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The Radiosensitizing Agent 7-Hydroxystaurosporine (UCN-01) Inhibits the DNA Damage Checkpoint Kinase hChk1¹

Division of Oncology Research, Mayo Clinic, Rochester, Minnesota 55905 [E. C. B., D. F. L., L. M. K., J. N. S.], and Department of Pharmacology and Cancer Biology, Duke University, Durham, North Carolina 27710 [R. T. A.]

	ABSTRACT

Top ABSTRACT Introduction Materials and Methods Results and Discussion Note Added in Proof REFERENCES

 
The investigational anticancer agent 7-hydroxystaurosporine (UCN-01) abrogates the G₂ checkpoint in tumor cells and sensitizes them to the lethal effects of genotoxic anticancer agents. On the basis of the role of the Cdc25C phosphatase in maintenance of this damage-inducible checkpoint, we hypothesized that UCN-01 inhibits a component of the signal transduction pathway that modulates Cdc25C phosphorylation. Of the three kinases known to phosphorylate Cdc25C on Ser²¹⁶, both checkpoint kinase 1 (hChk1) and Cdc25C-associated protein kinase 1 (cTAK1) were potently inhibited by UCN-01 with IC₅₀s of 11 and 27 nM, respectively. Treatment of K562 erythroblastoid leukemia cells with similar drug concentrations resulted in decreased levels of Ser²¹⁶ phosphorylation of Cdc25C and complete disruption of the -radiation-induced G₂ checkpoint. In contrast to hChk1, the hChk2 kinase was 100-fold more resistant to inhibition by UCN-01 (IC₅₀, 1040 nM). These results suggest that disruption of the DNA damage-induced G₂ checkpoint by UCN-01 is mediated through the inhibition of the Cdc25C kinases, hChk1 and cTAK1, and that hChk2 activity is not sufficient to enforce the G₂ checkpoint in cells treated with a pharmacological inhibitor of hChk1.

	Introduction

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DNA damage-inducible cell cycle checkpoints are complex signal transduction networks that integrate the cellular responses to genotoxic insults by arresting cell cycle progression during the repair of DNA damage or the induction of apoptosis. The integrity of these checkpoint pathways is critical for maintenance of genomic stability and cellular recovery from genotoxic damage. In genetic models, loss of the G₂-M checkpoint leads to an increased sensitivity to DNA-damaging agents, suggesting that small molecule inhibitors of the G₂-M checkpoint response might be useful in cancer therapy as radio- and chemosensitizing agents (1) . In fact, UCN-01³ is one of several compounds that abrogates the G₂-M checkpoint at concentrations associated with sensitization of cells to DNA-damaging agents (2 , 3) . Because UCN-01 inhibits multiple protein kinases (4) , the identification of the molecular target(s) responsible for UCN-01-mediated checkpoint abrogation could facilitate the identification of more selective second-generation checkpoint inhibitors.

The induction of a G₂ arrest after DNA damage depends, in part, on inhibition of cyclin B1/Cdk1 activity through phosphorylation of the Cdk1 subunit at Thr¹⁴ and Tyr¹⁵ (5) . The dynamic changes in the phosphorylation of Cdk1 during both the normal cell cycle and in response to DNA damage are controlled by an interplay between the enzymatic activities of the Wee1-like kinases and the Cdc25C phosphatase. In recent years, the signaling pathways controlling Cdc25C activation and subcellular localization have been partially elucidated. Current models suggest that the serine-threonine kinases hChk1 and hChk2 are activated by DNA damage signaling pathway(s) dependent on the ATM family of protein kinases (6, 7, 8) . Activated hChk1 and/or hChk2 then phosphorylate Cdc25C on Ser²¹⁶, which creates a consensus binding site for 14-3-3 proteins. In undamaged interphase cells, Ser²¹⁶ also is phosphorylated by a third, constitutively active kinase, the Cdc25C-associated protein kinase (cTAK1). Association with 14-3-3 proteins leads to sequestration of phosphorylated Cdc25C in the cytoplasm and prevents premature dephosphorylation of Cdk1 and activation of the cyclin B1/Cdk1 complex.

