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John S. Lazo - One of the best experts on this subject based on the ideXlab platform.

  • hypoxia mediated regulation of CDC25A phosphatase by p21 and mir 21
    Cell Cycle, 2009
    Co-Authors: Pierre Queiroz E De Oliveira, Lin Zhang, Zhenghe Wang, John S. Lazo
    Abstract:

    Hypoxia is a common feature of solid tumors and represents a critical factor in their progression and responsiveness to chemotherapy and radiotherapy. We now report that hypoxic exposure of colon cancer cells decreased the protein levels of the cell cycle-controlling phosphatase CDC25A. Hypoxia decreased the mitotic population and caused S-phase arrest in these cells. Suppression of CDC25A was phosphatase family member-specific, as a similar decrease was not observed with closely related Cdc25B or Cdc25C phosphatases. Pharmacological and genetic blockade of Chk1 and Chk2 failed to inhibit the hypoxia-mediated loss of CDC25A, indicating this process was not regulated by a traditional ATM/ATR checkpoint response. In addition, hypoxia did not affect ectopically expressed CDC25A levels suggesting independence from an increase in proteasomal degradation. CDC25A mRNA levels also decreased in human colon cancer cells 24 hr after hypoxia supporting a mechanistic role for decreased CDC25A expression or mRNA stability. The reduction in CDC25A mRNA and protein was dependent on the cyclin-dependent kinase inhibitor p21 and miR-21, which were upregulated in HCT116 colon cancer cells during hypoxia. These results reveal previously unknown mechanisms for the transient suppression of CDC25A, providing a coordinated and fundamental adaptive change that may be exploited by cancer cells conferring proliferative and survival advantages.

  • cdc25b dual specificity phosphatase inhibitors identified in a high throughput screen of the nih compound library
    Assay and Drug Development Technologies, 2009
    Co-Authors: Paul A. Johnston, Marni Brisson Tierno, Tong Ying Shun, Sunita N Shinde, William D Paquette, Kay M Brummond, Caleb Foster, Peter Wipf, John S. Lazo
    Abstract:

    Abstract The University of Pittsburgh Molecular Library Screening Center (Pittsburgh, PA) conducted a screen with the National Institutes of Health compound library for inhibitors of in vitro cell division cycle 25 protein (Cdc25) B activity during the pilot phase of the Molecular Library Screening Center Network. Seventy-nine (0.12%) of the 65,239 compounds screened at 10 μM met the active criterion of ≥50% inhibition of Cdc25B activity, and 25 (31.6%) of these were confirmed as Cdc25B inhibitors with 50% inhibitory concentration (IC50) values <50 μM. Thirteen of the Cdc25B inhibitors were represented by singleton chemical structures, and 12 were divided among four clusters of related structures. Thirteen (52%) of the Cdc25B inhibitor hits were quinone-based structures. The Cdc25B inhibitors were further characterized in a series of in vitro secondary assays to confirm their activity, to determine their phosphatase selectivity against two other dual-specificity phosphatases, mitogen-activated protein kin...

  • independent mechanistic inhibition of cdc25 phosphatases by a natural product caulibugulone
    Molecular Pharmacology, 2007
    Co-Authors: Marni Brisson, Robert J Tomko, Caleb Foster, Theresa Nguyen, Peter Wipf, John S. Lazo
    Abstract:

    Caulibugulones are novel but poorly characterized cytotoxic isoquinoline quinones and iminoquinones identified in extracts from the marine bryozoan Caulibugula intermis . We now report that the caulibugulones are selective in vitro inhibitors of the Cdc25 family of cell cycle-controlling protein phosphatases compared with either human vaccinia H1-related phosphatase (VHR) or tyrosine phosphatase 1B (PTP1B). The in vitro inhibition of Cdc25B by caulibugulone A was irreversible and attenuated by reducing agents or catalase, consistent with direct oxidation of the enzyme by reactive oxygen species. Mechanistically, caulibugulone A directly inhibited cellular Cdc25B activity, generated intracellular reactive oxygen species and arrested cells in both G1 and G2/M phases of the cell cycle. Caulibugulone A also caused the selective degradation of CDC25A protein by a process that was independent of reactive oxygen species production, proteasome activity, and the Chk1 signaling pathway. Instead, caulibugulone A stimulated the phosphorylation and subsequent activation of p38 stress kinase, leading to CDC25A degradation. Thus, caulibugulone inhibition of cellular CDC25A and B phosphatases occurred through at least two different mechanisms, leading to pronounced cell cycle arrest.

