Ubiquitin mediated protein degradation is crucial for regulation of cell signaling and protein quality control. Poly(ADP-ribose) (PAR) is a cell-signaling molecule that mediates changes in protein function through binding at PAR binding sites. Here we characterize the PAR binding protein, Iduna, and show that it is a PAR-dependent ubiquitin E3 ligase. Iduna’s E3 ligase activity requires PAR binding because point mutations at Y156A and R157A eliminate Iduna’s PAR binding and Iduna’s E3 ligase activity. Iduna’s E3 ligase activity also requires an intact really interesting new gene (RING) domain because Iduna possessing point mutations at either H54A or C60A is devoid of ubiquitination activity. Tandem affinity purification reveals that Iduna binds to a number of proteins that are either PARsylated or bind PAR including PAR polymerase-1, 2 (PARP1, 2), nucleolin, DNA ligase III, KU70, KU86, XRCC1, and histones. PAR binding to Iduna activates its E3 ligase function, and PAR binding is required for Iduna ubiquitination of PARP1, XRCC1, DNA ligase III, and KU70. Iduna’s PAR-dependent ubiquitination of PARP1 targets it for proteasomal degradation. Via PAR binding and ubiquitin E3 ligase activity, Iduna protects against cell death induced by the DNA damaging agent N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and rescues cells from G1 arrest and promotes cell survival after γ-irradiation. Moreover, Iduna facilitates DNA repair by reducing apurinic/apyrimidinic (AP) sites after MNNG exposure and facilitates DNA repair following γ-irradiation as assessed by the comet assay. These results define Iduna as a PAR-dependent E3 ligase that regulates cell survival and DNA repair.
Poly(ADP-ribosyl)ation is a post-translational modification that is instantly stimulated by DNA strand breaks creating a unique signal for the modulation of protein functions in DNA repair and cell cycle checkpoint pathways. Here we report that lack of poly(ADP-ribose) synthesis leads to a compromised response to DNA damage. Deficiency in poly(ADP-ribosyl)ation metabolism induces profound cellular sensitivity to DNA-damaging agents, particularly in cells deficient for the protein kinase ataxia telangiectasia mutated (ATM). At the biochemical level, we examined the significance of poly(ADP-ribose) synthesis on the regulation of early DNA damage-induced signaling cascade initiated by ATM. Using potent PARP inhibitors and PARP-1 knock-out cells, we demonstrate a functional interplay between ATM and poly(ADP-ribose) that is important for the phosphorylation of p53, SMC1, and H2AX. For the first time, we demonstrate a functional and physical interaction between the major DSB signaling kinase, ATM and poly(ADP-ribosyl)ation by PARP-1, a key enzyme of chromatin remodeling. This study suggests that poly(ADP-ribose) might serve as a DNA damage sensory molecule that is critical for early DNA damage signaling. Poly(ADP-ribosyl)ation is a post-translational modification that is instantly stimulated by DNA strand breaks creating a unique signal for the modulation of protein functions in DNA repair and cell cycle checkpoint pathways. Here we report that lack of poly(ADP-ribose) synthesis leads to a compromised response to DNA damage. Deficiency in poly(ADP-ribosyl)ation metabolism induces profound cellular sensitivity to DNA-damaging agents, particularly in cells deficient for the protein kinase ataxia telangiectasia mutated (ATM). At the biochemical level, we examined the significance of poly(ADP-ribose) synthesis on the regulation of early DNA damage-induced signaling cascade initiated by ATM. Using potent PARP inhibitors and PARP-1 knock-out cells, we demonstrate a functional interplay between ATM and poly(ADP-ribose) that is important for the phosphorylation of p53, SMC1, and H2AX. For the first time, we demonstrate a functional and physical interaction between the major DSB signaling kinase, ATM and poly(ADP-ribosyl)ation by PARP-1, a key enzyme of chromatin remodeling. This study suggests that poly(ADP-ribose) might serve as a DNA damage sensory molecule that is critical for early DNA damage signaling. Double-strand breaks (DSB) 5The abbreviations used are: DSB, DNA double-strand break; ATM, ataxia telangiectasia mutated; MRN, Mre11/Rad50/NBS1; SMC1, structural maintenance of chromosomes 1; IR, ionizing radiation; MNNG, N-methyl-N′-nitro-N-nitrosoguanidine; PAR, poly(ADP-ribose); PARP, poly(ADP-ribose) polymerase; PARG, poly(ADP-ribose) glycohydrolase; DNA-PK, DNA-dependent protein kinase; ATR, A-T and RAD-3-related; MEF, mouse embryonic fibroblast; A-T, ataxia-telangiectasia; Gy, Gray; GST, glutathione S-transferase. are potentially the most cytotoxic form of DNA damage in human cells because they lead to genomic rearrangements, cancer predisposition, and perhaps cell death if unrepaired or repaired incorrectly (1.O'Driscoll M. Jeggo P.A. Nat. Rev. Genet. 