Poly(ADP-ribose) polymerase activity is not affected in ataxia telangiectasia cells and knockout mice

Françoise Dantzer, Josiane Ménissier-de Murcia, Carrolee Barlow1,2, Anthony Wynshaw-Boris1 and Gilbert de Murcia3

Unité 9003 du Centre National de la Recherche Scientifique, Laboratoire conventionné avec le Commissariat à l'Energie Atomique, Ecole Supérieure de Biotechnologie de Strasbourg, boulevard Sébastien Brant, F-67400 Illkirch-Graffenstaden, France and
1 Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA


    Abstract
 Top
 Abstract
 References
 
Poly(ADP-ribose) polymerase (PARP) is a constitutive factor of the DNA damage surveillance network in dividing cells. Based on its capacity to bind to DNA strand breaks, PARP plays a regulatory role in their resolution in vivo. ATM belongs to a large family of proteins involved in cell cycle progression and checkpoints in response to DNA damage. Both proteins may act as sensors of DNA damage to induce multiple signalling pathways leading to activation of cell cycle checkpoints and DNA repair. To determine a possible relationship between PARP and ATM, we examined the PARP response in an ATM-null background. We demonstrated that ATM deficiency does not affect PARP activity in human cell lines or Atm-deficient mouse tissues, nor does it alter PARP activity induced by oxidative damage or {gamma}-irradiation. Our results support a model in which PARP and ATM could be involved in distinct pathways, both effectors transducing the damage signal to cell cycle regulators.

Abbreviations: AT, ataxia telangiectasia; BSA, bovine serum albumin; DSB, double-strand breaks; PARP, poly(ADP-ribose) polymerase; PBS, phosphate-buffered saline; SSB, single-strand breaks.

Ataxia telangiectasia (AT) is a rare human autosomal recessive disorder marked by progressive neurodegeneration, immunodeficiency, premature ageing and cancer predisposition. The gene responsible for AT, ATM (mutated in AT) encodes a putative lipid or protein kinase (1,2) similar to several yeast and mammalian proteins implicated in mitogenic signal transduction, meiotic recombination and cell cycle control, suggesting that ATM may be involved in a signal transduction pathway that responds to DNA damage. Most of the human AT patient phenotypes are recapitulated in Atm-deficient mice (3,4). Cell lines established from Atm-deficient mice, like those from AT patients, exhibit a defect in genomic stability as well as cell cycle checkpoint abnormalities after ionizing radiation. Both defects may contribute to the genomic instability and increased radiosensitivity observed in AT cells (5).

Poly(ADP-ribose) polymerase (PARP) is a zinc finger enzyme involved in the detection of DNA strand breaks in the nuclei of most actively dividing eukaryotic cells. In response to these breaks, PARP catalyses the immediate transfer of the ADP-ribose moiety from its substrate, NAD+, to a limited number of protein acceptors involved in the maintenance of chromatin architecture or in DNA metabolism, including PARP itself (for a review see ref. 6). A number of similarities between PARP and ATM suggest that both proteins may be components of a DNA damage signalling apparatus: (i) PARP and ATM are both nuclear proteins and show the same expression pattern, both proteins being highly expressed in the primary spermatocytes at the pachytene stage during meiosis (7,8); (ii) both activities are activated following DNA damage (6,9); and (iii) recently, PARP–/– mice and Atm–/– mice were observed to exhibit acute radiation sensitivity, manifested particularly in the gastrointestinal tract, leading to the death of the mutant animal in <7 days (3,10). Previous investigations sought to determine whether a difference in PARP activity could account for the enhanced sensitivity of AT cells to ionizing radiation; these studies, however, resulted in conflicting data (11,12). In this work we have re-evaluated the occurence of PARP activation in AT cells as well as in Atm-deficient mice.

To investigate in vivo PARP activity following DNA damage in AT cells, we examined poly(ADP-ribose) synthesis following {gamma}-irradiation and H2O2 treatment by immunofluorescence staining. EBV-transformed lymphoblastoid cell lines from AT patients (AT13) and normal individuals (1104) were irradiated with 10 Gy {gamma}-rays at 4°C or treated with 1 mM H2O2. Poly(ADP-ribosylation) activity was analysed by indirect immunofluorescence labelling of ADP-ribose polymers using a monoclonal antibody (13) precisely as described in Figure 1Go. Lymphoblastoid cell lines were grown in RPMI medium as described. Fibroblasts were grown in minimum essential medium supplemented with 15% fetal calf serum and 0.5% gentamicin.