  • discovery and characterization of novel small molecule inhibitors of human cdc25b dual specificity phosphatase
    Molecular Pharmacology, 2004
    Co-Authors: Marni Brisson, Jack C Yalowich, Angela Giorgianni, Dror Tobi, Corey R J Stephenson, Theresa Nguyen, Ivet Bahar, Andreas Vogt, Peter Wipf, John S. Lazo
    Abstract:

    CDC25A and Cdc25B dual-specificity phosphatases are key regulators of cell cycle transition and proliferation. They have oncogenic properties and are overexpressed in many human tumors. Because selective Cdc25 phosphatase inhibitors would be valuable biological tools and possible therapeutic agents, we have assayed a small molecule library for in vitro inhibition of Cdc25. We now report the identification of two new structurally distinct classes of Cdc25 inhibitors with cellular activity. The cyclopentaquinoline 3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-4,8-dicarboxylic acid (5661118) and the naphthofurandione 3-benzoyl-naphtho[1,2-b]furan-4,5-dione (5169131) had in vitro IC50 values of 2.5 to 11 M against recombinant Cdc25 and were less potent inhibitors of other phosphatases. Unlike 5661118, 5169131 caused reversible inhibition of Cdc25B and displayed competitive inhibitor kinetics. No growth inhibitory activity was seen with 5661118, whereas 10 to 30 M 5169131 caused G1/S and G2/M arrest. We also found that 5169131 inhibited human PC-3 prostate and MDAMB-435 breast cancer cell proliferation. Concentration-dependent Tyr15 hyperphosphorylation was seen on cyclin-dependent kinase with a 1-h 5169131 treatment, consistent with Cdc25 inhibition. Cells resistant to DNA toposiomerase II inhibitors were as sensitive to 5169131 as parental cells, indicating that this quinone compound does not inhibit topoisomerase II in vivo. Molecular modeling was used to predict a potential interaction site between the inhibitor and Cdc25B and to provide insights as to the molecular origins of the experimental observations. Based on its kinetic profile and cellular activity, we suggest that 5169131 could be an excellent tool for further studies on the cellular roles of Cdc25.

  • binding and inhibition of cdc25 phosphatases by vitamin k analogues
    Biochemistry, 2003
    Co-Authors: Iliya Lefterov, Colleen N Scott, Meifang Wang, John S. Lazo, Craig S Wilcox, Brian I. Carr
    Abstract:

    A synthetic K vitamin analogue, 2-(2-mercaptothenol)-3-methyl-1,4-naphthoquinone or Cpd 5, was previously found to be a potent inhibitor of cell growth [Nishikawa et al., (1995) J. Biol. Chem. 270, 28304−28310]. The mechanisms of cell growth were hypothesized to include the inactivation of cellular protein tyrosine phosphatases, especially the Cdc25 family [Tamura et al. (2000) Cancer Res. 60, 1317−1325]. In this study, we synthesized PD 49, a new biotin containing Cpd 5 derivative, to search for evidence of direct interaction of these arylating analogues with CDC25A, Cdc25B, and Cdc25C phosphatases. PD 49 was shown to directly bind to GST-CDC25A, GST-Cdc25B, their catalytic fragments, and GST-Cdc25C. The binding could be competed with excess glutathione or Cpd 5, and a cysteine-to-serine mutation of the catalytic cysteine abolished binding. This was consistent with an involvement in binding of cysteine in the catalytic domain. This interaction between PD 49 and Cdc25 also occurred in lysates of treated c...

Brian I. Carr - One of the best experts on this subject based on the ideXlab platform.

