7 Hydroxystaurosporine

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Yves Pommier - One of the best experts on this subject based on the ideXlab platform.

  • the intra s phase checkpoint affects both dna replication initiation and elongation single cell and dna fiber analyses
    Molecular and Cellular Biology, 2007
    Co-Authors: Jennifer A. Seiler, Mirit I Aladjem, Chiara Conti, Ali Syed, Yves Pommier

    To investigate the contribution of DNA replication initiation and elongation to the intra-S-phase checkpoint, we examined cells treated with the specific topoisomerase I inhibitor camptothecin. Camptothecin is a potent anticancer agent producing well-characterized replication-mediated DNA double-strand breaks through the collision of replication forks with topoisomerase I cleavage complexes. After a short dose of camptothecin in human colon carcinoma HT29 cells, DNA replication was inhibited rapidly and did not recover for several hours following drug removal. That inhibition occurred preferentially in late-S-phase, compared to early-S-phase, cells and was due to both an inhibition of initiation and elongation, as determined by pulse-labeling nucleotide incorporation in replication foci and DNA fibers. DNA replication was actively inhibited by checkpoint activation since 7-Hydroxystaurosporine (UCN-01), the specific Chk1 inhibitor CHIR-124, or transfection with small interfering RNA targeting Chk1 restored both initiation and elongation. Abrogation of the checkpoint markedly enhanced camptothecin-induced DNA damage at replication sites where histone γ-H2AX colocalized with replication foci. Together, our study demonstrates that the intra-S-phase checkpoint is exerted by Chk1 not only upon replication initiation but also upon DNA elongation.

  • The intra-S-phase checkpoint affects both DNA replication initiation and elongation: single-cell and -DNA fiber analyses
    Cancer Research, 2007
    Co-Authors: Yves Pommier, Jennifer A. Seiler, Chiara Conti, Mirit I Aladjem

    5227 To investigate the contribution of DNA replication initiation and elongation to the intra-S-phase checkpoint, we examined cells treated with the specific topoisomerase I inhibitor camptothecin. Camptothecin is a potent anticancer agent producing well-characterized replication DNA double-strand breaks through the collision of replication forks with topoisomerase I cleavage complexes. Following a short, non-toxic dose of camptothecin in human colon carcinoma HT29 cells, DNA replication was inhibited rapidly and did not recover for several hours following drug removal. That inhibition occurred preferentially in the late-S-, compared to early-S-phase cells, and was due to both an inhibition of initiation and elongation. DNA replication was actively inhibited by checkpoint activation since 7-Hydroxystaurosporine (UCN-01) or a specific Chk1 inhibitor restored both initiation and elongation. Abrogation of the checkpoint markedly enhanced CPT-induced DNA damage at replication foci sites where histone gamma-H2AX colocalized with replication foci. Together, our study demonstrates that the intra S-phase checkpoint is exerted not only upon replication initiation but also upon DNA elongation.

  • p21cdkn1a allows the repair of replication mediated dna double strand breaks induced by topoisomerase i and is inactivated by the checkpoint kinase inhibitor 7 Hydroxystaurosporine
    Oncogene, 2006
    Co-Authors: Takahisa Furuta, R L Hayward, Ling Hua Meng, Haruyuki Takemura, Gregory J Aune, William M Bonner, Mirit I Aladjem, Kurt W Kohn, Yves Pommier

