ARTICLE |
Correspondence to: László Virág, INOTEK Corporation, 100 Cummings Center, Suite 419E, Beverly, MA 01915. E-mail: viraglaszlo@hotmail.com
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Summary |
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Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme activated by DNA damage. Activated PARP cleaves NAD+ into nicotinamide and (ADP-ribose) and polymerizes the latter on nuclear acceptor proteins. Over-activation of PARP by reactive oxygen and nitrogen intermediates represents a pathogenetic factor in various forms of inflammation, shock, and reperfusion injury. Using a novel commercially available substrate, 6-biotin-17-nicotinamide-adenine-dinucleotide (bio-NAD+), we have developed three applications, enzyme cytochemistry, enzyme histochemistry, and cell ELISA, to detect the activation of PARP in oxidatively stressed cells and tissues. With the novel assay we were able to detect basal and hydrogen peroxide-induced PARP activity in J774 macrophages. We also observed that mitotic cells display remarkably elevated PARP activity. Hydrogen peroxide-induced PARP activation could also be detected in wild-type peritoneal macrophages but not in macrophages from PARP-deficient mice. Application of hydrogen peroxide to the skin of mice also induced bio-NAD+ incorporation in the keratinocyte nuclei. Hydrogen peroxide-induced PARP activation and its inhibition by pharmacological PARP inhibitors could be detected in J774 cells with the ELISA assay that showed good correlation with the traditional [3H]-NAD incorporation method. The bio-NAD+ assays represent sensitive, specific, and non-radioactive alternatives for detection of PARP activation.
(J Histochem Cytochem 50:9198, 2002)
Key Words: poly(ADP-ribose) polymerase, hydrogen peroxide, biotinylated NAD+, enzyme histochemistry, cell ELISA
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Introduction |
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Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme that becomes activated in response to DNA damage (
For measurement of PARP activity, the incorporation of radioactivity from isotope-labeled NAD+ into TCA-precipitable proteins is considered the gold standard (
Recently, a novel non-radioactive assay has been marketed by Trevigen (Gaithersburg, MD) for screening of potential PARP inhibitors. The assay utilizes a novel PARP substrate, 6-biotin-17-nicotinamide-adenine-dinucleotide (bio-NAD+), which was originally developed to detect and isolate mono-ADP-ribosylated proteins (
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Materials and Methods |
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Materials
Biotinylated NAD+ and the TACS-Saphire substrate were purchased from Trevigen. Streptavidinperoxidase was from Sigma (St Louis, MO). Peroxynitrite was from Cayman Chemical (Ann Arbor, MI). The PARP inhibitor PJ-34 was synthesized in our laboratory as described (
Application of Hydrogen Peroxide to the Skin
Animal experiments conform with the "Guide for the Care and Use of Laboratory Animals" published by the US National Institutes of Health and the treatment protocol was approved by the Institutional Animal Care and Use Committee. C57/BL6 mice were used in the experiments and were allowed free access to food and water. Hair was removed from the back of mice (n=4) by Veet creme. Next day, hydrogen peroxide (250 nmol/50 µl PBS, pH 7.4) was smeared onto the skin by a micropipette. Control animals (n=4) were treated the same way with PBS. After 30 min, mice were sacrificed and skin was excised. Samples were embedded in cryoembedding medium and immediately placed in a -70C freezer.
Isolation of Peritoneal Macrophages
PARP-proficient (PARP+/+) and PARP-deficient (PARP-/-) mice generated by
Cytochemical PARP Detection
J774.1 cells were cultured on coverslips in RPMI medium supplemented with 10% FCS. At 20 min after treatment with H2O2 (500 µM), the medium was removed and replaced with PARP reaction buffer (56 mM HEPES, 28 mM KCl, 28 mM NaCl, 2 mM MgCl2, pH 8.0, complemented with 0.01% digitonin, 12.5 µM biotinylated NAD+ immediately before use). Control reactions were carried out in the presence of the PARP inhibitors PJ34 (30 µM) or 3-aminobenzamide (5 mM). After 60-min incubation at 37C, the cells were fixed in 95% ethanol (10 min at -20C) followed by 10 min in 10% TCA (-20C). Coverslips were rinsed in PBS, pH 7.4 (10 min), and endogenous peroxidase was blocked by 0.5% H2O2/methanol for 15 min. After two 5-min rinses with PBS, pH 7.4, coverslips were blocked in 1% BSA/PBS for 30 min followed by two rinses in PBSTriton X-100 (0.1%). Incorporated biotin was detected by streptavidinperoxidase (diluted 1:100 in PBSTriton X-100 for 30 min at room temperature). Coverslips were washed four times for 5 min with PBS, pH 7.4Triton X-100 and color was developed with cobalt-enhanced nickelDAB substrate. Coverslips were mounted with glycerol on slides and viewed with a Zeiss Axiolab microscope. Pictures were taken with a Zeiss Axiocam digital camera.
