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Experimental Therapeutics |
Division of Oncology Research, Mayo Clinic, Rochester, Minnesota 55905 [J. N. S., E. C. B., L. M. K., R. S. T., P. R., R. T. A.], and National Cancer Research Institute, Biology Division, Tokyo, Japan 104-0045 [Y. T.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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- and UV radiation-induced phosphorylation of p53 on Ser15, a modification that may be directly mediated by the ATM and ATR kinases. DNA-dependent protein kinase, another ATM-related protein involved in DNA damage repair, was resistant to the inhibitory effects of caffeine. Likewise, the catalytic activity of the G2 checkpoint kinase, hChk1, was only marginally suppressed by caffeine but was inhibited potently by the structurally distinct radiosensitizer, UCN-01. These data suggest that the radiosensitizing effects of caffeine are related to inhibition of the protein kinase activities of ATM and ATR and that both proteins are relevant targets for the development of novel anticancer agents. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Accumulating data suggest that several members of the PIKK4 family play pivotal roles in cell cycle checkpoint functions in eukaryotic cells (5 , 6) . The PIKK family members share a COOH-terminal kinase domain bearing significant sequence homology to the catalytic domains of mammalian and yeast phosphoinositide 3-kinases. Genetic and biochemical evidence indicates that several of the PIKK family members, including yeast Rad3p and Mec1p, and the mammalian proteins, ATM and ATR, are proximal components of DNA damage-induced, cell cycle checkpoint pathways. The importance of ATM in the regulation of cell viability and genomic stability in humans is dramatically highlighted by the severe pathologies displayed by patients with the heritable disorder, A-T, which results from mutational inactivation of both ATM alleles. The A-T syndrome is characterized by progressive cerebellar ataxia and neurodegeneration, immunodeficiency, heightened cancer susceptibility, and a marked increase in cellular sensitivity to ionizing radiation (7 , 8) . Cells derived from A-T patients are defective in the activation of G1, S, and G2 cell cycle checkpoints after exposure to ionizing radiation. These pleiotropic checkpoint abnormalities presumably underlie the radiation hypersensitivity and chromosomal instability displayed by A-T cells (9 , 10) .
A second mammalian PIKK, ATR, carries out checkpoint-related functions that partially overlap with those performed by ATM. Both ATM and ATR contribute to the phosphorylation and accumulation of p53 after cellular exposure to - or UV-radiation. These DNA-damaging agents induce the phosphorylation of p53 on Ser15, and genetic manipulations that reduce the activity of either ATM or ATR decrease Ser15 phosphorylation in irradiated cells (11
, 12)
. Furthermore, both ATM and ATR phosphorylate p53 at Ser15 in immune complex kinase assays (11
, 13
, 14)
. Recent findings suggest that ATM may be the major Ser15 kinase in -irradiated cells, whereas ATR plays a more prominent role in the phosphorylation of p53 at Ser15 in response to UV light exposure (3
, 11)
.
Based in large part on studies of the homologous proteins in yeast, it is predicted that ATM and ATR function as proximal signal transducers in G1, S, and G2 checkpoint pathways. With the exception of p53, the downstream components of these pathways remain largely undefined. Studies in yeast and vertebrate cells have outlined a pathway whereby ATM and/or ATR might inhibit the passage of DNA-damaged cells from G2 into M phase. DNA damage leads to the activation of two related protein kinases, Chk1 and Chk2, which then phosphorylate the Cdc25C phosphatase on Ser-216. This modification creates a binding site for 14-3-3 proteins (15, 16, 17, 18) . The 14-3-3-bound phosphatase is then sequestered outside of the nucleus and is prohibited from dephosphorylating and activating the mitosis-promoting cyclin B-cdc2 complex. In Schizosaccharomyces pombe, the ATM/ATR homologue, Rad3p, is required for the activation of spChk1 in response to DNA damage, suggesting that ATM and/or ATR might carry out similar functions in mammalian cells (19) .
A normal function of cell cycle checkpoints is to protect cells and the host organism from the deleterious consequences of replicating or segregating damaged chromosomes. Therefore, it seems paradoxical that p53-deficient, and hence G1 checkpoint-compromised tumor cells, frequently show an elevated resistance to DNA-damaging therapeutic agents (20) . These cells retain a functional G2 checkpoint, and accumulating evidence suggests that this checkpoint, which is largely p53 independent, is critical for cellular recovery from DNA damage. Abrogation of the G2 checkpoint often leads to a marked increase in the sensitivity of cells to ionizing radiation and certain chemotherapeutic agents (21 , 22) . These observations have provoked speculation that inhibitors of the G2 checkpoint signaling pathway might effectively sensitize cancer cells to the lethal effects of ionizing radiation or DNA-damaging drugs. Indeed, two known inhibitors of cell cycle arrest at the G2 checkpoint, caffeine and UCN-01 (7-hydroxystaurosporine), preferentially radiosensitize cells that lack functional p53 (23, 24, 25, 26) . The molecular targets for both drugs remain unclear. Nonetheless, the sensitizing actions of caffeine and UCN-01 have raised hopes that adjunctive therapy with G2 checkpoint inhibitors will increase the therapeutic efficacies of radiation and other genotoxic therapies in the large population of cancer patients whose tumor cells lack functional p53.
The checkpoint defects induced by caffeine and other methylxanthines are reminiscent of the checkpoint defects seen in A-T cells. Exposure of cultured cells to caffeine results in a delayed and attenuated accumulation of wild-type p53 and abrogation of the G1 checkpoint in response to ionizing radiation (27) . Caffeine treatment also results in RDS, which represents a canonical radiation-response defect of A-T cells (28, 29, 30) . On the basis of these similarities, we hypothesized that inhibition of PIKK-dependent signaling pathways could be responsible for the checkpoint defects observed in caffeine-treated cells. In this study, we show that caffeine inhibits ATM and ATR kinase activities at drug concentrations similar to those shown previously to sensitize cells to killing by ionizing radiation. Furthermore, caffeine-treated cells display several of the phenotypic abnormalities reported previously in cells deficient in ATM or ATR function. These data identify ATM and ATR as relevant molecular targets for caffeine and suggest that more potent and specific inhibitors of these PIKK family members might be clinically useful radio- and chemosensitizing agents.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1, and phospho-Ser15-p53 were generated as described (11
, 31, 32, 33)
. Antibodies for ATM (Ab-3), DNA-PK (Ab-1), and p53 (Ab-6) were obtained from Oncogene Science-Calbiochem; phospho-specific mitogen-activated protein kinase (Erk-1 and Erk2) antibodies were obtained from New England Biolabs. The viral HA-specific mouse monoclonal antibody, HA.11, was purchased from Babco. For flow cytometry, a mouse anti-BrdUrd monoclonal antibody was obtained from Becton Dickinson, and a goat-anti-mouse antibody conjugated with FITC was obtained from Sigma. hChk1 was amplified by PCR from a human testes cDNA library (Clontech), and the full-length cDNA was cloned into the pEF-BOS plasmid with a COOH-terminal HA2 epitope tag. To generate a GST fusion protein containing amino acids 200–256 of Cdc25C (GST-Cdc25C200–256), the corresponding cDNA fragment was amplified by PCR from a human testes cDNA library and cloned into the pGEX-KG vector.