    This study provides evidence for the importance of p21 CDKN1A for the repair of replication-mediated DNA double-strand breaks (DSBs) induced by topoisomerase I. We report that defects of p21 CDKN1A and p53 enhance camptothecin-induced histone H2AX phosphorylation (yH2AX), a marker for DNA DSBs. In human colon carcinoma HCT116 cells with wild-type (wt) p53, yH2AX reverses after camptothecin removal. By contrast, yH2AX increases after camptothecin removal in HCT116 cells deficient for p53 (p53-/-) or p21 CDKN1A (p21-/-) as the cells reach the late-S and G2 phases. Since p21-/- cells exhibit similar S-phase arrest as wt cells in response to camptothecin and aphidicolin does not abrogate the enhanced yH2AX formation in p21-/- cells, we conclude that enhanced yH2AX formation in p21-/- cells is not due to re-replication. The cell cycle checkpoint abrogator and Chkl/Chk2 inhibitor 7-Hydroxystaurosporine (UCN-01) also increases camptothecin-induced yH2AX formation and inhibits camptothecin-induced p21 CDKN1A upregulation in HCT116 wt cells. TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling) assays demonstrate that yH2AX formation in late S and G2 cells following CPT treatment corresponds to DNA breaks. However, these breaks are not related to apoptotic DNA fragmentation. We propose that p21 CDKN1A prevents the collapse of replication forks damaged by stabilized topoisomerase I cleavage complexes.

  • Abrogation of Chk1-mediated S/G2 checkpoint by UCN-01 enhances ara-C-induced cytotoxicity in human colon cancer cells.
    Acta pharmacologica Sinica, 2004
    Co-Authors: Rong-guang Shao, Chun-xia Cao, Yves Pommier

    AIM: To investigate whether 7-Hydroxystaurosporine (UCN-01) affects cell cycle progression in arabinosylcytosine (ara-C) treated human colon carcinoma HT-29 cells. METHODS: Cytotoxicity, DNA synthesis, cell cycle distribution, protein level, and kinase activity were determined by clonogenic assay, flow cytometry, DNA synthesis assay, immunoblotting, and kinase assays, respectively. RESULTS: UCN-01 abrogated an S/G2-phase checkpoint in HT-29 cells treated with ara-C. When UCN-01 was added after treatment with ara-C, the rate of recovery of DNA synthesis was enhanced and colony-forming ability diminished. Thus, premature recovery of DNA synthesis was associated with increased cytotoxicity. Measurements of cyclin A and B protein levels, Cdk2 and Cdc2 kinase activities, Cdc25C phosphorylation, and Chk1 kinase activity were consistent with UCN-01-induced abrogation of the S/G2-phase checkpoint in ara-C treated cells. CONCLUSION: The abrogation of the S/G2 checkpoint may be due to inhibition of Chk1 kinase by UCN-01. The enhanced cytotoxicity produced when UCN-01 was combined with ara-C suggested a rationale for the use of this drug combination for tumors that might be susceptible to cell cycle checkpoint abrogation.

  • UCN-01 Inhibits p53 Up-Regulation and Abrogates γ-Radiation-induced G2-M Checkpoint Independently of p53 by Targeting Both of the Checkpoint Kinases, Chk2 and Chk1
    Cancer research, 2002
    Co-Authors: Jihyun La Rose, Kurt W Kohn, Hongliang Zhang, H. Takemura, Yves Pommier

    UCN-01 (7-Hydroxystaurosporine) is a cell-cycle checkpoint abrogator that sensitizes cells to ionizing radiation (IR) and chemotherapeutic agents. It has been shown previously that UCN-01 abrogates DNA-damage-induced G2 checkpoint most selectively in p53-defective cells, by primarily targeting Chk1. Here we show that UCN-01 prevented IR-induced p53 up-regulation and p53 phosphorylation on serine 20, a site previously identified for Chk2 (or/and Chk1) kinase. We found that in human colon carcinoma HCT116 cells, IR treatment enhanced Chk2 kinase activity, whereas Chk1 activity remained unchanged, which suggested that UCN-01 may interrupt IR-induced p53 response by inhibiting Chk2 kinase. This conclusion is supported by in vitro kinase assays, showing that UCN–01 inhibits Chk2 immunoprecipitated from HCT116 cells (IC50, ∼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.

William Plunkett - One of the best experts on this subject based on the ideXlab platform.

