blots, immunocytochemistry, and flow cytometry (Affar et al. 1998,1999). However, the use of the murine monoclonal anti-poly(ADP-ribose) antibody in mouse tissues, especially in inflamed tissues, often results in high background staining (unpub- lished observations). Moreover, the amount of the polymer synthesized does not necessarily reflect the degree of PARP activation, because in the cells and tissues poly(ADP-ribose) is rapidly metabolized by poly(ADP- ribose) glycohydrolase (PARG) (Ueda et al. 1972). Recently, a novel non-radioactive assay has been marketed by Trevigen (Gaithersburg, MD) for screen- ing of potential PARP inhibitors. The assay utilizes a novel PARP substrate, 6-biotin-17-nicotinamide-ade- nine-dinucleotide (bio-NAD ϩ ), which was originally developed to detect and isolate mono-ADP-ribosylated proteins (Zhang and Snyder 1993). In this assay, 96- well plates are coated with histones as acceptor proteins and biotinyl-ADP ribose is incorporated from bio-NAD ϩ into the histones by purified PARP. Biotin incorporation is then detected by streptavidin–peroxidase and a suitable peroxidase substrate. The potency of PARP inhibitors is assessed on the basis of their inhibition of biotinyl–ADP-ribose incorporation. We set out to investigate whether this commercially available novel PARP substrate, bio-NAD ϩ , can be used to detect cellular PARP activation. We have developed a cellular ELISA (CELISA) assay to quantify PARP activation in cultured cells and an enzyme cytochemical/ histochemical reaction to detect PARP activation in oxidatively stressed cells and tissues. J774 macrophages were stained for PARP activity with the bio-NAD ϩ substrate (Figure 1). All cells showed a predominantly nuclear staining (Figure 1A), indicating that the bio-NAD ϩ -metabolizing enzyme localizes in the nucleus. The intensity of nuclear staining in the vast majority of cells was moderate, reflecting basal PARP activity. In sharp contrast to interphase cells, mitotic cells displayed an intense nuclear staining (Figures 1A and 1B). These cells appeared to be in the metaphase or ana–telophase of the mitotic cycle. Treatment of J774 cells with 500 ␮ M H 2 O 2 induced a marked enhancement of ADP ribosylating activity, as indicated by the strong nuclear staining of H 2 O 2 -treated cells (Figure 1C). Pretreatment of cells with the novel potent PARP inhibitor PJ34 (5 ␮ M) (Abdelkarim et al. 2001; Soriano et al. 2001) 30 min before H 2 O 2 exposure prevented enhancement of bio- NAD ϩ staining (Figure 1D). Similar results were also obtained with other types of cells, including fibroblasts and human keratinocytes (data not shown). To prove the identity of the product synthesized by the cells from bio-NAD ϩ , we used wild-type (PARP ϩ / ϩ ) and PARP-deficient (PARP Ϫ / Ϫ ) macrophages (Figure 2). Exposure of cells to 200 ␮ M hydrogen peroxide induced bio-NAD ϩ incorporation into the nuclei of wild-type but not of PARP-deficient macrophages. Bio-NAD ϩ incorporation could be inhibited with PJ34 (Figure 2) or 3-aminobenzamide (not shown). These results indicate that PARP-1 is responsible for bio-NAD ϩ incorporation into hydrogen peroxide-treated macrophages. To demonstrate the ability of the bio-NAD method to detect PARP activation in tissues, we applied hydrogen peroxide (250 nmol/50 ␮ l) to the skin of mice for 30 min. Skin was excised and immediately frozen in cryoembedding medium to preserve enzyme activity. Frozen sections (10 ␮ m) permeabilized with Triton X-100 were incubated with the bio-NAD ϩ substrate followed by biotin detection with streptavidin–per- oxidase. In control (vehicle-treated) skin, no detect- able ADP ribosylation was found (Figure 3A). Peroxynitrite treatment activated PARP in the skin, as indicated by the appearance of darkly stained cells (Figure 3B). Staining was nuclear and was most intense in keratinocytes. However, some scattered cells in the dermis also showed nuclear PARP activity (Figure 3B). The presence of the PARP inhibitor PJ34 (5 ␮ M) (Figure 3C) or 3-aminobenzamide (5 mM) (not shown) abolished peroxynitrite-induced bio-ADP–ribose incorporation, demonstrating that PARP activation was responsible for the staining. A cellular ELISA method allows the quantification of PARP activity. Furthermore, the potency of pharmacological PARP inhibitors in cells can also be deter- mined in a CELISA. J774 macrophages seeded in 96-well plates were exposed to hydrogen peroxide (50–400 ␮ M) in the presence or absence of PJ34 (5 ␮ M). Hydrogen peroxide induced a dose-dependent PARP activation in J774 cells, and pretreatment with the PARP inhibitor PJ-34 suppressed hydrogen peroxide- induced PARP activation (Figure 4). As a reference method, [ 3 H]-NAD incorporation was also used to measure PARP activity and the two assays showed good correlation ( r 2 ϭ 0.92) with the bio-NAD ϩ method, giving higher induction results. We have demonstrated that bio-NAD can be used as a substrate for PARP in cells and tissues. In untreated J774 macrophage, bio-NAD ϩ metabolizing activity could be detected in the nuclei. The nuclear staining pattern indicates that bio-NAD is metabolized most likely by PARP, a nuclear enzyme polymerizing ADP- ribose units on nuclear acceptor proteins. It has been shown previously that bio-NAD ϩ can also serve as a substrate for mono-ADP ribosylation in vitro (Zhang and Snyder 1993). However, this G-protein-coupled process takes place in the plasma membrane. Because bio-NAD staining is localized to the nuclei, mono- ADP ribosylation is not likely to contribute to bio- NAD ϩ metabolism in our system. Furthermore, the polymeric nature of the PARP product, as opposed to mono-ADP-ribose, may also be responsible for its good detectability with streptavidin–peroxidase. We observed an interesting phenomenon in untreated J774 cells. Mitotic cells displayed strong nuclear posi- tivity compared to interphase cells. Many cells repre- senting various stages of mitosis could be found with strong reactivity localized to the condensed chromatin but not to the interchromatin areas. This observation, however, is not surprising in light of previous observations linking PARP to the process of replication. PARP has been shown to associate with and to regulate the activity of topoisomerase I, an enzyme-uncoiling DNA, by temporarily cutting into one of the DNA strands (Ferro et al. 1984; Bauer et al. 2000; Bauer and Kun 2000). Association of PARP with other proteins in- volved in replication, such as the DNA polymerase I–pri- mase complex, has also been demonstrated (Dantzer et al. 1998). Furthermore, it has also been reported that in PARP knockout cells, but not in ...