Immune Complex Kinase Assays.
The ATM and DNA-PK proteins were immunoprecipitated from 0.2% Tween 20 extracts prepared from A549 cells (34)
. The modifications to the procedure reported previously were that the lysis buffer was modified by addition of 10 mM sodium fluoride and 20 mM ß-glycerophosphate, and that, prior to immunoprecipitation of ATM, the cells were subjected to two freeze-thaw cycles in a dry ice-ethanol bath. In the indicated experiments, immunoprecipitated proteins were incubated for 15 min on ice with 2x final concentrations of caffeine or other checkpoint inhibitors diluted in kinase base buffer (10 mM HEPES, 50 mM NaCl, and 10 mM MgCl2, pH 7.4). The kinase reactions were then performed as detailed previously (34)
. Kinase reactions were terminated by the addition of 4x SDS sample buffer, and reaction products were resolved by SDS-PAGE. Incorporation of 32Pi into the PHAS-I substrate was quantitated by phosphorimaging.
ATR and mTOR immunoprecipitations were performed as described (11 , 32) . ATR was immunoprecipitated from bull testis extract, and mTOR was immunoprecipitated from rat brain extract. The immune complexes were incubated with graded concentrations of caffeine as described above. The kinase reactions were then performed using kinase reaction components and conditions that were identical to those used in the ATM kinase reactions. All kinase reactions were performed under linear reaction conditions.
The hChk1 kinase assays were performed with recombinant epitope-tagged hChk1. K562 cells were transfected with 5 µg of pEF-BOS-hChk1-HA2 by electroporation using conditions described previously (11)
. After 18–20 h, the transfected cells were lysed in buffer containing 50 mM HEPES (pH 7.6), 150 mM sodium chloride, 10 mM sodium fluoride, 30 mM sodium PPi, 1 mM EDTA, 10 mM ß-glycerophosphate, 1 mM sodium orthovanadate, 1 mM DTT, 10 µg/ml aprotinin, 5 µg/ml pepstatin, 5 µg/ml leupeptin, 20 nM microcystin, and 1% Triton X-100. After removal of insoluble material by centrifugation, cell extracts were pooled and aliquoted. The extracts were rotated with 0.2 µl of HA.11 ascites fluid and a secondary rabbit anti-mouse antibody bound to protein A-Sepharose beads. Immunoprecipitates were washed three times in wash buffer [50 mM HEPES (pH 7.6), 1 M sodium chloride, 10% glycerol, and 1% Triton X-100] and three times in kinase base buffer [50 mM Tris (pH 7.4), 10 mM MgCl2]. Drug incubations were performed as described above. The kinase reaction mix was then added to yield final concentrations of 50 mM Tris (pH 7.4), 10 mM MgCl2, 1 mM DTT, 10 µM [-32P]ATP (specific activity, 50 Ci/mmol; ICN), and 25 ng/µl recombinant GST-Cdc25C200–256 in a total volume of 40 µl. The phosphorylation reactions were incubated for 15 min at 30°C, and the incorporation of radioactive phosphate into GST-Cdc25C200–256 was determined as described above.
p53 Phosphorylation.
Subconfluent cultures of A549 cells were treated with 50 µM LLnL and the indicated concentrations of caffeine or wortmannin for 15 min prior to -irradiation. Cells were exposed to 0 or 20 Gy from a 137Cs source at a dose rate of 6.4 Gy/min and then returned to 37°C. After 3 h, the cells were washed with PBS (pH 7.4) and lysed on ice in TNE buffer [50 mM Tris (pH 8.0), 150 mM sodium chloride, 5 mM EDTA, 1% NP40, 0.1% SDS, 1 mM DTT, 10 µg/ml aprotinin, 5 µg/ml pepstatin, 5 µg/ml leupeptin, 20 nM microcystin, and 10 mM ß-glycerophosphate]. Insoluble material was cleared by centrifugation, and soluble proteins (40 µg per sample lane) were resolved by SDS-PAGE. The levels of Ser15-phosphorylated and nonphosphorylated p53 were determined as described (11)
.
A similar protocol was used to examine p53 phosphorylation following UV irradiation. A549 cells were incubated with 50 µM LLnL for 2 h before addition of the indicated concentrations of caffeine or wortmannin. After an additional 15 min, the culture medium was removed and replaced with HBSS (pH 7.4). The cells were exposed immediately to 0 or 50 J·m-2 UV radiation, and then the HBSS was replaced with fresh medium. The starting concentrations of LLnL, caffeine, and wortmannin were maintained throughout these manipulations by re-addition of the appropriate compounds following each change of the incubation medium. After 4 h, the cells were lysed and processed as described above.
Mitogen-activated Protein Kinase Phosphorylation.
Subconfluent cultures of A549 cells were serum starved for 16 h and then were pretreated for 45 min with the MEK inhibitor, PD098059, or for 15 min with caffeine. The pretreated cells were stimulated with 100 ng per ml PMA for 5 min and then were lysed in TNE buffer. The detergent-soluble proteins were processed for immunoblotting with phospho-Erk-specific antibodies. The protein blots were simultaneously probed with PLC-1-specific antibodies to insure that equal amounts of extract protein were loaded in each sample lane.
RDS.
Inhibition of DNA synthesis after -irradiation of A549 cells was measured using a modification of thymidine incorporation assays described previously (28
, 34)
. Subconfluent A549 cells were plated into 96-well plates (2000 cells per 100 µl/well), and the intracellular DNA pool was prelabeled with 1.5 nCi/well [methyl-14C]thymidine (specific activity, 59 mCi/mmol; ICN). After 72 h, the indicated concentrations of caffeine or wortmannin were added to the cells. After 15 min, cells were exposed to 0 or 30 Gy -radiation as described above. Twenty min after irradiation, the cells were pulsed for 40 min with 2 µCi/well [methyl-3H]thymidine (specific activity, 5 Ci/mmol; Amersham). Cells were harvested by trypsinization, transferred onto glass filters, and lysed in distilled water. Filter-bound radioactivity was determined by liquid scintillation counting. The relative DNA synthesis rate for a given drug treatment after radiation was calculated using the equation 
Each treatment condition was tested in six replicate wells.
Flow Cytometry.
Subconfluent cultures of A549 cells were treated with 5 µM BrdUrd and the indicated concentrations of caffeine 15 min prior to -irradiation. Cells were exposed to 0 or 5 Gy and then returned to 37°C. After 6 h, cells were trypsinized and fixed in 70% ice-cold ethanol/30% PBS. Cells were processed for flow cytometry essentially as described previously (35)
. Briefly, following RNaseA treatment, pepsin digestion, and acid denaturation, samples were serially incubated with a primary anti-BrdUrd antibody and then a secondary FITC-conjugated antibody. Samples were resuspended in 20 µg/ml propidium iodide prior to analysis.