  • Inhibition of topoisomerase IIα and G2 cell cycle arrest by NK314, a novel benzo[c]phenanthridine currently in clinical trials
    Molecular cancer therapeutics, 2007
    Co-Authors: Lei Guo, Xiaojun Liu, Kiyohiro Nishikawa, William Plunkett

    NK314 is a novel synthetic benzo[c]phenanthridine alkaloid that has recently entered clinical trials as an antitumor compound, based on impressive activities in preclinical models. The present investigations were directed at determining the mechanism of action of this agent. NK314 induced significant G(2) cell cycle arrest in several cell lines, independent of p53 status, suggesting the existence of a common mechanism of checkpoint activation. The Chk1-Cdc25C-Cdk1 G(2) checkpoint pathway was activated in response to 100 nmol/L NK314 in ML-1 human acute myeloid leukemia cells. This was associated with the phosphorylation of the histone variant H2AX, an action that was predominant in the G(2) population, suggesting that double-strand DNA breaks caused cells to activate the checkpoint pathway. Double-strand DNA breaks were visualized as chromosomal aberrations when the G(2) checkpoint was abrogated by 7-Hydroxystaurosporine. In vitro assays showed that NK314 inhibited the ability of topoisomerase IIalpha to relax supercoiled DNA and trapped topoisomerase IIalpha in its cleavage complex intermediate. CEM/VM1 cells, which are resistant to etoposide due to mutations in topoisomerase IIalpha, were cross-resistant to NK314. However, CEM/C2 cells, which are resistant to camptothecin due to mutations in topoisomerase I, retained sensitivity. These findings support the conclusion that the major mechanism of NK314 is to inhibit topoisomerase IIalpha, an action that leads to the generation of double-strand DNA breaks, which activate the G(2) DNA damage checkpoint pathway.

  • Pharmacodynamics of cytarabine alone and in combination with 7-Hydroxystaurosporine (UCN-01) in AML blasts in vitro and during a clinical trial.
    Blood, 2005
    Co-Authors: Deepa Sampath, Zheng Shi, Jorge E. Cortes, Zeev Estrov, Michael Andreeff, Varsha Gandhi, William Plunkett

    Chk1 and Akt signaling facilitate survival of cells treated with nucleoside analogues. Activation of Chk1 in response to cytarabine (ara-C) induced an S-phase checkpoint characterized by the inhibition of Cdk2, cell cycle arrest, no change in constitutively active Akt, or low-stress kinase signaling in ML-1 cells. However, inhibition of Chk1 by UCN-01 in S-phase-arrested cells resulted in an abrogation of the checkpoint, inhibition of Akt, activation of JNK, and a rapid induction of apoptosis. Similarly, primary acute myelogenous leukemia (AML) blasts exposed to ara-C and UCN-01 demonstrated a selective loss in cloning potential when compared with normal progenitors. Therefore, we evaluated a pilot clinical trial of ara-C in combination with UCN-01 in patients with relapsed AML. Blasts from some patients demonstrated a previously activated Chk1-Cdk2 DNA damage response pathway that decreased during therapy. Constitutively phosphorylated Akt kinase declined on addition of UCN-01 to the ara-C infusion, an action accompanied by an activation of JNK and reduction in absolute AML blast counts. Thus, use of UCN-01 in combination with ara-C decreases Chk1 phosphorylation, inhibits the Akt survival pathway, and activates JNK during the course of therapy, offering a rationale for the cytotoxic action of this combination during AML treatment. (Blood. 2006;107:2517-2524)

  • Cisplatin-induced ubiquitination of RNA polymerase II large subunit and suppression of induction by 7-Hydroxystaurosporine (UCN-01).
    Oncology reports, 2003
    Co-Authors: Li Ying Yang, Hong Jiang, Kelly M. Rangel, William Plunkett

