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Experimental Therapeutics |
-Radiation-induced G2-M Checkpoint Independently of p53 by Targeting Both of the Checkpoint Kinases, Chk2 and Chk1
Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20892-4255
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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10 nM). In addition, UCN-01 efficiently abrogated both the initiation and maintenance of IR-induced G2 arrest in HCT116 cells and their isogenic p53 (-/-) derivative, indicating that G2 checkpoint abrogation by UCN-01 is p53 independent. In the p53 (-/-) cells, there was no p21Waf1/Cip1 induction nor UCN-01-induced apoptosis. Taken together, these observations indicate that UCN-01 can modulate both Chk1 and Chk2 in intact cells and enhance IR-induced apoptosis in p53-deficient, and consequently p21-deficient, cells. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chk1 and Chk2 (also named hCds1) are the human homologues of the fission yeast checkpoint kinases Chk1 and Cds1, respectively. Studies in mammalian cells suggest that the two checkpoint kinases have distinct roles. Chk2 activation is most pronounced in response to DNA damage caused by DNA double-strand breaks such as those produced by IR. This activation pathway requires the kinase activity of the ATM gene product (8 , 9) . By contrast, Chk1 activation requires the kinase activity of ATR protein and is primarily elicited by DNA-replication-blocking agents such as hydroxyurea (HU) or UV (10) . The Chk1 and Chk2 pathways probably overlap and cooperate with each other to enforce the checkpoints after DNA damage (11) . In Chk2-/- mouse embryonic stem cells (12) as well as in human HEK-293 cells that are induced to express antisense Chk2 (13) , Chk2 is required for the maintenance of DNA damage-induced G2 arrest (12) and S-phase checkpoint (13) . In contrast, Chk1 is required for the initiation of G2 arrest in response to DNA damage (10) . Therefore, Chk1 and Chk2 might jointly enforce the G2 checkpoint response.
Both Chk1 and Chk2 phosphorylate Cdc25C on serine 216 (8 , 9 , 14) , an event proposed to be critical for G2 checkpoint regulation. Phosphorylation of serine 216 inactivates Cdc25C directly after DNA damage (15) and creates a binding site for 14-3-3 (16 , 17) . The 14-3-3-bound Cdc25C phosphatase is then sequestered in the cytoplasm and prevented from activating its nuclear substrate Cdc2 (Cdk1), a critical kinase for driving cells through G2 (16) . Recently, Chk2 and Chk1 were also found to phosphorylate p53 in vitro on serine 20 (12 , 18 , 19) , a site implicated in p53 stabilization by regulation of the p53-MDM2 interactions (20 , 21) . Moreover, Chk2 is required for IR-induced p53 stabilization but not required for UV-induced response, indicating the key role of Chk2 in connecting p53 to the cellular response to IR-mediated DNA double-strand breaks (12) . In contrast, p53 activation by UV has been associated with Chk1 (12) . Recently, Chk2 has also been implicated in the S-phase checkpoint through phosphorylation and degradation of Cdc25A, thereby preventing the activation of the Cdk2/Cyclin A kinase (22) .
7-hydroxystaurosporine (UCN-01) is an anticancer agent in Phase II clinical trials. UCN-01 enhances the sensitivity of cancer cells to radiation and chemotherapeutic agents by abrogating DNA damage-induced checkpoints (3 , 4 , 7) . UCN-01 abrogates the G2 checkpoint by targeting the Cdc25C-Cdc2 regulatory pathway (23 , 24) . In vitro assays performed with recombinant protein kinases led to the conclusion that UCN-01 selectively inhibits Chk1 rather than Chk2 (24 , 25) .
In this study, we report that UCN-01 inhibits both IR-induced p53 up-regulation and p53 phosphorylation on serine 20. We propose that this effect is related to Chk2 inhibition because pharmacological concentrations of UCN-01 inhibited both of the immunoprecipitated Chk1 and Chk2 kinase activities, and IR activated only Chk2, whereas Chk1 activity remained unchanged. We also report that UCN-01 abrogates both the initiation and maintenance of IR-induced G2 arrest in both p53 wild-type and p53-null HCT116 cells, and that UCN-01 induces apoptosis selectively in the p53-null cells that fail to up-regulate p21Waf1/Cip1 in response to IR.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression and Purification of Recombinant Proteins.