Staurosporine and UCN-01 are capable of inhibiting the phosphotransferase activities of several serine-threonine protein kinases (4) . Because the kinase activity of Wee1 is resistant to UCN-01 (9) , we hypothesized that the mechanism of UCN-01-mediated checkpoint abrogation might involve inhibition of one or more of the kinases that regulate Cdc25C. In this study, we show that UCN-01 inhibits hChk1 and cTAK1 at drug concentrations associated with abrogation of the G₂ checkpoint. In contrast, hChk2 was relatively resistant to checkpoint inhibitory concentrations of UCN-01. These data identify hChk1 as a relevant molecular target for UCN-01 and suggest that specific hChk1 inhibitors will efficiently disrupt the G₂ checkpoint.

	Materials and Methods

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Cell Culture, Antibodies, and Plasmid Constructs.
The K562 erythroblastoid leukemia cell line was maintained in RPMI 1640 (Life Technologies, Inc.) containing 10% fetal bovine serum. UCN-01 (generously provided by Dr. Edward Sausville, National Cancer Institute) was dissolved in DMSO and stored at -20°C. The viral HA-specific mouse monoclonal antibody, HA.11, was purchased from Babco, and a Cdc25C-specific polyclonal rabbit antisera (C-20) was purchased from Santa Cruz Biotechnology. hChk1 and hChk2 were amplified by PCR from a human testes cDNA library (Clontech), and the respective full-length cDNA was cloned into the pEF-BOS plasmid with COOH-terminal tandem HA epitope tags (HA₂). cTAK1 was PCR amplified from the same cDNA library and cloned into the pcDNA3.1+ plasmid with an NH₂-terminal tandem HA epitope tag. The coding sequence for cTAK1 differed from that published previously and contained a single amino acid substitution (G443S; Ref. 10 ). This difference was present in all five cDNA clones sequenced and in nine human sequences from the dbEST database (GenBank), which suggests that this variant represents a polymorphism of the cTAK1 sequence. The kinase dead point-mutants, Chk1 (D130A), Chk2 (D347A), and cTAK1 (D196N), were generated by site-directed mutagenesis using the GeneEditor kit from Promega Corp. The GST-Cdc25C^200–256 fusion protein was generated as described previously (11) .

Immune Complex Kinase Assays.
The hChk1 and hChk2 kinase assays were performed as described previously for hChk1 (11) . Kinase assays for cTAK were performed essentially as described for hChk1 except for minor changes in the immunoprecipitation conditions. K562 cells transiently expressing epitope-tagged cTAK1 were lysed in buffer (20 mM HEPES, 0.15 M NaCl, 1.5 mM MgCl₂, and 1 mM EGTA, pH 7.4) containing 1 mM DTT, 10 µg/ml aprotinin, 5 µg/ml pepstatin, 5 µg/ml leupeptin, 20 nM microcystin, 10 mM ß-glycerophosphate, and 0.5% Triton X-100. After immunoprecipitation with HA.11 and protein A-Sepharose beads, immunoprecipitates were washed twice in lysis buffer, twice in high-salt buffer [0.1 M Tris-HCl (pH 7.4), 0.6 M NaCl], and twice in kinase buffer [50 mM Tris (pH 7.4), 10 mM MgCl₂]. Kinase assays were then performed as outlined previously for hChk1 (11) . All kinase reactions were performed under linear reaction conditions.

Flow Cytometry.
Ethanol-fixed cells were stained with propidium iodide and analyzed by flow cytometry as described previously (12) .

Cdc25C Mobility.
K562 cells were treated with graded concentrations of UCN-01 and then lysed in TNE buffer [50 mM Tris (pH 8.0), 150 mM sodium chloride, 5 mM EDTA, 1% NP40, and 0.1% SDS] containing 0.1 mM sodium orthovanadate, 10 µg/ml aprotinin, 5 µg/ml pepstatin, 5 µg/ml leupeptin, 20 nM microcystin, 200 µM dephostatin, 10 µM cypermethrin, 200 nM okadaic acid, and 25 nM tautomycin. The detergent-soluble proteins were then processed for immunoblotting with Cdc25C-specific antisera.

hChk1/hChk2 Mobility Shift.
K562 cells were lysed in TNE buffer containing 1 mM DTT, 10 µg/ml aprotinin, 5 µg/ml pepstatin, 5 µg/ml leupeptin, 20 nM microcystin, and 10 mM ß-glycerophosphate. The detergent-soluble proteins were then processed for immunoblotting with the HA.11 monoclonal antibody.