    Exposure of cells to DNA-damaging agents induces hyperphosphorylation of the C-terminal domain (CTD) of mammalian RNA polymerase II (RNAP II) large subunit (LS); the hyperphosphorylated RNAP II is then ubiquitinated. The purpose of this study was to verify that cisplatin-induced RNAP II ubiquitination is transcription dependent in living cells and to determine whether 7-Hydroxystaurosporine (UCN-01) inhibits the ubiquitination induced by cisplatin. Cisplatin at clinically achievable concentrations (2.5-10 micro M) induced the ubiquitination of RNAP II in exponentially growing A2780 human ovarian tumor cells; the effect was drug-dose and exposure-time dependent. Such induction, however, was not observed in colcemid-selected mitotic cells. When detergent extraction was applied, the ubiquitinated RNAP II was recovered in the detergent-insoluble fraction, indicating that the protein was tightly bound to DNA. In an in vitro transcription reaction that consists of nuclear extracts and an immobilized DNA template containing a site-specific cisplatin lesion, the elongating RNAP II that was stalled at a cisplatin lesion site on the template was targeted by ubiquitins. Together, our results indicate that the ubiquitination is associated with transcription-coupled repair. We previously showed that the Ser/The kinase-inhibitor UCN-01 inhibits nucleotide excision repair. Here, we further determined the effect of UCN-01 on the phosphorylation and ubiquitination of RNAP II LS in a whole-cell system. Immunoblotting results showed that UCN-01 suppressed the cisplatin-induced ubiquitination and the cisplatin-induced shift from the hypophosphorylated IIa to the hyperphosphorylated IIo, without affecting the basal levels of the IIo and IIa forms of the RNAP II CTD, suggesting that UCN-01 acts by suppressing cisplatin-mediated induction of the one or more kinases that is responsible for the conversion of the IIo that is important for ubiquitination.

  • Inhibition of cyclin-dependent kinase 2 by the Chk1-Cdc25A pathway during the S-phase checkpoint activated by fludarabine: Dysregulation by 7-Hydroxystaurosporine
    Molecular pharmacology, 2002
    Co-Authors: Deepa Sampath, Zheng Shi, William Plunkett

    Human myeloid leukemia ML-1 cells responded to cytostatic concentrations of fludarabine nucleoside (F-ara-A) by instituting an arrest in S-phase that involved the inhibition of cyclin-dependent kinase 2 (Cdk2). This seemed to be mediated by 1) persistent phosphorylation on the Tyr15 residue of Cdk2 and 2) an increased association of Cdk2 with p21. S-phase arrest was also associated with an increase in Chk1 kinase activity. Concomitantly, the activity of Cdc25A phosphatase was decreased. Immunoprecipitation studies demonstrated complexes of Cdk2, Cdc25A, and Chk1. The addition of the Chk1 kinase inhibitor 7-Hydroxystaurosporine (UCN-01) to F-ara-A–arrested S-phase cells resulted in a rapid decrease in the fraction of cells with an S-phase DNA content and a corresponding increase in the fraction of apoptotic cells. Under these conditions, the kinase activity of Chk1 was reduced, Cdc25A phosphatase activity was increased, the level of Tyr15 phosphorylation of Cdk2 was reduced, and the kinase activity associated with immunoprecipitates of Cdk2 and cyclin A was reactivated. UCN-01 also had no effect on the association of p21 with Cdk2. Lastly, cells incubated with UCN-01 before F-ara-A addition did not arrest in S-phase. Thus, the DNA damage induced by F-ara-A initiated a hierarchical regulatory cascade through Chk1 and Cdc25A that resulted in Cdk2 inhibition, affecting an S-phase checkpoint that was dysregulated by UCN-01. These results suggest a mechanism by which UCN-01 enhances the cytotoxicity of agents that cause an S-phase arrest.