Total RNA was extracted from HT29 cells with RNeasy (Qiagen, Valencia, CA). Reverse-transcription was performed according to the manufacturer’s instruction (Perkin-Elmer, Branchburg, NJ). A cDNA- encoding full-length Chk2/hCds1 was PCR amplified using the 5' primer 5'-CAT ATG TCT CGG GAG TG GAT GTT GAG G-3') and 3' primer (5'-CTC GAG GAC ATT TCT TTC GTG TTC AAA C-3') according to the published sequence (8)
. The PCR product was inserted into plasmid pET-15b (Novagen, Madison, WI) and expressed in BL-21 (DE3) bacteria after IPTG induction. The cell pellet from a 1-liter culture was washed in ice-cold 20 mM Tris-HCl (pH 7.9) buffer, sonicated, and then centrifuged at 14,000 x g for 30 min. Recombinant Chk2 was purified from the supernatants over nickel-column chromatography according to the manufacturer’s instruction (Novagen, Madison, WI). To generate a Cdc25C fragment as a GST fusion protein, the cDNA fragment encoding amino acids 200–256 of Cdc25C was amplified by PCR (26)
from a human cDNA library and cloned into the pGEX-2T vector.
Protein Analyses.
Cell lysates were prepared as reported previously (7)
. Briefly, cells were lysed with cell lysis buffer [0.3% NP40, 1 mM EDTA, 50 mM Tris-HCl (pH 7.4), 2 mM EGTA, 1% Triton X-100, 150 mM NaCl, 25 mM NaF, 1 mM Na3VO4, 2 mM AEBSF, 5 µg/ml aprotinin, 1 µg/ml leupeptin] for 30 min on ice, and the lysates were clarified by centrifugation at 12,000 xg for 15 min at 4°C. Protein concentration was quantified (Bio-Rad, Hercules, CA) and protein samples (100 µg) were separated by SDS/PAGE and transferred onto immobilon membranes (Millipore, Bedford, MA). Chk2, p53, and p21Waf1/Cip1 proteins were identified using anti-Chk2, anti-p53, and anti-p21Waf1/Cip1 primary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), and reactive bands were visualized using the enhanced chemiluminescence detection system (NEN Life Science Products, Boston, MA).
To study the effect of UCN-01 on p53 phosphorylation, HCT116 cells were treated with 50 µM proteasome inhibitor LLnL (Sigma) for 30 min and UCN-01 for 15 min before 20-Gy IR (27) . After 1 h incubation, cell extracts were processed as described above and analyzed with anti-p53 (Santa Cruz), anti-phospho-serine 15 (New England Biolab), and anti-phospho-serine 20 antibodies (kindly provided by Dr. Yoichi Taya, National Cancer Center Research Institute, Tokyo, Japan).
Protein Kinase Assays.
The Chk1 and Chk2 proteins were immunoprecipitated from HCT116 cells by anti-Chk2 or anti-Chk1 antibody (Santa Cruz Biotechnology). Immunoprecipitated samples were incubated with 1 µg of GST-Cdc25C in reaction buffer [50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 10 µM ATP, and 10 µCi [
-32P]-ATP] in a total volume of 20 µl and incubated at 30°C for 30 min. For the drug inhibition experiments, samples were coincubated with drug during the reactions. Five µl of 5x sample buffer were added, and samples were boiled for 5 min and separated by 12% SDS-PAGE. Chk2 protein kinase activity, measured as 32P incorporation into GST-Cdc25C, was determined using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Assays performed with recombinant Chk2 was assayed in the same conditions as described above except using 200 ng of recombinant Chk2.
Cell Cycle Analyses.
Cell cycle assays were performed as described previously (7)
. Briefly, cells were harvested and fixed in 70% ethanol. The fixed cells were then stained with propidium iodide (50 µg/ml) after treatment with RNase (5 µg/ml). The stained cells were analyzed for DNA content by fluorescence-activated cell sorting (FACS) in a FACScan (SOBR model, Becton Dickinson Instrument, San Jose, CA). Cell cycle fractions were quantified with CellQuest (Becton Dickinson).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Lane 2) was markedly reduced by treatment with UCN-01 in a dose-dependent manner (Fig. 1A
, Lanes 3–6). This effect could be observed at 10 nM UCN-01 (additional data not shown). Recently, Chk2 has been shown to be directly responsible for the p53 stabilization after cellular exposure to IR (12
, 19) . This effect has been related to Chk2-mediated phosphorylation of p53 on serine 20 (12
, 19
, 20
, 28)
. Chk1 has also been shown to phosphorylate recombinant p53 in vitro on serine 20 (18)
.
