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Department of Biochemistry and Molecular Biology, Southern Research Institute (M.J.C., X.-L.C., B.X.) and Comprehensive Cancer Center (M.J.C., X.-L.C., B.X.), University of Alabama at Birmingham, Birmingham, Alabama; and Department of Genetics, Louisiana State University Sciences Center, New Orleans, Louisiana (X.T.)
Received April 3, 2007; accepted May 16, 2007
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Abstract |
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-H2AX and NBS1 focus formation compared with cells treated with control peptides, demonstrating that wtNIP possesses a strong inhibitory effect on ATM. The inhibitory effect of wtNIP also leads to a significant decrease in clonogenic survival in response to IR. Furthermore, wtNIP does not radiosensitize cells with defective ATM, suggesting a specific inhibition of ATM. Together, these data provide a proof of principle for the use of NBS1 C-terminal small peptides as specific ATM inhibitors and radiosensitizers.
). A-T is characterized by progressive neurodegeneration, variable immunodeficiency, an extremely high predisposition to the development of lymphoid malignancies, and a hypersensitivity to IR. Cells derived from patients with A-T show a variety of abnormalities, including cell cycle checkpoint defects, chromosomal instability, and hypersensitivity in response to IR. ATM is remarkable for its large size and the existence of a sequence in its carboxyl terminus similar to phosphatidylinositol 3-kinases. A family of genes, including Tel1, Mec1, and Rad3 in yeast, Mei-41 in Drosophila melanogaster, and ATR and DNA-PK in vertebrates, are similar in size and presence of the carboxyl terminal kinase sequence and are all involved in controlling DNA damage response (Abraham, 2001). The functional domains of the ATM protein include several HEAT repeats that act as scaffolding for assembly of molecular components, a phosphatidylinositol 3-like kinase domain that can phosphorylate serine/threonine followed by glutamine (the S/T-Q consensus sequence), and a FAT carboxyl-terminal domain that may regulate protein activity and stability (Perry and Kleckner, 2003). ATM activation requires functional NBS1 (Cerosaletti and Concannon, 2004; Difilippantonio et al., 2005; Falck et al., 2005; Cerosaletti et al., 2006). Mutations in the NBS1 gene are responsible for Nijmegen breakage syndrome (NBS), a hereditary disorder that imparts an increased predisposition to development of malignancy and a hypersensitivity to IR (Shiloh, 1997). NBS1 forms a complex with Mre11 and Rad50 to be called the MRN complex. MRN is highly conserved, and it influences each aspect of chromosome break metabolism (Varon et al., 1998). Studies have shown that the MRN complex can detect DNA double-strand breaks and recruit ATM to damaged DNA molecules (Lee and Paull, 2004, 2005). The C terminus motif of NBS1 contains a conserved sequence motif that binds to two of the HEAT repeats (2 and 7) of ATM. This interaction is essential to activate the kinase (Falck et al., 2005). Because the binding of NBS1 is critical for ATM to be functioning in response to DNA damage, we hypothesized that interfering with the NBS1-ATM interaction may block ATM activation and confer radiosensitization. To test this hypothesis, we developed several small peptides containing the conserved C-terminal sequence motif of NBS1 and fused them to a polyarginine internalization sequence. Herein, we describe the characterization of the C-terminal NBS1 inhibitory peptide in terms of internalization, half-life, cellular cytotoxicity, effects on the DNA damage response, and radiosensitivity. Together, these data may lead to a better understanding of the mechanisms that could be used to increase the radiosensitivity of cancer and provide data that could be rapidly translated into the development of novel radiosensitizing drugs.
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Materials and Methods |
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Peptide Synthesis. All peptides were synthesized by Abgent (San Diego, CA) and labeled with a biotin tag at their N termini for detection in vitro. Three peptides were produced: 1) one containing the polyarginine (R9) internalization sequence alone, 2) a wild-type NBS1 inhibitory peptide (wtNIP) corresponding to amino acids 735 to 744 of human NBS1, and 3) a random sequence peptide in which amino acids 735 to 744 of human NBS1 were scrambled (scNIP). The peptides were dissolved in dimethyl sulfoxide, stored at -20°C, and reconstituted in DMEM/10% FBS before use.