PARP Enzyme Histochemistry
Cryosections (10 µm) were fixed for 10 min in 95% ethanol at -20C and then rinsed in PBS. Sections were permeabilized by 1% Triton X-100 in 100 mM Tris, pH 8.0, for 15 min. Reaction mixture (10 mM MgCl2, 1 mM dithiothreitol, 30 µM biotinylated NAD+, in 100 mM Tris, pH 8.0) was then applied to the sections for 30 min at 37C. Reaction mix containing PARP inhibitors (30 µM PJ34 or 5 mM 3-aminobenzamide) or biotinylNAD+-free reaction mix were used as controls. After three washes in PBS, incorporated biotin was detected by peroxidase-conjugated streptavidin (1:100, 30 min, RT). After three 10-min washes in PBS, color was developed with cobalt-enhanced nickelDAB substrate: 4 min incubation in nickelDAB solution (95 mg DAB, 1.6 g NaCl, 2 g nickel sulfate and 25 µl of 30% hydrogen peroxide in 0.1 M acetate buffer pH 6.0) followed by 5 min incubation in TRIScobalt solution (1.2 g TRIS base, 1 g cobalt chloride in 200 ml distilled water, pH 7.2). Sections were counterstained in Nuclear Fast Red, dehydrated, and mounted in Vectamount.
CELISA Method for Detection of PARP Activation
J774.1 cells were seeded in 96-well plates in RPMI/10% fetal bovine serum. Next day, cells were treated with the PARP inhibitor PJ34 (5 µM) for 30 min and then stimulated with hydrogen peroxide (100400 µM). Medium was then replaced by PARP reaction buffer (56 mM HEPES, 28 mM KCl, 28 mM NaCl, 2 mM MgCl2) containing 0.01% digitonin and 10 µM biotinylated NAD+. Plates were incubated for 30 min at 37C. Buffer was then aspirated and cells were fixed by the addition of 200 µl/well pre-chilled 95% ethanol at -20C for 10 min. Endogenous peroxidase activity was blocked by 15-min incubation in 0.5% hydrogen peroxide/methanol. Wells were washed once with 300 µl/well PBS and then blocked by 1% BSA in PBS (200 µl/well) for 30 min at 37C. BSA solution was then aspirated and replaced by 50 µl/well peroxidase-labeled streptavidin (diluted 1:500 in 1% BSAPBS). After incubation (30 min at 37C), plates were washed three times with PBS and reaction was developed with TACS-Saphire (Trevigen) substrate (100 µl/well). The optical density was measured with a microplate spectrophotometer (Molecular Devices; Sunnyvale, CA). Data were expressed as mean ± SD of quadruplicate samples.
[3H]-NAD Incorporation Assay
The assay was carried out as described previously (
Statistical Analysis
PARP activity in the CELISA and [3H]-NAD assays was determined from quadruplicate samples. Data are given as mean ± SD of quadruplicate samples. Experiments were repeated three times. Statistical significance was calculated by unpaired Student's t-test.
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Results |
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Enzyme Cytochemical Detection of PARP Activity in J774 Cells
J774 macrophages were stained for PARP activity with the bio-NAD+ substrate (Fig 1). All cells showed a predominantly nuclear staining (Fig 1A), indicating that the bio-NAD+-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 (Fig 1A and Fig 1B). These cells appeared to be in the metaphase or anatelophase of the mitotic cycle. Treatment of J774 cells with 500 µM H2O2 induced a marked enhancement of ADP ribosylating activity, as indicated by the strong nuclear staining of H2O2-treated cells (Fig 1C). Pretreatment of cells with the novel potent PARP inhibitor PJ34 (5 µM) (
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Lack of Hydrogen Peroxide-induced Bio-NAD+ Incorporation into PARP-1-deficient Macrophages
To prove the identity of the product synthesized by the cells from bio-NAD+, we used wild-type (PARP+/+) and PARP-deficient (PARP-/-) macrophages (Fig 2). Exposure of cells to 200 µM hydrogen peroxide induced bio-NAD+ incorporation into the nuclei of wild-type but not of PARP-deficient macrophages. Bio-NAD+ incorporation could be inhibited with PJ34 (Fig 2) or 3-aminobenzamide (not shown). These results indicate that PARP-1 is responsible for bio-NAD+ incorporation into hydrogen peroxide-treated macrophages.