  • fluorinated cpd 5 a pure arylating k vitamin derivative inhibits human hepatoma cell growth by inhibiting cdc25 and activating mapk
    Biochemical Pharmacology, 2006
    Co-Authors: Meifang Wang, Brian I. Carr
    Abstract:

    Abstract We previously synthesized several K-vitamin derivatives, which are potent growth inhibitors of human tumor cells, including Hep3B human hepatoma cells. Among these, Cpd 5 was the most potent. However, being a quinone derivative, Cpd 5 has the potential for generating toxic reactive oxygen species (ROS). We therefore synthesized a fluorinated derivative of Cpd 5, F-Cpd 5. The calculated reduction potential of F-Cpd 5 was much higher than that for Cpd 5 and it was not predicted to generate ROS. This was supported by our observation that F-Cpd 5 generated significantly lower ROS than Cpd 5. F-Cpd 5 was three times more potent than Cpd 5 in inhibiting Hep3B cell growth. Interestingly, under identical culture conditions, F-Cpd 5 inhibited mitogen-induced DNA synthesis in normal rat hepatocytes 12-fold less potently than Hep3B cells. F-Cpd 5 was found to induce caspase-3 cleavage and nuclear DNA laddering, evidences for apoptosis. It preferentially inhibited the activities of the cell cycle controlling phosphatases CDC25A and Cdc25B, by binding to their catalytic cysteines. Consequently, inhibitory tyrosine phosphorylation of the Cdc25 substrate kinases Cdk2 and Cdk4 were induced. F-Cpd 5 also induced phosphorylation of the MAPK proteins ERK1/2, JNK1/2 and p38 in Hep3B cells and the MAPK inhibitors (U0126, JNKI-II, and SB 203580) antagonized its growth inhibition. F-Cpd 5 inhibited the action of cytosolic ERK phosphatase activity, which likely caused the ERK phosphorylation. F-Cpd 5 thus differentially inhibited growth of normal and tumor cells by preferentially inhibiting the actions of CDC25A and Cdc25B phosphatases and inducing MAPK phosphorylation.

  • pm 20 a novel inhibitor of CDC25A induces extracellular signal regulated kinase 1 2 phosphorylation and inhibits hepatocellular carcinoma growth in vitro and in vivo
    Molecular Cancer Therapeutics, 2006
    Co-Authors: Siddhartha Kar, Meifang Wang, Wei Yao, Christopher J Michejda, Brian I. Carr
    Abstract:

    We have synthesized several new phenyl maleimide compounds, which are potent growth inhibitors of several human tumor cell lines. Among these, PM-20 was the most potent with an IC50 of 700 nmol/L for Hep3B human hepatoma cell growth. Two other derivatives, PM-26 and PM-38, did not inhibit Hep3B cell growth even at 100 micromol/L. Interestingly, under identical experimental conditions, PM-20 inhibited DNA synthesis of primary cultures of normal hepatocytes at a 10-fold higher concentration than that needed to inhibit the DNA synthesis of the Hep3B hepatoma cells. PM-20 affected two cellular signaling pathways in Hep3B cells: Cdc25 phosphatase and extracellular signal-regulated kinase (ERK) 1/2. It competitively inhibited the activity of Cdc25 (preferentially CDC25A) by binding to the active site, likely through the catalytic cysteine, but did not inhibit PTP1B, CD45, or MKP-1 phosphatases. As a result of its action, tyrosine phosphorylation of the cellular CDC25A substrates Cdk2 and Cdk4 was induced. It also induced strong and persistent phosphorylation of the CDC25A substrate ERK1/2. Hep3B cell lysates were found to contain ERK2 phosphatase(s) activity, which was inhibited by the actions of PM-20. However, activity of exogenous dual-specificity ERK2 phosphatase MKP1 was not inhibited. Induction of ERK1/2 phosphorylation correlated with the potency of growth inhibition in tumor cell lines and inhibition of ERK1/2 phosphorylation by the mitogen-activated protein kinase (MAPK)/ERK kinase 1/2 inhibitor U0126 or overexpression of the CDC25A gene in Hep3B cells antagonized the growth inhibitory actions of PM-20. Growth of transplantable rat hepatoma cells in vivo was also inhibited by PM-20 action with a concomitant induction of pERK in the tumors. The mechanism(s) of growth inhibition of Hep3B hepatoma cells by the phenyl maleimide PM-20 involves prolonged ERK1/2 phosphorylation, likely resulting from inhibition of the ERK phosphatase CDC25A. PM-20 thus represents a novel class of tumor growth inhibitor that inhibits mainly CDC25A, is dependent on ERK activation, and has a considerable margin of selectivity for tumor cells compared with normal cells.