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. As expected, phosphorylation on both serine 15 and serine 20 residues was increased in irradiated cells, as detected by immunoblotting of cellular extracts with phosphoserine 15- or phosphoserine 20-specific antibodies. UCN-01 treatment strongly inhibited IR-induced serine 20 phosphorylation, whereas serine 15 phosphorylation remained elevated in UCN-01-treated cells (Fig. 1B)
.
Because both Chk2 and Chk1 can phosphorylate p53 on serine 20 (12
, 18
, 19
, 32)
, we examined Chk1 and Chk2 kinase activity in HCT116 cells in response to IR. Irradiated cells exhibited a significant increase in Chk2 activity, whereas Chk1 activity displayed no detectable change (Fig. 1C)
. These observations are consistent with previous reports indicating that Chk2 rather than Chk1 is a primary checkpoint kinase responsive to IR treatment (8
, 9
, 33)
. These data suggested that UCN-01 prevents Chk2-mediated p53 phosphorylation on serine 20 by inhibiting Chk2. The lack of inhibition of serine 15 phosphorylation by UCN-01 indicates that UCN-01 has no detectable effect on ATM.
UCN-01 Inhibits the Kinase Activities of Both Chk1 and Chk2 Immunoprecipitated from HCT116 Cells.
UCN-01 has recently been shown to inhibit recombinant Chk1 rather than Chk2, as determined by in vitro assays with recombinant Chk1 and Chk2 proteins (24
, 25)
. To examine whether endogenous Chk2 was sensitive to UCN-01, Chk1 or Chk2 were immunoprecipitated from HCT116 cells treated with IR. Kinase assays were performed with the immune complexes in the absence or presence of UCN-01. A GST-Cdc25C fusion protein was used as substrate (26)
because serine 216 in GST-Cdc25C has been described as a major regulatory phosphorylation site of Cdc25C for both Chk1 and Chk2 (8
, 9)
. The specificity of the anti-Chk2 immunoprecipitates was first confirmed by immunoblotting as shown in Fig. 2A
.
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. Similar inhibition was also observed from immunoprecipitates of untreated cells (data not shown). We also verified the optimal ATP concentrations for Chk2 and Chk1 kinase activities in our assay condition. As seen in Fig. 2C
, a low ATP concentration (approximately 20 µM) is required for maximal Chk2 activity, whereas a much higher ATP concentration (
100 µM) is required for Chk1 (Fig. 2C)
. Because UCN-01 is a competitive inhibitor of ATP (34)
, the 10 µM of ATP used in our assay is predicted to be within the optimal ranges for Chk2 kinase inhibition assay.
To exclude the possibility that Chk2 might interact with Chk1 physically in vivo and that Chk1 might be coimmunoprecipitated by anti-Chk2 antibody, we performed immunoblotting of Chk2 or Chk1 immunoprecipitates with anti-Chk1 antibody. Under our experimental conditions, we did not detect Chk1 in the Chk2 immunoprecipitates. As a control, Chk1 was readily detected in Chk1 immunoprecipitates (Fig. 2A
, right panel). We also could not detect Chk2 in the Chk1 immunoprecipitates (not shown). Therefore, we concluded that kinase inhibition by UCN-01 in the Chk2 immunoprecipitates was not caused by the presence of coimmunoprecipitated Chk1. These findings suggest that UCN-01 can inhibit cellular Chk2.
Recombinant Chk2 Is Resistant to UCN-01 Inhibition and Is Expressed as Multimer.
To test the sensitivity of recombinant Chk2 to UCN-01, we subcloned the Chk2 full-length cDNA into pET-15b containing a polyhistidine tag for subsequent purification of recombinant His-Chk2 using nickel-column chromatography. As expected, purified recombinant His-Chk2 ran as a Mr 66,000 protein in SDS-PAGE (Fig. 3A
, left panel). Native gel electrophoresis demonstrated that recombinant Chk2 exists in multiple forms, with bands corresponding to monomer, dimer, and tetramer (Fig. 3A
, right panel).
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. Consistent with a recent report (24)
, we observed that recombinant Chk2 was relatively resistant to inhibition by UCN-01, with IC50 around 500 nM (Fig. 3C)
.
UCN-01 Abrogates the Initiation and Maintenance of the IR-induced G2 Checkpoint Independently of p53.
