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1 KuDOS Pharmaceuticals Ltd., Cambridge Science Park, Milton Road, Cambridge; 2 Northern Institute for Cancer Research, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne; and 3 Wellcome Trust and Cancer Research UK Gurdon Institute and Department of Zoology, Cambridge University, Cambridge, United Kingdom
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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H2AX, NBS1, and SMC1. KU-55933 did not show inhibition of UV light DNA damage induced cellular phosphorylation events. Exposure of cells to KU-55933 resulted in a significant sensitization to the cytotoxic effects of ionizing radiation and to the DNA double-strand break-inducing chemotherapeutic agents, etoposide, doxorubicin, and camptothecin. Inhibition of ATM by KU-55933 also caused a loss of ionizing radiation-induced cell cycle arrest. By contrast, KU-55933 did not potentiate the cytotoxic effects of ionizing radiation on ataxia-telangiectasia cells, nor did it affect their cell cycle profile after DNA damage. We conclude that KU-55933 is a novel, specific, and potent inhibitor of the ATM kinase. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The increased sensitivity of A-T cells to ionizing radiation is not associated with defective repair of DNA single-strand breaks or in the removal of base damage from DNA (17 , 18) . Instead, after exposure to ionizing radiation, A-T cells have been reported to display residual chromosomal breaks (19) , slower DNA DSB rejoining than wild-type cells (20) , and a failure to repair all DNA DSBs (21) , which could account for the radiosensitivity and genome instability associated with A-T (5) . In light of these findings, it has been proposed that inhibition of ATM may give rise to radiosensitization and that ATM therefore represents an attractive target for the development of new radiosensitizing agents (e.g., ref. 22 ).
To date, studies designed to test the effects of ATM inhibition on radiosensitivity have mainly used the relatively nonspecific PIKK and PI3k inhibitors wortmannin and caffeine (e.g., ref. 23 ). Although the ATM-directed p53 response has been shown to be diminished in these experiments, it is most likely that the increased sensitivity to ionizing radiation observed was manifested through the ability of wortmannin and caffeine to inhibit not only ATM but a number of PIKKs, including ataxia-telangiectasia and Rad3-related kinase (ATR), the DNA-dependent protein kinase (DNA-PK), as well as PI3K (e.g., refs. 23, 24, 25, 26, 27, 28 ). These small molecule approaches have demonstrated the feasibility of achieving sensitization to ionizing radiation but have not proven that such sensitization could be achieved by inhibition of ATM alone. Furthermore, wortmannin probably hits too many targets to be a viable clinical agent for the sensitization of tumors to ionizing radiation because the drug covalently modifies large numbers of proteins and is consequently unlikely to be tolerated in vivo at radiosensitizing concentrations. In the case of caffeine, the sensitization is by the nonspecific targeting of ATM, ATR, and DNA-PK and the effective concentration is clinically prohibitive. Indeed, serum concentrations of 1 mmol/L, which are required to achieve radiosensitization, are associated with fatal tachyarrythmias (22) .
By screening a combinatorial library based around the nonspecific PI3K and DNA-PK inhibitor LY294002 (24 , 28) , we have identified a novel, specific and very potent small molecule inhibitor of ATM, 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one, termed KU-55933. In this article, we describe the characterization of KU-55933 by biochemical means and demonstrate the biological effects of KU-55933 in cell culture model systems to determine the function and specificity of the molecule. Our data not only provide characterization of a novel compound that acts as a radio- and chemosensitizor but also describe a molecular tool that can be used to study the many signaling facets of ATM in the cellular responses to DNA damage. The data also provide a starting point for the development of more potent and pharmaceutically acceptable ATM inhibitors that may ultimately be put to use in clinical settings.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Lines and Culture.
The human tumor cell lines U2OS, LoVo, SW620, and HeLa B, the A-T fibroblast cell line AT4, and the normal human fibroblast 1BR were cultured as monolayers in DMEM (supplemented with 10% v/v FCS, 100 units/mL penicillin, and 100 µg/mL streptomycin). Glutamine was added to a final concentration of 2 mmol/L. The Chinese hamster cell lines V3 and V3YAC (a kind gift from Penny Jeggo, University of Sussex, Brighton, United Kingdom; ref. 29
) were grown in RPMI 1640 containing glutamine (Sigma) and 10% FCS (Sigma). The V3YAC cell line was maintained under Geneticin selection at a final concentration of 500 µg/mL to ensure retention of the YAC.
Purified Enzyme Assays.
ATM for use in the in vitro assay was obtained from HeLa nuclear extract (Computer Cell Culture Centre, Mons, Belgium) by immunoprecipitation with rabbit polyclonal antiserum raised to the COOH-terminal 400 amino acids of ATM in buffer containing 25 mmol/L HEPES (pH 7.4), 2 mmol/L MgCl2, 250 mmol/L KCl, 500 µmol/L EDTA, 100 µmol/L Na3VO4, 10% v/v glycerol, and 0.1% v/v Igepal. ATM-antibody complexes were isolated from nuclear extract by incubating with protein A-Sepharose beads for 1 hour and then through centrifugation to recover the beads. In the well of a 96-well plate, ATM-containing Sepharose beads were incubated with 1 µg of substrate glutathione S-transferase–p53N66 (NH2-terminal 66 amino acids of p53 fused to glutathione S-transferase) in ATM assay buffer [25 mmol/L HEPES (pH 7.4), 75 mmol/L NaCl, 3 mmol/L MgCl2, 2 mmol/L MnCl2, 50 µmol/L Na3VO4, 500 µmol/L DTT, and 5% v/v glycerol] at 37°C in the presence or absence of inhibitor. After 10 minutes with gentle shaking, ATP was added to a final concentration of 50 µmol/L and the reaction continued at 37°C for an additional 1 hour. The plate was centrifuged at 250 x g for 10 minutes (4°C) to remove the ATM-containing beads, and the supernatant was removed and transferred to a white opaque 96-well plate and incubated at room temperature for 1.5 hours to allow glutathione S-transferase–p53N66 binding. This plate was then washed with PBS, blotted dry, and analyzed by a standard ELISA technique with a phospho-serine 15 p53 antibody (Cell Signaling Technology, Inc., Beverly, MA). The detection of phosphorylated glutathione S-transferase–p53N66 substrate was performed in combination with a goat antimouse horseradish peroxidase-conjugated secondary antibody (Pierce, Rockford, IL). Enhanced chemiluminescence solution (NEN, Boston, MA) was used to produce a signal and chemiluminescent detection was carried out via a TopCount (Packard, Meriden, CT) plate reader.