  • UCN-01 (7-Hydroxystaurosporine) inhibits DNA repair and increases cytotoxicity in normal lymphocytes and chronic lymphocytic leukemia lymphocytes.
    Molecular cancer therapeutics, 2002
    Co-Authors: Takahiro Yamauchi, Michael J. Keating, William Plunkett

    Elevated DNA repair processes represent resistance mechanisms to the treatment of malignancies with alkylating agents. Recently, the cell cycle checkpoint abrogator, UCN-01, was reported to inhibit nucleotide excision repair in cell-free systems. We hypothesized that if UCN-01 was combined with DNA-damaging agents, UCN-01 might inhibit the damage repair processes, thereby enhancing cytotoxicity in quiescent cells. Here, we investigated the effect of UCN-01 on DNA repair and viability of quiescent normal lymphocytes and chronic lymphocytic leukemia lymphocytes treated with UV or the cyclophosphamide prodrug 4-hydroperoxycyclophosphamide (4-HC). DNA damage repair kinetics were determined as DNA single strand breaks by the alkaline single cell gel electrophoresis (comet) assay and by [3H]thymidine incorporation. Pretreatment with UCN-01 inhibited DNA repair initiated by UV or 4-HC in normal lymphocytes as well as chronic lymphocytic leukemia lymphocytes in a concentration-dependent manner at clinically relevant levels (50–300 nm). This inhibition was demonstrated by the decreases in incision capability, DNA resynthesis, and in rejoining, suggesting that UCN-01 inhibits the multiple sites of the repair processes. The higher UCN-01 concentration (300 nm) maximized the inhibitory effects and enhanced the UV- or 4-HC-induced cytotoxicity, as determined by annexin V binding or Hoechst 33342 staining. This enhancement was not obtained by the lower concentrations that incompletely inhibited the repair, suggesting the close association between the inhibition of the repair and the enhancement of the cytotoxicity. Our findings suggest that UCN-01 may be a good candidate for combination strategies of cancer treatment.

Takashi Tsuruo - One of the best experts on this subject based on the ideXlab platform.

  • Survival-signaling pathway as a promising target for cancer chemotherapy
    Cancer Chemotherapy and Pharmacology, 2003
    Co-Authors: Naoya Fujita, Takashi Tsuruo

    The serine/threonine kinase AKT, also known as PKB or RAC-PK, is a key molecule for protecting cells from undergoing apoptosis. Several studies have suggested that the AKT-mediated survival-signaling pathway is an attractive target for cancer chemotherapy: (1) the AKT pathway is relatively inactive in resting cells; (2) amplification of the AKT gene occurs in some tumors; (3) loss of the tumor suppressor gene PTEN (phosphatase and tensin homolog deleted on chromosome 10) is common in tumors and its loss constitutively activates AKT; (4) AKT is activated at the cancer invasion front. To clarify which drugs exhibit their cytotoxicity by inhibiting the AKT pathway, we screened anticancer drugs that could downregulate phospho-AKT levels and AKT kinase activity. We found that UCN-01 (7-Hydroxystaurosporine), heat-shock protein 90 (HSP90) inhibitors, and topotecan (10-hydroxy-9-dimethylaminomethyl-(S)-camptothecin) possessed the ability to interfere with the AKT pathway. UCN-01 directly suppressed upstream AKT kinase 3-phosphoinositide-dependent protein kinase-1 (PDK1) (IC_50

  • Interference with PDK1-Akt survival signaling pathway by UCN-01 (7-Hydroxystaurosporine)
    Oncogene, 2002
    Co-Authors: Saori Sato, Naoya Fujita, Takashi Tsuruo

    3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in activating the AGC subfamily of protein kinases. In particular, PDK1 plays an important role in the regulation of Akt/PKB survival pathway by phosphorylating Akt on Thr308. Here we show that UCN-01 (7-Hydroxystaurosporine), a drug now in clinical trials and with a unique fingerprint pattern, induced dephosphorylation and inactivation of Akt, resulting in the turn-off of the survival signals and the induction of apoptosis. Further analysis revealed that UCN-01-mediated Akt inactivation was caused by inhibiting upstream Akt kinase PDK1 (IC50=33 nM) both in vitro and from cells, but not by suppressing Akt itself or phosphatidylinositide-3-OH kinase. UCN-01-induced PDK1 inhibition was also observed in in vivo murine and human tumor xenografts. Overexpression of active form of Akt diminished the cytotoxic effects of UCN-01, suggesting that UCN-01 may in part exert its cytotoxicity by inhibiting PDK1-Akt survival pathway. Because UCN-01 has already proved to have potent anti-tumor activity in vivo, PDK1-Akt survival pathway is a new, attractive target for cancer chemotherapy.