Irradiation. An X-RAD 320 Irradiation Cabinet (Precision X-Ray, East Haven, CT) was employed at 320 kV and 160 mA, with a 0.8-mm Sn + 0.25-mm Cu + 1.5-mm Al (half-value layer 
3.7 Cu) filter at a target-to-source distance of 20 cm and a dose rate of 3.4 Gy/min. All irradiations were conducted under normal atmospheric pressure and temperature.
Immunoprecipitation and Western Blotting. For coimmunoprecipitation of ATM, NBS1, and MRE11, cells were lysed for 1 h in ice-cold lysis buffer, which consisted of 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 5 mM Na3VO4,1mM NaF, and 1 mM phenylmethylsulfonyl fluoride. After centrifugation, supernatants were incubated with antibodies. After extensive washing with the lysis buffer, immunoprecipitates were analyzed by immunoblot using specific antibodies. For Western blotting analysis, samples (cell lysates or immunoprecipitates) were separated on to 2% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and probed with various antibodies.
Immunofluorescence Microscopy. Exponentially growing cultures of cells were plated on sterile 22-cm2 coverslips and incubated for 24 h at 37°C in 5% CO2 humidified air before they were treated with the NIP peptides at room temperature. Coverslips were washed with phosphate-buffered saline and fixed with 4% paraformaldehyde and 0.25% Triton X-100 for 15 min at room temperature, blocked for 30 min at room temperature, and incubated with fluorescein isothiocyanate-conjugated streptavidin or anti- H2AX and phospho-NBS1 antibodies (Rockland Immunochemicals, Gilbertsville, PA) for 1 h at room temperature. Coverslips were then mounted with Vectashield Elite (Vector Labs, Burlingame, CA) and observed with a Leica fluorescence microscope. Images were captured at 40x magnification using a Retiga EXi digital camera (QImaging, Surrey, BC, Canada) and analyzed with Image-Pro Plus software (ver. 4.1; Media Cybernetics, Inc., Bethesda, MD).
MTT Assay. For cytotoxicity studies, exponentially growing cultures of HeLa or DU-145 cells were harvested, plated in 96-well plates (5000 cells/well) in complete media, and incubated overnight. On the following day, cells were treated with the NIP peptides (0, 5, 10, 20, 50, or 100 µM) or paclitaxel (Taxol; 0, 10, 50 or 100 µM) as a positive control. At the end of the time course, an MTT cell viability assay (Promega Corp., Madison, WI) was used according to the manufacturer's guidelines to determine peptide cytotoxicity.
Colony Formation Assays. To determine radiosensitivity, the colony-forming assay was incorporated. Cells were harvested with 0.125% trypsin/0.05% EDTA, pelleted, and resuspended in 1 ml of fresh media with a 22-gauge needle to disperse clumps before hemocytometer counting in trypan blue. Cells were then plated at limiting dilutions in six-well plates and allowed to adhere overnight. Cultures were treated with phosphate-buffered saline, R9, wtNIP, or scNIP for 1 h and irradiated (0-6Gy). Fresh peptides were added every 4 h until 24 h after IR, when the medium was replaced with peptide-free medium. Cultures were incubated for 1 to 2 days, harvested, and stained with 0.5% crystal violet in methanol. Colony number was determined with a dissecting microscope. A population of >50 cells was counted as one colony, and the number of colonies was expressed as a percentage of the value for untreated mock-irradiated control cells. The surviving curves were plotted by linear regression analyses, and the D0 value represents the radiation dose that leads to 37% of survival. To determine the radiosensitizing potential of the peptides compared with other small-molecule inhibitors, we calculated the sensitizing enhancement ratio (SER) based on the dose of radiation required to reduce survival to 37% in the presence of scNIP or wtNIP. The following formula was used:
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Statistics. To establish statistical significance, Student's t test was incorporated. The data were first fit to each experimental group over a dose range of 0 to 6 Gy. Significant differences were established at p < 0.05.