ATR kinase activity was determined as for ATM except that the kinase was extracted by immunoprecipitation with antiserum raised to amino acids 400–480 of ATR (30) . mTOR protein was isolated from HeLa cell cytoplasmic extract by immunoprecipitation and activity determined essentially as previously described (31) with recombinant PHAS-1 as a substrate. The DNA-PK (24 , 32) , PI3K (28) , and PI4K (33) assays were performed essentially as described previously.
The concentration of inhibitor that achieved 50% inhibition of the enzyme (IC50) for KU-55933 (and KU-58050 for ATM) in all assays was derived from sigmoidal plots with GraphPad Prism version 3.03 for Windows (GraphPad Software, San Diego, CA). Enzyme activity was plotted against concentration of inhibitor compound, and the data were calculated from the means of at least three independent experiments.
Kinetic Analysis.
The activity of ATM was assayed using the ELISA technique described above. For inhibition experiments, KU-55933 was added to the reaction mixture and preincubated for 10 minutes before addition of ATP. Inhibition was performed at 0, 1, 5, 10, 20, or 40 nmol/L KU-55933 in varying concentrations of ATP (0.5 to 100 µmol/L). Ki was determined following standard Michaelis-Menten kinetics and with the Lineweaver Burke double reciprocal plot.
Cell-based Determinations of ATM Inhibition.
U2OS cells [which have a robust p53 response (34)
] were exposed to ionizing radiation (3, 5, or 15 Gy) or UV (5 or 50 J/m2) and the ATM response determined by Western blot analysis of p53 serine 15 phosphorylation and stabilization of wild-type p53. Whole cell extracts were obtained from each time point, proteins separated by SDS-PAGE, and the ATM-specific increase in phosphorylated serine 15 measured with a p53 phospho-serine 15 specific antibody (Cell Signaling Technology, Inc.). Overall p53 stabilization with time was also observed with a p53-specific antibody (DO-1; Santa Cruz Biotechnology, Santa Cruz, CA). Similarly, for studying ATM-dependent phosphorylations on H2AX, CHK1, NBS1, and SMC1, the following antibodies were used: CHK1 phospho-serine 345 and NBS1 phospho-serine 343 antibodies were purchased from Cell Signaling Technology, Inc. Histone H2A (H-124) and CHK1 antibodies were from Santa Cruz Biotechnology. SMC1 and SMC1 phospho-serine 966 antibodies were from Bethyl Laboratories (Montgomery, TX). NBS1 antibody was from Oncogene (Cambridge, MA), and H2AX phospho-serine 139 (JBW301) was from Upstate Technology (Charlottesville, VA). For determination of a cellular IC50 for KU-55933, the peak response time for p53 serine 15 phosphorylation of 2 hours was used to monitor inhibition of ATM. The compounds were titrated onto cells and preincubated for 1 hour before ionizing radiation. Using scanning densitometry, the percentage inhibition relative to vehicle control was calculated, and the IC50 value was calculated as for the in vitro determinations.
Cytotoxicity Studies.
The effect of ATM inhibition on cellular survival was measured by clonogenic assays. For HeLa cells, tissue culture treated 6-well plates were seeded at an appropriate concentration to give 100 to 200 colonies per well and returned to the incubator to allow the cells to attach. Four hours later, compound or vehicle control was added to the cells. For the standard clonogenic survival studies, the cells were incubated for 1 hour in the presence of inhibitor before irradiation or the addition of chemotherapeutic compounds. The cells were then incubated for 16 hours before the media were replaced with fresh DMEM in the absence of drugs. After 7 to 14 days (depending on cell type), colonies formed were fixed and stained with Giemsa (Sigma) and scored with a ColCount-automated colony counter (Oxford Optronics Ltd., Oxford, United Kingdom). The data were calculated as surviving fractions with respect to vehicle controls ± SE. For the analysis of the cytotoxic effects of etoposide on LoVo, SW620, V3, and V3YAC cells, the clonogenic assay was performed as described previously (32)
: briefly, exponentially growing cells in 6-well plates were exposed to drugs for 16 hours, harvested by trypsinization, counted, and seeded into 10-cm Petri dishes at densities varying from 100 to 100,000 cells per dish in fresh medium for colony formation. Colonies were stained with crystal violet after 7 to 14 days, counted as above and the data were normalized to vehicle controls. The sensitizer enhancement ratio was calculated as the surviving fraction of cells in the absence of KU-55933 divided by the surviving fraction of cells in the presence of KU-55933 for any given dose or concentration of cytotoxic insult.
Cell Cycle Analysis.
1BR or AT4 cells were seeded in 10-cm Petri dishes and treated on day 2 (80 to 90% confluence). Cells were preincubated for 1 hour with KU-55933 or vehicle control and then exposed to 5 Gy of ionizing radiation (Faxitron 43855D, dose rate 1 Gy/min). Time courses of cell cycle distribution were performed (data not shown), and the optimal time for discrimination of populations was selected as 16 hours. All subsequent experiments were performed at the 16-hour time point. Cells were stained with propidium iodide according to standard protocols (35)
and analyzed by FACS with a FACScalibur (BD Biosciences, Franklin Lakes, NJ). Exponentially growing (50–70% confluent) SW620 cells in 60 mm dishes were exposed to KU-55933 or DMSO for 1 h before addition of etoposide (final concentration of 0.1 and 1 µmol/L) for 16 h before harvesting, propidium iodide staining and analysis as above.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. By analysis of concentration versus percentage inhibition curves, it was determined that the IC50 for KU-55933 was 12.9 ± 0.1 nmol/L. Counterscreening this molecule against other members of the PIKK family (Table 1)
demonstrated that the compound exhibited at least a 100-fold differential in selectivity. Furthermore, in screening a commercially available panel of 60 kinases (Upstate Biotechnology, Lake Placid, NY), it was found that at a single point concentration of 10 µmol/L, KU-55933 did not significantly inhibit any kinase tested (data not shown). By contrast, the related molecule KU-58050 (Fig. 1)
, which has a piperidine replacement to the morpholine moiety of KU-55933, had a calculated IC50 against ATM of only 2.96 ± 0.44 µmol/L. This result highlights the importance of the morpholine oxygen for ATM inhibition. The importance of a hydrogen bond mediated between the morpholine oxygen of LY294002 and the hinge region of PI3k p110 was highlighted in a recent crystal structure determination (36)
. KU-58050 thus serves as a useful negative control compound for the cellular studies described below because its IC50 for ATM is 
230 times higher than that for KU-55933.
