Skip to main content

Advertisement

SpringerLink
Log in

PARP-2 depletion results in lower radiation cell survival but cell line-specific differences in poly(ADP-ribose) levels

  • Research Article
  • Published:
Cellular and Molecular Life Sciences Aims and scope Submit manuscript

Abstract

Poly(ADP-ribose) polymerase-2 (PARP-2) activity contributes to a cells’ poly(ADP-ribosyl)ating potential and like PARP-1, has been implicated in several DNA repair pathways including base excision repair and DNA single strand break repair. Here the consequences of its stable depletion in HeLa, U20S, and AS3WT2 cells were examined. All three PARP-2 depleted models showed increased sensitivity to the cell killing effects on ionizing radiation as reported in PARP-2 depleted mouse embryonic fibroblasts providing further evidence for a role in DNA strand break repair. The PARP-2 depleted HeLa cells also showed both higher constitutive and DNA damage-induced levels of polymers of ADP-ribose (PAR) associated with unchanged PARP-1 protein levels, but higher PARP activity and a concomitant lower PARG protein levels and activity. These changes were accompanied by a reduced maximal recruitment of PARP-1, XRCC1, PCNA, and PARG to DNA damage sites. This PAR-associated phenotype could be reversed in HeLa cells on re-expression of PARP-2 and was not seen in U20S and AS3WT2 cells. These results highlight the complexity of the relationship between different members of the PARP family on PAR metabolism and suggest that cell model dependent phenotypes associated with the absence of PARP-2 exist within a common background of radiation sensitivity.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Ame JC, Rolli V, Schreiber V, Niedergang C, Apiou F, Decker P, Muller S, Hoger T, Menissier-de Murcia J, de Murcia G (1999) PARP-2, A novel mammalian DNA damage-dependent poly(ADP-ribose) polymerase. J Biol Chem 274(25):17860–17868

    Article  CAS  PubMed  Google Scholar 

  2. Shieh WM, Ame JC, Wilson MV, Wang ZQ, Koh DW, Jacobson MK, Jacobson EL (1998) Poly(ADP-ribose) polymerase null mouse cells synthesize ADP-ribose polymers. J Biol Chem 273(46):30069–30072

    Article  CAS  PubMed  Google Scholar 

  3. Menissier de Murcia J, Ricoul M, Tartier L, Niedergang C, Huber A, Dantzer F, Schreiber V, Ame JC, Dierich A, LeMeur M, Sabatier L, Chambon P, de Murcia G (2003) Functional interaction between PARP-1 and PARP-2 in chromosome stability and embryonic development in mouse. EMBO J 22(9):2255–2263

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Schreiber V, Ame JC, Dolle P, Schultz I, Rinaldi B, Fraulob V, Menissier-de Murcia J, 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(25):23028–23036

    Article  CAS  PubMed  Google Scholar 

  5. Beck C, Robert I, Reina-San-Martin B, Schreiber V, Dantzer F (2014) Poly(ADP-ribose) polymerases in double-strand break repair: focus on PARP1, PARP2 and PARP3. Exp Cell Res. doi:10.1016/j.yexcr.2014.07.003

    PubMed  Google Scholar 

  6. Yelamos J, Schreiber V, Dantzer F (2008) Toward specific functions of poly(ADP-ribose) polymerase-2. Trends in mol med 14(4):169–178

    Article  CAS  Google Scholar 

  7. Mortusewicz O, Ame JC, Schreiber V, Leonhardt H (2007) Feedback-regulated poly(ADP-ribosyl)ation by PARP-1 is required for rapid response to DNA damage in living cells. Nucleic Acids Res 35(22):7665–7675

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. Althaus FR, Kleczkowska HE, Malanga M, Muntener CR, Pleschke JM, Ebner M, Auer B (1999) Poly ADP-ribosylation: a DNA break signal mechanism. Mol Cell Biochem 193(1–2):5–11

    Article  CAS  PubMed  Google Scholar 

  9. Malanga M, Althaus FR (2004) Poly(ADP-ribose) reactivates stalled DNA topoisomerase I and Induces DNA strand break resealing. J Biol Chem 279(7):5244–5248

    Article  CAS  PubMed  Google Scholar 

  10. Schreiber V, Dantzer F, Ame JC, de Murcia G (2006) Poly(ADP-ribose): novel functions for an old molecule. Nat Rev 7(7):517–528

