Nonisotopic Methods for Determination of Poly(ADP‐Ribose) Levels and Detection of Poly(ADP‐Ribose) Polymerase

Jean François Haince1, Guy G. Poirier1, Jim B. Kirkland2

1 Laval University Medical Research Center, Ste.‐Foy, Quebec, 2 University of Guelph, Guelph, Ontario
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 18.7
DOI:  10.1002/0471143030.cb1807s21
Online Posting Date:  February, 2004
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Poly(ADP‐ribosyl)ation is a post‐translational modification catalyzed mostly by the 116‐kDa enzyme poly(ADP‐ribose) polymerase‐1 (PARP‐1), a nuclear enzyme that transfers an ADP‐ribose moiety onto a limited number of nuclear proteins, including itself. When cells are exposed to environmental stresses such as alkylating agents or free radicals, there is up to a 500‐fold increase in net poly(ADP‐ribose) synthesis in response to DNA strand breaks. The enzyme responsible for 80% to 90% of this stimulated poly(ADP‐ribose) synthesis is PARP‐1, while other PARPs are responsible for the remaining 10% to 20%. The physiological meaning of these phenomena is not clear; however, it can be interpreted as a way of translating an event occurring on DNA to the nucleus by protein modification and finally to the cytoplasm via NAD+ depletion. It has also been proposed that the presence of negatively charged poly(ADP‐ribose) at the site of DNA damage may play several roles in regulation of base excision repair, p53 functions, and apoptosis. This unit describes protocols for measuring the levels of poly(ADP‐ribose) in cells using nonisotopic reagents and for identifying the poly(ADP‐ribose) polymerase enzymes present in cells.

Keywords: poly(ADP‐ribose); poly(ADP‐ribose)polymerase; apoptosis; DNA damage

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Nonisotopic Methods for Determination of Poly(ADP‐Ribose) Levels in Intact Cells
  • Basic Protocol 1: Poly(ADP‐Ribose) Preparation from Cell Cultures
  • Basic Protocol 2: Quantification of pADPr by Immunodot Blot
  • Alternate Protocol 1: Quantification of pADPr by Enzyme‐Linked Immunosorbant Assay
  • Basic Protocol 3: High‐Resolution Gel Electrophoresis and Immunodetection of pADPr
  • Basic Protocol 4: Detection of Poly(ADP‐Ribosyl)ated Proteins by Immunoblotting
  • Basic Protocol 5: Immunocytochemical Detection of pADPr
  • Detection of Poly(ADP‐Ribose) Polymerase Family Members in Cells
  • Basic Protocol 6: Detection of Poly(ADP‐Ribose) Polymerase Enzymes by Immunoblot
  • Alternate Protocol 2: Detection of PARP‐1 Cleavage During Cell Death
  • Basic Protocol 7: Nonisotopic‐Activity Immunoblot Detection of Poly(ADP‐Ribose) Polymerases
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Poly(ADP‐Ribose) Preparation from Cell Cultures

  Materials
  • Cells of interest in appropriate medium
  • DNA‐damaging agent
  • PBS, pH 7.4 ( appendix 2A), ice cold
  • 20% (w/v) trichloroacetic acid (TCA), ice cold
  • 100% ethanol, ice cold
  • 1 M KOH/50 mM EDTA
  • AAGE9 buffer (see recipe)
  • HCl, concentrated
  • 50% (w/v) dihydroxyboryl Bio‐Rex 70 (DHBB) affinity resin (Alvarez‐Gonzalez et al., )
  • 1 M ammonium acetate, pH 9.0
  • H 2O, 37°C
  • Rubber policeman
  • 37° and 60°C water baths or heat blocks
  • 10‐ml Econocolumn (Bio‐Rad)

Basic Protocol 2: Quantification of pADPr by Immunodot Blot

  Materials
  • DHBB‐purified pADPr (see protocol 1)
  • 0.4 M NaOH/10 mM EDTA
  • 0.4 M NaOH
  • PBS‐MT (see recipe)
  • Anti‐pADPr primary antibody
  • Peroxidase‐conjugated secondary antibody
  • PBS‐T: PBS containing 0.1% (v/v) Tween 20
  • PBS ( appendix 2A)
  • Chemiluminescence reagents for HRP‐conjugated secondary antibodies
  • 7.8 × 11.5–cm Hybond N+ nylon membrane (Amersham Bioscience)
  • Dot blot manifold system (Life Technologies)
  • Orbital platform shaker
  • Cooled CCD camera with ChemiImager 4000 and Digital Imaging and Analysis System (Alpha Innotech) or X‐ray film
  • Additional reagents and equipment for autoradiography and densitometry (unit 6.3; optional)