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Inhibition of ATM-dependent Cellular Phosphorylation Events.
To assess the function of ATM at the cellular level, we looked at the phosphorylation status of serine 15 of p53, a marker of ATM activity (10
, 11)
after treating U2OS osteosarcoma cells with ionizing radiation. Western blot analysis with a phospho-serine 15 specific antibody showed that the predicted response was evident after 10 minutes and was maintained up to 4 hours after ionizing radiation induction [Fig. 2A(i)
, top panel]. The classic stabilization of the p53 protein after ionizing radiation was also clearly observed [Fig. 2A(i)
, bottom panel]. Strikingly, when this experiment was carried out in the presence of 10 µmol/L KU-55933, the ionizing radiation-induced p53 serine 15 phosphorylation was totally absent [Fig. 2A(ii)
, top panel]. Notably, in this experiment, the stabilization of the wild-type p53 protein was evident after ionizing radiation treatment but with somewhat delayed kinetics compared with that observed in the control cells [Fig. 2A(ii)
, bottom panel). This proved that KU-55933 was active at the cellular level in ablating a well-characterized ATM-dependent phosphorylation event. By monitoring p53 serine 15 phosphorylation at the 2-hour time point after ionizing radiation, we went on to analyze effective concentrations of KU-55933 and KU-58050 required to inhibit cellular ATM function. Fig. 2B
reveals that KU-55933 has a dose-dependent effect in inhibiting this ATM-dependent phosphorylation event with an estimated IC50 of 300 nmol/L. Furthermore, and consistent with results obtained in the enzymatic assays, the data in Fig. 2B
show that KU-58050 does not prevent the ATM-dependent phosphorylation of p53 serine 15 until a dose of 30 µmol/L.
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shows that the addition of KU-55933 has no appreciable effects on UV-induced phosphorylation of H2AX on serine 139 (37)
, NBS1 on serine 343 (38)
, CHK1 on serine 345 (39)
, and SMC1 on serine 966 (40)
. In stark contrast to the UV responses, KU-55933 ablates the ionizing radiation-induced phosphorylation of these ATM substrates. Similar data (not shown) were also obtained for other ATM-mediated phosphorylation events on CHK2, MDC1, TopBP1, and BRCA1 in response to ionizing radiation but not to UV-mediated DNA damage. Taken together, these results additionally highlight the specific nature of the inhibitor and reveal that at the dose used (10 µmol/L), the molecule is highly specific at inhibiting ATM-dependent ionizing radiation-mediated DNA damage responses in living cells in line with our findings in the biochemical enzymatic assays (Table 1)
. These results also clearly demonstrate that KU-55933 does not appreciably inhibit the cellular activities of the most closely related enzyme to ATM, ATR, which is a known early responder to UV-induced DNA damage.
Cellular Radiosensitization by KU-55933.
The effects of KU-55933 and KU-58050 on ionizing radiation-induced cytotoxicity were studied by in vitro clonogenic survival assays on the human tumor cell line HeLa (Fig. 3A)
and on A-T fibroblasts (Fig. 3B)
. KU-55933 was found to maximally potentiate ionizing radiation-induced cell killing at a concentration of 10 µmol/L and was hence used at this dose in these and later experiments (data not shown). Note, that at a concentration of 1 µmol/L the sensitizer enhancement ratio at 2 Gy was 1.8 on HeLa cells (data not shown). It should be noted that neither of the two compounds alone, at concentrations up to 10 µmol/L, were found to significantly reduce the clonogenic survival of the cell lines tested. Fig. 3A
clearly reveals that KU-55933 sensitizes HeLa cells to a range of ionizing radiation doses whereas KU-58050 does not. In the case of HeLa cells, the sensitizer enhancement ratio at 2 Gy, for 10 µmol/L of KU-55933 was 2.6 ± 0.6. Similar data has been obtained with the LoVo and SW620 cell lines (data not shown). Highlighting the specific nature of ATM inhibition, we found that A-T fibroblasts were not sensitized to the effects of KU-55933 in combination with ionizing radiation (Fig. 3B)
. This is consistent with KU-55933 specifically inhibiting ATM but not the other DNA damage-activated PIKKs, ATR, and DNA-PK (Table 1)
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. Through clonogenic survival assays we found that KU-55933 sensitizes HeLa cells to the cytotoxic effects of the topoisomerase II inhibitors etoposide, doxorubicin, amsacrine (Fig. 4A–C)
, and the topoisomerase I inhibitor camptothecin (Fig. 4D)
. The negative control molecule KU-58050 had no impact on cellular survival in response to either etoposide (Fig. 4A)
or camptothecin (Fig. 4D)
, again highlighting the specific nature of KU-55933. To additionally investigate the sensitization of cells to the cytotoxic effects of etoposide by KU-55933, equipotent doses of etoposide were administered to LoVo (1 µmol/L) and SW620 (1 µmol/L) cells, as well as to the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) –/– V3 (0.3 µmol/L) and the DNA-PKcs complemented derivative V3-YAC (1 µmol/L) cells (Table 2)
. The DNA-PKcs–deficient V3 line was greatly sensitized by the ATM inhibitor, thus indicating the cumulative effects of the loss of a key DNA damage signaling protein and a key component of a major DNA double-strand break repair pathway, nonhomologous end joining.
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Effects of KU-55933 on the Cell Cycle.
One of the hallmarks of A-T cells is their inability to mediate the G1-S–, intra-S–, and G2-M–phase DNA damage checkpoints in response to ionizing radiation (7
, 9)
. Ultimately, in response to DNA double-strand breaks, A-T cells will arrest at the G2 stage of the cell cycle (9)
. To study the effects of ATM inhibition via KU-55933 on cell cycle distribution, we have analyzed asynchronous cells pre- and postexposure to 5 Gy of ionizing radiation or to etoposide. There is no effect on the cell cycle distributions from exposure to KU-55933 alone (Fig. 5A–C)
. After exposure to ionizing radiation, the distribution of the cells shifts, as shown in Fig. 5A
. For 1BR cells, this shift is manifest as clear G1 and G2 peaks in the absence of KU-55933, consistent with the cells accumulating at the G1-S and G2-M checkpoints in response to ionizing radiation induced DNA damage. In the presence of KU-55933 however, these responses of 1BR cells are significantly altered (Fig. 5A)
, with the distribution showing a large accumulation of cells in G2-M. The cell cycle distribution of SW620 cells after exposure to etoposide was also affected by KU-55933 (Fig. 5C)
, again with a significant increase in the G2-M population. These data are therefore consistent with KU-55933 preventing the normal ATM-dependent cell cycle checkpoint responses to ionizing radiation, leading to the eventual activation of an ATM-independent arrest at the G2-M stage.