2006; 7: 45-54Crossref PubMed Scopus (463) Google Scholar). Consequently, the DNA damage response involves parallel modulation of redundant signaling pathways leading to lesion detection, processing, and repair. Ataxia telangiectasia mutated (ATM) is a DNA damage-responding kinase that is rapidly activated after the induction of DSB (2.Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2703) Google Scholar). Within minutes of DNA damage induction, ATM is recruited and activated in the vicinity of DSBs, where it induces the phosphorylation of a number of proteins required for DNA damage response and repair, including proteins of MRN (Mre11/Rad50/NBS1) complex, p53, SMC1 and histone variant H2AX (3.Lavin M.F. Birrell G. Chen P. Kozlov S. Scott S. Gueven N. Mutat. Res. 2005; 569: 123-132Crossref PubMed Scopus (170) Google Scholar). However, the detailed mechanisms of how ATM is activated and regulates its downstream effectors are not fully understood. Although ATM activation is mainly associated with DSB formation as part of the damage detection mechanism following ionizing radiation (IR), several studies indicate that the signaling kinase ATM is also activated in response to the environmental carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) (4.Adamson A.W. Kim W.J. Shangary S. Baskaran R. Brown K.D. J. Biol. Chem. 2002; 277: 38222-38229Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 5.Beardsley D.I. Kim W.J. Brown K.D. Mol. Pharmacol. 2005; 68: 1049-1060Crossref PubMed Scopus (16) Google Scholar, 6.Stojic L. Cejka P. Jiricny J. Cell Cycle. 2005; 4: 473-477Crossref PubMed Scopus (43) Google Scholar). Poly(ADP-ribose) polymerases (PARPs) are also constitutive factors of the DNA damage surveillance network, acting through a DNA break sensor function (7.Bouchard V.J. Rouleau M. Poirier G.G. Exp. Hematol. 2003; 31: 446-454Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). Several observations indicate that poly(ADP-ribosyl)ation also plays an early role in DSB signaling and repair pathways (8.Bryant H.E. Schultz N. Thomas H.D. Parker K.M. Flower D. Lopez E. Kyle S. Meuth M. Curtin N.J. Helleday T. Nature. 2005; 434: 913-917Crossref PubMed Scopus (3706) Google Scholar, 9.Farmer H. McCabe N. Lord C.J. Tutt A.N. Johnson D.A. Richardson T.B. Santarosa M. Dillon K.J. Hickson I. Knights C. Martin N.M. Jackson S.P. Smith G.C. Ashworth A. Nature. 2005; 434: 917-921Crossref PubMed Scopus (4751) Google Scholar, 10.McCabe N. Turner N.C. Lord C.J. Kluzek K. Bialkowska A. Swift S. Giavara S. O'Connor M.J. Tutt A.N. Zdzienicka M.Z. Smith G.C.M. Ashworth A. Cancer Res. 2006; 66: 8109-8115Crossref PubMed Scopus (1029) Google Scholar, 11.Andrabi S.A. Kim N.S. Yu S.W. Wang H. Koh D.W. Sasaki M. Klaus J.A. Otsuka T. Zhang Z. Koehler R.C. Hurn P.D. Poirier G.G. Dawson V.L. Dawson T.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 18308-18313Crossref PubMed Scopus (520) Google Scholar). PARP-1 and PARP-2 are highly activated upon binding to DNA strand interruptions and synthesize, within seconds, large amounts of the negatively charged polymer of ADP-ribose (PAR) on several nuclear proteins including themselves, histones, topoisomerase I, and DNA-dependent protein kinase (DNA-PK) (12.Kim M.Y. Zhang T. Kraus W.L. Genes Dev. 2005; 19: 1951-1967Crossref PubMed Scopus (660) Google Scholar). The immediate activation of PARP-1 by DNA breaks and the resulting PAR build-up, which appears minutes after DNA damage induction, are among the earliest cell responses to DNA damage (13.Schultz N. Lopez E. Saleh-Gohari N. Helleday T. Nucleic Acids Res. 2003; 31: 4959-4964Crossref PubMed Scopus (250) Google Scholar, 14.Hochegger H. Dejsuphong D. Fukushima T. Morrison C. Sonoda E. Schreiber V. Zhao G.Y. Saberi A. Masutani M. Adachi N. Koyama H. de Murcia G. Takeda S. 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Furthermore, multiple reports show that PARP inhibitors increase the cytotoxicity of DNA-damaging agents and IR (19.Calabrese C.R. Almassy R. Barton S. Batey M.A. Calvert A.H. Canan-Koch S. Durkacz B.W. Hostomsky Z. Kumpf R.A. Kyle S. Li J. Maegley K. Newell D.R. Notarianni E. Stratford I.J. Skalitzky D. Thomas H.D. Wang L.Z. Webber S.E. Williams K.J. Curtin N.J. J. Natl. Cancer Inst. 2004; 96: 56-67Crossref PubMed Scopus (430) Google Scholar, 20.Haince J.F. Rouleau M. Hendzel M.J. Masson J.Y. Poirier G.G. Trends Mol. Med. 2005; 11: 456-463Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 21.Plummer E.R. Curr. Opin. Pharmacol. 