Alternate Protocol 1: Quantification of pADPr by Enzyme‐Linked Immunosorbant Assay

  • 1 µg/ml poly‐L‐lysine in PBS‐T 2
  • PBS ( appendix 2A)
  • DHBB‐purified pADPr samples (see protocol 1) in PBS‐T 2
  • PBS‐T 2: PBS ( appendix 2A)/0.05% (w/v) Tween 20
  • PBS‐MT 2 (see recipe)
  • Anti‐pADPr primary antibody diluted in PBS‐MT 2
  • Peroxidase‐conjugated secondary antibody
  • ABTS/H 2O 2 solution (see recipe)
  • 96‐well microtiter plate (Falcon 3912)
  • Microtiter plate reader

Basic Protocol 3: High‐Resolution Gel Electrophoresis and Immunodetection of pADPr

  Materials
  • 20% (w/v) polyacrylamide gel (see recipe)
  • Electrode buffer: 54 mM Tris‐borate buffer (pH 8.3)/1.2 mM EDTA
  • Alkali‐digested pADPr sample isolated from cell culture (see protocol 1, step ) or lyophilized DHBB‐purified sample (see protocol 1)
  • Modified Laemmli sample buffer (see recipe)
  • Transfer buffer: 35 mM Tris‐borate buffer (pH 8.3)/1 mM EDTA
  • PBS‐MT (see recipe)
  • Anti‐pADPr primary antibody
  • Peroxidase‐conjugated secondary antibody
  • PBS‐T: PBS containing 0.1% (v/v) Tween 20
  • PBS ( appendix 2A)
  • Chemiluminescence reagents for HRP‐conjugated secondary antibodies
  • Purified pADPr standard (see protocol 1)
  • Nitrocellulose Hybond N+ membrane (Amersham Bioscience)
  • Hair dryer
  • 312‐nm UV source
  • X‐Omat blue film (Kodak)
  • Additional reagents and equipment for preparing and running acrylamide gels (unit 6.1), immunoblot detection (see protocol 2, steps to , and unit 6.2), and autoradiography and densitometry (unit 6.3)

Basic Protocol 4: Detection of Poly(ADP‐Ribosyl)ated Proteins by Immunoblotting

  Materials
  • Cells in appropriate medium or pretreated anesthetized animal
  • DNA‐damaging agents, oxidative stress, or apoptosis‐inducing stimulus
  • HeBS ( appendix 2A), ice cold
  • HeBS containing 1 mM PMSF and Complete protease inhibitor cocktail (Roche), ice cold
  • Extraction buffer (see recipe)
  • 2× SDS sample buffer ( appendix 2A)
  • Reducing sample buffer (see recipe)
  • Ponceau S dye (see recipe)
  • PBS‐MT (see recipe)
  • Anti‐pADPr primary antibody
  • Peroxidase‐conjugated secondary antibody
  • PBS‐T: PBS containing 0.1% (v/v) Tween 20
  • PBS ( appendix 2A)
  • Chemiluminescence reagents for HRP‐conjugated secondary antibodies
  • Prestained molecular weight standards (optional)
  • Cell scraper (optional)
  • 15‐ml conical centrifuge tube
  • Sonicator
  • 65°C water bath
  • 0.45‐µm nitrocellulose membrane (Amersham Bioscieince)
  • Orbital platform shaker
  • X‐Omat blue film (Kodak)
  • Additional regents and equipment for cell trypsinization (unit 1.1; optional), gel electrophoresis (unit 6.1), electrotransfer (unit 6.2), and immunoblotting (see protocol 2, steps to )

Basic Protocol 5: Immunocytochemical Detection of pADPr

  Materials
  • Cells of interest in appropriate medium
  • DNA‐damaging stimulus (e.g., 100 µM MNNG, 200 µM H 2O 2)
  • PBS ( appendix 2A), ice cold
  • 70:30 (v/v) methanol/acetone, ice cold
  • PBS containing 1% (w/v) Triton X‐100 and 10% (v/v) FBS
  • Anti‐pADPr primary antibody
  • Fluorophore‐conjugated secondary antibody
  • Fluoromount‐G (Southern Biotech)
  • Coverslips and slides
  • Humid chamber (i.e., a petri dish lined with a wet paper towel)
  • Additional reagents and equipment for fluorescence microscopy (unit 4.2)

