ARTICLE |
Correspondence to: R. Eric Davis, Metabolism Branch, Bldg 10, Rm 5A02, NIH, 10 Center Drive, MSC 1374, Bethesda, MD 20892-1374..
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Summary |
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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. EthenoNAD 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. (J Histochem Cytochem 46:12791289, 1998)
Key Words: poly(ADP-ribose) polymerase, ADP-ribosyl transferase, EC 2.4.2.30, ethenoadenine, ethenoadenosine, 1,N6-ethenoNAD
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Introduction |
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Poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) is found in high abundance in the nuclei of virtually all mammalian cells. The biochemical functions of PARP in vitro are well known (
Extensive studies of the biological function and significance of PARP under normal and pathological conditions have suggested roles in many important biological processes, including differentiation, DNA replication, development, the response to DNA damage, apoptosis, gene expression, immunoglobulin heavy chain class switching, and regulation of chromatin structure and enzyme activity. However, more recent studies of two PARP "knockout" mouse models (
There are a number of means for detecting and measuring PARP activity, defined here as the synthesis of poly(ADP-ribose) (
Here we describe a modified in situ method to measure PARP activity, based on immunodetection of etheno-modified adenine incorporated into poly(ADP-ribose). Chemical modification of adenine, even within NAD, can be accomplished using chloroacetaldehyde to deposit an etheno (vinyl) bridge across the 1 and N6 positions (-NAD, may serve as substrates for many enzymes, including PARP in one isolated report (
-adenine, and quantitative measurement of fluorescence (
-adenine within poly(ADP-ribose). Despite the high quantum yield and large Stokes' shift of
-adenine (peak emission 420 nm), the wavelength for its excitation (peak 294 nm) is below the range of standard fluorescent microscopes containing glass (
-adenine within poly(ADP-ribose) would be successful, because it has been shown that
-adenine fluorescence is highly quenched when it is part of a nucleic acid polymer, increasing with the proportion of adenine units that are etheno-modified (
-adenosine to detect its presence within poly(ADP-ribose) in an immunohistochemical method to detect PARP activity in situ.
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Materials and Methods |
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Reagents
1,N6-etheno-NAD (-NAD) was purchased from Sigma (St Louis, MO), as were all other reagents unless otherwise specified. Hybridoma supernatant of 1G4, a mouse IgG2a
antibody specific for
-adenosine (
Samples and Preparation
For stimulation of PARP activity, cultured cells were exposed to various DNA fragmenting agents while still in culture. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is a known strong stimulator of PARP activity (
An initial gentle fixation step, when employed, was chiefly performed with the crosslinking agent dimethylsuberimidate (DMS), freshly dissolved in ice-cold buffers at a concentration of 5.420 mg/ml, for 1030 min. Buffers employed were Na2HPO4 (pKa 9.0) or sodium bicine (pH adjusted to 8.5, also containing 1.8 mM CaCl2), both at 100 mM. Other crosslinking fixatives used, all from Pierce (Rockford, IL), were DTBP (dimethyl 3,3'-dithiobispropionimidate), an analogue of DMS containing a cleavable disulfide bridge, DSP [dithiobis(succinimidylpropionate)], an N-hydroxysuccinimide diester also containing a cleavable disulfide bridge, and DTSSP [dithiobis(sulfosuccinimidylpropionate)], a cell-impermeant analogue of DSP. To reduce nonspecific binding of primary antibody in some applications, unreacted functional groups of crosslinking fixatives were inactivated after fixation by exposure to a buffer containing protein or glycine (50100 mM).
Permeabilization was employed in all staining procedures, except with a transfection agent (see below), because NAD does not cross cell membranes. Permeabilization was achieved with cold ethanol, which also served as an initial fixative, or with hypotonic lysis or detergents, usually after fixation. Hypotonic lysis was accomplished as previously described (
Slices 750 µm thick were prepared from fresh whole rat brains with a tissue slicer (Stoelting; Wood Dale, IL) and were kept in artificial cerebrospinal fluid (aCSF) until fixation. To induce DNA fragmentation and stimulate PARP activity, some slices were treated with sodium nitroprusside (SNP, a source of nitric oxide) and pyrogallol (a source of superoxide anion), freshly dissolved in water and added to aCSF at final concentrations of 2 mM each, for 30 min at 37C. Slices were fixed with DMS (20 mg/ml) for 30 min and then frozen in Tissue-Tek/OCT (Miles; Elkhart, IN) for storage at -80C. For detection of PARP activity in situ, cryostat sections 5 µm thick were placed on glass slides, briefly incubated in hypotonic lysis buffer containing 0.2% Brij-58, and then taken to the -NAD incorporation step below.