    Article  CAS  Google Scholar 

  11. Caldecott KW (2003) XRCC1 and DNA strand break repair. DNA Repair 2(9):955–969

    Article  CAS  PubMed  Google Scholar 

  12. Keil C, Grobe T, Oei SL (2006) MNNG-induced cell death is controlled by interactions between PARP-1, poly(ADP-ribose) glycohydrolase, and XRCC1. J Biol Chem 281(45):34394–34405

    Article  CAS  PubMed  Google Scholar 

  13. Robert I, Karicheva O, Reina San Martin B, Schreiber V, Dantzer F (2013) Functional aspects of PARylation in induced and programmed DNA repair processes: preserving genome integrity and modulating physiological events. Mol Aspects Med 34(6):1138–1152. doi:10.1016/j.mam.2013.02.001

    Article  CAS  PubMed  Google Scholar 

  14. Woodhouse BC, Dianov GL (2008) Poly ADP-ribose polymerase-1: an international molecule of mystery. DNA Repair 7(7):1077–1086

    Article  CAS  PubMed  Google Scholar 

  15. Audebert M, Salles B, Weinfeld M, Calsou P (2006) Involvement of polynucleotide kinase in a poly(ADP-ribose) polymerase-1-dependent DNA double-strand breaks rejoining pathway. J Mol Biol 356(2):257–265

    Article  CAS  PubMed  Google Scholar 

  16. Mansour WY, Rhein T, Dahm-Daphi J (2010) The alternative end-joining pathway for repair of DNA double-strand breaks requires PARP1 but is not dependent upon microhomologies. Nucleic Acids Res 38(18):6065–6077

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Robert I, Dantzer F, Reina-San-Martin B (2009) Parp1 facilitates alternative NHEJ, whereas Parp2 suppresses IgH/c-myc translocations during immunoglobulin class switch recombination. J Exp Med 206(5):1047–1056. doi:10.1084/jem.20082468

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. Ghodgaonkar MM, Zacal N, Kassam S, Rainbow AJ, Shah GM (2008) Depletion of poly(ADP-ribose) polymerase-1 reduces host cell reactivation of a UV-damaged adenovirus-encoded reporter gene in human dermal fibroblasts. DNA Repair 7(4):617–632. doi:10.1016/j.dnarep.2008.01.001

    Article  CAS  PubMed  Google Scholar 

  19. Pines A, Vrouwe MG, Marteijn JA, Typas D, Luijsterburg MS, Cansoy M, Hensbergen P, Deelder A, de Groot A, Matsumoto S, Sugasawa K, Thoma N, Vermeulen W, Vrieling H, Mullenders L (2012) PARP1 promotes nucleotide excision repair through DDB2 stabilization and recruitment of ALC1. J cell Biol 199(2):235–249. doi:10.1083/jcb.201112132

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  20. Biard DS (2007) Untangling the relationships between DNA repair pathways by silencing more than 20 DNA repair genes in human stable clones. Nucleic Acids Res 35(11):3535–3550

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  21. Biard DS, Despras E, Sarasin A, Angulo JF (2005) Development of new EBV-based vectors for stable expression of small interfering RNA to mimick human syndromes: application to NER gene silencing. Mol Cancer Res 3(9):519–529

    Article  CAS  PubMed  Google Scholar 

  22. Le Chalony C, Hoffschir F, Gauthier LR, Gross J, Biard DS, Boussin FD, Pennaneach V (2012) Partial complementation of a DNA ligase I deficiency by DNA ligase III and its impact on cell survival and telomere stability in mammalian cells. Cell Mol Life Sci 69(17):2933–2949. doi:10.1007/s00018-012-0975-8

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  23. Favaudon V (1983) Gamma-radiolysis study of the reductive activation of neocarzinostatin by the carboxyl radical. Biochimie 65(11–12):593–607

    CAS  PubMed  Google Scholar 

  24. Fernet M, Megnin-Chanet F, Hall J, Favaudon V (2010) Control of the G2/M checkpoints after exposure to low doses of ionising radiation: implications for hyper-radiosensitivity. DNA Repair 9(1):48–57. doi:10.1016/j.dnarep.2009.10.006

    Article  CAS  PubMed  Google Scholar 

  25. Godon C, Cordelieres FP, Biard D, Giocanti N, Megnin-Chanet F, Hall J, Favaudon V (2008) PARP inhibition versus PARP-1 silencing: different outcomes in terms of single-strand break repair and radiation susceptibility. Nucleic Acids Res 36(13):4454–4464