2006; 6: 364-368Crossref PubMed Scopus (102) Google Scholar). This is manifested by a delay in the progression through the S phase and accumulation of cells in G2/M (22.Noel G. Godon C. Fernet M. Giocanti N. Megnin-Chanet F. Favaudon V. Mol. Cancer Ther. 2006; 5: 564-574Crossref PubMed Scopus (113) Google Scholar, 23.Jacobson E.L. Meadows R. Measel J. Carcinogenesis. 1985; 6: 711-714Crossref PubMed Scopus (41) Google Scholar). Although viable, both PARP-1 and PARP-2 knock-out mice display an acute sensitivity to IR and alkylating agents (24.Shall S. de Murcia G. Mutat. Res. 2000; 460: 1-15Crossref PubMed Scopus (477) Google Scholar, 25.Menissier de Murcia J. Ricoul M. Tartier L. Niedergang C. Huber A. Dantzer F. Schreiber V. Ame J.C. Dierich A. LeMeur M. Sabatier L. Chambon P. de Murcia G. EMBO J. 2003; 22: 2255-2263Crossref PubMed Scopus (497) Google Scholar). PARP-1/PARP-2 double knock-out mice die at early stages of embryogenesis, demonstrating the crucial functions of poly(ADP-ribosyl)ation in DNA damage signaling and the existence of functional redundancy between these two PARPs (25.Menissier de Murcia J. Ricoul M. Tartier L. Niedergang C. Huber A. Dantzer F. Schreiber V. Ame J.C. Dierich A. LeMeur M. Sabatier L. Chambon P. de Murcia G. EMBO J. 2003; 22: 2255-2263Crossref PubMed Scopus (497) Google Scholar). Interestingly, PARP-1/ATM double mutant mice are defective for both repair and signaling of DNA damage, leading to embryonic lethality because of their dramatic sensitivity to DNA damage (26.Menisser-de Murcia J. Mark M. Wendling O. Wynshaw-Boris A. de Murcia G. Mol. Cell Biol. 2001; 21: 1828-1832Crossref PubMed Scopus (86) Google Scholar, 27.Huber A. Bai P. Menissier-de Murcia J. de Murcia G. DNA Repair. (Amst.). 2004; 3: 1103-1108Crossref PubMed Scopus (191) Google Scholar). Recently, the notion that PAR mediates key events in cell cycle regulation such as mitotic spindle control through PAR binding interaction (28.Fang Y. Liu T. Wang X. Yang Y.M. Deng H. Kunicki J. Traganos F. Darzynkiewicz Z. Lu L. Dai W. Oncogene. 2006; 25: 3598-3605Crossref PubMed Scopus (68) Google Scholar, 29.Chang P. Coughlin M. Mitchison T.J. Nat. Cell Biol. 2005; 7: 1133-1139Crossref PubMed Scopus (226) Google Scholar, 30.Chang P. Jacobson M.K. Mitchison T.J. Nature. 2004; 432: 645-649Crossref PubMed Scopus (193) Google Scholar) stimulated our interest on the possible role of PAR-binding proteins in early DNA damage response and specifically the interplay between the signaling kinase ATM and PAR molecules. A number of proteins involved in DNA damage signaling contain modular domains that mediate specific protein-protein interactions. Consequently, a PAR-binding domain has been found in a number of proteins involved in DNA damage response pathways such as p53, p21, XRCC1, Ku70, topoisomerase I, and DNA ligase IIIα (31.El-Khamisy S.F. Masutani M. Suzuki H. Caldecott K.W. Nucleic Acids Res. 2003; 31: 5526-5533Crossref PubMed Scopus (527) Google Scholar, 32.Leppard J.B. Dong Z. Mackey Z.B. Tomkinson A.E. Mol. Cell Biol. 2003; 23: 5919-5927Crossref PubMed Scopus (190) Google Scholar, 33.Malanga M. Althaus F.R. J. Biol. Chem. 2004; 279: 5244-5248Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 34.Pleschke J.M. Kleczkowska H.E. Strohm M. Althaus F.R. J. Biol. Chem. 2000; 275: 40974-40980Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar). However, despite major advances primarily by biochemical and genetic studies, the precise role of poly(ADP-ribosyl)ation in the immediate DNA damage signaling remains to be elucidated. The finding that ATM is activated, at least in vitro, by PAR molecules (35.Goodarzi A.A. Lees-Miller S.P. DNA Repair. (Amst.). 2004; 3: 753-767Crossref PubMed Scopus (65) Google Scholar) raises the issue as to whether PAR influences the phosphorylation cascade initiated by the ATM protein kinase. In this study, we determined how PAR synthesis modulated the early DNA damage signaling response. We show that impaired PAR synthesis in cells treated with the PARP inhibitors is associated with delayed phosphorylation of p53, H2AX, and SMC1 after DNA damage caused by treatment with both MNNG and IR. The decreased phosphorylation and stabilization of p53 and delayed phosphorylation of SMC1 and histone H2AX were also confirmed in mouse embryonic fibroblasts (MEFs) derived from PARP-1-/- mice. In addition, we demonstrate that PAR interacts