Incorporation of -NAD ("PARP Reaction")
After permeabilization, cells were incubated with -NAD at a final concentration of 400 µM, which is the approximate nuclear concentration of NAD in vivo, under conditions previously described for the in situ incorporation of NAD into poly(ADP-ribose) (
-NAD (from a 16-mm stock solution in water, stored at 4C), and the rest water. As a negative control, some reaction mixtures also contained 2.5% volume of the PARP inhibitor benzamide in DMSO, to a final concentration of 10 mM, with correspondingly less water. Cells or sections on glass slides were rinsed with 1 x slide reaction buffer (100 mM Tris-HCl, pH 8, 1 mM dithiothreitol, and 10 mM MgCl2) at RT, then incubated with a reaction mixture containing similar amounts of
-NAD with or without benzamide. Reactions were performed at 37C for 20 min, followed by fixation with 4% paraformaldehyde (PFA) or 10% formalin. To establish that the reaction product was poly(ADP-ribose) containing
-adenosine, in some instances slides were subsequently digested with snake venom phosphodiesterase (from Crotalus adamanteus, type I) purchased from Worthington (Freehold, NJ), as described by
In limited numbers of experiments, two nonlethal approaches were used to introduce -NAD into living cells, so that
-NAD incorporation occurred "in vivo." Transfection of
-NAD was accomplished with Lipofectin (Gibco BRL/Life Technologies; Gaithersburg, MD), using protocols suggested by the manufacturer, at a final concentration of 0.51 mM
-NAD. In J774 cells grown on chamber slides,
-NAD was also introduced by reversible permeabilization with ATP (
-NAD (0.4 mM). Permeabilization was reversed by the addition of MgCl2 to 10 mM. With both approaches, subsequent steps included brief fixation with 95% ethanol, fixation in formalin or PFA (10 min), permeabilization with 0.1% Triton X-100, and immunostaining with antibody 1G4.
Immunodetection
For certain cell types, hydrogen peroxide (3% in water) was initially used for 5 min at RT to inactivate endogenous peroxidase. For cells not permeabilized with detergent or alcohol, slides were also incubated with PBS containing 0.1% Triton X-100 for 5 min at RT. Slides were blocked with normal goat serum (NGS, 5% in PBS) for 5 min at RT, then incubated with primary antibody 1G4, diluted 1:10 in PBS with 1% bovine serum albumin, for 30 min at RT in a humid chamber. For visible light microscopy, slides were then immunostained as previously described, using vats of biotinylated goat anti-mouse secondary antibody and avidinhorseradish peroxidase (both from Jackson Immunoresearch Laboratories; West Grove, PA), development with 3,3'-diaminobenzidine, intensification with CuCl2, and counterstaining with hematoxylin or Neutral Fast Red (
Colorimetric Quantitation of In Situ Assay
U251 glioblastoma cells were seeded in 96-well plates at various densities, allowed to adhere, and incubated under control conditions or with MNNG for 30 min. After fixation and permeabilization with cold ethanol, the PARP reaction was performed as described above for adherent cells, using various concentrations of -NAD, with or without 10 mM benzamide, in a volume of 50 µl/well. After paraformaldehyde fixation and NGS blocking, wells were incubated with immunodetection reagents as above, then developed with 3,3',5,5'-tetramethylbenzidine; absorbance was read at 650 nm.
Flow Cytometry
After the PARP reaction and paraformaldehyde fixation, suspended cells were incubated for 5 min with cold IFA medium: 10 mM HEPES (pH 7.4), 150 mM NaCl, 4% bovine calf serum, 0.1% Triton X-100, and 0.1% azide. Cells were resuspended at 107 cells/ml in 70% IFA medium, 10% NGS, 10% normal human serum (type AB), and 10% 1G4 supernatant (final dilution 1:10) and incubated on ice for 1 h or overnight. After washing in IFA medium, cells were incubated with fluorescein-conjugated goat anti-mouse secondary antibody (Pharmingen; San Diego, CA), washed, fixed again with paraformaldehyde, and analyzed with a FACStar flow cytometer (BectonDickinson; Mountain View, CA).