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, the cell cycle distribution of the AT4 cells was altered after exposure to ionizing radiation with the cells arresting predominantly in G2, consistent with published data for an asynchronous population of A-T cells (9)
. Significantly, this response to ionizing radiation was not altered in the presence of KU-55933 (Fig. 5B)
, revealing that the effects of the molecule on cell cycle distribution after ionizing radiation are ATM dependent. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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10 fold more potency against ATM in comparison to ATR (23)
and hence can be ineffectual in studies where discrimination between these related DNA damage-activated kinases is required.
As an initial study to understand the key attributes for the binding of KU-55933 to the ATP active site of ATM, we produced the molecule KU-58050. Similar in all respects to KU-55933 other than an oxygen in the cyclic amine side chain, KU-58050 was in comparison a very poor ATM inhibitor with an IC50 > 200 times than that seen for KU-55933. The chemical substitution of replacing the morpholine group with a piperidine highlights the importance of the morpholine oxygen. On the basis of the crystal structure of the porcine PI3K p110 subunit in complex with LY294002 (36)
, the morpholine is most probably hydrogen bonding within the hinge region of the ATP-binding site of ATM. This of course can only be fully established from a crystal structure determination of ATM bound with KU-55933. Additional structure-activity relationships surrounding KU-55933 are taking place at present to give a better understanding about other key features of the molecule required for its strong potency and selectivity toward ATM. Although a weakly active molecule against ATM, KU-58050 acts as a useful control molecule to study the specificity of KU-55933 as described in the work presented here.
The role of the ATM protein in the stabilization of p53 in response to DNA double-strand break-inducing agents is very well established (4 , 10 , 11 , 13 , 44) . The direct phosphorylation of p53 on serine 15 by ATM is a key early event in this process (10 , 11) . We have shown that KU-55933 prevents this cellular phosphorylation event in a concentration-dependent manner. Interestingly, in the U2OS cell line we have studied, the stabilization of p53 still occurs in the presence of KU-55933, albeit in a delayed fashion. This is similar to the suboptimal induction of p53 seen in some A-T cell lines (44) . It has been speculated that the eventual induction of p53 in such A-T cells occurs by the back-up or partially redundant action of ATR in phosphorylating p53 on serine 15 in the absence of ATM (3 , 4 , 30) . Notably, however, we did not see any serine 15 phosphorylation after ionizing radiation in the presence of KU-55933 suggesting that, over the time course of this experiment, ATR is not activated to a significant degree and that some other mechanism(s) is involved in the gross stabilization of p53. Another key phosphorylation event on p53, which is believed to be involved in disrupting the p53/Mdm2 interaction, leading to p53 stabilization, is on serine 20 (45) . This phosphorylation is thought to be mediated by the downstream target of ATM, CHK2. Consistent with KU-55933 efficiently preventing ATM activation, we have also been unable to see serine 20 phosphorylation of p53 in response to ionizing radiation in cells treated with the drug (data not shown).
A number of other substrates for ATM have been discovered and similar to p53, these have generally been found to also be cellular substrates for ATR in response to UV irradiation (3 , 4) . We have shown quite clearly that for a range of these substrates that we have tested (e.g., SMC-1, NBS-1, and CHK-1) in response to the ionizing radiation treatment of cells, KU-55933 almost completely ablates these phosphorylation events. Notably, however, is that the phosphorylation of H2AX on serine 139 is not completely inhibited by KU-55933 in response to ionizing radiation. This most probably reflects the fact that this site is also targeted by DNA-PK after ionizing radiation (46) . As one would expect from the in vitro-specific nature of KU-55933, the molecule did not prevent UV phosphorylation events on the proteins we studied, most probably reflecting the fact that the agent does not inhibit these ATR-dependent events. Through the use of KU-55933, it will be intriguing to study other ATM-dependent sites of phosphorylation, in particular, the serine 1981 autophosphorylation site (47)
A classic hallmark of cells derived from A-T patients is their extreme sensitivity to ionizing radiation (5 , 19 , 21) . We have shown that KU-55933 can recapitulate this phenotype in a range of cell lines and that it does not additionally sensitize A-T cells to the effects of ionizing radiation. By contrast, we were able to additionally sensitize DNA-PKcs–deficient cells to the effects of etoposide indicating the distinct nature of the ATM and DNA-PK pathways in response to DNA double-strand breaks. As it has been shown that certain cell types derived from A-T mice are not hypersensitive to ionizing radiation (48) , it will be interesting to evaluate the radiosensitizing effects of KU-55933 on a range of different cell types and tissues. For the ultimate development of a clinical radiosensitizor, the effects of ATM inhibition on normal tissues must also be evaluated and understood. KU-55933 will be a useful tool to address this key issue.
Hypersensitivity to DNA double-strand break-inducing agents is a characteristic of ATM-deficient cells (5) . In this study, we have shown that KU-55933 can sensitize ATM-proficient cells to DNA double-strand break-inducing agents other than ionizing radiation. The topoisomerase I inhibitor camptothecin, although initially producing a DNA single-strand break, ultimately produces a DNA double-strand break during replication, which is presumed to be the lethal lesion (49) . KU-55933 sensitized cells to camptothecin, consistent with the known sensitivity of A-T cells to this particular agent (50) . Similarly, the topoisomerase II inhibitors used had their lethal effects (51) enhanced in the presence of the ATM inhibitor. Again, this is in keeping with observations with cells defective in ATM (41, 42, 43) . One might have expected some differences between the phenotypes of A-T cells and ATM-inhibited cells because inhibited ATM could act in a dominant-negative fashion, whereas in A-T cells, the protein is generally not present. However in these initial studies, we did not observe anything strikingly different between the two scenarios. Finally, and in line with the fact that A-T cells are not markedly hypersensitive to DNA-alkylating agents or interstrand cross-linking agents (5 , 43 , 52 , 53) , we found that KU-55933 did not sensitize cells to such drugs.
ATM-deficient cells are well known as being defective in the major cell cycle checkpoints after DNA damage (4, 5, 6, 7, 8, 9) . Therefore, it would be expected that the addition of an ATM inhibitor to ATM-proficient cells would yield a similar phenotype. Indeed, we found that KU-55933 perturbed the checkpoint effects of 1BR cells in response to ionizing radiation and SW620 cells in response to etoposide, producing a similar shift in cell cycle distribution to that seen in irradiated A-T cells. Moreover, the molecule did not affect the ionizing radiation-induced cell cycle profile of A-T cells in response to ionizing radiation, once again highlighting the highly specific actions of this molecule. Clearly, additional studies to monitor the effects of KU-55933 at specific checkpoints are warranted.