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Results |
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, 2004; Falck et al., 2005; Cerosaletti et al., 2006). In addition, it has been shown that expression of an NBS1 transgene lacking the ATM binding domain in NBS cells leads to a dramatic reduction in ATM activation (Difilippantonio et al., 2005). Because inhibiting NBS1 association with ATM leads to suboptimal ATM activation after IR, the NBS1-ATM interaction can be a novel target for developing radiosensitizers. One approach to inhibiting NBS1-ATM interaction would be to use small peptides containing the conserved C-terminal sequence, which will presumably compete with endogenous NBS1-ATM interactions (Fig. 1A). Therefore, we designed peptides containing two functional domains: one an interfering domain that will inhibit the NBS1-ATM association, and the other an internalization domain that will transport the interfering peptides into cells. For the interfering domain, we used the amino acid sequences containing the conserved C-terminal motif of NBS1 as shown in Fig. 1B. This sequence contains the shortest ATM binding motif based on in vitro data (data not shown). For the internalization domain, we used a polyarginine sequence, which has been shown to have a significant efficiency of transporting small peptides and proteins across the plasma membrane (Fuchs and Raines, 2004; Deshayes et al., 2005) Three peptides were generated, including the R9-alone, and a wtNIP corresponding to amino acids 73to 44 of human NBS1. The third peptide was designed as a negative control, using a random sequence generator to produce a peptide in which amino acids 735 to 744 of NBS1 were scrambled (scNIP). These peptides were labeled with a biotin tag at their N termini for detection in vitro.
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wtNIP Abrogated the NBS1-ATM Interaction. To investigate whether R9-conjugated NIP peptides could inhibit NBS1-ATM interactions, we performed coimmunoprecipitation experiments in cells treated with the NIP peptides. Four hours after peptide treatment, HeLa cells were harvested and subjected to immunoprecipitation using an anti-NBS1 antibody. The immunoprecipitates were then blotted with anti-ATM, NBS1, and MRE11 antibodies. We observed a normal level of ATM-NBS1 association in R9-treated cells compared with control cells. However, in wtNIP-treated cells, NBS1 was no longer able to bring down ATM (Fig. 3). Furthermore, the wtNIP affected only the NBS1-ATM interaction and did not interfere with NBS1 binding to MRE11. In contrast, scNIP did not affect the NBS1-ATM interaction. In cells treated with IR, wtNIP showed an effect similar to that in unirradiated cells. These observations demonstrate that wtNIP can abrogate the NBS1-ATM interaction in the absence or the presence of DNA damage.
wtNIP Inhibits IR-Induced -H2AX and NBS1 pSer343 Focus Formation. One of the earliest responses to IR-induced DNA damage is the formation of -H2AX foci, which requires functional ATM (Burma et al., 2001; Furuta et al., 2003). Because wtNIP showed an inhibitory effect on the NBS1-ATM interaction, we investigated whether IR-induced -H2AX focus formation was inhibited by the peptide. Immunofluorescence microscopy was used to detect the presence of -H2AX foci in mock-irradiated or irradiated cells in the presence of R9, wtNIP or scNIP. The average number of -H2AX foci/nucleus in HeLa cells significantly increased after IR in cells treated with R9 (42 foci/nucleus) or scNIP (41 foci/nucleus), whereas cells treated with wtNIP displayed only an average of 6.9 -H2AX foci/nucleus, similar to that of mock-irradiated cells (Fig. 4). Similar results were observed in DU-145 cells, whereas R9 or scNIP exposure did not affect IR-induced focus formation, and wtNIP showed significantly reduced H2AX foci/nucleus (Supplemental Fig. 2). Therefore, IR-induced -H2AX focus formation can be inhibited by wt-NIP.