Basic Protocol 6: Detection of Poly(ADP‐Ribose) Polymerase Enzymes by Immunoblot

  Materials
  • Tissue, fresh
  • Extraction buffer (see recipe), ice cold
  • 2× SDS sample buffer ( appendix 2A)
  • HeBS ( appendix 2A), ice cold
  • Reducing sample buffer (see recipe)
  • Anti‐PARP primary antibody
  • PBS‐MT (see recipe) with and without 1 mM NaN 3
  • Horseradish peroxidase–conjugated secondary antibody
  • PBS‐T: PBS/0.1% (v/v) Tween 20
  • PBS ( appendix 2A)
  • Chemiluminescence reagents for HRP‐conjugated secondary antibodies
  • Glass homogenizer with Teflon pestle
  • 15‐ml conical centrifuge tube
  • Sonicator
  • 65°C water bath
  • 0.45‐µm nitrocellulose membrane
  • Orbital platform shaker
  • X‐Omat blue film (Kodak)
  • Additional reagents and equipment for preparation of cell extracts (see protocol 5, steps 2a to ), electrophoresis (unit 6.1), electrotransfer (unit 6.2), and autoradiography (unit 6.3)

Alternate Protocol 2: Detection of PARP‐1 Cleavage During Cell Death

  Materials
  • Tissue, fresh
  • Extraction buffer (see recipe), ice cold
  • 2× SDS sample buffer ( appendix 2A)
  • HeBS ( appendix 2A), ice cold
  • Reducing sample buffer (see recipe)
  • 6 × 8–cm 8% or 10% resolving SDS‐polyacrylamide minigel (unit 6.1)
  • Prestained or biotinylated molecular weight standards
  • Running buffer (unit 6.1) containing freshly added 0.7 M 2‐mercaptoethanol
  • Renaturation buffer (see recipe) with and without 100 µM NAD
  • Nonisotopic‐activity immunoblot reducing buffer (see recipe)
  • PBS‐MT (see recipe)
  • Anti‐pADPr primary antibody
  • Horseradish peroxidase–conjugated secondary antibody
  • PBS‐T: PBS/0.1% (v/v) Tween 20
  • PBS ( appendix 2A)
  • Chemiluminescence reagents for HRP‐conjugated secondary antibodies
  • Glass homogenizer with Teflon pestle
  • 15‐ml conical centrifuge tube
  • Sonicator
  • 65°C water bath or heat block
  • Orbital platform shaker
  • X‐Omat blue film (Kodak)
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1), immunoblotting (unit 6.2), and autoradiography (unit 6.3)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Aboul‐Ela, N., Jacobson, E.L., and Jacobson, M.K. 1988. Labeling methods for the study of poly‐ and mono(ADP‐ribose) metabolism in cultured cells. Anal. Biochem. 174:239‐250.
   Affar, E.B., Duriez, P.J., Shah, R.G., Sallmann, F.R., Bourassa, S., Kupper, J.H., Burkle, A., and Poirier, G.G. 1998. Immunodot blot method for the detection of poly(ADP‐ribose) synthesized in vitro and in vivo. Anal. Biochem. 259:280‐283.
   Affar, E.B., Duriez, P.J., Shah, R.G., Winstall, E., Germain, M., Boucher, C., Bourassa, S., Kirkland, J.B., and Poirier, G.G. 1999a. Immunological determination and size characterization of poly(ADP‐ribose) synthesized in vitro and in vivo. Biochim. Biophys. Acta 1428:137‐146.
   Affar, E.B., Shah, R.G., and Poirier, G.G. 1999b. Poly(ADP‐ribose) turnover in quail myoblast cells: Relation between the polymer level and its catabolism by glycohydrolase. Mol. Cell Biochem. 193:127‐135.
   Alvarez‐Gonzalez, R., Juarez‐Salinas, H., Jacobson, E.L., and Jacobson, M.K. 1983. Evaluation of immobilized boronates for studies of adenine and pyridine nucleotide metabolism. Anal. Biochem. 135:69‐77.
