Interaction between ATM and PARP-1 in response to DNA damage and sensitization of ATM deficient cells through PARP inhibition
© Aguilar-Quesada et al; licensee BioMed Central Ltd. 2007
Received: 27 October 2006
Accepted: 25 April 2007
Published: 25 April 2007
ATM and PARP-1 are two of the most important players in the cell's response to DNA damage. PARP-1 and ATM recognize and bound to both single and double strand DNA breaks in response to different triggers. Here we report that ATM and PARP-1 form a molecular complex in vivo in undamaged cells and this association increases after γ-irradiation. ATM is also modified by PARP-1 during DNA damage. We have also evaluated the impact of PARP-1 absence or inhibition on ATM-kinase activity and have found that while PARP-1 deficient cells display a defective ATM-kinase activity and reduced γ-H2AX foci formation in response to γ-irradiation, PARP inhibition on itself is able to activate ATM-kinase. PARP inhibition induced γ H2AX foci accumulation, in an ATM-dependent manner. Inhibition of PARP also induces DNA double strand breaks which were dependent on the presence of ATM. As consequence ATM deficient cells display an increased sensitivity to PARP inhibition. In summary our results show that while PARP-1 is needed in the response of ATM to gamma irradiation, the inhibition of PARP induces DNA double strand breaks (which are resolved in and ATM-dependent pathway) and activates ATM kinase.
The ATM protein kinase is centrally involved in the cellular response to ionizing radiation (IR) and other DNA double-strand-break-inducing insults. In persons affected with ataxia-telangiectasia (A-T), associated mutations in the ataxia-telangiectasia mutated (atm) gene render cells unable to cope with the genotoxic stresses from ionizing radiation and oxidative damage, thus resulting in a higher concentration of unrepaired DNA. Functional inactivation of the ATM gene product and Atm-null mice, which were created by disrupting the Atm locus, recapitulate the human A-T phenotype and display growth retardation, mild neurological dysfunction, male and female infertility, extreme predisposition to thymic lymphomas, and acute sensitivity to ionizing radiation [1–3].
ATM, the product of the ATM gene, is a member of a family of large proteins found in various organisms that share a COOH-terminal PI3 kinase-like domain. ATM has serine/threonine protein kinase activity and mediates the activation of multiple signal transduction pathways reviewed in [4–6].
Although it has been well established that IR exposure activates the ATM kinase domain, the actual mechanism by which ATM responds to damaged DNA has remained enigmatic until recently. Initial evidences indicated that ATM activation might involve autophosphorylation. A breakthrough in our understanding of this process came in a landmark publication by Bakkenist and Kastan . They found that ATM molecules are inactive in undamaged cells, being held as dimers or higher-order multimers. In this configuration, the kinase domain of each molecule is blocked by the FAT domain of the other. Following DNA damage, each ATM molecule phosphorylates the other on a serine residue at position 1981 within the FAT domain, a phosphorylation that releases the two molecules from each other's grip, turning them into fully active monomers.
Poly(ADP-ribose) polymerase (PARP-1) is a nuclear enzyme which is activated in response to genotoxic insults by binding damaged DNA and attaching polymers of ADP-ribose to nuclear proteins at the expense of its substrate NAD+. The protein respond to DNA damage by transferring 50 to 200 molecules of ADP-ribose to various nuclear proteins, including transcription factors, histones and PARP-1 itself . This poly(ADP-ribosyl)ation activity of PARP-1 appears to be important for maintaining genomic integrity  and it has been associated with longevity. Furthermore, PARP-1 is activated by agents infringing single stranded DNA damage such as alkylating agents, ionizing radiation, and oxidative damage.