To further support the idea that wtNIP can inhibit ATM-mediated DNA damage pathways, we investigated IR-induced NBS1 focus formation, an event considered to be an ATM-dependent process at the sites of DSBs (Lim et al., 2000). NBS1 foci are a result of ATM-mediated NBS1 phosphorylation on serine 343. Using an anti-phospho-Ser343 NBS1 antibody, we observed that NBS1 phosphorylation was significantly inhibited in cells treated with wtNIP compared with those treated with R9 or scNIP (Fig. 5A and supplemental Fig. 3A). The average number of foci in mock-irradiated HeLa cells was 6, 8, and 6 for R9, wtNIP, and scNIP, respectively. Cells treated with R9 or scNIP displayed 25 and 31 foci per nucleus, whereas cells treated with wtNIP showed only 6 foci per nucleus after treatment with 6-Gy IR (Fig. 5B).
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It is important to note that there was a low level of back-ground focus formation for both NBS1 and -H2AX phosphorylation, which has been correlated to mitosis in normally growing mammalian cell cultures (McManus and Hendzel, 2005).
wtNIP Increases Radiation Sensitivity. We then tested whether exposure to the NIP peptides will increase cellular radiosensitivity using the colony forming assay. Figure 6A depicts the survival curves for HeLa cells treated with R9, wtNIP, or scNIP over a dose range of 0 to 6 Gy. We found that neither R9 nor scNIP affects radiosensitivity, whereas wtNIP can significantly decrease IR-induced survival. Radiation survival curves were characterized based on D0 to define the effect of NIP effect on radiosensitivity. D0 represents the mean lethal dose required for 37% survival and is a measure of the intrinsic radiosensitivity of the cell. D0 values for HeLa treated with wtNIP were 1.9 compared with 3.0 for cells treated with scNIP. To establish the statistical significance of wtNIP-induced radiosensitivity, Student's t test (paired two-sample for means) was incorporated. The data were first fit to each experimental group over a dose range of 0 to 6 Gy. Significant differences (p < 0.05) in clonogenic survival were observed between cells treated with wtNIP and those treated with DMEM, R9, or scNIP. The SER was 1.58. This is comparable with other tested radiosensitizers, including gemcitabine, 5-fluorouracil, pentoxifylline, vinorelbine, and some ATM-specific radiosensitizers with SERs from 1.1 to 2.5 (Zhang et al., 1998; Lawrence et al., 2001; Robinson and Shewach, 2001; Strunz et al., 2002; Collis et al., 2003; Zhang et al., 2004). These observations have been confirmed in the prostate cancer cell line DU-145 (data not shown) with an SER of 1.46. Taken as a whole, they provide strong evidence for the radiosensitizing potential of the wtNIP peptide.
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). To test this possibility, we performed colony-forming assays in cell lines with defective ATM (GM9607) or DNA-PKcs (M059J). Although treatment with wtNIP led to an increase in radiosensitivity in M059J cells (Fig. 6C) with an SER of 1.83, GM9607 (Fig. 6D) displayed no change in radiosensitivity. Because GM9607 cells are ATM-deficient and have functional ATR and DNA-PKcs, our observations strongly suggest that wtNIP can specifically target ATM, but not ATR or DNA-PKcs, to achieve radiosensitization. |
Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). The development of siRNA also led to the generation of an siRNA that could inhibit ATM function in prostate cancer cells. Both DU-145 and PC-3 cells, when transfected with these plasmids, exhibited an increase in radiosensitivity at clinically relevant radiation doses (Collis et al., 2003). More recently, the use of high-throughput screening has provided a new generation of ATM inhibitors that can be quickly translated to clinical studies. By screening a combinatorial library of compounds around the DNA-PKcs inhibitor LY294002, Hickson et al. (2004) reported a compound (KU55933) to selectively inhibit the ATM kinase. Their studies have shown a significant increase in radiosensitivity in HeLa cells. However, the in vivo radiosensitization effect and the toxicity of the compounds have not been reported. Despite these promising findings, one of the major concerns of developing ATM inhibitors is the uncertainty of pleiotropic effects of such inhibitors. Due to the complex effects associated with malfunction of the protein kinase, the outcome of directly targeting ATM kinase activity can be complicated, in that it is unclear whether the only effect of these reagents will be to confer radiosensitization.