   Ame, J.C., Rolli, V., Schreiber, V., Niedergang, C., Apiou, F., Decker, P., Muller, S., Hoger, T., Menissier‐de Murcia, J., and de Murcia, G. 1999. PARP‐2, A novel mammalian DNA damage‐dependent poly(ADP‐ribose) polymerase. J. Biol. Chem. 274:17860‐17868.
   Benchoua, A., Couriaud, C., Guegan, C., Tartier, L., Couvert, P., Friocourt, G., Chelly, J., Menissier‐de Murcia, J., and Onteniente, B. 2002. Active caspase‐8 translocates into the nucleus of apoptotic cells to inactivate poly(ADP‐ribose) polymerase‐2. J. Biol. Chem. 277:34217‐34222.
   Boucher, C., Gobeil, S., Samejima, K., Earnshaw, W.C., and Poirier, G.G. 2001. Identification and analysis of caspase substrates: Proteolytic cleavage of poly(ADP‐ribose)polymerase and DNA fragmentation factor 45. Methods Cell. Biol. 66:289‐306.
   Boyonoski, A.C., Spronck, J.C., Gallacher, L.M., Jacobs, R.M., Shah, G.M., Poirier, G.G., and Kirkland, J.B. 2002a. Niacin deficiency decreases bone marrow poly(ADP‐ribose) and the latency of ethylnitrosourea‐induced carcinogenesis in rats. J. Nutr. 132:108‐114.
   Boyonoski, A.C., Spronck, J.C., Jacobs, R.M., Shah, G.M., Poirier, G.G., and Kirkland, J.B. 2002b. Pharmacological intakes of niacin increase bone marrow poly(ADP‐ribose) and the latency of ethylnitrosourea‐induced carcinogenesis in rats. J. Nutr. 132:115‐120.
   Burkle, A. 2001. Physiology and pathophysiology of poly(ADP‐ribosyl)ation. Bioessays 23:795‐806.
   Chang, H., Sander, C.S., Muller, C.S., Elsner, P., and Thiele, J.J. 2002. Detection of poly(ADP‐ribose) by immunocytochemistry: A sensitive new method for the early identification of UVB‐ and H2O2‐induced apoptosis in keratinocytes. Biol. Chem. 383:703‐708.
   Chiarugi, A. 2002. Poly(ADP‐ribose) polymerase: Killer or conspirator? The ‘suicide hypothesis’ revisited. Trends Pharmacol. Sci. 23:122‐129.
   Chiarugi, A. and Moskowitz, M.A. 2002. Cell biology. PARP‐1—a perpetrator of apoptotic cell death? Science 297:200‐201.
   Cook, B.D., Dynek, J.N., Chang, W., Shostak, G., and Smith, S. 2002. Role for the related poly(ADP‐Ribose) polymerases tankyrase 1 and 2 at human telomeres. Mol. Cell Biol. 22:332‐342.
   D'Amours, D., Germain, M., Orth, K., Dixit, V.M., and Poirier, G.G. 1998. Proteolysis of poly(ADP‐ribose) polymerase by caspase 3: Kinetics of cleavage of mono(ADP‐ribosyl)ated and DNA‐bound substrates. Radiat. Res. 150:3‐10.
   D'Amours, D., Desnoyers, S., D'Silva, I., and Poirier, G.G. 1999. Poly(ADP‐ribosyl)ation reactions in the regulation of nuclear functions. Biochem. J. 342:2492‐68.
   Davidovic, L., Vodenicharov, M., Affar, E.B., and Poirier, G.G. 2001. Importance of poly(ADP‐ribose) glycohydrolase in the control of poly(ADP‐ribose) metabolism. Exp. Cell Res. 268:7‐13.
   Donzelli, M., Negri, C., Mandarino, A., Rossi, L., Prosperi, E., Frouin, I., Bernardi, R., Burkle, A., and Scovassi, A.I. 1997. Poly(ADP‐ribose) synthesis: A useful parameter for identifying apoptotic cells. Histochem. J. 29:831‐837.
   Duriez, P.J. and Shah, G.M. 1997. Cleavage of poly(ADP‐ribose) polymerase: A sensitive parameter to study cell death. Biochem. Cell Biol. 75:337‐349.
   Germain, M., Affar, E.B., D'Amours, D., Dixit, V.M., Salvesen, G.S., and Poirier, G.G. 1999. Cleavage of automodified poly(ADP‐ribose) polymerase during apoptosis. Evidence for involvement of caspase‐7. J. Biol. Chem. 274:28379‐28384.
   Herceg, Z. and Wang, Z.Q. 2001. Functions of poly(ADP‐ribose) polymerase (PARP) in DNA repair, genomic integrity and cell death. Mutat. Res. 477:97‐110.