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  26. Zaremba T, Thomas HD, Cole M, Coulthard SA, Plummer ER, Curtin NJ (2011) Poly(ADP-ribose) polymerase-1 (PARP-1) pharmacogenetics, activity and expression analysis in cancer patients and healthy volunteers. Biochem J 436(3):671–679. doi:10.1042/BJ20101723

    Article  CAS  PubMed  Google Scholar 

  27. Virag L, Scott GS, Cuzzocrea S, Marmer D, Salzman AL, Szabo C (1998) Peroxynitrite-induced thymocyte apoptosis: the role of caspases and poly (ADP-ribose) synthetase (PARS) activation. Immunology 94(3):345–355

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  28. Erdelyi K, Bai P, Kovacs I, Szabo E, Mocsar G, Kakuk A, Szabo C, Gergely P, Virag L (2009) Dual role of poly(ADP-ribose) glycohydrolase in the regulation of cell death in oxidatively stressed A549 cells. FASEB J 23(10):3553–3563. doi:10.1096/fj.09-133264

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  29. Frizzell KM, Gamble MJ, Berrocal JG, Zhang T, Krishnakumar R, Cen Y, Sauve AA, Kraus WL (2009) Global analysis of transcriptional regulation by poly(ADP-ribose) polymerase-1 and poly(ADP-ribose) glycohydrolase in MCF-7 human breast cancer cells. J Biol Chem 284(49):33926–33938. doi:10.1074/jbc.M109.023879

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  30. Lan L, Nakajima S, Oohata Y, Takao M, Okano S, Masutani M, Wilson SH, Yasui A (2004) In situ analysis of repair processes for oxidative DNA damage in mammalian cells. Proc Natl Acad Sci USA 101(38):13738–13743

    Article  PubMed Central  PubMed  Google Scholar 

  31. Mortusewicz O, Fouquerel E, Ame JC, Leonhardt H, Schreiber V (2011) PARG is recruited to DNA damage sites through poly(ADP-ribose)- and PCNA-dependent mechanisms. Nucleic Acids Res 39(12):5045–5056

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Bolin C, Boudra MT, Fernet M, Vaslin L, Pennaneach V, Zaremba T, Biard D, Cordelieres FP, Favaudon V, Megnin-Chanet F, Hall J (2011) The impact of cyclin-dependent kinase 5 depletion on poly(ADP-ribose) polymerase activity and responses to radiation. Cell Mol Life Sci 69:951–962

    Article  PubMed Central  PubMed  Google Scholar 

  33. Slade D, Dunstan MS, Barkauskaite E, Weston R, Lafite P, Dixon N, Ahel M, Leys D, Ahel I (2011) The structure and catalytic mechanism of a poly(ADP-ribose) glycohydrolase. Nature 477(7366):616–620. doi:10.1038/nature10404

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  34. Fu KK, Phillips TL, Heilbron DC, Ross G, Kane LJ (1979) Relative biological effectiveness of low- and high-LET radiotherapy beams for jejunal crypt cell survival at low doses per fraction. Radiology 132(1):205–209. doi:10.1148/132.1.205

    Article  CAS  PubMed  Google Scholar 

  35. Nikjoo H, Lindborg L (2010) RBE of low energy electrons and photons. Phys Med Biol 55(10):R65–R109. doi:10.1088/0031-9155/55/10/R01

    Article  CAS  PubMed  Google Scholar 

  36. Noel G, Giocanti N, Fernet M, Megnin-Chanet F, Favaudon V (2003) Poly(ADP-ribose) polymerase (PARP-1) is not involved in DNA double-strand break recovery. BMC cell biol 4:7. doi:10.1186/1471-2121-4-7

    Article  PubMed Central  PubMed  Google Scholar 

  37. Ohtsuki K, Ishida N (1975) Neocarzinostatin-induced breakdown of deoxyribonucleic acid in HeLa-S3 cells. J antibiot 28(2):143–148

    Article  CAS  PubMed  Google Scholar 

  38. Ziv Y, Bar-Shira A, Pecker I, Russell P, Jorgensen TJ, Tsarfati I, Shiloh Y (1997) Recombinant ATM protein complements the cellular A-T phenotype. Oncogene 15(2):159–167. doi:10.1038/sj.onc.1201319

    Article  CAS  PubMed  Google Scholar 

  39. Mohapatra S, Kawahara M, Khan IS, Yannone SM, Povirk LF (2011) Restoration of G1 chemo/radioresistance and double-strand-break repair proficiency by wild-type but not endonuclease-deficient Artemis. Nucleic Acids Res 39(15):6500–6510. doi:10.1093/nar/gkr257