A function of PARP-1 as a nick sensor has been proposed . Its rapid activation upon DNA damage may result in poly (ADP-ribosyl)ation of key enzymes such as transducers of DNA damage, or alternatively, PARP-1 automodification could result in the recruitment of transducers to the damaged site. In this regard, a link between ATM and PARP-1 is supported by recent findings. One of these studies has demonstrated a sustained PARP-1 activation in ATM-/- cells due to the persistence of DNA damage [11, 12]. Moreover, Menissier-de Murcia et al. , have reported that ATM and PARP-1 double deficient mice have a severe synergistic phenotypes leading to early embryonic lethality due to the effects of these proteins on signalling DNA damage and/or on distinct pathways of DNA repair. Furthermore, a recent study has shown that in vitro, PARP-1 inhibited the activation a subset of ATM substrates such as phosphorylation of p53 on serine 15 .
The aim of this study has been to elucidate the interaction between PARP-1 and ATM and how this partnership is involved in regulation of DNA repair pathways. We present evidences showing a physical association between ATM and PARP-1 in response to DNA damage as well as a poly (ADP-ribosyl)ation of ATM. The biological consequence of this interaction is a diminished activation of ATM-kinase in the absence of PARP-1. Surprisingly, preventing poly(ADP-ribosyl)ation with PARP inhibitors results in an increased constitutive ATM-kinase related to the PARP inhibitor's ability to induce DNA double strand breaks (DSBs) which were resolved in an ATM-dependent manner.
PARP-1 interacts with ATM in vivo and ATM is modified by poly(ADP-ribosyl)ation
PARP-1 is needed for optimal activation of ATM
PARP inhibitors promote ATM activation through induction of DSBs
To gain insight about the increased basal ATM-kinase activity after the inhibition of PARP-1 with ANI (figure 2C), we performed an indirect inmunofluorescence against H2AX in its phosphorylated form, after the incubation with the PARP inhibitor in order to detect any DNA damage response. ATM wild type and deficient cells (G361 and HT144 respectively) were incubated with ANI at different times. Remarkably the sole fact of the incubation with ANI was able to elicits H2AX phosphorylation in wild type but not in ATM deficient cells. This effect was transient and reached a peak in 2 hours, declining afterwards (figure 3). γ-Irradiation was used as positive internal control (figure 3, upper panels). Previous results from our group have shown that PARP-1 null cells have a deficient p53 ser15 phosphorylation in response to ionising radiation , confirming with a different ATM substrate that ATM activation is compromised in the absence of PARP-1.
Cells have evolved various sophisticated pathways to sense and overcome DNA damage as a mechanism to preserve the integrity of the genome. Environmental attacks like radiations or toxins, or spontaneous DNA lesions, trigger checkpoint activation and consequent cell cycle arrest leading to DNA repair or apoptosis. Two key proteins that coordinate recognition of DNA damage and signal transduction to p53 are ATM and PARP-1. ATM and PARP-1 participate in distinct forms of DNA repair that partially compensate for each other. PARP-1 and ATM participate in base excision repair (BER) and homologous recombination (HR), respectively. It is normally assumed that ATM signals for double strand breaks while PARP-1 participates in signalling from single DNA strand lesions. Here we report that these two proteins form a molecular complex that co-localizes in DNA damage foci.
Considerable evidence from in vitro, cell culture and ex vivo studies shows that poly(ADP-ribosyl)ation plays a critical role in the survival and maintenance of genomic stability of proliferating cells exposed to low or moderate levels of DNA-damaging agents . The data presented in this study strongly support a role for PARP-1 and poly (ADP-ribose) in ATM activation: in the absence of PARP-1 there is a deficient ATM-kinase activation in response to ionizing radiation as measured by intrinsic kinase activity and H2AX phosphorylation. These results are in agreement with previous data showing that PARP-1 deficient mice are extremely sensitive to low doses of γ-radiation (as is the case for ATM-null mice), and this phenotype could be adscribed to a deficient ATM-kinase activation in tissues such as the intestine epithelium . Also, results from our group have shown that p53 accumulation and p53-dependent gene activation are compromised in parp-1 knockout cells after γ-irradiation . The insight of the consequences of the poly(ADP-ribosyl)ation of ATM are not clear yet since the inhibition of PARP induced indirectly DNA DSB, initiating new responses to DNA damage that interfere with elucidation of the activation of ATM.