Richard R. Drake - One of the best experts on this subject based on the ideXlab platform.

  • Two-drug combinations that increase apoptosis and modulate bak and bcl-X(L) expression in human colon tumor cell lines transduced with herpes simplex virus thymidine kinase.
    Cancer gene therapy, 2000
    Co-Authors: Robyn A. Mcmasters, Tasha N. Wilbert, Kelly E. Jones, Karen Pitlyk, Robert L. Saylors, Mary Pat Moyer, Timothy C. Chambers, Richard R. Drake

    Herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir (GCV) gene therapy can induce apoptosis in tumor cells that are normally resistant to this type of cell death, although the cellular mechanisms by which this occurs remain to be elucidated. Human colon tumor cell lines expressing HSV-TK were treated with GCV or four other inducers of apoptosis: butyrate, camptothecin (CPT), Taxol (paclitaxel), or 7-Hydroxystaurosporine (UCN-01). Over a 2–4 day treatment period with GCV or the other four drugs, protein levels of the apoptosis agonist Bak increased 1.5- to 3-fold, whereas a corresponding decrease in the levels of the apoptosis antagonist, Bcl-XL, was observed in butyrate-, CPT-, and 7-Hydroxystaurosporine (UCN-01)-treated cells. GCV and paclitaxel treatments resulted in increased levels of Bcl-XL. In two-drug combinations with GCV plus one of the four other drugs, increased tumor cell killing was found with GCV plus UCN-01 or with some GCV/butyrate combinations; the other two tested combinations were largely antagonistic. The GCV/UCN-01 and GCV/butyrate combinations resulted in increased Bak and decreased Bcl-XL protein levels, while the GCV/CPT and GCV/paclitaxel combinations resulted in increased levels of both proteins. The results highlight the potential for new combination therapies of HSV-TK/GCV and chemotherapeutic drugs that result in increased tumor cell apoptosis for future treatments of colon cancer.

Steven Grant - One of the best experts on this subject based on the ideXlab platform.

  • Simultaneous exposure of transformed cells to SRC family inhibitors and CHK1 inhibitors causes cell death.
    Cancer biology & therapy, 2011
    Co-Authors: Clint Mitchell, Yun Dai, Hossein A. Hamed, Nichola Cruickshanks, Yong Tang, M. Danielle Bareford, Nissan Hubbard, Gary W. Tye, Adly Yacoub, Steven Grant

    The present studies were initiated to determine in greater molecular detail the regulation of CHK1 inhibitor lethality in transfected and infected breast cancer cells and using genetic models of transformed fibrobalsts. Multiple MEK1/2 inhibitors (PD184352, AZD6244 [ARRY-142886]) interacted with multiple CHK1 inhibitors (UCN-01 [7-Hydroxystaurosporine], AZD7762) to kill mammary carcinoma cells and transformed fibroblasts. In transformed cells, CHK1 inhibitor-induced activation of ERK1/2 was dependent upon activation of SRC family non-receptor tyrosine kinases as judged by use of multiple SRC kinase inhibitors (PP 2, Dasatinib; AZD0530), use of SRC/FYN/YES deleted transformed fibroblasts or by expression of dominant negative SRC. Cell killing by SRC family kinase inhibitors and CHK1 inhibitors was abolished in BAX/BAK−/− transformed fibroblasts and suppressed by overexpression of BCL-XL. Treatment of cells with BCL-2/BCL-XL antagonists promoted SRC inhibitor + CHK1 inhibitor-induced lethality in a BAX/BAK-dependent fashion. Treatment of cells with [SRC + CHK1] inhibitors radio-sensitized tumor cells. These findings argue that multiple inhibitors of the SRC-RAS-MEK pathway interact with multiple CHK1 inhibitors to kill transformed cells.