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  40. Szanto M, Rutkai I, Hegedus C, Czikora A, Rozsahegyi M, Kiss B, Virag L, Gergely P, Toth A, Bai P (2011) Poly(ADP-ribose) polymerase-2 depletion reduces doxorubicin-induced damage through SIRT1 induction. Cardiovasc Res 92(3):430–438. doi:10.1093/cvr/cvr246

    Article  CAS  PubMed  Google Scholar 

  41. Ambrose HE, Willimott S, Beswick RW, Dantzer F, de Murcia JM, Yelamos J, Wagner SD (2009) Poly(ADP-ribose) polymerase-1 (Parp-1)-deficient mice demonstrate abnormal antibody responses. Immunology 127(2):178–186. doi:10.1111/j.1365-2567.2008.02921.x

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  42. Yelamos J, Monreal Y, Saenz L, Aguado E, Schreiber V, Mota R, Fuente T, Minguela A, Parrilla P, de Murcia G, Almarza E, Aparicio P, Menissier-de Murcia J (2006) PARP-2 deficiency affects the survival of CD4+CD8+ double-positive thymocytes. EMBO J 25(18):4350–4360. doi:10.1038/sj.emboj.7601301

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  43. Kutuzov MM, Khodyreva SN, Ame JC, Ilina ES, Sukhanova MV, Schreiber V, Lavrik OI (2013) Interaction of PARP-2 with DNA structures mimicking DNA repair intermediates and consequences on activity of base excision repair proteins. Biochimie 95(6):1208–1215. doi:10.1016/j.biochi.2013.01.007

    Article  CAS  PubMed  Google Scholar 

  44. Shirai H, Poetsch AR, Gunji A, Maeda D, Fujimori H, Fujihara H, Yoshida T, Ogino H, Masutani M (2013) PARG dysfunction enhances DNA double strand break formation in S-phase after alkylation DNA damage and augments different cell death pathways. Cell Death Dis 4:e656. doi:10.1038/cddis.2013.133

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  45. Uchiumi F, Watanabe T, Ohta R, Abe H, Tanuma S (2013) PARP1 gene expression is downregulated by knockdown of PARG gene. Oncol Rep 29(5):1683–1688. doi:10.3892/or.2013.2321

    PubMed Central  CAS  PubMed  Google Scholar 

  46. Huber A, Bai P, de Murcia JM, de Murcia G (2004) PARP-1, PARP-2 and ATM in the DNA damage response: functional synergy in mouse development. DNA Repair 3(8–9):1103–1108

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

Research in Inserm U612 is supported by funding from Institut Curie and Inserm. MTB had a PhD fellowship from the Presidency of the University of Paris-Sud XI, SC and AF have PhD fellowships from the French Ministry of Research. CB, TZ and MTB were supported by Institute Curie’s International Post-doctoral fellow program and TZ by a fellowship from the Fondation Pierre-Gilles de Gennes. The assistance of members of the Institut Curie Small Animal Radiation Platform is gratefully acknowledged.

Author information

Author notes

  1. Celeste Bolin

    Present address: Department of Biology, The College of Idaho, 2112 Cleveland Boulevard, Caldwell, ID, 83605, USA

  2. Tomasz Zaremba

    Present address: AstraZeneca Pharma Poland Sp. z o.o.ul., Postępu 18, 02-676, Warsaw, Poland

  3. Fabrice P. Cordelières

    Present address: Pôle d’imagerie photonique, Institut François Magendie, Bordeaux Imaging Center, UMS 3420 CNRS-Université de Bordeaux-US4 INSERM, 146 Rue Léo-Saignat, 33077, Bordeaux, France

  4. Frédérique Mégnin-Chanet

    Present address: Inserm U1030, Gustave Roussy Cancer Campus Grand Paris, 114 rue Edouard-Vaillant, 94805, Villejuif, France

Authors and Affiliations

  1. Institut Curie, Centre de Recherche, Bât. 110-112, Centre Universitaire, 91405, Orsay, France

    Mohammed-Tayyib Boudra, Celeste Bolin, Sara Chiker, Alexis Fouquin, Tomasz Zaremba, Laurence Vaslin, Fabrice P. Cordelières, Frédérique Mégnin-Chanet, Vincent Favaudon, Marie Fernet, Vincent Pennaneach & Janet Hall