Recent studies have shown that the DNA-damage-induced G2 checkpoint is established and maintained by differential signaling pathways. Chk2 has been implicated in the maintenance of the G2 arrest (12
, 13)
. To study the point at which UCN-01 interrupts the G2 checkpoint, we examined the effects of UCN-01 on the initiation and the maintenance of the IR-induced G2 arrest. As expected, 16 h after IR, the wild-type p53 HCT116 cells arrested both in G1 and G2 phases, whereas the HCT116 p53 (-/-) cells arrested only in G2 (Fig. 4, A and B)
. Treatment of cells with UCN-01 immediately (15 min) before exposure to IR greatly reduced the number of cells arrested in G2, indicating that UCN-01 prevented the G2 arrest in both p53 wild-type and p53-null cells (Fig. 4A)
.
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. UCN-01 was then added to the arrested cells for 8 h before harvesting the cells for cell cycle analysis. In the absence of UCN-01, the G2 arrest was maintained for at least up to 36 h after IR treatment (Fig. 4B)
. For both p53 wild-type and p53-null cells, the addition of UCN-01 to the G2-arrested cells caused a concentration-dependent decrease of cells in the G2 phase and a corresponding increase in the G1 (and sub-G1) populations (Fig. 4B)
, indicating that cells were unable to sustain the G2 arrest in the presence of UCN-01. Treatment of irradiated cells with nocodazole and UCN-01 arrested cells mostly in G2-M, excluding the possibility that cells were arrested in G1 (data not shown). These results demonstrate that UCN-01 effectively inhibits the maintenance of the G2 arrest in both p53 (+/+) and p53 (-/-) cells.
Inactivation of Cdc2 (= Cdk1) kinase is a well-characterized mechanism for checkpoint-mediated G2 arrest (26
, 35
, 36)
. This inactivation is attributable, at least in part, to the decreased activity of Cdc25C phosphatase, which results in an increased phosphorylation of Cdc2 on its T14- and Y-15-inhibitory residues. Inactive phosphorylated Cdc2 can be detected by SDS-PAGE as a band with slower electrophoretic mobility (37)
. Cdc2 phosphorylation was examined under the same conditions as described in Fig. 4B
. Consistent with the cell cycle analysis, UCN-01 inhibited IR-induced Cdc2 phosphorylation, as measured by Western blotting (Fig. 4C)
. Thus, UCN-01 may activate Cdc25C by inhibiting either Chk1 or Chk2. Removal of the inhibitory phosphorylations of Cdc2 by UCN-01 would activate the Cdc2 kinase and abrogate the sustained G2 arrest induced by IR.
Fig. 4C
shows that IR treatment of p53 wild-type (+/+) HCT116 cells led to an increase of p53 and p21Waf1/Cip1 that was sustained for at least 8 h (between time 24 and time 32 h after IR), and coincided with both the G1 and G2 arrests (Fig. 4B)
. UCN-01 inhibited the induction of p21Waf1/Cip1 in the HCT116 p53(+/+) cells in a dose-dependent manner. However, at the highest dose of UCN-01 (1 µM for 8 h), p21Waf1/Cip1 levels remained higher than in the untreated cells. Under these conditions, the p53 levels were only modestly reduced by UCN-01 (Fig. 4C)
, suggesting differential p53 regulation at early and late times after IR treatment. It is noticeable that UCN-01 can inhibit p21Waf1/Cip1 up-regulation independently of p53 down-regulation.
In HCT116 p53 (-/-) cells, no p21Waf1/Cip1 induction was observed after IR treatment, which is consistent with p53-dependent activation of p21Waf1/Cip1. UCN-01 preferentially increased the number of cells with sub-G1 DNA content in a dose-dependent manner (Fig. 4, B and D)
. This increase is in agreement with previous reports (3, 4, 5, 6, 7)
that UCN-01 preferentially enhances the DNA-damage-induced apoptosis in cells lacking p53 and defective in G1 arrest.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). However, Chk2 appears to be the essential cellular kinase for p53 phosphorylation on serine 20 in response to IR because Chk2-/- mouse embryonic stem cells display a completely defective p53 stabilization in response to IR (but not to UV; Ref. 12
). Compared with Chk2, the role of Chk1 in cellular response to IR is less well elucidated. Recent studies have shown that Chk1 activity is not increased by IR (22
, 33)
. Nevertheless, Chk1 phosphorylation has been reported at a moderate level after exposure to IR (10
, 26)
and to be more pronounced in response to UV (40)
. Furthermore, a recent study by Chen et al. (41)
indicated that phosphorylation of Chk1 is not correlated with its kinase activity. In accordance with these studies, we found no change in Chk1 activity by immunoprecipitation/kinase assays, whereas Chk2 activity was markedly increased in response to IR (Fig. 1C)
. Therefore, our observations are consistent with the possibility that Chk2 is the primary protein kinase that stabilizes and activates p53 by phosphorylating the serine 20 residue of p53 in IR-treated HCT116 cells (Fig. 5)
.