  • CHK1 Inhibitors in Combination Chemotherapy: Thinking Beyond the Cell Cycle
    Molecular interventions, 2011
    Co-Authors: Paul Dent, Yun Dai, Yong Tang, Adly Yacoub, Paul B. Fisher, Steven Grant

    Cellular sensing of DNA damage, along with concomitant cell cycle arrest, is mediated by a great many proteins and enzymes. One focus of pharmaceutical development has been the inhibition of DNA damage signaling, and checkpoint kinases (Chks) in particular, as a means to sensitize proliferating tumor cells to chemotherapies that damage DNA. 7-Hydroxystaurosporine, or UCN-01, is a clinically relevant and well-studied kinase activity inhibitor that exerts chemosensitizing effects by inhibition of Chk1, and a multitude of Chk1 inhibitors have entered development. Clinical development of UCN-01 has overcome many initial obstacles, but the drug has nevertheless failed to show a high level of clinical activity when combined with chemotherapeutic agents. One very likely reason for the lack of clinical efficacy of Chk1 inhibitors may be that the inhibition of Chk1 causes the compensatory activation of ATM and ERK1/2 pathways. Indeed, inhibition of many enzyme activities, not necessarily components of cell cycle regulation, may block Chk1 inhibitor–induced ERK1/2 activation and enhance the toxicity of Chk1 inhibitors. This review examines the rationally hypothesized actions of Chk1 inhibitors as cell cycle modulatory drugs as well as the impact of Chk1 inhibition upon other cell survival signaling pathways. An understanding of Chk1 inhibition in multiple signaling contexts will be essential to the therapeutic development of Chk1 inhibitors.

  • Statins synergistically potentiate 7-Hydroxystaurosporine (UCN-01) lethality in human leukemia and myeloma cells by disrupting Ras farnesylation and activation
    Blood, 2007
    Co-Authors: Yun Dai, Paul Dent, Xin-yan Pei, Payal Khanna, Shuang Chen, Steven Grant

    Interactions between UCN-01 and HMG-CoA reductase inhibitors (ie, statins) have been examined in human leukemia and myeloma cells. Exposure of U937 and U266 cells to minimally toxic concentrations of UCN-01 and various statins (eg, lovastatin, simvastatin, or fluvastatin) dramatically increased mitochondrial dysfunction, caspase activation, and apoptosis. Comparable effects were observed in other leukemia and myeloma cell lines as well as in primary acute myeloid leukemia (AML) blasts but not in normal hematopoietic cells. Potentiation of UCN-01 lethality by lovastatin was associated with disruption of Ras prenylation and activation. These events were significantly attenuated by farnesyl pyrophosphate (FPP) but not by geranylgeranyl pyrophosphate (GGPP), implicating perturbations in farnesylation rather than geranylgeranylation in synergistic interactions. Coexposure to statins and UCN-01 resulted in inactivation of ERK1/2 and Akt, accompanied by JNK activation. U266 cells ectopically expressing JNK1-APF, a dominant negative JNK1 mutant, displayed significantly reduced susceptibility to lovastatin/UCN-01–mediated lethality. Moreover, transfection of U266 cells with constitutively activated H-Ras (Q61L) attenuated ERK1/2 inactivation and dramatically diminished the lethality of this regimen. Collectively, these findings indicate that HMG-CoA reductase inhibitors act through a Ras farnesylation-associated mechanism to induce signaling perturbations, particularly prevention of Ras and ERK1/2 activation, in UCN-01–treated cells, resulting in the synergistic induction of cell death.