Simultaneous measurements of DNA content (red fluorescence) and BrdUrd content (green fluorescence) were obtained on all samples using a FACScan flow cytometer (Becton Dickinson). A minimum of 20,000 ungated events was recorded for each sample. Analyses of the data were performed using the WinMDI Version 2.7 software program.5
Doublets and clumps were excluded from the analysis by gating on the DNA pulse width versus area. The percentage of BrdUrd-negative cells remaining in G2 following DNA damage was determined by gating on the bivariate distribution of green height (BrdUrd-FITC) versus red area (propidium iodide; see Fig. 3B
, inset).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In parallel assays, we examined the sensitivity of ATM kinase activity to a second radiosensitizing agent, UCN-01. Interestingly, the protein kinase activity of immunoprecipitated ATM was not inhibited by 1 µM UCN-01 (Fig. 1A)
, despite the fact that this concentration of UCN-01 maximally sensitizes intact cells to ionizing radiation (23)
.
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ATM is a member of a family of proteins that contain a PI3K-like catalytic domain (5)
. To determine whether caffeine exerted differential inhibitory effects on the protein kinase activities of the mammalian PIKK family members, we developed caffeine concentration-inhibition curves for ATM, ATR, mTOR, and DNA-PK (Fig. 2A–D)
, as well as hChk1 (Fig. 2E)
. The PHAS-I-phosphorylating activities of ATM, mTOR, and ATR exhibited similar sensitivities to caffeine, with 50% inhibition of kinase activity (IC50) observed at drug concentrations of 0.2, 0.4, and 1.1 mM, respectively. The inhibitory potency of caffeine toward ATM was identical when a GST-p53 fusion protein was used as the substrate (data not shown). The latter substrate is phosphorylated at the Ser15 residue, which appears to be a physiological site for modification by ATM in DNA-damaged cells (12, 13, 14)
. On the other hand, the protein kinase activity of the DNA-PK heterotrimer was relatively resistant to caffeine (IC50, 10 mM), whereas hChk1 displayed an intermediate level of sensitivity (IC50, 5 mM). Because caffeine induces radiosensitization and inhibits checkpoint functions at concentrations of 0.5–2 mM, the results of our in vitro kinase assays suggest that DNA-PK and hChk1 are not relevant targets for the radiosensitizing effects of caffeine in intact cells (24, 25, 26
, 36)
. Furthermore, other studies have shown that rapamycin, which is a highly selective inhibitor of mTOR kinase activity, does not sensitize cells to killing by ionizing radiation (37)
. Thus, these observations directed our attention toward the possible roles of ATM and ATR inhibition in the disruption of checkpoint functions by caffeine.
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. As reported previously, A549 cells also displayed the RDS phenotype after treatment with the structurally distinct ATM inhibitor, wortmannin (34)
. The observed differences in DNA synthesis inhibition for the irradiated cells pretreated with ATM-inhibitory concentrations of caffeine or wortmannin, compared with no drug treatment, are similar to the studies reported previously of A-T versus wild-type fibro-blasts (28)
.
A second checkpoint defect characteristic of ATM-deficient cells is the failure of cells irradiated in the G2 phase of the cell cycle to arrest prior to mitosis (38)
. As with the S-phase checkpoint, we hypothesized that caffeine should abrogate this G2 checkpoint at concentrations associated with significant inhibition of intracellular ATM kinase activity. To examine the fate of cells irradiated in the G2 phase of the cell cycle, BrdUrd was added to the incubation medium just prior to drug treatment and irradiation. BrdUrd is a thymidine analogue that is incorporated into DNA during replication and can be recognized by a specific 
-BrdUrd antibody. The cell cycle distribution of a mixed population of BrdUrd-labeled and -unlabeled cells can be assessed using a bivariate flow cytometric analysis of DNA content versus BrdUrd intensity. Therefore, at time points following BrdUrd addition and irradiation, cells arrested in G2-M (i.e., those with 4N DNA content), which do not contain BrdUrd, must have been in G2-M at the time of irradiation. (Fig. 3B
, inset). Using this technique, A549 cells irradiated in G2-M remained arrested in that phase of the cell cycle 6 h later. However, pretreatment of these cells with caffeine at concentrations >0.3 mM significantly abrogated this checkpoint (Fig. 3B)
. The dose-response relationship for abrogation of this ATM-dependent G2-checkpoint response correlates closely with the range of caffeine concentrations associated with significant radiosensitization (25
, 26)
.
Recent studies have suggested that ATM and ATR are directly responsible for the phosphorylation of the NH2 terminus of p53 at Ser15 after cellular exposure to -radiation or UV light (11, 12, 13, 14)
. Furthermore, these studies provide indirect evidence that ATM plays a major role in the early phase of Ser15 phosphorylation in -irradiated cells, whereas ATR may be more important for UV light-induced phosphorylation of this site in p53. On the basis of the premise that caffeine treatment inhibits the protein kinase activities of both ATM and ATR, we predicted that the - and UV radiation-inducible phosphorylation of Ser15 would be blocked by caffeine. The intensity of the Ser15 phosphorylation response in the irradiated cells was monitored by immunoblotting of cellular extracts with phospho-Ser15-specific (-P-Ser15) antibodies (33)
. In these experiments, the cells were pretreated with the proteosome inhibitor, LLnL, to block the degradation of p53 in irradiated and unirradiated cells. LLnL treatment stabilizes the total cellular levels of p53, thereby facilitating the interpretation of changes in -P-Ser15 immunoreactivity after cellular exposure to DNA-damaging agents (33
, 39)
. Caffeine treatment strongly inhibited -radiation- and UV light-inducible phosphorylation of p53 at Ser15 (Fig. 3, C and D)
, at concentrations similar to those required for significant inhibition of ATM and ATR kinase activities, respectively. The phosphorylation of Ser15 in -irradiated cells was also suppressed by pretreatment of the cells with 30 µM wortmannin, a drug concentration known to inhibit the kinase activity of ATM, but not ATR, in intact cells (34)
. Interestingly, wortmannin treatment also inhibited Ser15 phosphorylation of p53 after UV radiation, which suggests that ATM or another wortmannin-sensitive protein kinase participates in UV light-induced p53 modification in A549 cells.
As a purine analogue, caffeine might interfere with checkpoint signaling functions simply by acting as a broad-spectrum kinase inhibitor. Although the relative resistance of hChk1 and DNA-PK activity to caffeine argues against this possibility, we wished to address this issue further by examining the effect of caffeine on the function of a well-studied cytoplasmic signaling cascade, the Ras to Erk pathway (40)
. Serum-starved A549 cells were stimulated with the phorbol ester PMA, and activation of Erk1 was determined with phospho-Erk1-specific antibodies (Fig. 3E)
. As expected, the Erk1 activation response was strongly suppressed by pretreatment of the cells with PD098059, an inhibitor of MEKs, the upstream activators of Erks. In contrast, caffeine pretreatment had no effect on PMA-induced Erk1 activation in A549 cells (Fig. 3E)
, indicating that concentrations of caffeine that interfere with checkpoint signaling functions do not cause a global inhibition of conventional protein kinase activities in these cells.