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10 nM; Fig. 2B
). UCN-01 has been shown previously to inhibit recombinant Chk1 rather than recombinant Chk2 kinases as determined by in vitro biochemical assays (24
, 25)
. It is not likely that the inhibition of immunoprecipitated Chk2 is caused by the unspecific immunoprecipitation because Chk2 antibody obtained from another source gave a similar result.3
Furthermore, this kinase would have to be active on GST-Cdc25C and to be sensitive to UCN-01. The apparent discrepancy between our Chk2 inhibition by UCN-01 data and the previous reports is probably attributable to the different ATP concentrations used. Under our assay conditions, we have determined that Chk2 requires low concentrations of ATP (
20 µM) for maximal kinase activity, whereas Chk1 requires much a higher concentration of ATP (100 µM; Fig. 2C
). Because UCN-01 is a competitive inhibitor of ATP (34)
, a high concentration of ATP (>20 µM), as used in the previous studies (24
, 25)
, will likely render Chk2 less sensitive to UCN-01 in the kinase assays (Fig. 2C)
.
We observed that our bacterially expressed Chk2 was markedly less sensitive to UCN-01(with IC50, 500 nM) than immunoprecipitated cellular Chk2 (IC50, 10 nM). One possible explanation for the differences in UCN-01 sensitivity between the endogenous and recombinant Chk2 might be that recombinant Chk2, when overexpressed, is folded differently from cellular Chk2 under physiological conditions. Indeed, we found that the recombinant Chk2 is expressed in multiple forms including dimers and tetramers. The UCN-01 sensitivities of immunoprecipitated Chk2 might also be dependent on associated cofactors that bind to Chk2. A recent study showed that endogenous Chk2 is a component of a large protein complex of Mr 200,000 or even Mr 600,000 (42)
. In any case, the differences in drug sensitivity of recombinant and immunoprecipitated Chk2 kinase raise some questions regarding the search for Chk2 inhibitors using only recombinant protein. UCN-01 has been reported to inhibit other protein kinases beside Chk1. UCN-01 inhibits C-TAK1 kinase (25
, 43)
, which phosphorylates Cdc25C on serine 216 and prevents Cyclin B-cdc2 activation during the normal cell cycle in the absence of DNA damage. Sato et al. also reported recently that UCN-01 inhibits the PDK1/Akt survival pathway (44)
.
Our experiments demonstrate that UCN-01 effectively abrogates not only the initiation but also the maintenance of the IR-induced G2 checkpoint, which is at least in part regulated by the Cdc25C-cdc2 pathway (Figs. 4
and 5
). Given that Chk1 and Chk2 are required for the initiation and maintenance of G2 checkpoint, respectively (10
, 12)
, these observation are consistent with the possibility that UCN-01 abrogates the G2 checkpoint by blocking both Chk1 and Chk2. Recently, a novel alkaloid G2 checkpoint inhibitor, debromohymenialdisine, isolated from a marine sponge has been reported to be a dual inhibitor of Chk2 and Chk1 (45)
. In contrast to the previous report that UCN-01 preferentially abrogates the IR-induced G2 checkpoint in cells with defective p53 function (4)
, we found that UCN-01 abrogated the IR-induced G2 phase accumulation effectively both in p53 wild-type and p53-null HCT116 cells (Fig. 4, A and B)
. These findings indicate that UCN-01 can abrogate the G2 checkpoint independently of p53.