physically with ATM, that this interaction is mediated by PAR-binding domains and that it has functional consequences. Cell Lines−The human lung adenocarcinoma cells (A549) (ATCC CCL-185) were maintained in Ham’s F12K medium with 2 mm glutamine containing 15% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 μg/ml). SV40-transformed normal skin fibroblasts (GM00637), and A-T fibroblasts (GM09607) as well as mouse embryonic fibroblasts derived from PARP-1+/+ (F20) and PARP-1-/- (A1) mice (36.Wang Z.Q. Auer B. Stingl L. Berghammer H. Haidacher D. Schweiger M. Wagner E.F. Genes Dev. 1995; 9: 509-520Crossref PubMed Scopus (715) Google Scholar) were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, glutamine (2 mm), penicillin (100 units/ml), and streptomycin (100 μg/ml). Alternatively, the EBV-immortalized normal lymphoblastoid cells (C3ABR) and A-T lymphoblastoid cells (GM03189) were used and maintained in RMPI 1640 medium supplemented with 15% fetal bovine serum, glutamine (2 mm), penicillin (100 units/ml), and streptomycin (100 μg/ml). Immunoprecipitation, Western Blotting, and Antibodies−We exposed control and A-T cells to genotoxic agents (MNNG and IR) and collected whole cell extracts at the indicated time points as described (37.Pandita T.K. Lieberman H.B. Lim D.S. Dhar S. Zheng W. Taya Y. Kastan M.B. Oncogene. 2000; 19: 1386-1391Crossref PubMed Scopus (135) Google Scholar). We performed precipitation of endogenous PARP-1 with an anti-PARP-1 monoclonal antibody (F1–23). Cells were resuspended in 25 mm NaPO4 buffer pH 8, 150 mm NaCl, 1 mm EDTA, 0.2% Nonidet P-40, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, and protease inhibitors Complete™ (Roche Applied Science) and kept on ice for 20 min. NaPO4 and NaCl concentration were then adjusted to 175 mm (150 μl/ml) and 0.3 m (30 μl/ml), respectively, and homogenized using a Dounce homogenizer with 30 strokes with the tight-fitting pestle. A suspension of magnetic protein G beads (Invitrogen) was washed twice with binding buffer (175 mm NaPO4, pH 8.0 containing 150 mm NaCl, 1 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, 1 mm dithiothreitol, and protease inhibitors) prior to immunoprecipitation. Cleared lysate was diluted to 1:2 (v/v) with binding buffer without NaCl, mixed with the antibody-coated beads, and incubated with rotation at 4 °C for 90–120 min. The beads were washed with the binding buffer. The beads containing the immunoprecipitated samples were collected, resuspended in 250 μl of SDS loading buffer and boiled 5 min at 100 °C. Whole cell extracts (10 μg) or immunoprecipitates were analyzed by SDS-PAGE and electrotransferred onto nitrocellulose membranes. Immunoblots were probed with antibodies directed against PAR (96–10 antisera), PARP-1 (clone C2–10), p53 protein, p53 phospho-S15, SMC1 phospho-S957, SMC1 (Cell Signaling), H2AX phospho-S139 (Upstate), and β-actin (Calbiochem). Immunoblots were developed using the Super Signal West Dura Extended Duration (Pierce) to allow quantitative analysis. Purification of GST-ATM Fragments and in Vitro Pull-down Assays−For in vitro PAR binding characterization, we prepared a series of GST-ATM constructs as described (38.Khanna K.K. Keating K.E. Kozlov S. Scott S. Gatei M. Hobson K. Taya Y. Gabrielli B. Chan D. Lees-Miller S.P. Lavin M.F. Nat. Genet. 1998; 20: 398-400Crossref PubMed Scopus (407) Google Scholar). For the GST pull-down, glutathione-Sepharose beads (80 μl) were washed with lysis buffer and then resuspended in 400 μl of lysis buffer containing either purified GST-tagged ATM domains (1 μg) or GST protein alone. For each condition, PARP-1 (1 μg), 32P-modified PARP-1 (100 nm), PARG (1 unit), or PAR (100 nm) were added to the reaction mixture. After a 2-h incubation at 4 °C with constant mixing, the beads were washed extensively in lysis buffer containing 500 mm NaCl. The beads were collected by centrifugation, resuspended in 250 μl of SDS loading buffer and boiled 5 min at 100 °C. After separation by SDS-PAGE, bound material was detected by autoradiography, immunoblotting with anti-PARP-1 (clone C2–10) or Coomassie Blue staining. Nitrocellulose PAR Binding Assay−Synthetic peptides or purified proteins were loaded onto a nitrocellulose membrane (0.1 μm pore size) using a dot blot manifold system. Membranes were incubated with 32P-labeled automodified PARP-1 or 32P-labeled purified PAR prepared as described (39.Shah G.M. Poirier D. Duchaine C. Brochu G. Desnoyers S. Lagueux J. Verreault A. Hoflack J.C. Kirkland J.B. Poirier G.G. Anal. Biochem. 