Statistics.
All statistical analyses were performed with the Sigma Plot 5.0 (SPSS) software package. The kinase inhibition data were fit with the Hill four-parameter model by the least-squares method. The concentrations of UCN-01 resulting in half-maximal inhibition (IC₅₀) for the respective kinases were calculated by solving the model for a relative activity of 0.5.

	Results and Discussion

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As a preliminary step toward identifying relevant molecular target(s) inhibited by UCN-01, we developed a UCN-01 concentration-inhibition relationship for disruption of the G₂ checkpoint in exponentially growing K562 erythroblastoid leukemia cells. K562 cells were treated with 8 Gy -radiation and graded concentrations of UCN-01. After 21 h, 80% of cells were arrested in G₂ after irradiation alone, which is consistent with the lack of a DNA damage-inducible G₁ checkpoint in this p53-null cell line. In contrast, treatment of cells with 100 nM UCN-01 completely abrogated the radiation-induced G₂ arrest (Fig. 1) . When parallel samples were treated with the microtubule inhibitor nocodazole, nearly all irradiated cells were arrested with a tetraploid DNA content with or without UCN-01 treatment (Fig. 1A and data not shown). These results demonstrate that the drug-treated cells traversed the G₂-M checkpoint and were not simply arrested in G₁. On the basis of these data, we predicted that one or more kinases in the G₂ checkpoint pathway would be inhibited by UCN-01 at concentrations of 100 nM.

View larger version (24K): [in this window] [in a new window]   Fig. 1. Effects of UCN-01 on the G2 checkpoint. A, K562 cells were treated with 0 or 8 Gy -radiation and graded concentrations of UCN-01. In the indicated samples, 0.3 µg/ml of the nocodazole (noc) was added at the time of irradiation. After 21 h, cells were fixed in 70% ethanol, and cell cycle distributions were determined by staining with propidium iodide and analysis with flow cytometry. Three independent trials were performed. Histograms of red fluorescence intensity (DNA content) from 20,000 ungated events are shown from a representative experiment. The percentage of cells arrested in G2 are listed on each histogram. B, the results are summarized graphically for cells treated with 8 Gy and graded concentrations of UCN-01. The data are presented as the means from three independent trials; bars, SE.

View larger version (29K): [in this window] [in a new window]   Fig. 2. Inhibition of hChk1, hChk2, and cTAK1 activities by UCN-01. K562 erythroblastoid leukemia cells were transfected with the indicated constructs for hChk1 (A), hChk2 (B), and cTAK1 (C), and cells were lysed 18–20 h later. Epitope-tagged kinases were immunoprecipitated with an HA-specific monoclonal antibody, and the immunoprecipitates were preincubated on ice with buffer alone or 2x final concentrations of UCN-01. Kinase reactions were initiated by addition of [-32P]ATP and the GST-Cdc25C200–256 kinase substrate. The reaction products were resolved by SDS-PAGE, and the incorporation of 32Pi into GST-Cdc25C200–256 was determined by phosphorimaging. Equal expression of epitope-tagged proteins was then confirmed by immunoblotting with an HA-specific antibody. The final concentration of UCN-01 used in the indicated samples was 300 nM. In experiments using graded concentrations of UCN-01, the kinase activity at a given drug concentration was normalized to that measured in the untreated control, and the results are summarized graphically. Data points represent the means from three independent experiments; bars, SE. The data were fit with a Hill four-parameter regression model, and IC50s were calculated.

The phosphorylation of Cdc25C on Ser²¹⁶ and the exclusion of Cdc25C from the nucleus are maintained throughout interphase. In unstressed cells, this phosphorylation is presumably maintained by the cTAK1 kinase (10) . We evaluated the sensitivity of cTAK1 to UCN-01 to complete our survey of known protein kinases that can phosphorylate Cdc25C on Ser²¹⁶. Interestingly, cTAK1 was nearly as sensitive as hChk1 to the inhibitory effects of UCN-01 in immune-complex kinase assays with an IC₅₀ of 27 nM (Fig. 2C) . Although cTAK1 has no known function in the DNA damage checkpoint responses, we cannot exclude the possibility that inhibition of cTAK1 kinase activity by UCN-01 could contribute to the checkpoint inhibitory effects of this drug.