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Results |
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The method to detect PARP activity reported here uses -NAD as a substrate and antibody detection techniques in a variety of formats. With all, the pattern of results was clearly consistent with the interpretation that
-NAD was utilized as a substrate by activated PARP in individual cells and incorporated into an analogue form of poly(ADP-ribose) containing
-adenosine. A strong positive result was consistently obtained in cultured cells incubated with MNNG, a known strong stimulator of PARP activity in vitro. As shown in Figure 1A, microscopic methods showed the reaction product to be exclusively nuclear, as expected for the localization of poly(ADP-ribose). Similar strong staining was also produced by UVB irradiation, but only in cells that had first been incubated with BrdU. It is known that UVB rapidly produces DNA strand breaks at sites of BrdU incorporation. The DNA-fragmenting effect of 312-nm light is increased 1000-fold by BrdU (
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When the PARP reaction was performed in the presence of benzamide, a PARP inhibitor, all signal was abrogated, even with MNNG or BrdU/UVB. DMSO, the solvent used for adding benzamide to the PARP reaction, had no effect by itself at the concentration used (2.5%). Negative results were also obtained on omission of any of the necessary components, such as -NAD or primary antibody, on addition of an excess of unlabeled NAD to the PARP reaction mixture, on replacing the primary antibody with a matched IgG2a
isotype, and when cells were not permeabilized before (or during) the PARP reaction. After the PARP reaction, signal was also eliminated by incubation of MNNG-treated cells with snake venom phosphodiesterase, which cleaves poly(ADP-ribose) into free monomeric units. No staining was observed in control cells, except those whose morphology suggested that they had undergone spontaneous apoptosis in culture (Figure 1B). Although the staining in such cells was not exclusively nuclear, it was eliminated by incubation with benzamide. These results were a mirror image of those from MNNG-treated cultures, in which apoptotic cells showed no reaction product (Figure 1A). The latter observation is not surprising, because it is known that PARP is cleaved and inactivated during apoptosis by activated caspase-3 (
Colorimetric measurement confirmed that staining results were indicative of the state of PARP activity, as defined by poly(ADP-ribose) synthesis. The aggregate signal produced by a fixed number of MNNG-treated U251 cells increased with the concentration of -NAD but showed saturation typical of an enzymatic reaction (Figure 2A). Addition of benzamide to the PARP reaction reduced the optical density almost to background levels. Aggregate signal increased in linear proportion to the number of MNNG-treated U251 cells per well, above a certain number, again as expected for an enzymatic reaction (Figure 2B).
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Flow cytometry also confirmed that the method could detect PARP activity in individual cells (Figure 3). After MNNG treatment and the complete PARP reaction, almost the entire population of cellular events was brightly fluorescent, shifted above the range of signal in MNNG-treated cells reacted in the presence of benzamide. In control cells, the complete PARP reaction produced a weak signal, as shown by the effect of adding benzamide to the PARP reaction mixture. Although qualitative microscopic evaluation of control cells found no definite staining (except in rare apoptotic cells), this weak signal with flow cytometry was consistent with the weak colorimetric signal produced by control U251 cells (Figure 2B). Conventional aggregate methods to measure PARP activity, using sensitive fluorimetric or radiometric methods to detect poly(ADP-ribose), also consistently show a weak but specific signal in control cells (
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Similar results were obtained in a wide variety of adherent and nonadherent cell types from both human and rodent sources, although not all were tested in all formats. Neoplastic cell lines examined included lymphoid (Jurkat and REH), myeloid (HL-60 and NB4), macrophage (J774), glial (U251), neural (PC-12 and SY5Y), squamous (SKOV-3), and melanoma (K1735). Positive results were obtained after induced differentiation using HL-60, NB4, and PC-12 cells, although this required stimulation by MNNG. PARP activity was also detected in non-neoplastic cells, i.e., in monolayer cultures of primary fetal rat brain (pure glial and mixed glial/neuronal).
As implied by its application to different cell types in various formats, the method was quite robust. In some cell types, PARP activity was successfully detected with many combinations of fixation and permeabilization steps, but other cell types had more specific requirements. Initial fixation was not absolutely required but was valuable according to the cell type and format involved. For microscopic methods, fixation was primarily important in the preservation of morphology. However, the effect of fixation on morphology varied considerably among cell types and was less if cells were immobilized on glass at an early point. DMS and related crosslinking fixatives were found to be the best overall, preserving PARP activity in all cell types. For some adherent cell types, fixation was important in preserving desired attachment to chamber slides, especially against subsequent permeabilization. Ethanol was found to be the best fixative in this respect, but it was also found to destroy PARP activity in certain cell types (HL-60). In PC-12 cells, no fixative was found to prevent detachment and preserve PARP activity before or after differentiation by nerve growth factor, but cells could still be stained after detachment, cytocentrifugation, and DMS fixation. In addition to ethanol and hypotonic buffer, various detergents were successful at permeabilization and subsequent PARP activity detection in one or more cell types. Those used were Triton X-100 (0.10.5%), Brij-58 (0.2%), digitonin (0.01%), lysolecithin (0.25 mg/ml), and saponin (0.10.5%).