1995; 227: 1-13Crossref PubMed Scopus (162) Google Scholar), washed extensively, and subjected to autoradiography. BioPorter-mediated Delivery of Peptides into Cells−Wild-type and mutant ATM N-terminal PAR-binding peptides were delivered into cells using the BioPorter reagent (Sigma) (40.Sawada M. Hayes P. Matsuyama S. Nat. Cell Biol. 2003; 5: 352-357Crossref PubMed Scopus (139) Google Scholar, 41.Zelphati O. Wang Y. Kitada S. Reed J.C. Felgner P.L. Corbeil J. J. Biol. Chem. 2001; 276: 35103-35110Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Purified peptides were diluted to the desired concentration in PBS. The diluted peptide solution (2.5 μg of total) was added to the dried BioPorter reagent and allowed to sit at room temperature for 5 min followed by gentle mixing. The suspensions were mixed with 250 μl of serum-free medium and were then added to cells (∼60–80% confluency) cultured in 6-well cell culture plates for 3–4 h at 37 °C. Cultures were subsequently used for experiments. Cytotoxicity Studies−Cells were suspended in RPMI 1640 medium and plated out at 2 × 105 cells/ml in 24-well cell culture plates. Cells were exposed for 1 h to a range of concentrations of the PARP inhibitor PJ-34 (Axxora) or a range of MNNG concentrations in growth medium. After treatment, fresh media with or without PJ-34 was added, and plates were incubated for 4–5 days at 37 °C in a 10% CO2 incubator until untreated control cells reached ∼2 × 106 cells/ml. Cell viability (triplicate wells for each drug concentration) was determined by adding 0.1 ml of 0.4% Trypan blue to a 0.5-ml cell suspension (42.Beamish H. Williams R. Chen P. Lavin M.F. J. Biol. Chem. 1996; 271: 20486-20493Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Viable cells were counted, and viabilities were expressed as the number of cells in drug-treated wells relative to cells in control wells (% of control). Immunofluorescence and Microscopy Analysis−A549 or GM00637 cells were fixed at the indicated time points following treatment with DNA-damaging agents (MNNG or IR). PAR synthesis was detected with the rabbit anti-PAR 96–10 antibody. Nuclear foci of DNA damage were visualized using anti-H2AX pS139 monoclonal antibody (Upstate). Cells were observed using a Zeiss microscope (Axioplan IIM) equipped with a CoolSnapHQ cooled CCD camera. The measurement of fluorescence intensity and colocalization was performed using MetaMorph 6.0 (Universal Imaging) software. Composite figures of collected images were assembled in Adobe Photoshop. Confocal Microscopy and Quantification of γ-H2AX Foci−To detect MNNG-induced γ-H2AX foci, PARP-1+/+ and PARP-1-/- MEF grown on coverslips were fixed at various time points after MNNG exposure and stained with anti-H2AX pS139 monoclonal antibody. Cells labeled as described above were placed on a Nikon inverted confocal laser-scanning microscope and images were captured using the LaserSharp software. The number of γ-H2AX foci per cell were manually counted in three separate fields (at least 50 cells/fields). Cells were classified into four different categories according to the number of γ-H2AX foci per cell (0–5, 5–15, 15–30, or more than 30 foci per cell). Cell Cycle Analysis−Cell cycle distribution after PJ-34 and MNNG treatments was determined by flow cytometry analysis using propidium iodine staining for DNA content. Cell death was characterized by the number of cells with fragmented DNA (sub-G1 population). Loss of PAR Synthesis Sensitizes Normal Cells to DNA-damaging Agents−Genetic disorders that cause deficiency in DNA damage signaling proteins are characterized by an increased sensitivity to a variety of DNA-damaging agents. Cells derived from patients with Ataxia-Telangiectasia (A-T) illustrate perfectly this paradigm showing a defective response to DNA damage (43.Shiloh Y. Trends in Biochemical Sciences. 2006; 31: 402-410Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar). Similarly, it has been demonstrated that disruption of PAR synthesis causes an increase in sensitivity to DNA-damaging agents (21.Plummer E.R. Curr. Opin. Pharmacol. 