  • binding and inhibition of cdc25 phosphatases by vitamin k analogues
    Biochemistry, 2003
    Co-Authors: Iliya Lefterov, Colleen N Scott, Meifang Wang, John S. Lazo, Craig S Wilcox, Brian I. Carr
    Abstract:

    A synthetic K vitamin analogue, 2-(2-mercaptothenol)-3-methyl-1,4-naphthoquinone or Cpd 5, was previously found to be a potent inhibitor of cell growth [Nishikawa et al., (1995) J. Biol. Chem. 270, 28304−28310]. The mechanisms of cell growth were hypothesized to include the inactivation of cellular protein tyrosine phosphatases, especially the Cdc25 family [Tamura et al. (2000) Cancer Res. 60, 1317−1325]. In this study, we synthesized PD 49, a new biotin containing Cpd 5 derivative, to search for evidence of direct interaction of these arylating analogues with CDC25A, Cdc25B, and Cdc25C phosphatases. PD 49 was shown to directly bind to GST-CDC25A, GST-Cdc25B, their catalytic fragments, and GST-Cdc25C. The binding could be competed with excess glutathione or Cpd 5, and a cysteine-to-serine mutation of the catalytic cysteine abolished binding. This was consistent with an involvement in binding of cysteine in the catalytic domain. This interaction between PD 49 and Cdc25 also occurred in lysates of treated c...

  • spatial analysis of key signaling proteins by high content solid phase cytometry in hep3b cells treated with an inhibitor of cdc25 dual specificity phosphatases
    Journal of Biological Chemistry, 2001
    Co-Authors: Andreas Vogt, Alexander P Ducruet, Takahito Adachi, Jon Chesebrough, Brian I. Carr, Kaoru Nemoto, John S. Lazo
    Abstract:

    Abstract Protein phosphorylation frequently results in the subcellular redistribution of key signaling molecules, and this spatial change is critical for their activity. Here we have probed the effects of a Cdc25 inhibitor, 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone, or Compound 5, on the spatial regulation and activation kinetics of tyrosine phosphorylation-dependent signaling events using two methods: (i) high-content, automated, fluorescence-based, solid-phase cytometry and (ii) a novel cellular assay for CDC25A activity in intact cells. Immunofluorescence studies demonstrated that Compound 5 produced a concentration-dependent nuclear accumulation of phospho-Erk and phospho-p38, but not nuclear factor κB. Immunoblot analysis confirmed Erk phosphorylation and nuclear accumulation, andin vitro kinase assays showed that Compound 5-activated Erk was competent to phosphorylate its physiological substrate, the transcription factor Elk-1. Pretreatment of cells with the MEK inhibitor U-0126 prevented the induction by Compound 5 of phospho-Erk (but not phospho-p38) nuclear accumulation and protected cells from the antiproliferative effects of Compound 5. Overexpression of CDC25A in whole cells caused dephosphorylation of Erk that was reversed by Compound 5. The data show that an inhibitor of Cdc25 increases Erk phosphorylation and nuclear accumulation and support the hypothesis that CDC25A regulates Erk phosphorylation status.

  • spatial analysis of key signaling proteins by high content solid phase cytometry in hep3b cells treated with an inhibitor of cdc25 dual specificity phosphatases
    Journal of Biological Chemistry, 2001
    Co-Authors: Andreas Vogt, Alexander P Ducruet, Takahito Adachi, Jon Chesebrough, Brian I. Carr, Kaoru Nemoto, John S. Lazo
    Abstract:

    Abstract Protein phosphorylation frequently results in the subcellular redistribution of key signaling molecules, and this spatial change is critical for their activity. Here we have probed the effects of a Cdc25 inhibitor, 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone, or Compound 5, on the spatial regulation and activation kinetics of tyrosine phosphorylation-dependent signaling events using two methods: (i) high-content, automated, fluorescence-based, solid-phase cytometry and (ii) a novel cellular assay for CDC25A activity in intact cells. Immunofluorescence studies demonstrated that Compound 5 produced a concentration-dependent nuclear accumulation of phospho-Erk and phospho-p38, but not nuclear factor κB. Immunoblot analysis confirmed Erk phosphorylation and nuclear accumulation, andin vitro kinase assays showed that Compound 5-activated Erk was competent to phosphorylate its physiological substrate, the transcription factor Elk-1. Pretreatment of cells with the MEK inhibitor U-0126 prevented the induction by Compound 5 of phospho-Erk (but not phospho-p38) nuclear accumulation and protected cells from the antiproliferative effects of Compound 5. Overexpression of CDC25A in whole cells caused dephosphorylation of Erk that was reversed by Compound 5. The data show that an inhibitor of Cdc25 increases Erk phosphorylation and nuclear accumulation and support the hypothesis that CDC25A regulates Erk phosphorylation status.

Ingrid Hoffmann - One of the best experts on this subject based on the ideXlab platform.

  • cdc25 phosphatases are required for timely assembly of cdk1 cyclin b at the g2 m transition
    Journal of Biological Chemistry, 2010
    Co-Authors: Oleg Timofeev, Florian Settele, Tore Kempf, Onur Cizmecioglu, Ingrid Hoffmann
    Abstract:

    Abstract Progression through mitosis requires the coordinated regulation of Cdk1 kinase activity. Activation of Cdk1 is a multistep process comprising binding of Cdk1 to cyclin B, relocation of cyclin-kinase complexes to the nucleus, activating phosphorylation of Cdk1 on Thr161 by the Cdk-activating kinase (CAK; Cdk7 in metazoans), and removal of inhibitory Thr14 and Tyr15 phosphorylations. This dephosphorylation is catalyzed by the dual specific Cdc25 phosphatases, which occur in three isoforms in mammalian cells, CDC25A, -B, and -C. We find that expression of CDC25A leads to an accelerated G2/M phase transition. In CDC25A-overexpressing cells, Cdk1 exhibits high kinase activity despite being phosphorylated on Tyr15. In addition, Tyr15-phosphorylated Cdk1 binds more cyclin B in CDC25A-overexpressing cells compared with control cells. Consistent with this observation, we demonstrate that in human transformed cells, CDC25A and Cdc25B, but not Cdc25C phosphatases have an effect on timing and efficiency of cyclin-kinase complex formation. Overexpression of CDC25A or Cdc25B promotes earlier assembly and activation of Cdk1-cyclin B complexes, whereas repression of these phosphatases by short hairpin RNA has a reverse effect, leading to a substantial decrease in amounts of cyclin B-bound Cdk1 in G2 and mitosis. Importantly, we find that CDC25A overexpression leads to an activation of Cdk7 and increase in Thr161 phosphorylation of Cdk1. In conclusion, our data suggest that complex assembly and dephosphorylation of Cdk1 at G2/M is tightly coupled and regulated by Cdc25 phosphatases.

  • the oncogenic serine threonine kinase pim 1 directly phosphorylates and activates the g2 m specific phosphatase cdc25c
    The International Journal of Biochemistry & Cell Biology, 2006
    Co-Authors: Malte Bachmann, Ingrid Hoffmann, Christian Kosan, Pei Xiang Xing, Mathias Montenarh, Tarik Möröy
    Abstract:

    The proto-oncogene Pim-1 encodes a serine-threonine kinase which is a downstream effector of cytokine signaling and can enhance cell cycle progression by altering the activity of several cell cycle regulators among them the G1 specific inhibitor p21(Waf), the phosphatase CDC25A and the kinase C-TAK1. Here, we demonstrate by using biochemical assays that Pim-1 can interact with the phosphatase Cdc25C and is able to directly phosphorylate the N-terminal region of the protein. Cdc25C is functionally related to CDC25A but acts specifically at the G2/M cell cycle transition point and can be inactivated by C-TAK1-mediated phosphorylation. Immuno-fluorescence experiments showed that Pim-1 and Cdc25C co-localize in the cytoplasm of both epithelial and myeloid cells. We find that phosphorylation by Pim-1 enhances the phosphatase activity of Cdc25C and in transfected cells that are arrested in G2/M by bleomycin, Pim-1 can enhance progression into G1. Therefore, we propose that Pim-1 activates Cdc25C by a direct phosphorylation and can thereby assume the function of a positive cell cycle regulator at the G2/M transition.