The discovery and characterization of KU-55933 as a specific and cellularly active ATM inhibitor raises the exciting prospect that derivatives of this molecule may serve as novel radio- and chemosensitizors in clinical settings. Recent reports have highlighted the use of antisense or small interfering RNAs to deplete ATM function in human tumor cells as a means of radiosensitization (54, 55, 56, 57) . To our knowledge, however, our work is the first to evaluate a small molecule approach to target ATM. Taken together, with data obtained through other approaches, our results reinforce the view that the ATM protein kinase represents a tractable and highly attractive target for the development of novel types of anticancer agent.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: I. Hickson and Y. Zhao contributed equally to this work.
Requests for reprints: Graeme C. M. Smith, KuDOS Pharmaceuticals Ltd., Cambridge Science Park, Milton Road, Cambridge CB4 0WG, United Kingdom. Phone: 44-0-1223-719719; Fax: 44-0-1223-719720; E-mail: gcmsmith{at}kudospharma.co.uk
Received 7/30/04. Revised 10/15/04. Accepted 10/22/04.
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H. Jiang, H. C. Reinhardt, J. Bartkova, J. Tommiska, C. Blomqvist, H. Nevanlinna, J. Bartek, M. B. Yaffe, and M. T. Hemann The combined status of ATM and p53 link tumor development with therapeutic response Genes & Dev., August 15, 2009; 23(16): 1895 - 1909. [Abstract] [Full Text] [PDF]
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Y.-W. Zhang, T. L. Jones, S. E. Martin, N. J. Caplen, and Y. Pommier Implication of Checkpoint Kinase-dependent Up-regulation of Ribonucleotide Reductase R2 in DNA Damage Response J. Biol. Chem., July 3, 2009; 284(27): 18085 - 18095. [Abstract] [Full Text] [PDF]
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D. Kumari, V. Somma, A. J. Nakamura, W. M. Bonner, E. D'Ambrosio, and K. Usdin The role of DNA damage response pathways in chromosome fragility in Fragile X syndrome Nucleic Acids Res., July 1, 2009; 37(13): 4385 - 4392. [Abstract] [Full Text] [PDF]
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 L. J. Martin, Z. Liu, J. Pipino, B. Chestnut, and M. A. Landek Molecular Regulation of DNA Damage-Induced Apoptosis in Neurons of Cerebral Cortex Cereb Cortex, June 1, 2009; 19(6): 1273 - 1293. [Abstract] [Full Text] [PDF]
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C.-Y. Tsai, A. S. Ray, D. B. Tumas, M. J. Keating, H. Reiser, and W. Plunkett Targeting DNA Repair in Chronic Lymphocytic Leukemia Cells with a Novel Acyclic Nucleotide Analogue, GS-9219 Clin. Cancer Res., June 1, 2009; 15(11): 3760 - 3769. [Abstract] [Full Text] [PDF]
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S. A. Stuart and J. Y. J. Wang Ionizing Radiation Induces ATM-independent Degradation of p21Cip1 in Transformed Cells J. Biol. Chem., May 29, 2009; 284(22): 15061 - 15070. [Abstract] [Full Text] [PDF]
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 S. Badie, C. Liao, M. Thanasoula, P. Barber, M. A. Hill, and M. Tarsounas RAD51C facilitates checkpoint signaling by promoting CHK2 phosphorylation J. Cell Biol., May 18, 2009; 185(4): 587 - 600. [Abstract] [Full Text] [PDF]
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I. R. Powley, A. Kondrashov, L. A. Young, H. C. Dobbyn, K. Hill, I. G. Cannell, M. Stoneley, Y.-W. Kong, J. A. Cotes, G. C.M. Smith, et al. Translational reprogramming following UVB irradiation is mediated by DNA-PKcs and allows selective recruitment to the polysomes of mRNAs encoding DNA repair enzymes Genes & Dev., May 15, 2009; 23(10): 1207 - 1220. [Abstract] [Full Text] [PDF]
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V. N. Ivanov, H. Zhou, M. A. Partridge, and T. K. Hei Inhibition of Ataxia Telangiectasia Mutated Kinase Activity Enhances TRAIL-Mediated Apoptosis in Human Melanoma Cells Cancer Res., April 15, 2009; 69(8): 3510 - 3519. [Abstract] [Full Text] [PDF]
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A. Sak, S. Grehl, M. Engelhard, A. Wierlemann, H.-P. Kaelberlah, P. Erichsen, C. Pottgen, M. Groneberg, and M. Stuschke Long-Term In vivo Effects of Cisplatin on {gamma}-H2AX Foci Signaling in Peripheral Lymphocytes of Tumor Patients After Irradiation Clin. Cancer Res., April 15, 2009; 15(8): 2927 - 2934. [Abstract] [Full Text] [PDF]
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 A. Soriani, A. Zingoni, C. Cerboni, M. L. Iannitto, M. R. Ricciardi, V. Di Gialleonardo, M. Cippitelli, C. Fionda, M. T. Petrucci, A. Guarini, et al. ATM-ATR-dependent up-regulation of DNAM-1 and NKG2D ligands on multiple myeloma cells by therapeutic agents results in enhanced NK-cell susceptibility and is associated with a senescent phenotype Blood, April 9, 2009; 113(15): 3503 - 3511. [Abstract] [Full Text] [PDF]
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 A. Kotnis, L. Du, C. Liu, S. W Popov, and Q. Pan-Hammarstrom Non-homologous end joining in class switch recombination: the beginning of the end Phil Trans R Soc B, March 12, 2009; 364(1517): 653 - 665. [Abstract] [Full Text] [PDF]
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R. A. Chanoux, B. Yin, K. A. Urtishak, A. Asare, C. H. Bassing, and E. J. Brown ATR and H2AX Cooperate in Maintaining Genome Stability under Replication Stress J. Biol. Chem., February 27, 2009; 284(9): 5994 - 6003. [Abstract] [Full Text] [PDF]
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S. Solier, O. Sordet, K. W. Kohn, and Y. Pommier Death Receptor-Induced Activation of the Chk2- and Histone H2AX-Associated DNA Damage Response Pathways Mol. Cell. Biol., January 1, 2009; 29(1): 68 - 82. [Abstract] [Full Text] [PDF]