2006; 6: 364-368Crossref PubMed Scopus (102) Google Scholar), suggesting that deficiency in PAR metabolism is linked to a defect in DNA damage signaling. Thus, to determine whether the effects of PARP inhibition impacts on the ATM signaling network, we compared the sensitivity to DNA alkylating agents in normal ATM-proficient lymphoblasts and in A-T patient cells. As shown in Fig. 1A, normal cells exposed for one hour to both MNNG and PARP inhibitors exhibit survival rates similar to A-T cells treated with MNNG alone, suggesting that the absence of PAR synthesis decreases the ability of normal cells to respond adequately to DNA damage to an extent similar to that observed in A-T cells. We next evaluated whether PJ-34 exhibits inhibitor-specific toxicity in the absence of treatment with exogenous cytotoxic drugs using survival as readout (Fig. 1B). We observed a relatively low toxicity in normal ATM-proficient cells when low doses of PJ-34 were added to growth medium (Fig. 1B). However, A-T cells are acutely sensitive to treatment with the same low doses of PJ-34 (Fig. 1B). This finding is consistent with previous reports showing that deficiency in DNA damage signaling proteins induces sensitivity to PARP inhibition (10.McCabe N. Turner N.C. Lord C.J. Kluzek K. Bialkowska A. Swift S. Giavara S. O'Connor M.J. Tutt A.N. Zdzienicka M.Z. Smith G.C.M. Ashworth A. Cancer Res. 2006; 66: 8109-8115Crossref PubMed Scopus (1029) Google Scholar, 26.Menisser-de Murcia J. Mark M. Wendling O. Wynshaw-Boris A. de Murcia G. Mol. Cell Biol. 2001; 21: 1828-1832Crossref PubMed Scopus (86) Google Scholar). We also established that the hypersensitivity of A-T cells to the suppression of PARP-1 function is due to the activation of apoptosis (supplemental Fig. S1). This is illustrated by the increased sub-G1 population (supplemental Fig. S1B) and the appearance of the specific PARP-1 cleavage product (supplemental Fig. S1C). Together, these results clearly show that disruption of PARP function by chemical inhibition in normal cells leads to the suppression of an important signaling event of the DNA damage response that is comparable to the cell survival defect observed in cells lacking ATM. MNNG Induces ATM-dependent Substrate Phosphorylation That Is Altered in the Absence of PAR Synthesis−The DNA damage-responsive kinase ATM is best known for its activation following IR (2.Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2703) Google Scholar). Besides, ATM has also been proven to be the main kinase responsible for the phosphorylation of p53 on serine 15 during MNNG-induced DNA damage (4.Adamson A.W. Kim W.J. Shangary S. Baskaran R. Brown K.D. J. Biol. Chem. 2002; 277: 38222-38229Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In addition, it was shown that NBS1, SMC1, and p53 were phosphorylated in an ATM-dependent manner at high doses of MNNG-induced DNA strand breaks, while ATR responds to the formation of DNA adducts and stalled replication induced by UV radiation (4.Adamson A.W. Kim W.J. Shangary S. Baskaran R. Brown K.D. J. Biol. Chem. 2002; 277: 38222-38229Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 5.Beardsley D.I. Kim W.J. Brown K.D. Mol. Pharmacol. 2005; 68: 1049-1060Crossref PubMed Scopus (16) Google Scholar, 6.Stojic L. Cejka P. Jiricny J. Cell Cycle. 2005; 4: 473-477Crossref PubMed Scopus (43) Google Scholar). We have analyzed this specific phosphorylation of p53 using a large panel of normal ATM-proficient and A-T cell lines. All ATM-proficient cells show induction of p53 phosphorylation 1 h after MNNG treatment (Fig. 2A). In contrast, negligible signal was detected in both A-T cell lines exposed to the same treatment (Fig. 2A). Taken together, these findings confirm that high MNNG doses activate the ATM kinase, which allow the direct and rapid phosphorylation of p53 on serine 15 (4.Adamson A.W. Kim W.J. Shangary S. Baskaran R. Brown K.D. J. Biol. Chem. 2002; 277: 38222-38229Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 6.Stojic L. Cejka P. Jiricny J. Cell Cycle. 2005; 4: 473-477Crossref PubMed Scopus (43) Google Scholar). Therefore, we used MNNG to activate both ATM protein kinase and PAR formation to establish the relationship between ATM and PARP-1. We hypothesized that PAR molecules might influence ATM function, either by promoting its kinase activity or by providing access to its downstream substrates. Recent studies have demonstrated that ATM kinase activity is significantly enhanced by PAR (35.Goodarzi A.A. Lees-Miller S.P. DNA Repair. (Amst.). 2004; 3: 753-767Crossref PubMed Scopus (65) Google Scholar). Because poly(ADP-ribosyl)ation occurs with kinetics similar to that of ATM activation during initial steps of the DNA damage signaling (27.Huber A. Bai P. Menissier-de Murcia J. de Murcia G. DNA Repair. (Amst.). 