View larger version (67K):
[in this window]
[in a new window]
 
Fig. 1. Indirect immunofluorescence labelling of poly(ADP-ribose) in normal and AT lymphoblastoid cells after damage. EBV-transformed lymphoblastoid cells (1104, normal cell line; AT13, AT cell line) were cultured in RPMI 1640 medium supplemented with 15% fetal calf serum (heat-inactivated) and 0.5% gentamicin in a humidified atmosphere of 5% CO2. Cells (5x104/sample) were grown on sterilized glass coverlips pretreated with 0.1% poly-L-lysine before processing. After 1 h, cells were mock-treated (A, B, G and H), treated with 1 mM H2O2 for 10 min at 37°C (C, D, I and J) or {gamma}-irradiated (10 Gy) at 4°C with a cobalt source and then incubated 20 min at 25°C before fixing to allow formation of poly(ADP-ribose) (E, F, K and L). After damage, cells were rinsed three times with ice-cold phosphate-buffered saline (PBS), fixed by immersion in ice-cold methanol:acetone (1:1) for 10 min, washed three times for 10 min with ice cold PBS-Tween 0.1% and stained overnight at 4°C in a humid chamber with 35 µl 10H monoclonal anti-poly(ADP-ribose) antibody (IgG3) diluted 1/100 in 1x PBS, 0.1% Tween, 0.1% bovine serum albumin (BSA) (B, D, F, H, J and L). The next day, cells were washed as before and stained for 3 h at 25°C in a humid chamber with 35 µl fluorescein-labelled goat anti-mouse secondary antibody diluted 1/200. DNA was stained using 35 µl 50 ng/ml DAPI for 10 min at 25°C (A, C, E, G, I and K). Slides are mounted with Mowiol 4-88. Images were captured with a Zeiss immunofluorescence microscope.

 
The normal cell line 1104 and the AT homozygote cell lines AT11 and AT13 were provided by Dr G.Lenoir (CIRC, Lyon, France). The heterozygote cell line GM 3188 and the AT homozygote cell line GM 3189 were provided by E.Moustacchi (Institut Curie, Paris, France). The normal cell line GM 546 and the T homozygote cell line GM 1525 were obtained from the National Institute of General Medical Science Mutant Cell Repository (Camden, NJ). The normal FM2S SV40-transformed fibroblast line and the homozygote AT1 SV40-transformed fibroblast line were provided by C.Arlett (University of Sussex, Brighton, UK).

The results displayed in Figure 1Go show a strong green nuclear pattern corresponding to poly(ADP-ribose) synthesis which was detected at the same extent in normal (Figure 1D and FGo) as well as in AT cells (Figure 1J and LGo) after DNA damage induced by H2O2 and {gamma}-irradiation, respectively. This signal was absent in undamaged control cells (Figure 1B and HGo). A total of four AT, one heterozygote and two normal lymphoblastoid EBV-transformed cell lines, as well as one AT and one normal SV40-transformed fibroblast lines, were subsequently analysed for induction of poly(ADP-ribose) synthesis following damage. As shown in Table IGo, all the cell lines tested showed induction of polymer formation following 0.3 mM H2O2 treatment or after {gamma}-irradiation with 10 Gy. No statistically significant difference between normal and AT cell lines could be observed. These results are at odds with those of Edwards and Taylor (11) describing defective induction of polymer synthesis in GM1525/AT2BI cells compared with wild-type cells following {gamma}-ray irradiation in the range 0–10 Gy. However, the data obtained by Edwards and Taylor do not allow a direct comparison with our data since the method used by these authors to measure poly(ADP-ribose) formation included an additional step of permeabilization which could have enhanced the amount of [14C]NAD+ incorporation in undamaged cells. The present results obtained with EBV-transformed lymphoblastoid and SV40-transformed fibroblast cell lines revealed no visible deficiency in {gamma}-ray- and H2O2-stimulated poly(ADP-ribose) synthesis in any of the AT cell lines tested.