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Enzyme Histochemical Detection of PARP Activation in Hydrogen Peroxide-treated Mouse Skin
To demonstrate the ability of the bio-NAD+ method to detect PARP activation in tissues, we applied hydrogen peroxide (250 nmol/50 µl) to the skin of mice for 30 min. Skin was excised and immediately frozen in cryoembedding medium to preserve enzyme activity. Frozen sections (10 µm) permeabilized with Triton X-100 were incubated with the bio-NAD+ substrate followed by biotin detection with streptavidinperoxidase. In control (vehicle-treated) skin, no detectable ADP ribosylation was found (Fig 3A). Peroxynitrite treatment activated PARP in the skin, as indicated by the appearance of darkly stained cells (Fig 3B). Staining was nuclear and was most intense in keratinocytes. However, some scattered cells in the dermis also showed nuclear PARP activity (Fig 3B). The presence of the PARP inhibitor PJ34 (5 µM) (Fig 3C) or 3-aminobenzamide (5 mM) (not shown) abolished peroxynitrite-induced bio-ADPribose incorporation, demonstrating that PARP activation was responsible for the staining.
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Determination of Cellular PARP Activation with Bio-NAD+ Substrate in a CELISA Method
A cellular ELISA method allows the quantification of PARP activity. Furthermore, the potency of pharmacological PARP inhibitors in cells can also be determined in a CELISA. J774 macrophages seeded in 96-well plates were exposed to hydrogen peroxide (50400 µM) in the presence or absence of PJ34 (5 µM). 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 (Fig 4). As a reference method, [3H]-NAD incorporation was also used to measure PARP activity and the two assays showed good correlation (r2 = 0.92) with the bio-NAD+ method, giving higher induction results.
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Discussion |
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We have demonstrated that bio-NAD+ can be used as a substrate for PARP in cells and tissues. In untreated J774 macrophage, bio-NAD+ 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+ can also serve as a substrate for mono-ADP ribosylation in vitro (
In line with previous data reporting PARP activation in oxidatively stressed cells (
In situ immunohistochemical demonstration of PARP activation in tissues by detecting poly(ADP-ribose) was successfully demonstrated in some cases by our group (
Another technical difficulty that must be considered is the removal of poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase (PARG). This could theoretically also occur with biotinylpoly(ADP-ribose). Bürkle and coworkers have previously reported that PARG can be inactivated by treating cells with trichloroacetic acid (
For a long time the only assay in which PARP inhibitors could be tested was a radioactive method using either 32P- or 3H-labeled NAD+. This assay could be used both with purified PARP and in cellular systems. The appearance of the bio-NAD+ assay on the market provided a non-radioactive alternative to assess the efficacy of potential PARP inhibitors in a cell-free system. Our CELISA protocol has now extended the applicability of the bio-NAD+ substrate to the measurement of cellular PARP activity. The CELISA assay showed good correlation with the [3H]-NAD+ method. It is also important to emphasize here that, according to our experience, because of differences in cell permeability of different compounds the IC50 values may dramatically differ in a cellular PARP assay compared to cell-free assays. Therefore, we believe that elaboration of a CELISA method for quantitation of cellular PARP activity represents an important advancement aiding more successful research in the PARP field.
In summary, we have developed three applications to detect or to measure PARP activation in cells and tissues. The assays are based on the use of biotinylated NAD+ as a commercially available PARP substrate. The straightforward protocols described here allow a simple, cost-effective two-step detection of activated PARP in oxidatively stressed tissues and cells.
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Acknowledgments |
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Supported by grants from the Hungarian Ministry of Health (ETT 104/99) and from the Hungarian National Science Research Fund (OTKA T 035182) to LV, by a grant from the National Institutes of Health to CS (R01GM60915), and by a grant from the Health Science Center of the University of Debrecen to ES (Mec 10/99). LV was supported by a Bolyai fellowship from the Hungarian Academy of Sciences.
Received for publication April 23, 2001; accepted July 27, 2001.
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