With transfection or ATP permeabilization, the incorporation of -NAD into poly(ADP-ribose), i.e., the PARP reaction, could be studied while cells were still in culture. With variation of the time of application of the stimulus to PARP activity, these approaches provided additional information about the metabolism of
-NAD, i.e., its duration as a substrate, its competition with native NAD, and the stability of the
-adenosine-labeled poly(ADP-ribose) in living cells. Furthermore, these methods also had the advantage that analysis was quite simple. At a given point, cells were simply harvested from culture, fixed, and immunostained with the antibody to
-adenosine. With Lipofectin, a cationic surfactant most commonly used for liposomal transfection of nucleic acid, we detected very strong nuclear labeling in a subset of HL-60 (Figure 4A) or U251 cells (Figure 4B) subsequently treated with MNNG. There was no staining of control cells or of cells cultured with benzamide during treatment with MNNG. The low frequency of staining was presumably due to low efficiency of
-NAD introduction. Similar "metabolic labeling" of cultured cells with nonradioactive nucleotide analogues by liposomal transfection has previously been described, e.g., RNA synthesis was detected in situ after introduction of bromo-UTP (
-NAD by PARP (and perhaps other routes) takes place in a near-physiological manner. Subsequent culture showed no toxicity due to
-NAD transfection alone, and after 24 h this approach could not detect MNNG-stimulated PARP activity.
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J774, a mouse macrophage cell line, can be permeabilized by ATP in a manner that is reversible and is related to the relative concentrations of ATP4- and divalent cations (Ca++ and Mg++). Dyes up to 831 Da, exceeding the molecular weight of -NAD (687.5 Da), can be rapidly introduced (
-NAD), or the permeabilizing agent (ATP), and by the addition of benzamide. Staining was most intense when the medium used for ATP permeabilization was a simple salt solution, either one found to be most effective for the introduction of dye into J774 cells (135 mM NaCl, 5 mM KCl, and 25 mM Tris, pH 8.0) (
-NAD) were simply added to the medium in which the cells were growing (DMEM with 10% fetal calf serum and antibiotics). The intensity of staining was also related to the time of addition of MNNG, being greatest when MNNG was added 3060 min before ATP permeabilization and
-NAD introduction. When the latter processes were stopped by adding MgCl2 before MNNG, staining was barely detectable, even for a comparable duration of MNNG exposure. This result may be evidence that
-NAD incorporation into poly(ADP-ribose) is reduced by competition with native NAD in cells. The dose of MNNG used was one that reduces NAD levels to <10% of normal in 30 min (unpublished results, and
-NAD is metabolized during the period before MNNG treatment. Normal mouse macrophages possess high levels of an NADase ectoenzyme (
-NAD (
-NAD, and then left in culture after addition of MgCl2 (to stop further ATP-mediated introduction of
-NAD) and benzamide (to prevent further
-NAD incorporation). Within 30 min, staining was considerably reduced (Figure 5B), which we interpret as evidence that
-adenosine-labeled poly(ADP-ribose) is susceptible to degradation in vivo. Native poly(ADP-ribose) is rapidly degraded by a specific enzyme, poly(ADP-ribose) glycohydrolase, which opposes PARP and is activated by the same conditions that activate PARP (
The method was also able to detect PARP activity in frozen sections prepared from slices of rat brain. Slices that had been incubated with SNP and pyrogallol between harvesting and fixation were found to have strong nuclear reactivity (Figure 6A). SNP and pyrogallol together generate peroxynitrite, a DNA-fragmenting agent and PARP stimulator believed to mediate some of the injury of ischemia/reperfusion (
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Discussion |
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In this report we describe a novel method for the in situ detection of PARP activity, defined as the capacity for poly(ADP-ribose) synthesis. The method is based on two essential phenomena: (a) the ability of PARP to utilize -NAD, an analogue of its normal substrate (NAD), and (b) the ability of a specific antibody to recognize the
-adenosine moiety after incorporation into an analogue form of poly(ADP-ribose). The method is derived from earlier in situ methods to detect PARP activity, which use antibodies to poly(ADP-ribose) and immunofluorescent detection. The original method used rabbit antibody in rat cells or frozen sections after ethanol fixation alone (
Although the present method similarly detects PARP activity on the basis of poly(ADP-ribose) production, the different substrate and antibody used may provide greater sensitivity. The epitope of unlabeled poly(ADP-ribose) bound by antibody 10H, most commonly used in previous in situ studies, appears to be a linear region of several monomeric units (-adenosine with exquisite sensitivity and specificity (
-adenosine moities incorporated into poly(ADP-ribose), independent of polymer configuration.