View this table:
[in this window]
[in a new window]
 
Table I. Poly(ADP-ribosylation) activity in normal and AT cell lines after ionizing radiation and H2O2 treatment
 
The translational expression of PARP in AT cells versus normal cells was assessed by western blotting of protein extracts from normal human lymphoblastoid cell lines (1104) and AT (AT11 and AT13) cell lines. As shown in Figure 2A, Goa single band with an apparent molecular weight of 116 000 was immunostained by a monoclonal anti-PARP antibody, in AT cells as well as in normal cells. PARP was also detected by its catalytic activity using an activity blot technique (14). Again, no significant difference could be detected between normal and AT cell lines (Figure 2BGo). Therefore, the AT cell lines tested in this study are deficient neither in PARP expression nor in poly(ADP-ribosylation) activity.



View larger version (39K):
[in this window]
[in a new window]
 
Fig. 2. Translational expression of PARP in normal and AT lymphoblastoid cell lines. Samples of 2x106 1104 (normal cell line), AT11 or AT13 (AT cell lines) cells were used per sample, collected by centrifugation at 250 g for 5 min at 25°C, washed twice with ice-cold PBS, pelleted again at 4°C and used for western blot or activity blot experiments as described by Simonin et al. (14). (A) Western blot analysis using a monoclonal anti-PARP antibody. (B) Activity blot analysis.

 
The involvement of ATM in cell cycle checkpoints (2) prompted us to monitor the distribution of PARP activity in the cell cycle of AT cells following damage. PARP activity was detected using a monoclonal anti-poly(ADP-ribose) antibody (13) by flow cytometric analysis; DNA content was measured by propidium iodide incorporation (Figure 3Go). A fluorescein-marked signal of polymer formation was observed after 10 Gy {gamma}-ray irradiation or after 1 mM H2O2 treatment in AT13 as well as in normal 1104 cells, in agreement with the results displayed above, obtained by immunofluorescence staining. The profile of poly(ADP-ribose) synthesis in the cell cycle was the same in AT as in normal cells following {gamma}-irradiation and H2O2 treatment. Interestingly, an increase in polymer synthesis was observed in the G2/M phases of the cell cycle following both immediate irradiation and H2O2 treatment and during the S phase following {gamma}-irradiation, thus confirming the importance of PARP activation specifically during DNA synthesis and before cell division.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 3. Flow cytometric analysis of PARP activity as a function of the cell cycle in normal and AT lymphoblastoid cells. The cells in suspension were damaged with 1 mM H2O2 or 10 Gy {gamma}-irradiation as described in the legend to Figure 1Go, washed twice with ice-cold PBS and fixed in PBS containing 90% methanol for 30 min at –20°C. The cells were washed again twice with ice-cold PBS and blocked by incubation (15 min, 25°C) in 1x PBS, 2% BSA. Cells were incubated overnight at 4°C with 100 µl 10H antibody diluted at 1/10 in 1x PBS, 2% BSA, washed once with ice-cold PBS and incubated again (30 min, 25°C) with 100 µl fluorescein-labelled goat anti-mouse antibody diluted 1/200 in 1x PBS, 2% BSA, 0.5% Tween. DNA content was determined using 10 µg/ml propidium iodide, 100 µg/ml RNase A. Flow cytometric analysis was carried out using an Epics Elite (Coulter). (A) Cell cycle distribution of the wild-type (1104) and AT (AT13) lymphoblastoid cells. (B) Distribution of PARP activity in the cell cycle revealed by the monoclonal anti-poly(ADP-ribose) antibody 10H.

 
To further examine a possible defect in poly(ADP-ribose) formation associated with AT, we measured, under standard conditions, the global poly(ADP-ribosylation) activity of cellular PARP in whole testis and in spleen cell extracts obtained from Atm-deficient mice (3) and from the wild-type counterpart. The results shown in Figure 4Go indicate that no statistical difference in PARP activity could be observed between Atm+/+ and Atm–/– mice in these tissues, thus confirming that poly(ADP-ribosylation) activity is not affected by the absence of ATM protein. In support of this, we have measured PARP activity in situ in wild-type and mutant mice. There may be small (<2-fold) regional differences in certain mouse tissues such as brain and thymus (data not shown). Overall, though, there are no large differences between wild-type and mutant mouse tissues.