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M. Hingorani, C. L. White, A. Merron, I. Peerlinck, M. E. Gore, A. Slade, S. D. Scott, C. M. Nutting, H. S. Pandha, A. A. Melcher, et al. Inhibition of Repair of Radiation-Induced DNA Damage Enhances Gene Expression from Replication-Defective Adenoviral Vectors Cancer Res., December 1, 2008; 68(23): 9771 - 9778. [Abstract] [Full Text] [PDF]
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 A. L. Medhurst, D. O. Warmerdam, I. Akerman, E. H. Verwayen, R. Kanaar, V. A. J. Smits, and N. D. Lakin ATR and Rad17 collaborate in modulating Rad9 localisation at sites of DNA damage J. Cell Sci., December 1, 2008; 121(23): 3933 - 3940. [Abstract] [Full Text] [PDF]
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 J. Blickwedehl, M. Agarwal, C. Seong, R. K. Pandita, T. Melendy, P. Sung, T. K. Pandita, and N. Bangia Role for proteasome activator PA200 and postglutamyl proteasome activity in genomic stability PNAS, October 21, 2008; 105(42): 16165 - 16170. [Abstract] [Full Text] [PDF]
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A. Kinner, W. Wu, C. Staudt, and G. Iliakis {gamma}-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin Nucleic Acids Res., October 1, 2008; 36(17): 5678 - 5694. [Abstract] [Full Text] [PDF]
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M. D. Rainey, M. E. Charlton, R. V. Stanton, and M. B. Kastan Transient Inhibition of ATM Kinase Is Sufficient to Enhance Cellular Sensitivity to Ionizing Radiation Cancer Res., September 15, 2008; 68(18): 7466 - 7474. [Abstract] [Full Text] [PDF]
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S. Burdak-Rothkamm, K. Rothkamm, and K. M. Prise ATM Acts Downstream of ATR in the DNA Damage Response Signaling of Bystander Cells Cancer Res., September 1, 2008; 68(17): 7059 - 7065. [Abstract] [Full Text] [PDF]
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C. Valli, G. Paroni, A. M. Di Francesco, R. Riccardi, M. Tavecchio, E. Erba, A. Boldetti, M. Gianni', M. Fratelli, C. Pisano, et al. Atypical retinoids ST1926 and CD437 are S-phase-specific agents causing DNA double-strand breaks: significance for the cytotoxic and antiproliferative activity Mol. Cancer Ther., September 1, 2008; 7(9): 2941 - 2954. [Abstract] [Full Text] [PDF]
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J. Guirouilh-Barbat, C. Redon, and Y. Pommier Transcription-coupled DNA Double-Strand Breaks Are Mediated via the Nucleotide Excision Repair and the Mre11-Rad50-Nbs1 Complex Mol. Biol. Cell, September 1, 2008; 19(9): 3969 - 3981. [Abstract] [Full Text] [PDF]
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B. A. Stohr and E. H. Blackburn ATM Mediates Cytotoxicity of a Mutant Telomerase RNA in Human Cancer Cells Cancer Res., July 1, 2008; 68(13): 5309 - 5317. [Abstract] [Full Text] [PDF]
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T. T. Koll, S. S. Feis, M. H. Wright, M. M. Teniola, M. M. Richardson, A. I. Robles, J. Bradsher, J. Capala, and L. Varticovski HSP90 inhibitor, DMAG, synergizes with radiation of lung cancer cells by interfering with base excision and ATM-mediated DNA repair Mol. Cancer Ther., July 1, 2008; 7(7): 1985 - 1992. [Abstract] [Full Text] [PDF]
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 E. Soutoglou and T. Misteli Activation of the Cellular DNA Damage Response in the Absence of DNA Lesions Science, June 13, 2008; 320(5882): 1507 - 1510. [Abstract] [Full Text] [PDF]
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 X. Zhao, R. J. Madden-Fuentes, B. X. Lou, J. M. Pipas, J. Gerhardt, C. J. Rigell, and E. Fanning Ataxia Telangiectasia-Mutated Damage-Signaling Kinase- and Proteasome-Dependent Destruction of Mre11-Rad50-Nbs1 Subunits in Simian Virus 40-Infected Primate Cells J. Virol., June 1, 2008; 82(11): 5316 - 5328. [Abstract] [Full Text] [PDF]
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 M. Yamaguchi, N. Fujimori-Tonou, Y. Yoshimura, T. Kishi, H. Okamoto, and I. Masai Mutation of DNA primase causes extensive apoptosis of retinal neurons through the activation of DNA damage checkpoint and tumor suppressor p53 Development, April 1, 2008; 135(7): 1247 - 1257. [Abstract] [Full Text] [PDF]
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E. Crescenzi, G. Palumbo, J. de Boer, and H. J.M. Brady Ataxia Telangiectasia Mutated and p21CIP1 Modulate Cell Survival of Drug-Induced Senescent Tumor Cells: Implications for Chemotherapy Clin. Cancer Res., March 15, 2008; 14(6): 1877 - 1887. [Abstract] [Full Text] [PDF]
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M. El-Shemerly, D. Hess, A. K. Pyakurel, S. Moselhy, and S. Ferrari ATR-dependent pathways control hEXO1 stability in response to stalled forks Nucleic Acids Res., February 2, 2008; 36(2): 511 - 519. [Abstract] [Full Text] [PDF]
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V. Stagni, M. G. di Bari, S. Cursi, I. Condo, M. T. Cencioni, R. Testi, Y. Lerenthal, E. Cundari, and D. Barila ATM kinase activity modulates Fas sensitivity through the regulation of FLIP in lymphoid cells Blood, January 15, 2008; 111(2): 829 - 837. [Abstract] [Full Text] [PDF]
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G. P. Shouse, X. Cai, and X. Liu Serine 15 Phosphorylation of p53 Directs Its Interaction with B56{gamma} and the Tumor Suppressor Activity of B56{gamma}-Specific Protein Phosphatase 2A Mol. Cell. Biol., January 1, 2008; 28(1): 448 - 456. [Abstract] [Full Text] [PDF]
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N. K. Kolas, J. R. Chapman, S. Nakada, J. Ylanko, R. Chahwan, F. D. Sweeney, S. Panier, M. Mendez, J. Wildenhain, T. M. Thomson, et al. Orchestration of the DNA-Damage Response by the RNF8 Ubiquitin Ligase Science, December 7, 2007; 318(5856): 1637 - 1640. [Abstract] [Full Text] [PDF]