   Kaufmann, S.H., Desnoyers, S., Ottaviano, Y., Davidson, N.E., and Poirier, G.G. 1993. Specific proteolytic cleavage of poly(ADP‐ribose) polymerase: An early marker of chemotherapy‐induced apoptosis. Cancer Res. 53:3976‐3985.
   Kickhoefer, V.A., Siva, A.C., Kedersha, N.L., Inman, E.M., Ruland, C., Streuli, M., and Rome, L.H. 1999. The 193‐kD vault protein, VPARP, is a novel poly(ADP‐ribose) polymerase. J. Cell. Biol. 146:917‐928.
   Lankenau, S., Burkle, A., and Lankenau, D.H. 1999. Detection of poly(ADP‐ribose) synthesis in Drosophila testes upon gamma‐irradiation. Chromosoma 108:44‐51.
   Lindahl, T., Satoh, M.S., Poirier, G.G., and Klungland, A. 1995. Post‐translational modification of poly(ADP‐ribose) polymerase induced by DNA strand breaks. Trends Biochem. Sci. 20:405‐411.
   Malanga, M. and Althaus, F.R. 1994. Poly(ADP‐ribose) molecules formed during DNA repair in vivo. J. Biol. Chem. 269:17691‐17696.
   Malanga, M., Bachmann, S., Panzeter, S., Zweifel, B., and Althaus, F.R. 1995. Poly(ADP‐ribose) quantification at the femtomole level in mammalian cells. Anal. Biochem. 228:245‐251.
   Oikawa, A., Tohda, H. Kanai, M., Miwa, M., and Sugimura, T. 1980. Inhibitors of poly(adenosine diphosphate ribose) polymerase induce sister chromatid exchanges. Biochem. Biophys. Res. Commun. 97:1311‐1316.
   Pieper, A.A., Verma, A., Zhang, J., and Snyder, S.H. 1999. Poly (ADP‐ribose) polymerase, nitric oxide and cell death. Trends Pharmacol. Sci. 20:171‐181.
   Pleschke, J.M., Kleczkowska, H.E., Strohm, M., and Althaus, F.R. 2000. Poly(ADP‐ribose) binds to specific domains in DNA damage checkpoint proteins. J. Biol. Chem. 275:40974‐40980.
   Schreiber, V., Ame, J.C., Dolle, P., Schultz, I., Rinaldi, B., Fraulob, V., Menissier‐de Murcia, J., and de Murcia, G. 2002. Poly(ADP‐ribose) polymerase‐2 (PARP‐2) is required for efficient base excision DNA repair in association with PARP‐1 and XRCC1. J. Biol. Chem. 277:23028‐23036.
   Scovassi, A.I. and Poirier, G.G. 1999. Poly(ADP‐ribosylation) and apoptosis. Mol. Cell Biochem. 199:125‐137.
   Shah, G.M., Poirier, D., Duchaine, C., Brochu, G., Desnoyers, S., Lagueux, J., Verreault, A., Hoflack, J.C., Kirkland, J.B., and Poirier, G.G. 1995. Methods for biochemical study of poly(ADP‐ribose) metabolism in vitro and in vivo. Anal. Biochem. 227:1‐13.
   Shall, S. 2002. Poly (ADP‐ribosylation)—a common control process? Bioessays 24:197‐201.
   Shall, S. and de Murcia, G. 2000. Poly(ADP‐ribose) polymerase‐1: What have we learned from the deficient mouse model? Mutat. Res. 460:1‐15.
   Soldani, C. and Scovassi, A.I. 2002. Poly(ADP‐ribose) polymerase‐1 cleavage during apoptosis: An update. Apoptosis 7:321‐328.
   Virag, L. and Szabo, C. 2002. The therapeutic potential of poly(ADP‐Ribose) polymerase inhibitors. Pharmacol. Rev. 54:375‐429.
Internet Resources
  http://parplink.u-strasbg.fr/index2.html
  The PARP Link Web Resource contains useful biological resources and pertinent information to the poly(ADP‐ribose) polymerases (PARP) family of proteins and to their role in maintaining the integrity of the eukaryotic genome. This site offers instant access to the latest information and data on the PARP proteins and poly(ADP‐ribosyl)ation and provides a valuable resource to the scientific community.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library