Finally, we found that UCN-01 preferentially potentiated IR-induced apoptosis in p53-null HCT116 cells. However, this potentiation was not simply caused by the loss of G2 arrest because both the p53 wild-type and p53-null cells demonstrated abrogation of the IR-induced G2 arrest. Consistently, UCN-01 has recently been shown to enhance the toxicity of several clinical agents in p53-defective human cancer cell lines independently of its ability to abrogate G2 arrest (46 , 47) . Similarly, Riberio et al. (48) demonstrated that radiosensitization by caffeine is not dependent on loss of the G2 checkpoint. Thus, G2 checkpoint abrogation by UCN-01 might be necessary but is not sufficient for the enhancement of IR-induced cell death. In the HCT116 p53 wild-type cells, the G2 and G1 arrests were associated with elevation of p21Waf1/Cip1. This p21Waf1/Cip1 up-regulation was partially suppressed by UCN-01. Thus, the lack of apoptosis in the p53 (+/+) cells might be related, at least in part, to p21Waf1/Cip1 induction, which has been shown to elicit G1 arrest and confer resistance to apoptosis (43 , 49, 50, 51) . By contrast, in HCT116 p53 (-/-) cells, we did not observe p21Waf1/Cip1 induction after IR treatment. Thus, the lack of p21Waf1/Cip1 induction might be responsible, at least in part, for the enhanced apoptosis in the HCT116 p53 (-/-) cells treated with UCN-01.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at Laboratory of Molecular Pharmacology, Center for Cancer Research, Building 37, Room 5068, NIH, Bethesda, MD 20892-4255. Fax: (301) 402-0752; E-mail: pommier{at}nih.gov 
2 The abbreviations used are: IR, ionizing radiation; ATM, ataxia telangiectasia mutated; ATR, ATM- and Rad 3-related; IPTG, isopropylthio-ß-D-galactoside; GST, glutathion-S-transferase; AEBSF, 4-(2-aminoethyl)-benzenesulfonylfluoride.
3 J. Chen, personal communication.
Received 5/11/01. Accepted 8/20/02.
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G. K. Schwartz Development of Cell Cycle Active Drugs for the Treatment of Gastrointestinal Cancers: A New Approach to Cancer Therapy J. Clin. Oncol., July 10, 2005; 23(20): 4499 - 4508. [Abstract] [Full Text] [PDF]
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P. N. Lara Jr, P. C. Mack, T. Synold, P. Frankel, J. Longmate, P. H. Gumerlock, J. H. Doroshow, and D. R. Gandara The Cyclin-Dependent Kinase Inhibitor UCN-01 Plus Cisplatin in Advanced Solid Tumors: A California Cancer Consortium Phase I Pharmacokinetic and Molecular Correlative Trial Clin. Cancer Res., June 15, 2005; 11(12): 4444 - 4450. [Abstract] [Full Text] [PDF]
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E. A. Sausville Indifferently Pursued or Unowned Drugs: Who Should Lead Where Companies Do Not Tread? J. Clin. Oncol., March 20, 2005; 23(9): 1796 - 1798. [Full Text] [PDF]
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A. W. Adamson, D. I. Beardsley, W.-J. Kim, Y. Gao, R. Baskaran, and K. D. Brown Methylator-induced, Mismatch Repair-dependent G2 Arrest Is Activated through Chk1 and Chk2 Mol. Biol. Cell, March 1, 2005; 16(3): 1513 - 1526. [Abstract] [Full Text] [PDF]
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M. Katsuki, V. T. G. Chuang, K. Nishi, K. Kawahara, H. Nakayama, N. Yamaotsu, S. Hirono, and M. Otagiri Use of Photoaffinity Labeling and Site-directed Mutagenesis for Identification of the Key Residue Responsible for Extraordinarily High Affinity Binding of UCN-01 in Human {alpha}1-Acid Glycoprotein J. Biol. Chem., January 14, 2005; 280(2): 1384 - 1391. [Abstract] [Full Text] [PDF]
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S. B. Kondapaka, M. Zarnowski, D. R. Yver, E. A. Sausville, and S. W. Cushman 7-Hydroxystaurosporine (UCN-01) Inhibition of Akt Thr308 but not Ser473 Phosphorylation: A Basis for Decreased Insulin-Stimulated Glucose Transport Clin. Cancer Res., November 1, 2004; 10(21): 7192 - 7198. [Abstract] [Full Text] [PDF]
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E. M. Hammond, M. J. Dorie, and A. J. Giaccia Inhibition of ATR Leads to Increased Sensitivity to Hypoxia/Reoxygenation Cancer Res., September 15, 2004; 64(18): 6556 - 6562. [Abstract] [Full Text] [PDF]