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 B. A. Olofsson, C. M. Kelly, J. Kim, S. M. Hornsby, and J. Azizkhan-Clifford Phosphorylation of Sp1 in Response to DNA Damage by Ataxia Telangiectasia-Mutated Kinase Mol. Cancer Res., December 1, 2007; 5(12): 1319 - 1330. [Abstract] [Full Text] [PDF]
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E. W. Verschuren, K. H. Ban, M. A. Masek, N. L. Lehman, and P. K. Jackson Loss of Emi1-Dependent Anaphase-Promoting Complex/Cyclosome Inhibition Deregulates E2F Target Expression and Elicits DNA Damage-Induced Senescence Mol. Cell. Biol., November 15, 2007; 27(22): 7955 - 7965. [Abstract] [Full Text] [PDF]
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D. J. Chen and C. S. Nirodi The Epidermal Growth Factor Receptor: A Role in Repair of Radiation-Induced DNA Damage Clin. Cancer Res., November 15, 2007; 13(22): 6555 - 6560. [Abstract] [Full Text] [PDF]
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B. E. Lally, G. A. Geiger, S. Kridel, A. E. Arcury-Quandt, M. E. Robbins, N. D. Kock, K. Wheeler, P. Peddi, A. Georgakilas, G. D. Kao, et al. Identification and Biological Evaluation of a Novel and Potent Small Molecule Radiation Sensitizer via an Unbiased Screen of a Chemical Library Cancer Res., September 15, 2007; 67(18): 8791 - 8799. [Abstract] [Full Text] [PDF]
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I. M. Munoz, P. A. Jowsey, R. Toth, and J. Rouse Phospho-epitope binding by the BRCT domains of hPTIP controls multiple aspects of the cellular response to DNA damage Nucleic Acids Res., August 13, 2007; 35(16): 5312 - 5322. [Abstract] [Full Text] [PDF]
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 M. J. Cariveau, X. Tang, X.-L. Cui, and B. Xu Characterization of an NBS1 C-Terminal Peptide That Can Inhibit Ataxia Telangiectasia Mutated (ATM)-Mediated DNA Damage Responses and Enhance Radiosensitivity Mol. Pharmacol., August 1, 2007; 72(2): 320 - 326. [Abstract] [Full Text] [PDF]
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M. S. Bentle, K. E. Reinicke, Y. Dong, E. A. Bey, and D. A. Boothman Nonhomologous End Joining Is Essential for Cellular Resistance to the Novel Antitumor Agent, {beta}-Lapachone Cancer Res., July 15, 2007; 67(14): 6936 - 6945. [Abstract] [Full Text] [PDF]
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C. Cerboni, A. Zingoni, M. Cippitelli, M. Piccoli, L. Frati, and A. Santoni Antigen-activated human T lymphocytes express cell-surface NKG2D ligands via an ATM/ATR-dependent mechanism and become susceptible to autologous NK- cell lysis Blood, July 15, 2007; 110(2): 606 - 615. [Abstract] [Full Text] [PDF]
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Y.-C. Tsai, H. Qi, and L. F. Liu Protection of DNA Ends by Telomeric 3' G-Tail Sequences J. Biol. Chem., June 29, 2007; 282(26): 18786 - 18792. [Abstract] [Full Text] [PDF]
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L. F. Povirk, R.-Z. Zhou, D. A. Ramsden, S. P. Lees-Miller, and K. Valerie Phosphorylation in the serine/threonine 2609-2647 cluster promotes but is not essential for DNA-dependent protein kinase-mediated nonhomologous end joining in human whole-cell extracts Nucleic Acids Res., June 9, 2007; 35(12): 3869 - 3878. [Abstract] [Full Text] [PDF]
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T. Kiziltepe, T. Hideshima, L. Catley, N. Raje, H. Yasui, N. Shiraishi, Y. Okawa, H. Ikeda, S. Vallet, S. Pozzi, et al. 5-Azacytidine, a DNA methyltransferase inhibitor, induces ATR-mediated DNA double-strand break responses, apoptosis, and synergistic cytotoxicity with doxorubicin and bortezomib against multiple myeloma cells Mol. Cancer Ther., June 1, 2007; 6(6): 1718 - 1727. [Abstract] [Full Text] [PDF]
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N. D. Fernandes, Y. Sun, and B. D. Price Activation of the Kinase Activity of ATM by Retinoic Acid Is Required for CREB-dependent Differentiation of Neuroblastoma Cells J. Biol. Chem., June 1, 2007; 282(22): 16577 - 16584. [Abstract] [Full Text] [PDF]
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 S. Difilippantonio, A. Celeste, M. J. Kruhlak, Y. Lee, M. J. Difilippantonio, L. Feigenbaum, S. P. Jackson, P. J. McKinnon, and A. Nussenzweig Distinct domains in Nbs1 regulate irradiation-induced checkpoints and apoptosis J. Exp. Med., May 14, 2007; 204(5): 1003 - 1011. [Abstract] [Full Text] [PDF]
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H. L. Ball, M. R. Ehrhardt, D. A. Mordes, G. G. Glick, W. J. Chazin, and D. Cortez Function of a Conserved Checkpoint Recruitment Domain in ATRIP Proteins Mol. Cell. Biol., May 1, 2007; 27(9): 3367 - 3377. [Abstract] [Full Text] [PDF]
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C. A. MacDougall, T. S. Byun, C. Van, M.-c. Yee, and K. A. Cimprich The structural determinants of checkpoint activation Genes & Dev., April 15, 2007; 21(8): 898 - 903. [Abstract] [Full Text] [PDF]
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 J. Bagley, G. Singh, and J. Iacomini Regulation of Oxidative Stress Responses by Ataxia-Telangiectasia Mutated Is Required for T Cell Proliferation J. Immunol., April 15, 2007; 178(8): 4757 - 4763. [Abstract] [Full Text] [PDF]
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N. Tomimatsu, C. G. T. Tahimic, A. Otsuki, S. Burma, A. Fukuhara, K. Sato, G. Shiota, M. Oshimura, D. J. Chen, and A. Kurimasa Ku70/80 Modulates ATM and ATR Signaling Pathways in Response to DNA Double Strand Breaks J. Biol. Chem., April 6, 2007; 282(14): 10138 - 10145. [Abstract] [Full Text] [PDF]
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Y. Shi, G. E. Dodson, P. S. Mukhopadhyay, N. P. Shanware, A. T. Trinh, and R. S. Tibbetts Identification of Carboxyl-terminal MCM3 Phosphorylation Sites Using Polyreactive Phosphospecific Antibodies J. Biol. Chem., March 23, 2007; 282(12): 9236 - 9243. [Abstract] [Full Text] [PDF]