2004; 3: 1103-1108Crossref PubMed Scopus (191) Google Scholar), it is therefore conceivable that PAR could trigger ATM phosphorylation of a subset of its targets within the first hour after DNA breaks formation. To understand the mechanism of PARP-1 activation in response to MNNG, we first determined the time-course of PARP activation in normal and A-T cells as well as in PARP-inhibited cells exposed to 50 μm MNNG (Fig. 2B). Exposure of both normal and A-T cells to MNNG results in an immediate synthesis of PAR that peaks at 30 min post-treatment, and rapidly decreases after 1 h, demonstrating how fast the PAR-dependent response can be (Fig. 2, B and C). However, in cells exposed to a 5 μm dose of PJ-34, we observed a striking inhibition (98.5%) of PAR synthesis following MNNG exposure (Fig. 2, B and C). Having observed that DNA damage-dependent PAR synthesis follows rapid kinetics in both normal and A-T cells, we next examined the ATM-dependent response within the same time frame following treatment with both MNNG and PJ-34. We performed biochemical analysis on the well-established ATM substrates p53, SMC1, and H2AX using phosphospecific antibodies. Importantly, cells treated with both MNNG and PJ-34 present a 5-fold decrease of p53 phosphorylation at 30 min and 1 h post-treatment (Fig. 2B-C). Pretreatment of the cells with PJ-34 significantly reduced the MNNG-induced phosphorylation of SMC1 on serine 957 throughout the treatment (Fig. 2, B and C). Next we used an ATM-deficient cell line to determine whether the phosphorylation cascade induced by MNNG is dependent of the protein kinase ATM. Although these cells demonstrate normal level of PAR synthesis following MNNG exposure, they exhibit a defective phosphorylation of ATM-dependent substrates p53, SMC1 and H2AX (Fig. 2, B and C). Besides, no phosphorylation of p53 and SMC1 was observed in the ATM-deficient cells when PAR synthesis peaks (>1 h). This indicates that PAR could not signal directly to other members of the PI3K kinase family such as ATR and DNA-PK. Importantly, we found that treatment of A-T lymphoblasts with MNNG greatly attenuated the phosphorylation of p53 and SMC1 in initial time points, which demonstrated that the protein kinase ATM is responsible phosphorylation of these effectors following MNNG exposure. Together these results suggest that the absence of PAR modulates the ATM-dependent phosphorylation of numerous downstream effectors including p53 and SMC1 immediately after MNNG exposure. Similar effects were also observed with another PARP inhibitor, DPQ (supplemental Fig. S2). In these cells, over 90% of the PAR synthesis was inhibited 1 h after treatment (data not shown) and the MNNG-induced phosphorylation of p53 is about 60% of that measured when PAR is fully synthesized (supplemental Fig. S2). These results indicate that the reduced phosphorylation of p53 is due to the absence of PAR rather than a PJ-34 specific effect. The ability of γ-H2AX to form nuclear foci upon DNA damage has long been recognized as a sensitive marker of DSB formation (44.Takahashi A. Ohnishi T. Cancer Letters. 2005; 229: 171-179Crossref PubMed Scopus (185) Google Scholar, 45.Yu Y. Zhu W. Diao H. Zhou C. Chen F.F. Yang J. Toxico
Apoptosis is a morphologically and biochemically distinct form of cell death that occurs under a variety of physiological and pathological conditions. In the present study, the proteolytic cleavage of poly(ADP-ribose) polymerase (pADPRp) during the course of chemotherapy-induced apoptosis was examined. Treatment of HL-60 human leukemia cells with the topoisomerase II-directed anticancer agent etoposide resulted in morphological changes characteristic of apoptosis. Endonucleolytic degradation of DNA to generate nucleosomal fragments occurred simultaneously. Western blotting with epitope-specific monoclonal and polyclonal antibodies revealed that these characteristic apoptotic changes were accompanied by early, quantitative cleavage of the M(r) 116,000 pADPRp polypeptide to an M(r) approximately 25,000 fragment containing the amino-terminal DNA-binding domain of pADPRp and an M(r) approximately 85,000 fragment containing the automodification and catalytic domains. Activity blotting revealed that the M(r) approximately 85,000 fragment retained basal pADPRp activity but was not activated by exogenous nicked DNA. Similar cleavage of pADPRp was observed after exposure of HL-60 cells to a variety of chemotherapeutic agents including cis-diaminedichloroplatinum(II), colcemid, 1-beta-D-arabinofuranosylcytosine, and methotrexate; to gamma-irradiation; or to the protein synthesis inhibitors puromycin or cycloheximide. Similar changes were observed in MDA-MB-468 human breast cancer cells treated with trifluorothymidine or 5-fluoro-2'-deoxyuridine and in gamma-irradiated or glucocorticoid-treated rat thymocytes undergoing apoptosis. Treatment with several compounds (tosyl-L-lysine chloromethyl ketone, tosyl-L-phenylalanine chloromethyl ketone, N-ethylmaleimide, iodoacetamide) prevented both the proteolytic cleavage of pADPRp and the internucleosomal fragmentation of DNA. The results suggest that proteolytic cleavage of pADPRp, in addition to being an early marker of chemotherapy-induced apoptosis, might reflect more widespread proteolysis that is a critical biochemical event early during the process of physiological cell death.