The results presented, to this point, support the hypothesis that the checkpoint inhibitory effects of UCN-01 are related to the inhibition of hChk1 but not hChk2 kinase activities. Both hChk1 and hChk2 are inducibly phosphorylated in response to genotoxic agents, and these modifications are presumed to be important in checkpoint activation (6 , 16) . Therefore, a UCN-01-induced block in an upstream event modulating hChk1 or hChk2 activation also might lead to abrogation of the G₂ checkpoint. To evaluate the effects of UCN-01 on the integrity of these upstream signaling pathways, we assessed whether UCN-01 affected the DNA damage-induced phosphorylation of transiently expressed hChk1 and hChk2 in K562 cells treated with 10 mM HU or 20 Gy -radiation. The mobility of hChk1 on SDS-PAGE was appreciably retarded 1 h after irradiation or continuous exposure to HU (data not shown), and pretreatment with 1 µM UCN-01 had no effect on this hChk1 mobility shift (Fig. 3A) . Likewise, the radiation-induced mobility shift of hChk2 also was unchanged by UCN-01 pretreatment (Fig. 3B) . Consistent with this observation, we have shown previously that the ATM kinase, which functions directly upstream of hChk2, also is resistant to inhibition by UCN-01 (11) . Thus, in contrast to the protein kinase activity of hChk1 itself, the pathways leading to hChk1 and hChk2 activation in damaged cells are not sensitive to UCN-01.

View larger version (31K): [in this window] [in a new window]   Fig. 3. Effects of UCN-01 on hChk1, hChk2, and Cdc25C phosphorylation. K562 cells were transiently transfected with constructs for hChk1(A) and hChk2 (B). After 18 h, cells were incubated with 1 µM UCN-01 for 15 min before exposure to 10 mM HU, 20 Gy -radiation () or control treatment (C). Cells were lysed after 60 min, and soluble proteins were resolved by SDS-PAGE. Proteins were transferred onto polyvinylidene difluoride membranes and then immunoblotted with an HA-specific monoclonal antibody. The results shown are representative of those obtained in three independent trials. C, Cdc25C mobility shift. K562 cells were exposed to 12 Gy -radiation. After 16 h, cells were treated with graded concentrations of UCN-01. At this time, 95% of cells were arrested in G2-M, as determined by flow cytometry (data not shown). Cells were lysed 60 min after drug treatment, and soluble proteins were resolved by SDS-PAGE. Proteins were transferred onto a polyvinylidene difluoride membrane and then immunoblotted with Cdc25C antiserum. The results shown are representative of those obtained in three independent trials.

The ATM and ATR proteins appear to control downstream signaling events in the G₂ checkpoint pathway (19, 20, 21) . We have shown previously that the checkpoint inhibitory effects of the radiosensitizing agent caffeine were related to the inhibition of ATM and ATR kinase activities (11) . Both epistasis experiments in yeast and genetic studies in mammalian cells suggest that these protein kinases participate in the activation of hChk1 and hChk2, which can then phosphorylate downstream targets such as Cdc25C. The present findings suggest that inhibition of hChk1 and cTAK1 kinase activities is sufficient to abrogate the G₂ checkpoint. Strikingly, the relative resistance of the hChk2 kinase to inhibition by UCN-01 suggests that activation of the hChk2 kinase, in the absence of hChk1 and cTAK1 kinase activities, is insufficient to maintain the -radiation-induced G₂ arrest. This implies either that phosphorylation of Cdc25C by hChk2 alone is insufficient to arrest cell cycle progression or that hChk2 is not a physiologically relevant protein kinase for Cdc25C in DNA-damaged cells. Although we cannot exclude the potential role for cTAK1 in the induction and/or maintenance of the checkpoint, our results reinforce the notion that a specific inhibitor of hChk1 would be an effective inhibitor of the G₂ checkpoint.