Effect of Caffeine on Radiation-induced ATM Activation.
The results presented to this point support the hypothesis that the radiosensitizing effects of caffeine are related to the inhibition of ATM and ATR kinase activities. However, because cellular exposure to -radiation increases the in vitro kinase activity of ATM (13
, 14)
, a caffeine-induced block in an upstream event leading to ATM activation might also confer multiple checkpoint defects in drug-exposed cells. To examine this possibility, A549 cells were pretreated with 3 mM caffeine and then irradiated with 20 Gy -radiation. Cells were then lysed, and the kinase activity of immunoprecipitated ATM was assessed. Because caffeine presumably functions as a reversible inhibitor of ATM kinase activity, we expected to recover catalytically active ATM when caffeine was removed during the process of immunoprecipitation. In fact, the protein kinase activity of ATM was increased by 
2-fold after cellular irradiation, and this increase was not affected by pretreatment of the cells with caffeine (Fig. 4)
. Thus, in contrast to the protein kinase activity of ATM itself, the pathway leading to ATM activation in irradiated cells is not sensitive to caffeine. Moreover, these results suggest that the mechanism of ATM activation by -radiation does not involve either auto- or trans-phosphorylation of ATM by either of the caffeine-sensitive PIKKs, ATM or ATR.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In previous studies, we demonstrated that the protein kinase activities of mTOR, ATM, and DNA-PK were inhibited by wortmannin (IC50s, 16–250 nM), whereas ATR activity was at least 10-fold more resistant to this drug (32
, 34)
. On the basis of the observation that the specific mTOR inhibitor, rapamycin, does not confer a radiosensitive phenotype (37)
, then ATM and DNA-PK emerge as the candidate molecular targets for wortmannin-mediated radiosensitization. In the present studies, we observed that caffeine, although not a global inhibitor of conventional protein kinase activities (Fig. 3E)
, effectively inhibited the phosphorylation of protein substrates by all four mammalian PIKKs. In contrast to the results obtained with wortmannin, the catalytic activity of DNA-PK was the most resistant to inhibition by caffeine. On the basis of the in vitro potency data for ATR, ATM, and DNA-PK, we surmise that inhibition of ATR and ATM correlates most closely with radiosensitization and the G2 checkpoint bypass observed in cells treated with low millimolar concentrations of caffeine. This conclusion is supported by the earlier finding that the intracellular concentration of caffeine rapidly equilibrates with that present in the extracellular medium (42)
. Hence, the potencies of caffeine as an inhibitor of ATM and ATR kinase activities in intact cells should approximate those obtained in the immune complex kinase assays. Although the contribution of other protein targets cannot be excluded, our results suggest that ATM is a common target for both wortmannin- and caffeine-mediated radiosensitization, whereas the concomitant inhibition of ATR activity by caffeine may contribute to the distinct radiosensitizing properties of this drug.
The disruptive effects of caffeine on the G1 and S-phase checkpoints are consistent with a drug-induced loss of ATM function in treated cells. A central regulator of the G1 checkpoint is p53, and studies in ATM-deficient cells indicate that ATM is required for the timely phosphorylation and activation of p53 after cellular exposure to ionizing radiation or radiomimetic agents (12)
. Indeed, ATM may be directly responsible for the phosphorylation of p53 at Ser15 in -irradiated cells (13
, 14)
. Our results show that caffeine treatment blocks -radiation-induced Ser15 phosphorylation at drug concentrations that inhibit the in vitro protein kinase activity of ATM by >80%. The idea that caffeine confers an A-T-like phenotype on ATM-positive cells is further substantiated by the observation that similar concentrations of caffeine abrogate S- and G2-phase cell cycle checkpoints. Loss of these checkpoints is a characteristic abnormality of cells derived from A-T patients. Interestingly, we have reported that treatment of A549 cells with ATM-inhibitory concentrations of wortmannin also abrogates the S-phase checkpoint, which results in an RDS phenotype (34)
. It is conceivable that the induction of this RDS phenotype might form the basis for a convenient cell-based screen for additional small molecule inhibitors of the S-phase checkpoint pathway governed by ATM.
The spectrum of checkpoint defects induced by caffeine treatment suggests that ATM is not the only checkpoint component affected by this drug:
(a) ATM-deficient cells display a normal level of sensitivity to UV radiation, whereas caffeine treatment renders cells hypersensitive to this DNA-damaging agent. Interestingly, recent results from our laboratory suggest that ATR, rather than ATM, may be the principle regulator of p53 activation after UV light exposure (11) . Consistent with this observation, we now show that caffeine inhibits the phosphorylation of p53 on Ser15 after UV light exposure at concentrations associated with significant inhibition of ATR kinase activity in vitro. The inhibition of ATR-dependent checkpoint functions by caffeine is also consistent with the sensitization of p53-null cells by UV light (43) . Collectively, these results suggest that ATR is a primary participant in both p53-dependent and -independent checkpoint pathways triggered by UV light exposure.
(b) -irradiation of A-T cells during G1 or S phase results in a protracted arrest in G2-phase. Caffeine shortens or eliminates the -radiation-induced G2 arrest in ATM-positive cells and abrogates the prolonged G2 arrest seen in -irradiated A-T fibroblasts (36
, 44
, 45)
. These results suggest that an ATM-independent pathway enforces the G2 checkpoint in A-T cells and that this alternative pathway is disrupted by caffeine. We propose that this alternative checkpoint pathway is mediated through ATR, and that the abrogation of the radiation-induced G2 arrest by caffeine reflects the concomitant inhibition of ATM and ATR kinase activities by this drug. In support of this notion, we observed that wortmannin-treated cells, like A-T cells, accumulate in G2 phase after exposure to -radiation. This phenotype is consistent with the fact that wortmannin inhibits the protein kinase activity of ATM, but not ATR, at drug concentrations that induce maximal radiosensitization (34)
. These observations imply that caffeine inhibits not only ATM but at least one additional checkpoint protein involved in the control of the G2 checkpoint and the cellular response to UV damage. Our pharmacological data suggest that this target may be ATR.
An important issue with respect to ATM and ATR inhibitors as potential chemotherapeutic agents is whether this class of drugs will show selectivity for tumor versus normal tissue. A frequent distinction between normal cells and cancer cells is that the latter have lost p53-dependent checkpoint controls, often through mutation of p53 itself (46) . The loss of p53 function abrogates the G1 checkpoint and may compromise a G2 checkpoint pathway that operates in parallel with those governed by ATR and/or ATM (47, 48, 49, 50, 51) . Recent evidence suggests that the therapeutic efficacies of certain DNA-damaging anticancer drugs are causally related to the loss of normal DNA damage checkpoint controls during the process of carcinogenesis (52 , 53) . These studies suggest that agents that interfere with checkpoint-related proteins may show selectivity for tumor cells bearing intrinsic defects in specific checkpoint pathways. Indeed, this prediction is supported by the observation that p53-deficient cells are preferentially sensitized to radiation-induced killing by caffeine (24, 25, 26) , which, based on the present findings, targets ATM and ATR. Similarly, the potent inhibition of Chk1 kinase activity by UCN-01 may explain the increased efficacy of this drug as a radiosensitizing agent in p53-mutated cells (23) .