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D. Deckbar, J. Birraux, A. Krempler, L. Tchouandong, A. Beucher, S. Walker, T. Stiff, P. Jeggo, and M. Lobrich Chromosome breakage after G2 checkpoint release J. Cell Biol., March 12, 2007; 176(6): 749 - 755. [Abstract] [Full Text] [PDF]
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B. Gabrielli, Y. Q. Chau, N. Giles, A. Harding, F. Stevens, and H. Beamish Caffeine Promotes Apoptosis in Mitotic Spindle Checkpoint-arrested Cells J. Biol. Chem., March 9, 2007; 282(10): 6954 - 6964. [Abstract] [Full Text] [PDF]
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B. P. C. Chen, N. Uematsu, J. Kobayashi, Y. Lerenthal, A. Krempler, H. Yajima, M. Lobrich, Y. Shiloh, and D. J. Chen Ataxia Telangiectasia Mutated (ATM) Is Essential for DNA-PKcs Phosphorylations at the Thr-2609 Cluster upon DNA Double Strand Break J. Biol. Chem., March 2, 2007; 282(9): 6582 - 6587. [Abstract] [Full Text] [PDF]
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N. P. Shanware, A. T. Trinh, L. M. Williams, and R. S. Tibbetts Coregulated Ataxia Telangiectasia-mutated and Casein Kinase Sites Modulate cAMP-response Element-binding Protein-Coactivator Interactions in Response to DNA Damage J. Biol. Chem., March 2, 2007; 282(9): 6283 - 6291. [Abstract] [Full Text] [PDF]
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K. Valerie, A. Yacoub, M. P. Hagan, D. T. Curiel, P. B. Fisher, S. Grant, and P. Dent Radiation-induced cell signaling: inside-out and outside-in Mol. Cancer Ther., March 1, 2007; 6(3): 789 - 801. [Abstract] [Full Text] [PDF]
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L. Carlessi, G. Buscemi, G. Larson, Z. Hong, J. Z. Wu, and D. Delia Biochemical and cellular characterization of VRX0466617, a novel and selective inhibitor for the checkpoint kinase Chk2 Mol. Cancer Ther., March 1, 2007; 6(3): 935 - 944. [Abstract] [Full Text] [PDF]
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S. E. Golding, E. Rosenberg, S. Neill, P. Dent, L. F. Povirk, and K. Valerie Extracellular Signal-Related Kinase Positively Regulates Ataxia Telangiectasia Mutated, Homologous Recombination Repair, and the DNA Damage Response Cancer Res., February 1, 2007; 67(3): 1046 - 1053. [Abstract] [Full Text] [PDF]
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Y. Pereg, S. Lam, A. Teunisse, S. Biton, E. Meulmeester, L. Mittelman, G. Buscemi, K. Okamoto, Y. Taya, Y. Shiloh, et al. Differential Roles of ATM- and Chk2-Mediated Phosphorylations of Hdmx in Response to DNA Damage. Mol. Cell. Biol., September 1, 2006; 26(18): 6819 - 6831. [Abstract] [Full Text] [PDF]
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N. McCabe, N. C. Turner, C. J. Lord, K. Kluzek, A. Bialkowska, S. Swift, S. Giavara, M. J. O'Connor, A. N. Tutt, M. Z. Zdzienicka, et al. Deficiency in the Repair of DNA Damage by Homologous Recombination and Sensitivity to Poly(ADP-Ribose) Polymerase Inhibition Cancer Res., August 15, 2006; 66(16): 8109 - 8115. [Abstract] [Full Text] [PDF]
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A. J. Deans, K. K. Khanna, C. J. McNees, C. Mercurio, J. Heierhorst, and G. A. McArthur Cyclin-Dependent Kinase 2 Functions in Normal DNA Repair and Is a Therapeutic Target in BRCA1-Deficient Cancers Cancer Res., August 15, 2006; 66(16): 8219 - 8226. [Abstract] [Full Text] [PDF]
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C. J. Lord, M. D. Garrett, and A. Ashworth Targeting the Double-Strand DNA Break Repair Pathway as a Therapeutic Strategy Clin. Cancer Res., August 1, 2006; 12(15): 4463 - 4468. [Abstract] [Full Text] [PDF]
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S. Biton, I. Dar, L. Mittelman, Y. Pereg, A. Barzilai, and Y. Shiloh Nuclear Ataxia-Telangiectasia Mutated (ATM) Mediates the Cellular Response to DNA Double Strand Breaks in Human Neuron-like Cells J. Biol. Chem., June 23, 2006; 281(25): 17482 - 17491. [Abstract] [Full Text] [PDF]
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Y. Zhao, H. D. Thomas, M. A. Batey, I. G. Cowell, C. J. Richardson, R. J. Griffin, A. H. Calvert, D. R. Newell, G. C.M. Smith, and N. J. Curtin Preclinical Evaluation of a Potent Novel DNA-Dependent Protein Kinase Inhibitor NU7441. Cancer Res., May 15, 2006; 66(10): 5354 - 5362. [Abstract] [Full Text] [PDF]
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H. E. Bryant and T. Helleday Inhibition of poly (ADP-ribose) polymerase activates ATM which is required for subsequent homologous recombination repair Nucleic Acids Res., March 23, 2006; 34(6): 1685 - 1691. [Abstract] [Full Text] [PDF]
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N. Levy, A. Martz, A. Bresson, C. Spenlehauer, G. de Murcia, and J. Menissier-de Murcia XRCC1 is phosphorylated by DNA-dependent protein kinase in response to DNA damage Nucleic Acids Res., January 5, 2006; 34(1): 32 - 41. [Abstract] [Full Text] [PDF]
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Y. Pereg, D. Shkedy, P. de Graaf, E. Meulmeester, M. Edelson-Averbukh, M. Salek, S. Biton, A. F. A. S. Teunisse, W. D. Lehmann, A. G. Jochemsen, et al. Phosphorylation of Hdmx mediates its Hdm2- and ATM-dependent degradation in response to DNA damage PNAS, April 5, 2005; 102(14): 5056 - 5061. [Abstract] [Full Text] [PDF]
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), 10 µmol/L KU-55933 (
), or 10 µmol/L KU-58050 (
) before irradiation, then allowed to grow for 16 hours before media replacement and colony formation. B. AT4 fibroblasts were treated for 1 hour with vehicle or 10 µmol/L KU-55933 before irradiation and allowed to grow for 16 hours before media replacement and colony formation; AT4 cells in the presence (
) of 10 µmol/L KU-55933. Data are the mean of at least three independent experiments ± SE.
; and G2-M,