PARP inhibitors have been used as radio and chemo-sensitizers in a number of experimental settings and a mechanism for DSB induction through the collision of unrepaired single DNA strand lesions with replication forks has been suggested [19, 20]. Early reports claimed that the PARP inhibitor 3-aminobenzamide was a radio-sensitizer only in rodent cells  however more recently ANI (1000-fold more potent at inhibiting PARP activity compared with 3-aminobenzamide (3-ABA)) has been found to be radiation sensitizer to both rodent and human tumor cells [9, 15, 22]. The novel PARP inhibitor AG14361 has shown to increase the specificity and in vivo activity to enhance radiation therapy of human cancer through vasoactive effects and not directly in the cells in culture . Therefore the question still remains open as to how human tumors could benefit from PARP inhibition during radiotherapy.
The second main conclusion in this study is that inhibition of PARP-1 activity leads to DSBs induction and activation of ATM and, at the same time, prevents IR-induced ATM-kinase activity. From the results presented here, there is a clear duality in the effect of PARP inhibition on ATM: while the lack of response to IR in ANI treated cells indicates that poly (ADP-rybosylation) of ATM is probably needed for optimal ATM activation, long term exposure to PARP inhibitor results in the generation of DSBs and secondarily in the activation of ATM kinase. DSBs generated in this way are due to stalled replication fork  and they are resolved by homologous recombination (HR), providing a therapeutic opportunity to specifically kill HR deficient tumor cells, as has been previously shown by different laboratories [25, 26].
In summary, our study demonstrates a strong association between ATM and PARP-1 during the response to ionizing radiation, being PARP-1 and its activity needed for optimal activation of ATM kinase. On the other hand, inhibition of PARP leads to the activation of ATM kinase as result of the generation of DSBs, making ATM deficient cells particularly sensitive to PARP inhibitors.
In this study we demonstrate the physical interaction between PARP-1 and ATM in response to ionizing radiation, the modification of ATM by poly(ADP-ribose) and the functional consequences of this interaction in PARP-1 deficient cells, where the activation of ATM kinase is compromised in response to IR. Additionally, PARP inhibition induces DNA double strand breaks who are resolved in an ATM-dependent manner. As result of that, ATM kinase is activated by PARP inhibition and ATM deficient cells are much more sensitive to PARP inhibition than ATM proficient cells.
Cell culture and treatments
We have used immortalised (3T3) murine embryonic fibroblasts expressing or lacking PARP-1 from parp-1 +/+ and -/- mice. G361 and HT144 are respectively wild type and A-T cell lines from a melanoma patient  (a kind gift from Dr. Y. Shiloh, Sackler School of Medicine, Tel Aviv).
Cells were in exponential growth at the time of IR treatment and the PARP inhibitor ANI was dissolved in culture medium at a concentration of 10 μM. It was added 60 min prior to IR. Irradiations, with or without ANI co-treatment, were performed using a 60Co source at a dose rate of 1.67 Gy/min. The used in all experiments was 10 Gy, unless otherwise stated.