The notion that p53-null tumor cells are "marked" for sensitization by inhibitors of ATM, ATR, hChk1, or other checkpoint control proteins is clearly provocative from the viewpoint of cancer therapy. The results of the present studies suggest that the archetypal radiosensitizing agent, caffeine, inhibits the protein kinase activities of ATM and ATR at concentrations overlapping those that cause moderate radiosensitization in p53-null tumor cells. However, whether suppression of ATM and/or ATR kinase activities will actually increase the therapeutic indices of existing anticancer therapies in patients awaits the development of more potent and specific inhibitors of these PIKK family members.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by the Mayo Foundation, Mayo Cancer Center; by Grants CA52995, CA76193 (both to R. T. A.), CA69709, and CA80829 (both to J. N. S.) from the NIH; and by a grant from the Admadjaja Thymoma Research Fund (to R. T. A.). R. S. T. is a recipient of a Special Fellow Award from the Leukemia Society of America. 
2 To whom requests for reprints should be addressed, at Mayo Foundation, Room 1325, Guggenheim Building, 200 First Street, S.W., Rochester, MN 55905. Phone: (507) 266-5232; Fax: (507) 284-3906; E-mail: sarkaria.jann{at}mayo.edu
3 Present address: Department of Pharmacology and Cancer Biology, Duke University, Box 3813, Durham, NC 27710.
4 The abbreviations used are: PIKK, phosphatidylinositol 3-kinase related kinase; ATM, ataxia-telangiectasia mutated; ATR, ATM- and Rad3-related; A-T, ataxia-telangiectasia; RDS, radioresistant DNA synthesis; PLC, phospholipase C; HA, hemagglutinin; BrdUrd, bromodeoxyuridine; DNA-PK, DNA-dependent protein kinase; LLnL, N-acetyl-Leu-Leu-Norleucinal; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase; PMA, phorbol myristate acetate.
5 J. Trotter, http://facs.scripps.edu.
Received 2/23/99. Accepted 7/ 7/99.
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 G. J. Nichols, J. Schaack, and D. A. Ornelles Widespread Phosphorylation of Histone H2AX by Species C Adenovirus Infection Requires Viral DNA Replication J. Virol., June 15, 2009; 83(12): 5987 - 5998. [Abstract] [Full Text] [PDF]
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 B. Cui, S. P. Johnson, N. Bullock, F. Ali-Osman, D. D. Bigner, and H. S. Friedman Bifunctional DNA Alkylator 1,3-Bis(2-chloroethyl)-1-nitrosourea Activates the ATR-Chk1 Pathway Independently of the Mismatch Repair Pathway Mol. Pharmacol., June 1, 2009; 75(6): 1356 - 1363. [Abstract] [Full Text] [PDF]
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M. Fragkos, J. Jurvansuu, and P. Beard H2AX Is Required for Cell Cycle Arrest via the p53/p21 Pathway Mol. Cell. Biol., May 15, 2009; 29(10): 2828 - 2840. [Abstract] [Full Text] [PDF]
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 R. J. Katzenberger, M. S. Marengo, and D. A. Wassarman Control of Alternative Splicing by Signal-dependent Degradation of Splicing-regulatory Proteins J. Biol. Chem., April 17, 2009; 284(16): 10737 - 10746. [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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J. Lee, J. A. Kim, V. Barbier, A. Fotedar, and R. Fotedar DNA Damage Triggers p21WAF1-dependent Emi1 Down-Regulation That Maintains G2 Arrest Mol. Biol. Cell, April 1, 2009; 20(7): 1891 - 1902. [Abstract] [Full Text] [PDF]
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J. Hein, S. Boichuk, J. Wu, Y. Cheng, R. Freire, P. S. Jat, T. M. Roberts, and O. V. Gjoerup Simian Virus 40 Large T Antigen Disrupts Genome Integrity and Activates a DNA Damage Response via Bub1 Binding J. Virol., January 1, 2009; 83(1): 117 - 127. [Abstract] [Full Text] [PDF]
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 D. A. Mordes, E. A. Nam, and D. Cortez Dpb11 activates the Mec1-Ddc2 complex PNAS, December 2, 2008; 105(48): 18730 - 18734. [Abstract] [Full Text] [PDF]
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Y. Auclair, R. Rouget, E. B. Affar, and E. A. Drobetsky ATR kinase is required for global genomic nucleotide excision repair exclusively during S phase in human cells PNAS, November 18, 2008; 105(46): 17896 - 17901. [Abstract] [Full Text] [PDF]
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 J. M. Leonard, H. Ye, C. Wetmore, and L. M. Karnitz Sonic Hedgehog signaling impairs ionizing radiation-induced checkpoint activation and induces genomic instability J. Cell Biol., November 3, 2008; 183(3): 385 - 391. [Abstract] [Full Text] [PDF]
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Y.-w. Chen, J. E. Cleaver, Z. Hatahet, R. E. Honkanen, J.-Y. Chang, Y. Yen, and K.-m. Chou Human DNA polymerase {eta} activity and translocation is regulated by phosphorylation PNAS, October 28, 2008; 105(43): 16578 - 16583. [Abstract] [Full Text] [PDF]
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V. M. Campa, R. Gutierrez-Lanza, F. Cerignoli, R. Diaz-Trelles, B. Nelson, T. Tsuji, M. Barcova, W. Jiang, and M. Mercola Notch activates cell cycle reentry and progression in quiescent cardiomyocytes J. Cell Biol., October 6, 2008; 183(1): 129 - 141. [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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S. S. Lakdawala, R. A. Schwartz, K. Ferenchak, C. T. Carson, B. P. McSharry, G. W. Wilkinson, and M. D. Weitzman Differential Requirements of the C Terminus of Nbs1 in Suppressing Adenovirus DNA Replication and Promoting Concatemer Formation J. Virol., September 1, 2008; 82(17): 8362 - 8372. [Abstract] [Full Text] [PDF]
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A. Restle, M. Farber, C. Baumann, M. Bohringer, K. H. Scheidtmann, C. Muller-Tidow, and L. Wiesmuller Dissecting the role of p53 phosphorylation in homologous recombination provides new clues for gain-of-function mutants Nucleic Acids Res., September 1, 2008; 36(16): 5362 - 5375. [Abstract] [Full Text] [PDF]
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J. R. Pomerening, J. A. Ubersax, and J. E. Ferrell Jr. Rapid Cycling and Precocious Termination of G1 Phase in Cells Expressing CDK1AF Mol. Biol. Cell, August 1, 2008; 19(8): 3426 - 3441. [Abstract] [Full Text] [PDF]
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S. J. Haring, A. C. Mason, S. K. Binz, and M. S. Wold Cellular Functions of Human RPA1: MULTIPLE ROLES OF DOMAINS IN REPLICATION, REPAIR, AND CHECKPOINTS J. Biol. Chem., July 4, 2008; 283(27): 19095 - 19111. [Abstract] [Full Text] [PDF]