  • cell cycle regulation by the cdc25 phosphatase family
    Progress in cell cycle research, 2000
    Co-Authors: Ida Nilsson, Ingrid Hoffmann
    Abstract:

    Activation of cyclin-dependent kinases in higher eukaryotic cells can be achieved through dephosphorylation by members of the Cdc25 phosphatase family, CDC25A, Cdc25B and Cdc25C. CDC25A plays an important role at the G1/S-phase transition. Cdc25B undergoes activation during S-phase and plays a role in activating the mitotic kinase Cdkl/cyclin B in the cytoplasm. Active Cdkl/cyclin B then phosphorylates and activates Cdc25C leading to a positive feedback mechanism and to entry into mitosis. CDC25A and B are potential human oncogenes. In addition, Cdc25 is a main player of the G2 arrest caused by DNA damage or in the presence of unreplicated DNA.

  • ectopic expression of CDC25A accelerates the g1 s transition and leads to premature activation of cyclin e and cyclin a dependent kinases
    Molecular and Cellular Biology, 1999
    Co-Authors: Ida Blomberg, Ingrid Hoffmann
    Abstract:

    Human Cdc25 phosphatases play important roles in cell cycle regulation by removing inhibitory phosphates from tyrosine and threonine residues of cyclin-dependent kinases. Three human Cdc25 isoforms, A, B, and C, have been discovered. Cdc25B and Cdc25C play crucial roles at the G2/M transition. In the present study, we have investigated the function of human CDC25A phosphatase. Cell lines that express human CDC25A in an inducible manner have been generated. Ectopic expression of CDC25A accelerates the G1/S-phase transition, indicating that CDC25A controls an event(s) that is rate limiting for entry into S phase. Furthermore, we carried out a detailed analysis of the expression and activation of human CDC25A. Activation of endogenous CDC25A occurs during late G1 phase and increases in S and G2 phases. We further demonstrate that CDC25A is activated at the same time as cyclin E- and cyclin A-dependent kinases. In vitro, CDC25A dephosphorylates and activates the cyclin-Cdk complexes that are active during G1. Overexpression of CDC25A in the inducible system, however, leads to a premature activation of both cyclin E-Cdk2 and cyclin A-Cdk2 complexes, while no effect of cyclin D-dependent kinases is observed. Furthermore, CDC25A overexpression induces a tyrosine dephosphorylation of Cdk2. These results suggest that CDC25A is an important regulator of the G1/S-phase transition and that cyclin E- and cyclin A-dependent kinases act as direct targets.

  • ectopic expression of CDC25A accelerates the g 1 s transition and leads to premature activation of cyclin e and cyclin a dependent kinases
    Molecular and Cellular Biology, 1999
    Co-Authors: Ida Blomberg, Ingrid Hoffmann
    Abstract:

    Human Cdc25 phosphatases play important roles in cell cycle regulation by removing inhibitory phosphates from tyrosine and threonine residues of cyclin-dependent kinases. Three human Cdc25 isoforms, A, B, and C, have been discovered. Cdc25B and Cdc25C play crucial roles at the G2/M transition. In the present study, we have investigated the function of human CDC25A phosphatase. Cell lines that express human CDC25A in an inducible manner have been generated. Ectopic expression of CDC25A accelerates the G1/S-phase transition, indicating that CDC25A controls an event(s) that is rate limiting for entry into S phase. Furthermore, we carried out a detailed analysis of the expression and activation of human CDC25A. Activation of endogenous CDC25A occurs during late G1 phase and increases in S and G2 phases. We further demonstrate that CDC25A is activated at the same time as cyclin E- and cyclin A-dependent kinases. In vitro, CDC25A dephosphorylates and activates the cyclin-Cdk complexes that are active during G1. Overexpression of CDC25A in the inducible system, however, leads to a premature activation of both cyclin E-Cdk2 and cyclin A-Cdk2 complexes, while no effect of cyclin D-dependent kinases is observed. Furthermore, CDC25A overexpression induces a tyrosine dephosphorylation of Cdk2. These results suggest that CDC25A is an important regulator of the G1/S-phase transition and that cyclin E- and cyclin A-dependent kinases act as direct targets.