  • Dissecting the Roles of Checkpoint Kinase 1/CDC2 and Mitogen-Activated Protein Kinase Kinase 1/2/Extracellular Signal-Regulated Kinase 1/2 in Relation to 7-Hydroxystaurosporine-Induced Apoptosis in Human Multiple Myeloma Cells
    Molecular pharmacology, 2006
    Co-Authors: Xin-yan Pei, Paul Dent, Yun Dai, Steven Grant

    The functional roles of Cdc2 and checkpoint kinase 1 (Chk1) in synergistic interactions between 7-Hydroxystaurosporine (UCN-01) and mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitors [e.g., 2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide (PD184352)] were examined in human multiple myeloma cells in relation to MEK1/2/ERK1/2 activation and lethality. Time course studies revealed that MEK1/2/extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation preceded Cdc2 dephosphorylation (Tyr15) after UCN-01 exposure. Furthermore, enforced expression of Cdc2 or small inducible RNA (siRNA)-mediated Cdc2 knockdown failed to modify ERK1/2 activation status in either the presence or absence of UCN-01, arguing against a causal relationship between these events. However, ectopic expression of Cdc2 sensitized cells to the lethality of UCN-01/MEK inhibitor regimen, whereas Cdc2 knockdown by siRNA significantly diminished the lethal effects of this combination. Conversely, Chk1 knockdown by siRNA enhanced lethality mediated by UCN-01/PD184352. It is interesting that Chk1 knockdown reduced basal ERK1/2 activation and antagonized the ability of UCN-01 to activate ERK1/2. Finally, ectopic expression of constitutively active MEK1 significantly protected cells from the UCN-01/MEK1/2 inhibitor regimen without modifying Cdc2 activation status. Together, these findings indicate that although UCN-01-mediated Chk1 inhibition and Cdc2 activation are unlikely to be responsible for MEK1/2/ERK1/2 activation, both of these events contribute functionally to enhanced lethality in cells coexposed to MEK inhibitors. They also suggest a role for Chk1 in UCN-01-induced ERK1/2 activation, implying the existence of a heretofore unrecognized link between Chk1 and ERK1/2 signaling.

  • Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) promotes mitochondrial dysfunction and apoptosis induced by 7-Hydroxystaurosporine and mitogen-activated protein kinase kinase inhibitors in human leukemia cells that ectopically express
    Molecular pharmacology, 2003
    Co-Authors: Yun Dai, Paul Dent, Steven Grant

    Previous studies have demonstrated that cotreatment with mitogen activated-protein kinase kinase (MEK) 1/2 inhibitors (e.g., PD184352) and the checkpoint abrogator 7-Hydroxystaurosporine (UCN-01) dramatically induces apoptosis in a variety of human leukemia and multiple myeloma cell types. The purpose of this study was to evaluate the roles of Bcl-2 family members and the relative contribution of the intrinsic mitochondrial versus the extrinsic receptor-related apoptotic pathways to MEK inhibitors/UCN-01-induced leukemic cell death. Cotreatment of U937 cells with PD184352 and UCN-01 resulted in the activation of procaspase-3, -9, and -8 as well as Bid cleavage. PD184352/UCN-01-induced mitochondrial dysfunction and apoptosis were both substantially attenuated in cells ectopically expressing Bcl-2, an N-terminal phosphorylation loop-deleted mutant Bcl-2, or Bcl-xL, but not in cells expressing dominant-negative (DN) caspase-8, cytokine response modifier A (cowpox virus-encoded antiapoptotic protein), or DN Fas-associated death domain. Coadministration of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) or TNF-alpha substantially increased MEK inhibitors (e.g., PD184352 or U0126)/UCN-01-induced mitochondrial dysfunction, activation of procaspase-8 and Bid, and apoptosis in Bcl-2- and Bcl-xL-overexpressing cells but not in those in which the extrinsic pathway was interrupted. Together, these findings suggest that the MEK inhibitors/UCN-01 regimen primarily induces leukemic cell apoptosis by engaging the intrinsic, mitochondrial apoptotic pathway and that resistance to these events conferred by increased expression of certain antiapoptotic Bcl-2 family members can be overcome, at least in part, by coadministration of TRAIL and other agents that activate the extrinsic apoptotic cascade.