View larger version (46K):
[in this window]
[in a new window]
 
Fig. 4. PARP activity assay in wild-type (+/+) and Atm-deficient (–/–) mice. Cells derived from testis and spleen of wild-type and Atm–/– mice were harvested, washed with PBS and lysed in 20 mM Tris–HCl, pH 7.5, 0. 4 M KCl, 5 mM dithiothreitol, 20% glycerol, 0.1 mM phenylmethylsulfonylfluoride. After one freeze–thawing step in liquid nitrogen, the resulting cell lysate was cleared by centrifugation for 20 min at 10 000 g. Samples corresponding to 25 µg protein were incubated for 10 min at 25°C in assay buffer (500 µl) consisting of 50 mM Tris–HCl, pH 8, 4 mM MgCl2, 0.2 mM dithiothreitol, 2 µg/ml total histones, 2 µg/ml DNase I-activated calf thymus DNA, 200 µM NAD+ and 5 µCi [{alpha}-32P]NAD+. The reaction was stopped by addition of 125 µl 100% trichloroacetic acid, 1% inorganic pyrophosphate and the acid-insoluble radioactivity was counted.

 
In conclusion, in this work we present evidence that PARP activity is normal in AT cells and tissues from Atm-deficient mice. The affinity of PARP for single-strand breaks (SSB) induced by both oxidative damage and {gamma}-irradiation imply an important role of the enzyme in repair of these breaks (6,15). In AT cells containing a functional PARP, SSB are repaired in a few minutes, as in normal cells, and are thus probably not responsible for their radiosensitivity and chromosomal instability. AT cells have impaired DNA double-strand break (DSB) repair, suggesting that these are the critical lesions caused by irradiation and oxidizing chemicals in these cells (16,17). This is supported by studies which demonstrate a defect in the illegitimate recombination pathway associated with AT (18). Illegitimate recombination is an important mechanism for rejoining of DSB induced by oxidative damage in human cells (19). One can imagine that the ATM gene product mainly participates in sensing of DSB, whereas PARP is involved in SSB sensing (6). Both proteins may trigger a separate signal transduction pathway that ensures the fidelity of strand break repair and arrest of the cell cycle after damage. In line with this hypothesis, it is interesting to note that the additional loss of PARP in an atm-null background is embryonic lethal (J.Ménissier-de Murcia, manuscript in preparation), thus confirming the importance of maintaining at least one of the two pathways.


    Notes
 
2 Present address: Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA Back

3 To whom correspondence should be adressed Email: demurcia{at}esbs.u-strsbg.fr Back


    Acknowledgments
 
We are indebted to E.Flatter for animal care and C.Waltzinger for FACS analysis. We also thank G.Lenoir for the generous gift of wt1104, AT11 and AT13 cell lines, E.Moustacchi for GM3188 and GM 3189 cell lines and C.Arlett for FM2S and AT1 cell lines. This work was supported by the Association pour la Recherche contre le Cancer, Electricité de France, the Commissariat à l'Energie Atomique and Fondation pour la Recherche Médicale. F.D. was supported by the Ligue Nationale Contre le Cancer.