ARTD1 (PARP1) is a key enzyme involved in DNA repair through the synthesis of poly(ADP-ribose) (PAR) in response to strand breaks, and it plays an important role in cell death following excessive DNA damage. ARTD1-induced cell death is associated with NAD(+) depletion and ATP loss; however, the molecular mechanism of ARTD1-mediated energy collapse remains elusive. Using real-time metabolic measurements, we compared the effects of ARTD1 activation and direct NAD(+) depletion. We found that ARTD1-mediated PAR synthesis, but not direct NAD(+) depletion, resulted in a block to glycolysis and ATP loss. We then established a proteomics-based PAR interactome after DNA damage and identified hexokinase 1 (HK1) as a PAR binding protein. HK1 activity is suppressed following nuclear ARTD1 activation and binding by PAR. These findings help explain how prolonged activation of ARTD1 triggers energy collapse and cell death, revealing insight into the importance of nucleus-to-mitochondria communication via ARTD1 activation.
<p>PDF file - 70KB, Figure S1: The specificity of the BAP1 antibody used in this study. (A) Immunostaining of U2OS cells transfected with control (Ctr) or BAP1 specific shRNA (BAP1 shRNA). Cells were transfected with the indicated shRNA for 48 hr and immunostained with BAP1 antibody and counterstained with DAPI to reveal DNA. (B) Transfection of the FokI nuclease fusion construct into U2OS cells containing the stably integrated reporter leads to local accumulation of damage response proteins γH2AX and GFP-BAP1. 24 hours after co-transfection of GFP-BAP1 and Fok1 constructs, cells were immunostained with γH2AX antibody and counterstained with DAPI to reveal DNA. GFP-BAP1 enriches at the sites of DSBs. Arrows indicate the enrichment of proteins at sites of DSBs. Bars, 5 microm.</p>
The nicotinamide analogue 6-aminonicotinamide (6AN) is presently undergoing evaluation as a potential modulator of the action of various antineoplastic treatments. Most previous studies of this agent have focused on a three-drug regimen of chemical modulators that includes 6AN. In the present study, the effect of single-agent 6AN on the efficacy of selected antineoplastic drugs was assessed in vitro. Colony-forming assays using human tumor cell lines demonstrated that pretreatment with 30-250 microM 6AN for 18 h resulted in increased sensitivity to the DNA cross-linking agent cisplatin, with 6-, 11-, and 17-fold decreases in the cisplatin dose that diminishes colony formation by 90% being observed in K562 leukemia cells, A549 non-small cell lung cancer cells, and T98G glioblastoma cells, respectively. Morphological examination revealed increased numbers of apoptotic cells after treatment with 6AN and cisplatin compared to cisplatin alone. 6AN also sensitized cells to melphalan and nitrogen mustard but not to chlorambucil, 4-hydroperoxycyclophosphamide, etoposide, or daunorubicin. In additional studies undertaken to elucidate the mechanism underlying the sensitization to cisplatin, atomic absorption spectroscopy revealed that 6AN had no effect on the rate of removal of platinum (Pt) adducts from DNA. Instead, 6AN treatment was accompanied by an increase in Pt-DNA adducts that paralleled the degree of sensitization. This effect was not attributable to 6AN-induced decreases in glutathione or NAD+, because other agents that depleted these detoxification cofactors (buthionine sulfoximine and 3-acetylpyridine, respectively) did not increase Pt-DNA adducts. On the contrary, 6AN treatment increased cellular accumulation of cisplatin. Further experiments revealed that 6AN was metabolized to 6-aminonicotinamide adenine dinucleotide (6ANAD+). Concurrent administration of nicotinamide and 6AN had minimal effect on cellular 6AN accumulation but abolished the formation of 6ANAD+, the increase in Pt-DNA adducts, and the sensitizing effect of 6AN in clonogenic assays. These observations identify 6AN as a potential modulator of cisplatin sensitivity and suggest that the 6AN metabolite 6ANAD+ exerts this effect by increasing cisplatin accumulation and subsequent formation of Pt-DNA adducts.
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