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Y. Yan, C. P. Black, P. T. Cao, J. L. Haferbier, R. H. Kolb, R. S. Spieker, A. M. Ristow, and K. H. Cowan {gamma}-Irradiation-Induced DNA Damage Checkpoint Activation Involves Feedback Regulation between Extracellular Signal-Regulated Kinase 1/2 and BRCA1 Cancer Res., July 1, 2008; 68(13): 5113 - 5121. [Abstract] [Full Text] [PDF]
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S. Ashwell and S. Zabludoff DNA Damage Detection and Repair Pathways--Recent Advances with Inhibitors of Checkpoint Kinases in Cancer Therapy Clin. Cancer Res., July 1, 2008; 14(13): 4032 - 4037. [Abstract] [Full Text] [PDF]
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H. Huang, L. Fletcher, N. Beeharry, R. Daniel, G. Kao, T. J. Yen, and R. J. Muschel Abnormal Cytokinesis after X-Irradiation in Tumor Cells that Override the G2 DNA Damage Checkpoint Cancer Res., May 15, 2008; 68(10): 3724 - 3732. [Abstract] [Full Text] [PDF]
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 T. Muramoto and J. R. Chubb Live imaging of the Dictyostelium cell cycle reveals widespread S phase during development, a G2 bias in spore differentiation and a premitotic checkpoint Development, May 1, 2008; 135(9): 1647 - 1657. [Abstract] [Full Text] [PDF]
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 M. Malmlof, G. Paajarvi, J. Hogberg, and U. Stenius Mdm2 as a Sensitive and Mechanistically Informative Marker for Genotoxicity Induced by Benzo[a]pyrene and Dibenzo[a,l]pyrene Toxicol. Sci., April 1, 2008; 102(2): 232 - 240. [Abstract] [Full Text] [PDF]
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 S. A. Gatz, M. Keimling, C. Baumann, T. Dork, K.-M. Debatin, S. Fulda, and L. Wiesmuller Resveratrol modulates DNA double-strand break repair pathways in an ATM/ATR-p53- and -Nbs1-dependent manner Carcinogenesis, March 1, 2008; 29(3): 519 - 527. [Abstract] [Full Text] [PDF]
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Y.-W. Zhang, Z.-X. Zhang, Z.-H. Miao, and J. Ding The Telomeric Protein TRF2 Is Critical for the Protection of A549 Cells from Both Telomere Erosion and DNA Double-Strand Breaks Driven by Salvicine Mol. Pharmacol., March 1, 2008; 73(3): 824 - 832. [Abstract] [Full Text] [PDF]
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 S. Sivasubramaniam, X. Sun, Y.-R. Pan, S. Wang, and E. Y.-H.P. Lee Cep164 is a mediator protein required for the maintenance of genomic stability through modulation of MDC1, RPA, and CHK1 Genes & Dev., March 1, 2008; 22(5): 587 - 600. [Abstract] [Full Text] [PDF]
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L. Zheng, N. Asprodites, A. H. Keene, P. Rodriguez, K. D. Brown, and E. Davila TLR9 engagement on CD4 T lymphocytes represses {gamma}-radiation-induced apoptosis through activation of checkpoint kinase response elements Blood, March 1, 2008; 111(5): 2704 - 2713. [Abstract] [Full Text] [PDF]
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 A. A. Levesque, A. A. Fanous, A. Poh, and A. Eastman Defective p53 signaling in p53 wild-type tumors attenuates p21waf1 induction and cyclin B repression rendering them sensitive to Chk1 inhibitors that abrogate DNA damage-induced S and G2 arrest Mol. Cancer Ther., February 1, 2008; 7(2): 252 - 262. [Abstract] [Full Text] [PDF]
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B. Muller, J. Blackburn, C. Feijoo, X. Zhao, and C. Smythe DNA-activated protein kinase functions in a newly observed S phase checkpoint that links histone mRNA abundance with DNA replication J. Cell Biol., January 28, 2008; 179(7): 1385 - 1398. [Abstract] [Full Text] [PDF]
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A. Z. Al-Minawi, N. Saleh-Gohari, and T. Helleday The ERCC1/XPF endonuclease is required for efficient single-strand annealing and gene conversion in mammalian cells Nucleic Acids Res., January 17, 2008; 36(1): 1 - 9. [Abstract] [Full Text] [PDF]
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X. Liu, A. Matsuda, and W. Plunkett Ataxia-telangiectasia and Rad3-related and DNA-dependent protein kinase cooperate in G2 checkpoint activation by the DNA strand-breaking nucleoside analogue 2'-C-cyano-2'-deoxy-1- -D-arabino-pentofuranosylcytosine Mol. Cancer Ther., January 1, 2008; 7(1): 133 - 142. [Abstract] [Full Text] [PDF]
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S. Jorgensen, I. Elvers, M. B. Trelle, T. Menzel, M. Eskildsen, O. N. Jensen, T. Helleday, K. Helin, and C. S. Sorensen The histone methyltransferase SET8 is required for S-phase progression J. Cell Biol., December 31, 2007; 179(7): 1337 - 1345. [Abstract] [Full Text] [PDF]
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 X. H. Zhang, C. Zhao, and Z. A. Ma The increase of cell-membranous phosphatidylcholines containing polyunsaturated fatty acid residues induces phosphorylation of p53 through activation of ATR J. Cell Sci., December 1, 2007; 120(23): 4134 - 4143. [Abstract] [Full Text] [PDF]
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S. Hanasoge and M. Ljungman H2AX phosphorylation after UV irradiation is triggered by DNA repair intermediates and is mediated by the ATR kinase Carcinogenesis, November 1, 2007; 28(11): 2298 - 2304. [Abstract] [Full Text] [PDF]
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M.G. Luciani, C. Campregher, and C. Gasche Aspirin blocks proliferation in colon cells by inducing a G1 arrest and apoptosis through activation of the checkpoint kinase ATM Carcinogenesis, October 1, 2007; 28(10): 2207 - 2217. [Abstract] [Full Text] [PDF]
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D. L. Bolton and M. J. Lenardo Vpr Cytopathicity Independent of G2/M Cell Cycle Arrest in Human Immunodeficiency Virus Type 1-Infected CD4+ T Cells J. Virol., September 1, 2007; 81(17): 8878 - 8890. [Abstract] [Full Text] [PDF]
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C.-H. Wu, J. van Riggelen, A. Yetil, A. C. Fan, P. Bachireddy, and D. W. Felsher Cellular senescence is an important mechanism of tumor regression upon c-Myc inactivation PNAS, August 7, 2007; 104(32): 13028 - 13033. [Abstract] [Full Text] [PDF]