Instead of directly inhibiting the ATM kinase activity to increase radiosensitivity, an alternative approach is to target IR-induced ATM activation, because this will directly lead to an increase in radiosensitivity without interfering with other important functions of ATM in the absence of DNA damage. Because the NBS1-ATM interaction is important for IR-induced activation of ATM, selectively disrupting the signaling pathway would be a novel approach for developing radiosensitizers. Furthermore, because the C-terminal of NBS1 association with ATM is necessary for ATM activation, we reasoned that a small peptide containing a portion of this conserved C-terminal domain (i.e., KEESLADDL) would compete with the NBS1-ATM association in vivo and sensitize tumor cells to radiation. Our data demonstrate that the wild-type NBS1 peptide can be used to inhibit ATM activation and induce radiosensitization.
Because the wtNIP peptide contains the conserved sequence among the phosphatidylinositol 3-kinase interacting proteins, such as ATR-interacting protein and Ku80 (Falck et al., 2005), we further reasoned that wtNIP could possibly interfere with ATR and DNA-PKcs activation. We tested the radiosensitizing effect of the peptides in cells with deficient ATM or DNA-PKcs. If the wtNIP could inhibit ATR or DNA-PKcs, then the ATM-deficient cells should be sensitized. However, the radiosensitivity of GM9607, which lacks ATM but has functional ATR and DNA-PKcs, was not affected by the peptide. In contrast, the DNA-PKcs mutant cells showed an increased radiosensitivity similar to that of HeLa and DU-145 cells treated with wtNIP. These observations therefore demonstrate specific ATM inhibition by the wtNIP peptide.
In summary, we have established a proof of principle in vitro, with results that may lend insight into a novel approach to the development of powerful radiosensitizers for clinical cancer therapy and use the peptides as specific ATM inhibitors for further elucidation of signaling pathways involved in the DNA damage response. However, the use of the polyarginine-mediated NBS1 peptide as a therapeutic agent still faces challenges such as peptide stability, toxicity, tumor specific targeting, and immunogenic effects, etc. Using the current concept to establish an assay for high-throughput screening to identify small molecules that can target the NBS1-ATM interaction will eventually lead to novel radiosensitizers usable for clinical settings. Future studies are also necessary to determine the structure of the NBS1-ATM interaction complex and how wtNIP competes with the interaction.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: DSB, double-strand break; A-T, ataxia-telangiectasia; IR, ionizing radiation; HEAT, huntingtin/elongation factor 3/the 65 kDa 
-regulatory subunit of protein phosphatase 2A/yeast PI-3K TOR1; MRN, Mre11-Rad50-NBS1 complex; NBS1, Nijmegan breakage syndrome; ATM, ataxia telangiectasia mutated; DMEM, Dulbecco's modified Eagle's media; FBS, fetal bovine serum; NIP, NBS1 inhibitory peptide; wtNIP, wild-type NBS1 inhibitory peptide; scNIP, scrambled NBS1 inhibitory peptide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; SER, sensitizing enhancement ratio; ATR, ataxia telangiectasia related; DNA-PKcs, DNA-dependent protein kinase catalytic subunit; siRNA, small interfering RNA; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; KU55933, 2-morpholin-4-yl-6 thianthren-1-yl-pyran-4-one.
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material. 
Address correspondence to: Bo Xu, MD, PhD, Southern Research Institute, Department of Biochemistry and Molecular Biology, 2000 9th Avenue South, Birmingham, AL 35205. E-mail: xu{at}sri.org32
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