    References
 Top
 Abstract
 References
 

  1. Savitsky,K., Bar-Shira,A., Gilad,S. et al. (1995) A single ataxia telangiectasia gene with a product similar to PI-3 kinase. Science, 268, 1749–1753.[ISI][Medline]
  2. Zakian,V.A. (1995) ATM-related genes: what do they tell us about functions of the human gene? Cell, 82, 685–687.[ISI][Medline]
  3. Barlow,C., Hirotsune,S., Paylor,R. et al. (1996) Atm-deficient mice: a paradigm of ataxia telangiectasia. Cell, 86, 159–171.[ISI][Medline]
  4. Xu,Y., Ashley,T., Brainerd,E.E., Bronson,R.T., Meyn,M.S. and Baltimore,D. (1996) Targeted disruption of ATM leads to growth retardation, chromosomal fragmentation during meiosis, immune defects and thymic lymphoma. Genes Dev., 10, 2411–2422.[Abstract]
  5. Lavin,M.F. (1993) Ataxia Telangiectasia. Springer-Verlag, Berlin, Germany.
  6. de Murcia,G. and Ménissier de Murcia,J. (1994) Poly(ADP-ribose) polymerase: a molecular nick-sensor. Trends Biochem. Sci., 19, 172–176.[ISI][Medline]
  7. Chen,G. and Lee,E.Y.H.P. (1996) The product of the ATM gene is a 370 kDa nuclear phosphoprotein. J. Biol. Chem., 271, 33693–33697.[Abstract/Free Full Text]
  8. Concha,I.I., Figueroa,J., Concha,M.I., Ueda,K. and Burzio,L.O. (1989) Intracellular distribution of poly(ADP-ribose) synthetase in rat spermatogenic cells. Exp. Cell Res., 180, 353–366.[ISI][Medline]
  9. Baskaran,R., Wood,L.D., Whitaker,L.L. et al. (1997) Ataxia telangiectasia mutant protein activates c-Abl tyrosine kinase in response to ionizing radiation. Nature, 387, 516–519.[ISI][Medline]
  10. Ménissier de Murcia,J., Niedergang,C., Trucco,C. et al. (1997) Requirement of poly(ADP-ribose) polymerase in recovery from DNA damage in mice and in cells. Proc. Natl Acad. Sci. USA, 94, 7303–7307.[Abstract/Free Full Text]
  11. Edwards,M.J. and Taylor,A.M.R. (1980) Unusual levels of (ADP-ribose)n and DNA synthesis in ataxia telangiectasia cells following {gamma}-ray irradiation. Nature, 287, 745–747.[ISI][Medline]
  12. Zwelling,L.A., Kerrigan D. and Mattern M.R. (1983) Ataxia-telangiectasia cells are not uniformly deficient in poly(ADP-ribose) synthesis following X-irradiation. Mutat. Res., 120, 69–78.[ISI][Medline]
  13. Masson,M., Niedergang,C., Schreiber,V., Muller,S., Ménissier-de Murcia,J. and de Murcia,G. (1998) XRCC1 is specifically associated with poly(ADP-ribose) polymerase and negatively regulates its activity following DNA damage. Mol. Cell. Biol., 18, 3563–3571.[Abstract/Free Full Text]
  14. Simonin,F., Briand,J.P., Muller,S. and de Murcia,G. (1991) Detection of poly(ADP ribose) polymerase in crude extracts by activity-blot. Anal. Biochem., 195, 226–231.[ISI][Medline]
  15. Trucco,C., Oliver,F.J., de Murcia,G. and Ménissier-de Murcia,J. (1998) DNA repair defect in poly(ADP-ribose) polymerase-deficient cell lines. Nucleic Acids Res., 26, 2644–2649.[Abstract/Free Full Text]
  16. Cox,R., Debenham,P.G., Masson,W.K. and Webb,M.B.T. (1986) Ataxia-telangiectasia: a human mutation giving high-frequency misrepair of DNA double-stranded scissions. Mol. Biol. Med., 3, 229–244.[ISI][Medline]
  17. Powell,S.N., Whitaker,S., Peacock,J. and McMillan,T. (1993) Ataxia-telangiectasia: an investigation of the repair defect in the cell line AT5BIVA by plasmid reconstruction. Mutat. Res., 294, 9–20.[ISI][Medline]
  18. Dar,M.E., Winters,A.A. and Jorgensen,T.J. (1997) Identification of defective illegitimate recombinational repair of oxidatively-induced DNA double-strand breaks in ataxia-telangiectasia cells. Mutat. Res., 384, 169–179.[ISI][Medline]
  19. Dar,M.E. and Jorgensen,T.J. (1995) Deletions at short direct repeats and base substitutions are characteristic mutations for bleomycin-induced double and single-strand breaks, respectively, in human shuttle vector system. Nucleic Acids Res., 23, 3224–3230.[Abstract]
Received August 10, 1998; revised October 1, 1998; accepted October 2, 1998.





This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (5)
Request Permissions
Google Scholar
Articles by Dantzer, F.
Articles by de Murcia, G.
PubMed
PubMed Citation
Articles by Dantzer, F.
Articles by de Murcia, G.