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A. Marusyk, L. J. Wheeler, C. K. Mathews, and J. DeGregori p53 Mediates Senescence-Like Arrest Induced by Chronic Replicational Stress Mol. Cell. Biol., August 1, 2007; 27(15): 5336 - 5351. [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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P. Jung, B. Verdoodt, A. Bailey, J. R. Yates III, A. Menssen, and H. Hermeking Induction of Cullin 7 by DNA damage attenuates p53 function PNAS, July 3, 2007; 104(27): 11388 - 11393. [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. 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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L. Geng, X. Zhang, S. Zheng, and R. J. Legerski Artemis Links ATM to G2/M Checkpoint Recovery via Regulation of Cdk1-Cyclin B Mol. Cell. Biol., April 1, 2007; 27(7): 2625 - 2635. [Abstract] [Full Text] [PDF]
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A. N. Tse, R. Carvajal, and G. K. Schwartz Targeting Checkpoint Kinase 1 in Cancer Therapeutics Clin. Cancer Res., April 1, 2007; 13(7): 1955 - 1960. [Abstract] [Full Text] [PDF]
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B. Ewald, D. Sampath, and W. Plunkett H2AX phosphorylation marks gemcitabine-induced stalled replication forks and their collapse upon S-phase checkpoint abrogation Mol. Cancer Ther., April 1, 2007; 6(4): 1239 - 1248. [Abstract] [Full Text] [PDF]
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D. Stauffer, B. Chang, J. Huang, A. Dunn, and M. Thayer p300/CREB-binding Protein Interacts with ATR and Is Required for the DNA Replication Checkpoint J. Biol. Chem., March 30, 2007; 282(13): 9678 - 9687. [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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M. C. Caino, J. L. Oliva, H. Jiang, T. M. Penning, and M. G. Kazanietz Benzo[a]pyrene-7,8-dihydrodiol Promotes Checkpoint Activation and G2/M Arrest in Human Bronchoalveolar Carcinoma H358 Cells Mol. Pharmacol., March 1, 2007; 71(3): 744 - 750. [Abstract] [Full Text] [PDF]
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H. Zhu, M. Huang, F. Yang, Y. Chen, Z.-H. Miao, X.-H. Qian, Y.-F. Xu, Y.-X. Qin, H.-B. Luo, X. Shen, et al. R16, a novel amonafide analogue, induces apoptosis and G2-M arrest via poisoning topoisomerase II Mol. Cancer Ther., February 1, 2007; 6(2): 484 - 495. [Abstract] [Full Text] [PDF]
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A. A. Levesque and A. Eastman p53-based cancer therapies: is defective p53 the Achilles heel of the tumor? Carcinogenesis, January 1, 2007; 28(1): 13 - 20. [Abstract] [Full Text] [PDF]
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R. J. Katzenberger, M. S. Marengo, and D. A. Wassarman ATM and ATR Pathways Signal Alternative Splicing of Drosophila TAF1 Pre-mRNA in Response to DNA Damage Mol. Cell. Biol., December 15, 2006; 26(24): 9256 - 9267. [Abstract] [Full Text] [PDF]
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D. E. White, D. Negorev, H. Peng, A. V. Ivanov, G. G. Maul, and F. J. Rauscher III KAP1, a Novel Substrate for PIKK Family Members, Colocalizes with Numerous Damage Response Factors at DNA Lesions Cancer Res., December 15, 2006; 66(24): 11594 - 11599. [Abstract] [Full Text] [PDF]
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C. Agarwal, A. Tyagi, and R. Agarwal Gallic acid causes inactivating phosphorylation of cdc25A/cdc25C-cdc2 via ATM-Chk2 activation, leading to cell cycle arrest, and induces apoptosis in human prostate carcinoma DU145 cells Mol. Cancer Ther., December 1, 2006; 5(12): 3294 - 3302. [Abstract] [Full Text] [PDF]
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A. J. Fikaris, A. E. Lewis, A. Abulaiti, O. M. Tsygankova, and J. L. Meinkoth Ras Triggers Ataxia-telangiectasia-mutated and Rad-3-related Activation and Apoptosis through Sustained Mitogenic Signaling J. Biol. Chem., November 17, 2006; 281(46): 34759 - 34767. [Abstract] [Full Text] [PDF]
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Y. Liu, A. Rusinol, M. Sinensky, Y. Wang, and Y. Zou DNA damage responses in progeroid syndromes arise from defective maturation of prelamin A J. Cell Sci., November 15, 2006; 119(22): 4644 - 4649. [Abstract] [Full Text] [PDF]
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C. L. Sansam, J. L. Shepard, K. Lai, A. Ianari, P. S. Danielian, A. Amsterdam, N. Hopkins, and J. A. Lees DTL/CDT2 is essential for both CDT1 regulation and the early G2/M checkpoint Genes & Dev., November 15, 2006; 20(22): 3117 - 3129. [Abstract] [Full Text] [PDF]
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C. C. S. Chini and J. Chen Repeated Phosphopeptide Motifs in Human Claspin Are Phosphorylated by Chk1 and Mediate Claspin Function J. Biol. Chem., November 3, 2006; 281(44): 33276 - 33282. [Abstract] [Full Text] [PDF]
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G. Buscemi, L. Carlessi, L. Zannini, S. Lisanti, E. Fontanella, S. Canevari, and D. Delia DNA Damage-Induced Cell Cycle Regulation and Function of Novel Chk2 Phosphoresidues Mol. Cell. Biol., November 1, 2006; 26(21): 7832 - 7845. [Abstract] [Full Text] [PDF]
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A. Tyagi, R. P. Singh, C. Agarwal, and R. Agarwal Silibinin activates p53-caspase 2 pathway and causes caspase-mediated cleavage of Cip1/p21 in apoptosis induction in bladder transitional-cell papilloma RT4 cells: evidence for a regulatory loop between p53 and caspase 2 Carcinogenesis, November 1, 2006; 27(11): 2269 - 2280. [Abstract] [Full Text] [PDF]
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D. J. Chang, P. J. Lupardus, and K. A. Cimprich Monoubiquitination of Proliferating Cell Nuclear Antigen Induced by Stalled Replication Requires Uncoupling of DNA Polymerase and Mini-chromosome Maintenance Helicase Activities J. Biol. Chem., October 27, 2006; 281(43): 32081 - 32088. [Abstract] [Full Text] [PDF]
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A. Reinke, J. C.-Y. Chen, S. Aronova, and T. Powers Caffeine Targets TOR Complex I and Provides Evidence for a Regulatory Link between the FRB and Kinase Domains of Tor1p J. Biol. Chem., October 20, 2006; 281(42): 31616 - 31626. [Abstract] [Full Text] [PDF]
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H. Yajima, K.-J. Lee, and B. P. C. Chen ATR-Dependent Phosphorylation of DNA-Dependent Protein Kinase Catalytic Subunit in Response to UV-Induced Replication Stress Mol. Cell. Biol., October 15, 2006; 26(20): 7520 - 7528. [Abstract] [Full Text] [PDF]
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L. Wang, C. J. Rhodes, and J. C. Lawrence Jr. Activation of Mammalian Target of Rapamycin (mTOR) by Insulin Is Associated with Stimulation of 4EBP1 Binding to Dimeric mTOR Complex 1 J. Biol. Chem., August 25, 2006; 281(34): 24293 - 24303. [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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S. O. Boe, M. Haave, A. Jul-Larsen, A. Grudic, R. Bjerkvig, and P. E. Lonning Promyelocytic leukemia nuclear bodies are predetermined processing sites for damaged DNA J. Cell Sci., August 15, 2006; 119(16): 3284 - 3295. [Abstract] [Full Text] [PDF]