  2. Inserm, U612, Bât. 110-112, Centre Universitaire, 91405, Orsay, France

    Mohammed-Tayyib Boudra, Celeste Bolin, Sara Chiker, Alexis Fouquin, Tomasz Zaremba, Laurence Vaslin, Frédérique Mégnin-Chanet, Vincent Favaudon, Marie Fernet, Vincent Pennaneach & Janet Hall

  3. Faculté de Médecine, Université Paris-XI, 94270, Le Kremlin Bicêtre, France

    Mohammed-Tayyib Boudra, Sara Chiker & Alexis Fouquin

  4. Commissariat à l’Energie Atomique, DSV-iMETI-SEPIA, 92265, Fontenay Aux Roses, France

    Denis Biard

  5. CNRS, UMR3348, Bât. 110-112, Centre Universitaire, 91405, Orsay, France

    Fabrice P. Cordelières

  6. Plateforme IBiSA d’Imagerie Cellulaire et Tissulaire, Institut Curie, Bât. 110-112, Centre Universitaire, 91405, Orsay, France

    Fabrice P. Cordelières

  7. Inserm U612, Institut Curie-Recherche, Bât. 110-112, Centre Universitaire, 91405, Orsay, France

    Janet Hall

Authors
  1. Mohammed-Tayyib Boudra
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Celeste Bolin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sara Chiker
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alexis Fouquin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tomasz Zaremba
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Laurence Vaslin
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Denis Biard
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Fabrice P. Cordelières
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Frédérique Mégnin-Chanet
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Vincent Favaudon
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Marie Fernet
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Vincent Pennaneach
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Janet Hall
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Janet Hall.

Electronic supplementary material

Below is the link to the electronic supplementary material.

18_2014_1765_MOESM1_ESM.tif

Supplementary Fig. 1 Impact of PARP-2 depletion in HeLa cells on mRNA expression profiles and cell survival. The depletion of PARP-2 was associated with (a) a reduction in parg mRNA in the three HeLa PARP-2KD clones. Data represents mean ± SD from at least three independent experiments. (b) Lower clonogenic survival was observed in HeLa PARP-2KD cells compared to Control cells (Control cells—open symbols, PARP-2KD cells—closed symbols) after exposure to increasing doses of γ-rays in S-phase (b-left panel) or neocarzinostatin in asynchronous cultures (b-right panel). Data represents mean ± SD from at least two independent experiments in triplicate.(c) Clonogenic survival of HeLa PARP-1KD cells compared to Control cells (Control cells—open symbols, PARP-1KD cells—filled symbols) after exposure to increasing doses of γ-rays in S-phase (c-left panel) and in asynchronous cultures (c-right panel). Data represents mean ± SD from one experiment in triplicate. In panels b and c, the solid lines represent the curves drawn for best fit of data to the linear-quadratic equation (see Materials and Methods). The dotted lines mark the upper and lower limits of the confidence interval drawn from the SDs calculated for the α and β parameters of the linear-quadratic model (TIFF 46907 kb)

18_2014_1765_MOESM2_ESM.tif

Supplementary Fig. 2 Restoring PARP-2 protein expression leads to restoration of PARG protein expression. The expression of PARP-1, PARG, and PARP-2 protein for HeLa PARP-2KD depleted cells (clones 1,470/1, 1,470/7, and 1,471/13) and the Control cells grown in the presence on hygromycin B (a-upper panel) were compared the protein expression to the same clones grown for 20 days in the absence of hygromycin B (a-lower panel). Growth in the absence of hygromycin B resulted in a concomitant increase in the expression of the PARP-2 and PARG proteins (a), a lowering of PARP activity to levels seen in Control cells as assessed by the incorporation of isotope from 32P-NAD+ into trichloroacetic acid-precipitable proteins (b) and levels of polymers that were similar to that seen in Control cells as assessed by western blotting from untreated cell cultures (c). (d) Quantitative analysis of the relative spot intensity with time for the recruitment of EGFP-PARP-1 to sites of laser-induced damage in Control cells (closed circles), the PARP-2KD clone 1,470/7 grown in the presence of hygromycin (open triangles) or the absence of hygromycin (closed triangles). Data represents mean relative spot intensity ± SEM, n = 40–57 individual cells (TIFF 46,903 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Boudra, MT., Bolin, C., Chiker, S. et al. PARP-2 depletion results in lower radiation cell survival but cell line-specific differences in poly(ADP-ribose) levels . Cell. Mol. Life Sci. 72, 1585–1597 (2015). https://doi.org/10.1007/s00018-014-1765-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00018-014-1765-2

Keywords

Search

Navigation