The different substrate and antibody used in the present method may also yield greater specificity in the detection of poly(ADP-ribose) synthetic capacity, i.e., PARP activity, at a particular point or period of interest. Even though most linear poly(ADP-ribose) is rapidly degraded by poly(ADP-ribose) glycohydrolase after MNNG exposure, the rate of degradation declines dramatically as chains become shorter, and there is a baseline of short polymers attached to PARP and other nuclear proteins (-adenine is not a normal cell constituent. Although
-adenine can be produced by vinyl chloride exposure and ethenoadenine binding intracellular proteins have been reported (
-NAD serves as a novel substrate for assay of PARP activity. (We found no evidence that such proteins interfered with the specificity of our method.) Accordingly, antibody 1G4 should not bind to preexisting poly(ADP-ribose) or to other cell constituents, regardless of whether or not
-adenine is incorporated into newly synthesized poly(ADP-ribose).
These attributes also allow -NAD to serve as a nonradioactive label in various studies of PARP activity and related metabolism in living cells, although special means to introduce
-NAD are required. Compared to transfection, ATP permeabilization is simple and inexpensive, and in our experiments produced a much higher proportion of labeled cells. However, it can be applied only to a limited number of cell types. Because radiolabeled adenine (
-adenosine. However, no positive results could be obtained. In living cells, radiolabeled adenine and adenosine are converted to ATP, which can then be incorporated into NAD by the enzyme NAD pyrophosphorylase (NMN adenylyltransferase; EC 2.7.7.1). This enzyme appears to channel NAD to PARP in the nucleus, such that radioactive ATP is rapidly incorporated into poly(ADP-ribose) (
-ATP directly to permeabilized cells in vitro or in vivo but, again, no positive results were produced. This suggests that e-ATP cannot serve as a substrate for NAD pyrophosphorylase. Other nucleotides containing
-adenine, including
-adenosine (
-NAD (
-NAD can serve as a substrate for the poly(ADP-ribose) synthetic function of PARP and suggest that
-adenine in poly(ADP-ribose) does not prevent its enzymatic degradation, but
-NAD cannot serve as a substrate for the NAD glycohydrolase function of PARP in human HL-60 cells (
Even though this method is an assay of enzyme activity, we found that initial fixation was a critical variable for most cell types. DMS is a homobifunctional crosslinking agent which forms imido diesters with free amino groups, principally at lysine residues. DMS has previously been shown to provide excellent fixation of ultrastructure in tissues, with remarkable preservation of enzyme activity by cytochemical staining (
As an in situ technique, this method can provide information about PARP activity in individual cells, at the same time allowing correlation with other features of the cells, e.g., morphology in microscopic formats and other parameters in flow cytometry. The basic method uses simple solutions, standard immunostaining reagents, and a commercially available nucleotide analogue (-NAD) which is relatively inexpensive. [A recent report suggests that biotinylated or digoxigenin-labeled NAD can be used in a similar approach (
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Acknowledgments |
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RED was supported by an Institutional Research Grant from the American Cancer Society. JCB was supported by the Pfeiffer Foundation Minority Summer Research Program and the Stanford Medical Scholars Program. JCH was supported by Dr Richard Zare and by an undergraduate research award from the Camille and Henry Dreyfus Foundation. Q-ATL and PDK were supported by NIH grant CA 42509 to Leonard A. Herzenberg.
We gratefully acknowledge the support of the following individuals: for cell lines, Michael Hsaio, Philipp Kahle, Eric Shooter, Esther Chang, Kevin Smith, Yacop Jacobs, Louie Naumovski, and Doug Ross; for primary cultures, Robert Sapolsky, Rona Giffard, and Michelle Emond; for brain samples, Brie Linkenhofer, Eric Schaible, and Daniel Madison; for preparation of frozen sections and assistance with immunostaining, Eva Pfendt; for expert secretarial and photographic assistance, Eileen Maisen and Phil Verzola; and for sponsorship and critical reading of the manuscript, Roger Warnke, Leonard Herzenberg, and Michael Cleary.
Received for publication February 2, 1998; accepted July 8, 1998.
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