Immunostaining forATM, γ-H2AX, PARP-1 and PAR (poly(ADP-ribose)) were performed on cells plated onto coverslips and grown for 24 h before treatments. The medium was removed, the coverslips rinsed twice in PBS (37°C) and fixed in ice-cold methanol-acetone (1:1) for 10 minutes in the experiments with ATM, γ-H2AX, and in formaldehyde 4% for 10 minutes in the experiments with PARP-1 or PAR. The coverslips were rinsed twice in PBS-T (PBS containing 0,1% Tween-20) prior to incubation with primary antibody for 16 h at 4°C. The primary antibodies used in these experiments were: rabbit polyclonal IgG anti-phospho-H2AX (Ser139) (Upstate, Lake Placid, NY), mouse monoclonal IgG anti-PARP (Ab-2, Oncogene). anti-ATM mouse monoclonal antibody SYR 10G3/1 was a kind gift from Y. Shiloh (Tel Aviv University); anti-PAR rabbit polyclonal antibody was purchased from Biomol, Plymouth Meeting, PA. The coverslips were rinsed 3 times in PBS-T followed by a 45 min incubation at room temperature in the dark and then rinsed 4 × 5 min in PBS-T. The secondary antibodies used in this study were FITC-conjugated goat anti-rabbit IgG (Sigma, St. Louis, MO) and Cy3-conjugated sheep anti-mouse IgG (Sigma, St. Louis, MO) at a concentration of 1:400. Antibodies were diluted in PBS containing 1% bovine serum albumin and 5% goat serum. Nuclear counterstaining with 4',6'-diamidino-2-phenylindole dihydrochloride (DAPI) was performed after removal of excess secondary antibody. Slides were prepared using the Vectashield mounting medium (Vector Lab., Inc., Burlingame, CA 94010), coverslipped and stored in the dark at 4°C. Immunofluorescence images were obtained linear range of detection to avoid signal saturation using a Leica confocal microscopy.
Immunoprecipitation and western blotting assay
Cell extracts, SDS-PAGE electrophoresis and western blotting were performed as previously described . For immunoprecipitation, cell lysates were precleared by constant mixing for 2 hours with protein A-Sepharose (Pharmacia). The beads were removed by centrifugation, and the supernatant was mixed constantly overnight with a monoclonal antibody against ATM (SYR 10G3/1) or PARP-1 (Anti-PARP-1 Ab-2, Oncogene). Immune complex were adsorbed onto protein A-Sepharose, boiled and electrophoresed on polyacrylamide gels. The membranes were probed with antibodies directed against ATM (SYR 10G3/1, Tel Aviv University), PARP-1 (Anti-PARP-1 Ab-2, Oncogene) and Poli (ADP-Ribose) (Biomol).
In Vitro ATM-kinase assay
ATM kinase assays were conducted using the protocol described by Sarkaria et al. . ATM was immunoprecipitated from G361 cell extracts and from parp-1 +/+ and parp-1 -/- murine spleen extracts. Briefly cell extracts were prepared by resuspending cells in lysis buffer (20 mM HEPES pH = 7.4, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, with protease inhibitors and 0.2% Tween during 20 minutes at 4°C. The lysates were clarified by centrifugation and immunoprecipitations were carried out with SYR 10G3/1 anti-ATM mouse monoclonal antibody. Then immune complex were adsorbed onto protein A-Sepharose and washed twice with kinase buffer (10 mM HEPES pH = 7.4, 50 mM NaCl, 10 mM MgCl2) and once with a high salt buffer (0.1 M Tris-HCl pH = 7.4, 0.6 M NaCl). Kinase reactions were initiated with the addition of an equal volume of kinase buffer containing PHAS-I (20 ng/ml), 10 mM MnCl2, 1 mM DTT and 10 mM [32P]ATP. Kinase reactions were performed at 30°C during 20 minutes and terminated by the addition of 6×SDS loading buffer (1:1), and reaction products were resolved by SDS-PAGE. Incorporation of 32P into the PHAS-I substrate was evaluated by phosphorimaging. All kinase reactions were performed under linear reaction conditions. Equal loading in each lane was guaranteed by coomasie blue staining.
base excision repair
double strand break
ethylene glycol tetraacetic acid
sodium dodecyl sulphate-polyacrylamide gel electrophoresis
We would like to acknowledge to the staff of the Radiotherapy Unit of the San Cecilio University Hospital, and particularly to Damian Guirado, for their help in the treatment of the cells and to Dr. Abelardo Lopez-Rivas (CABIMER, CSIC, Seville) for helpful discussion. This work has been supported by grants SAF 2003-01217, RNIHG c03/02, PI050972, SAF2006-01089 and FIS G03/152 to FJO, grants CICYT SAF:2001–3533 and SAF2004-00889 to JMRdA; grant FIS c03/02 to RQP.
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