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D. R. Borger and J. A. DeCaprio Targeting of p300/CREB Binding Protein Coactivators by Simian Virus 40 Is Mediated through p53 J. Virol., May 1, 2006; 80(9): 4292 - 4303. [Abstract] [Full Text] [PDF]
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C. M. Sturgeon, Z. A. Knight, K. M. Shokat, and M. Roberge Effect of combined DNA repair inhibition and G2 checkpoint inhibition on cell cycle progression after DNA damage. Mol. Cancer Ther., April 1, 2006; 5(4): 885 - 892. [Abstract] [Full Text] [PDF]
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P. J. Lupardus and K. A. Cimprich Phosphorylation of Xenopus Rad1 and Hus1 Defines a Readout for ATR Activation That Is Independent of Claspin and the Rad9 Carboxy Terminus Mol. Biol. Cell, April 1, 2006; 17(4): 1559 - 1569. [Abstract] [Full Text] [PDF]
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X. Wu, S. M. Shell, Z. Yang, and Y. Zou Phosphorylation of Nucleotide Excision Repair Factor Xeroderma Pigmentosum Group A by Ataxia Telangiectasia Mutated and Rad3-Related-Dependent Checkpoint Pathway Promotes Cell Survival in Response to UV Irradiation. Cancer Res., March 15, 2006; 66(6): 2997 - 3005. [Abstract] [Full Text] [PDF]
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Y. Ariumi and D. Trono Ataxia-Telangiectasia-Mutated (ATM) Protein Can Enhance Human Immunodeficiency Virus Type 1 Replication by Stimulating Rev Function J. Virol., March 1, 2006; 80(5): 2445 - 2452. [Abstract] [Full Text] [PDF]
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P. Petersen, D. M. Chou, Z. You, T. Hunter, J. C. Walter, and G. Walter Protein Phosphatase 2A Antagonizes ATM and ATR in a Cdk2- and Cdc7-Independent DNA Damage Checkpoint. Mol. Cell. Biol., March 1, 2006; 26(5): 1997 - 2011. [Abstract] [Full Text] [PDF]
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 R. Sakasai, K. Shinohe, Y. Ichijima, N. Okita, A. Shibata, K. Asahina, and H. Teraoka Differential involvement of phosphatidylinositol 3-kinase-related protein kinases in hyperphosphorylation of replication protein A2 in response to replication-mediated DNA double-strand breaks Genes Cells, March 1, 2006; 11(3): 237 - 246. [Abstract] [Full Text] [PDF]
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S. J. H. Arlander, S. J. Felts, J. M. Wagner, B. Stensgard, D. O. Toft, and L. M. Karnitz Chaperoning Checkpoint Kinase 1 (Chk1), an Hsp90 Client, with Purified Chaperones J. Biol. Chem., February 3, 2006; 281(5): 2989 - 2998. [Abstract] [Full Text] [PDF]
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T. Hiyama, M. Katsura, T. Yoshihara, M. Ishida, A. Kinomura, T. Tonda, T. Asahara, and K. Miyagawa Haploinsufficiency of the Mus81-Eme1 endonuclease activates the intra-S-phase and G2/M checkpoints and promotes rereplication in human cells Nucleic Acids Res., February 2, 2006; 34(3): 880 - 892. [Abstract] [Full Text] [PDF]
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Y. Seo, T. Yan, J. E. Schupp, K. Yamane, T. Radivoyevitch, and T. J. Kinsella The Interaction between Two Radiosensitizers: 5-Iododeoxyuridine and Caffeine Cancer Res., January 1, 2006; 66(1): 490 - 498. [Abstract] [Full Text] [PDF]
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R. A. Greenberg, B. Sobhian, S. Pathania, S. B. Cantor, Y. Nakatani, and D. M. Livingston Multifactorial contributions to an acute DNA damage response by BRCA1/BARD1-containing complexes Genes & Dev., January 1, 2006; 20(1): 34 - 46. [Abstract] [Full Text] [PDF]
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W. Zhang, L. Fletcher, and R. J. Muschel The Role of Polo-like Kinase 1 in the Inhibition of Centrosome Separation after Ionizing Radiation J. Biol. Chem., December 30, 2005; 280(52): 42994 - 42999. [Abstract] [Full Text] [PDF]
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G. I. Terzoudi, K. N. Manola, G. E. Pantelias, and G. Iliakis Checkpoint Abrogation in G2 Compromises Repair of Chromosomal Breaks in Ataxia Telangiectasia Cells Cancer Res., December 15, 2005; 65(24): 11292 - 11296. [Abstract] [Full Text] [PDF]
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Y. Lubelsky, N. Reuven, and Y. Shaul Autorepression of Rfx1 Gene Expression: Functional Conservation from Yeast to Humans in Response to DNA Replication Arrest Mol. Cell. Biol., December 1, 2005; 25(23): 10665 - 10673. [Abstract] [Full Text] [PDF]
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C.-G. Jung, H.-J. Kim, M. Kawaguchi, K. K. Khanna, H. Hida, K. Asai, H. Nishino, and Y. Miura Homeotic factor ATBF1 induces the cell cycle arrest associated with neuronal differentiation Development, December 1, 2005; 132(23): 5137 - 5145. [Abstract] [Full Text] [PDF]
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P. Lebrun, M. R. Montminy, and E. Van Obberghen Regulation of the Pancreatic Duodenal Homeobox-1 Protein by DNA-dependent Protein Kinase J. Biol. Chem., November 18, 2005; 280(46): 38203 - 38210. [Abstract] [Full Text] [PDF]
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K. Okamoto, K. Kashima, Y. Pereg, M. Ishida, S. Yamazaki, A. Nota, A. Teunisse, D. Migliorini, I. Kitabayashi, J.-C. Marine, et al. DNA Damage-Induced Phosphorylation of MdmX at Serine 367 Activates p53 by Targeting MdmX for Mdm2-Dependent Degradation Mol. Cell. Biol., November 1, 2005; 25(21): 9608 - 9620. [Abstract] [Full Text] [PDF]
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A. Tyagi, R. P. Singh, C. Agarwal, S. Siriwardana, R. A. Sclafani, and R. Agarwal Resveratrol causes Cdc2-tyr15 phosphorylation via ATM/ATR-Chk1/2-Cdc25C pathway as a central mechanism for S phase arrest in human ovarian carcinoma Ovcar-3 cells Carcinogenesis, November 1, 2005; 26(11): 1978 - 1987. [Abstract] [Full Text] [PDF]
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P. Kramata, Y.-P. Lu, Y.-R. Lou, J. L. Cohen, M. Olcha, S. Liu, and A. H. Conney Effect of administration of caffeine or green tea on the mutation profile in the p53 gene in early mutant p53-positive patches of epidermal cells induced by chronic UVB-irradiation of hairless SKH-1 mice Carcinogenesis, November 1, 2005; 26(11): 1965 - 1974. [Abstract] [Full Text] [PDF]
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