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 (22 , 23) . These studies suggest that agents that interfere with checkpoint-related proteins may show selectivity for tumor cells bearing intrinsic defects in specific checkpoint pathways. One of the most common mutations that affects checkpoint integrity is inactivation of the p53 tumor suppressor protein. Because p53 functions in several checkpoint pathways (19) , the loss of p53 might leave tumor cells more vulnerable to pharmacological inhibition of components of the remaining checkpoints in these cells. The expected outcome of checkpoint inhibitor treatment in this setting would be a selective increase in the sensitivities of cancer cells to conventional genotoxic cancer therapies. In fact, both caffeine and UCN-01 selectively abrogate the G₂ checkpoint in tumor cells that have lost p53-dependent checkpoint controls (2 , 24, 25, 26) . The recent demonstration that homozygous deletion of 14-3-3 in HCT116 colon cancer cells compromises the G₂ checkpoint provides novel insight into a potential mechanism for the selective pharmacological inhibition of the G₂ checkpoint (Fig. 4) . In normal cells, DNA damage leads to a p53-dependent accumulation of 14-3-3, which binds to and sequesters cyclin B1/Cdk1 complexes in the cytoplasm (27) . With cyclin B1/Cdk1 excluded from the nucleus, pharmacological disruption of the hChk1-dependent checkpoint pathway controlling Cdc25C localization will not lead to premature entry into mitosis. However, in tumor cells deficient in p53 function, the integrity of the G₂ arrest is solely dependent on the hChk1-dependent pathway, and disruption of this pathway could then lead to checkpoint abrogation.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Model for pharmacological G2 checkpoint abrogation. Two parallel signal transduction pathways prevent premature progression from G2 into mitosis in the face of damaged DNA. DNA damage induces the accumulation of 14-3-3 in a p53-dependent manner and leads to exclusion of cyclin B1/Cdk1 complexes from the nucleus. Unrepaired damage also leads to activation of hChk1, which then phosphorylates Cdc25C on Ser216. Phosphorylated Cdc25C is then sequestered in the cytoplasm after association with 14-3-3. Pharmacological inhibition of the hChk1-dependent checkpoint pathway will lead to loss of checkpoint integrity only if the p53-dependent signaling pathway has been compromised previously during the process of oncogenesis.

	Note Added in Proof

Top ABSTRACT Introduction Materials and Methods Results and Discussion Note Added in Proof REFERENCES

	ACKNOWLEDGMENTS

 
We thank Drs. Scott Kaufmann and Junjie Chen for helpful advice and discussions and James Tarara and the other members of the Mayo Cancer Center Flow Cytometry Laboratory for expert technical assistance.

	FOOTNOTES

 
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.

¹ This work was supported by the Mayo Foundation, Mayo Cancer Center, and by grants from the Fraternal Order of Eagles and the NIH CA52995 (to R. T. A.), CA69709 (to J. N. S.), and CA80829 (to J. N. S.).

² To whom requests for reprints should be addressed, at Mayo Foundation, Guggenheim Building, Room 1301, 200 First Street S.W., Rochester, MN 55905. Phone: (507) 266-5232; Fax: (507) 284-3906; E-mail: sarkaria.jann{at}mayo.edu

³ The abbreviations used are: UCN-01, 7-hydroxystaurosporine; Cdk, cyclin-dependent kinase; ATM, ataxia telangiectasia mutated; HA, hemagglutinin; PKC, protein kinase C; HU, hydroxyurea; hChk, checkpoint kinase; cTAK1, Cdc25C-associated protein kinase 1; ATR, AT and Rad3-related.

⁴ J. N. Sarkaria and D. F. Leistritz, unpublished data.

Received 12/ 9/99. Accepted 3/ 2/00.

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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]

				M. Kedde, C. le Sage, A. Duursma, E. Zlotorynski, B. van Leeuwen, W. Nijkamp, R. Beijersbergen, and R. Agami Telomerase-independent Regulation of ATR by Human Telomerase RNA J. Biol. Chem., December 29, 2006; 281(52): 40503 - 40514. [Abstract] [Full Text] [PDF]

				R. P. Perez, L. D. Lewis, A. P. Beelen, A. J. Olszanski, N. Johnston, C. H. Rhodes, B. Beaulieu, M. S. Ernstoff, and A. Eastman Modulation of Cell Cycle Progression in Human Tumors: A Pharmacokinetic and Tumor Molecular Pharmacodynamic Study of Cisplatin Plus the Chk1 Inhibitor UCN-01 (NSC 638850) Clin. Cancer Res., December 1, 2006; 12(23): 7079 - 7085. [Abstract] [Full Text] [PDF]