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A. N. Tse and G. K. Schwartz Potentiation of Cytotoxicity of Topoisomerase I Poison by Concurrent and Sequential Treatment with the Checkpoint Inhibitor UCN-01 Involves Disparate Mechanisms Resulting in Either p53-Independent Clonogenic Suppression or p53-Dependent Mitotic Catastrophe Cancer Res., September 15, 2004; 64(18): 6635 - 6644. [Abstract] [Full Text] [PDF]
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T. Yan, A. B. Desai, J. W. Jacobberger, R. M. Sramkoski, T. Loh, and T. J. Kinsella CHK1 and CHK2 are differentially involved in mismatch repair-mediated 6-thioguanine-induced cell cycle checkpoint responses Mol. Cancer Ther., September 1, 2004; 3(9): 1147 - 1157. [Abstract] [Full Text] [PDF]
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G. P. Dasmahapatra, P. Didolkar, M. C. Alley, S. Ghosh, E. A. Sausville, and K. K. Roy In vitro Combination Treatment with Perifosine and UCN-01 Demonstrates Synergism against Prostate (PC-3) and Lung (A549) Epithelial Adenocarcinoma Cell Lines Clin. Cancer Res., August 1, 2004; 10(15): 5242 - 5252. [Abstract] [Full Text] [PDF]
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M.-B. Yin, Z.-R. Li, S. Cao, F. A. Durrani, R. G. Azrak, C. Frank, and Y. M. Rustum Enhanced 7-Ethyl-10-hydroxycamptothecin (SN-38) Lethality by Methylselenocysteine Is Associated with Chk2 Phosphorylation at Threonine-68 and Down-Regulation of Cdc6 Expression Mol. Pharmacol., July 1, 2004; 66(1): 153 - 160. [Abstract] [Full Text] [PDF]
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P. S. Kho, Z. Wang, L. Zhuang, Y. Li, J.-L. Chew, H.-H. Ng, E. T. Liu, and Q. Yu p53-regulated Transcriptional Program Associated with Genotoxic Stress-induced Apoptosis J. Biol. Chem., May 14, 2004; 279(20): 21183 - 21192. [Abstract] [Full Text] [PDF]
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T. Kawabe G2 checkpoint abrogators as anticancer drugs Mol. Cancer Ther., April 1, 2004; 3(4): 513 - 519. [Abstract] [Full Text] [PDF]
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X. Meng, Y. Yuan, A. Maestas, and Z. Shen Recovery from DNA Damage-induced G2 Arrest Requires Actin-binding Protein Filamin-A/Actin-binding Protein 280 J. Biol. Chem., February 13, 2004; 279(7): 6098 - 6105. [Abstract] [Full Text] [PDF]
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B. J. Barnes, M. J. Kellum, K. E. Pinder, J. A. Frisancho, and P. M. Pitha Interferon Regulatory Factor 5, a Novel Mediator of Cell Cycle Arrest and Cell Death Cancer Res., October 1, 2003; 63(19): 6424 - 6431. [Abstract] [Full Text] [PDF]
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P. V. Jallepalli, C. Lengauer, B. Vogelstein, and F. Bunz The Chk2 Tumor Suppressor Is Not Required for p53 Responses in Human Cancer Cells J. Biol. Chem., May 30, 2003; 278(23): 20475 - 20479. [Abstract] [Full Text] [PDF]
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T. Furuta, H. Takemura, Z.-Y. Liao, G. J. Aune, C. Redon, O. A. Sedelnikova, D. R. Pilch, E. P. Rogakou, A. Celeste, H. T. Chen, et al. Phosphorylation of Histone H2AX and Activation of Mre11, Rad50, and Nbs1 in Response to Replication-dependent DNA Double-strand Breaks Induced by Mammalian DNA Topoisomerase I Cleavage Complexes J. Biol. Chem., May 23, 2003; 278(22): 20303 - 20312. [Abstract] [Full Text] [PDF]
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E. A. Kohn, C. J. Yoo, and A. Eastman The Protein Kinase C Inhibitor Go6976 Is a Potent Inhibitor of DNA Damage-induced S and G2 Cell Cycle Checkpoints Cancer Res., January 1, 2003; 63(1): 31 - 35. [Abstract] [Full Text] [PDF]
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) and Chk2 (
) activity. The Chk1/2 kinase assays were performed in the presence of various concentrations of ATP as indicated. Kinase activity is presented as percentages of maximal activity.
, protein degradation; (
), activation; 
, inhibition.