				B. Jaklevic, L. Uyetake, W. Lemstra, J. Chang, W. Leary, A. Edwards, S. Vidwans, O. Sibon, and T. Tin Su Contribution of Growth and Cell Cycle Checkpoints to Radiation Survival in Drosophila Genetics, December 1, 2006; 174(4): 1963 - 1972. [Abstract] [Full Text] [PDF]

				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]

				V. Leung-Pineda, C. E. Ryan, and H. Piwnica-Worms Phosphorylation of Chk1 by ATR Is Antagonized by a Chk1-Regulated Protein Phosphatase 2A Circuit Mol. Cell. Biol., October 15, 2006; 26(20): 7529 - 7538. [Abstract] [Full Text] [PDF]

				C. P. Fanton, M. W. Rowe, E. J. Moler, M. Ison-Dugenny, S. K. De Long, K. Rendahl, Y. Shao, T. Slabiak, T. G. Gesner, and M. L. MacKichan Development of a screening assay for surrogate markers of chk1 inhibitor-induced cell cycle release. J Biomol Screen, October 1, 2006; 11(7): 792 - 806. [Abstract] [PDF]

				K. M. Comess, J. D. Trumbull, C. Park, Z. Chen, R. A. Judge, M. J. Voorbach, M. Coen, L. Gao, H. Tang, P. Kovar, et al. Kinase Drug Discovery by Affinity Selection/Mass Spectrometry (ASMS): Application to DNA Damage Checkpoint Kinase Chk1 J Biomol Screen, October 1, 2006; 11(7): 755 - 764. [Abstract] [PDF]

				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]

				C. A. Granville, R. M. Memmott, J. J. Gills, and P. A. Dennis Handicapping the Race to Develop Inhibitors of the Phosphoinositide 3-Kinase/Akt/Mammalian Target of Rapamycin Pathway Clin. Cancer Res., February 1, 2006; 12(3): 679 - 689. [Abstract] [Full Text] [PDF]

				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]

				G. K. Schwartz and M. A. Shah Targeting the Cell Cycle: A New Approach to Cancer Therapy J. Clin. Oncol., December 20, 2005; 23(36): 9408 - 9421. [Abstract] [Full Text] [PDF]

				C. A. L. Clarke, L. N. Bennett, and P. R. Clarke Cleavage of Claspin by Caspase-7 during Apoptosis Inhibits the Chk1 Pathway J. Biol. Chem., October 21, 2005; 280(42): 35337 - 35345. [Abstract] [Full Text] [PDF]

				X. Liu, Y. Guo, Y. Li, Y. Jiang, S. Chubb, A. Azuma, P. Huang, A. Matsuda, W. Hittelman, and W. Plunkett Molecular Basis for G2 Arrest Induced by 2'-C-Cyano-2'-Deoxy-1-{beta}-D-Arabino-Pentofuranosylcytosine and Consequences of Checkpoint Abrogation Cancer Res., August 1, 2005; 65(15): 6874 - 6881. [Abstract] [Full Text] [PDF]

				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]

				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]

				K. Flatten, N. T. Dai, B. T. Vroman, D. Loegering, C. Erlichman, L. M. Karnitz, and S. H. Kaufmann The Role of Checkpoint Kinase 1 in Sensitivity to Topoisomerase I Poisons J. Biol. Chem., April 8, 2005; 280(14): 14349 - 14355. [Abstract] [Full Text] [PDF]

				U. K. Mukhopadhyay, A. M. Senderowicz, and G. Ferbeyre RNA Silencing of Checkpoint Regulators Sensitizes p53-Defective Prostate Cancer Cells to Chemotherapy while Sparing Normal Cells Cancer Res., April 1, 2005; 65(7): 2872 - 2881. [Abstract] [Full Text] [PDF]

				S. L. Maude and G. H. Enders Cdk Inhibition in Human Cells Compromises Chk1 Function and Activates a DNA Damage Response Cancer Res., February 1, 2005; 65(3): 780 - 786. [Abstract] [Full Text] [PDF]

				M. Urist, T. Tanaka, M. V. Poyurovsky, and C. Prives p73 induction after DNA damage is regulated by checkpoint kinases Chk1 and Chk2 Genes & Dev., December 15, 2004; 18(24): 3041 - 3054. [Abstract] [Full Text] [PDF]

				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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