Helen Piwnicaworms - One of the best experts on this subject based on the ideXlab platform.

  • contributions made by cdc25 phosphatases to proliferation of intestinal epithelial stem and progenitor cells
    PLOS ONE, 2011
    Co-Authors: Sofia Origanti, Lynn S White, Thaddeus S Stappenbeck, Helen Piwnicaworms
    Abstract:

    The CDC25 protein phosphatases drive cell cycle advancement by activating cyclin-dependent protein kinases (CDKs). Humans and mice encode three family members denoted CDC25A, -B and -C and genes encoding these family members can be disrupted individually with minimal phenotypic consequences in adult mice. However, adult mice globally deleted for all three phosphatases die within one week after Cdc25 disruption. A severe loss of absorptive villi due to a failure of crypt epithelial cells to proliferate was observed in the small intestines of these mice. Because the Cdc25s were globally deleted, the small intestinal phenotype and loss of animal viability could not be solely attributed to an intrinsic defect in the inability of small intestinal stem and progenitor cells to divide. Here, we report the consequences of deleting different combinations of Cdc25s specifically in intestinal epithelial cells. The phenotypes arising in these mice were then compared with those arising in mice globally deleted for the Cdc25s and in mice treated with irinotecan, a chemotherapeutic agent commonly used to treat colorectal cancer. We report that the phenotypes arising in mice globally deleted for the Cdc25s are due to the failure of small intestinal stem and progenitor cells to proliferate and that blocking cell division by inhibiting the cell cycle engine (through Cdc25 loss) versus by inducing DNA damage (via irinotecan) provokes a markedly different response of small intestinal epithelial cells. Finally, we demonstrate that CDC25A and CDC25B but not CDC25C compensate for each other to maintain the proliferative capacity of intestinal epithelial stem and progenitor cells.

  • response of small intestinal epithelial cells to acute disruption of cell division through cdc25 deletion
    Proceedings of the National Academy of Sciences of the United States of America, 2009
    Co-Authors: Lynn S White, Thaddeus S Stappenbeck, Kristen E Hurov, Helen Piwnicaworms
    Abstract:

    The CDC25 protein phosphatases (CDC25A, B, and C) drive cell cycle transitions by activating key components of the cell cycle engine. CDC25A and CDC25B are frequently overproduced in human cancers. Disruption of Cdc25B or Cdc25C individually or in combination has no effect on mouse viability. Here we report that CDC25A is the only family member to provide an essential function during early embryonic development, and that other family members compensate for its loss in adult mice. In contrast, conditional disruption of the entire family is lethal in adults due to a loss of small intestinal epithelial cell proliferation in crypts of Lieberkuhn. Cdc25 loss induced Wnt signaling, and overall crypt structures were preserved. In the face of continuous Wnt signaling, nearly all crypt epithelial progenitors differentiated into multiple cell lineages, including crypt base columnar cells, a proposed stem cell. A small population of Musashi/Dcamkl-1/nuclear β-catenin–positive epithelial cells was retained in these crypts. These findings have implications for the development of novel, less cytotoxic cancer chemotherapeutic drugs that specifically target the cell cycle.

  • normal cell cycle and checkpoint responses in mice and cells lacking cdc25b and cdc25c protein phosphatases
    Molecular and Cellular Biology, 2005
    Co-Authors: Angela M Ferguson, Peter J Donovan, Lynn S White, Helen Piwnicaworms
    Abstract:

    The Cdc25 family of protein phosphatases positively regulates cell division by activating cyclin-dependent protein kinases (CDKs). In humans and rodents, there are three Cdc25 family members—denoted CDC25A, Cdc25B, and Cdc25C—that can be distinguished based on their subcellular compartmentalizations, their abundances and/or activities throughout the cell cycle, the CDKs that they target for activation, and whether they are overexpressed in human cancers. In addition, murine forms of Cdc25 exhibit distinct patterns of expression throughout development and in adult tissues. These properties suggest that individual Cdc25 family members contribute distinct biological functions in embryonic and adult cell cycles of mammals. Interestingly, mice with Cdc25C disrupted are healthy, and cells derived from these mice exhibit normal cell cycles and checkpoint responses. Cdc25B−/− mice are also generally normal (although females are sterile), and cells derived from Cdc25B−/− mice have normal cell cycles. Here we report that mice lacking both Cdc25B and Cdc25C are obtained at the expected Mendelian ratios, indicating that Cdc25B and Cdc25C are not required for mouse development or mitotic entry. Furthermore, cell cycles, DNA damage responses, and CDC25A regulation are normal in cells lacking Cdc25B and Cdc25C. These findings indicate that CDC25A, or possibly other phosphatases, is able to functionally compensate for the loss of Cdc25B and Cdc25C in mice.

  • arsenite induced cdc25c degradation is through the ken box and ubiquitin proteasome pathway
    Proceedings of the National Academy of Sciences of the United States of America, 2002
    Co-Authors: Fei Chen, Helen Piwnicaworms, Zhuo Zhang, Jacquelyn J. Bower, Stephen S. Leonard, Min Ding, Vince Castranova, Xianglin Shi
    Abstract:

    Arsenite is a known human carcinogen that induces tumorigenesis through either a genotoxic or an epigenetic mechanism. In this study, the effect of arsenite on cell cycle regulation and the mechanisms that contribute to this effect were investigated. Treatment of the cells with arsenite suppressed cell proliferation and reduced cell viability in a dose- or time-dependent manner. Analysis of cell cycle profile and cell cycle regulatory proteins indicated that arsenite arrested the cell cycle at G2/M phase, partially through induction of cell division cycle 25 (Cdc25) isoform C (Cdc25C) degradation via ubiquitin–proteasome pathways. Mutation of the putative KEN box within the region 151 to 157 of human Cdc25C or treatment of the cells with a peptide competitor encompassing the KEN box partially inhibited arsenite-induced ubiquitination of Cdc25C. Thus, these results indicate that the regulated ubiquitination of Cdc25C may be involved in the arsenite-induced proteolytic down-regulation of Cdc25C activity in the G2/M phase of the cell cycle and suggest a link between cell cycle and the carcinogenic effects of arsenite.

  • the chk1 protein kinase and the cdc25c regulatory pathways are targets of the anticancer agent ucn 01
    Journal of Biological Chemistry, 2000
    Co-Authors: Paul R Graves, Julie K Schwarz, Janis Gales, Edward A Sausville, Patrick M Oconnor, Helen Piwnicaworms
    Abstract:

    Abstract A checkpoint operating in the G2 phase of the cell cycle prevents entry into mitosis in the presence of DNA damage. UCN-01, a protein kinase inhibitor currently undergoing clinical trials for cancer treatment, abrogates G2 checkpoint function and sensitizes p53-defective cancer cells to DNA-damaging agents. In most species, the G2 checkpoint prevents the Cdc25 phosphatase from removing inhibitory phosphate groups from the mitosis-promoting kinase Cdc2. This is accomplished by maintaining Cdc25 in a phosphorylated form that binds 14-3-3 proteins. The checkpoint kinases, Chk1 and Cds1, are proposed to regulate the interactions between human Cdc25C and 14-3-3 proteins by phosphorylating Cdc25C on serine 216. 14-3-3 proteins, in turn, function to keep Cdc25C out of the nucleus. Here we report that UCN-01 caused loss of both serine 216 phosphorylation and 14-3-3 binding to Cdc25C in DNA-damaged cells. In addition, UCN-01 potently inhibited the ability of Chk1 to phosphorylate Cdc25C in vitro. In contrast, Cds1 was refractory to inhibition by UCN-01in vitro, and Cds1 was still phosphorylated in irradiated cells treated with UCN-01. Thus, neither Cds1 nor kinases upstream of Cds1, such as ataxia telangiectasia-mutated, are targets of UCN-01 action in vivo. Taken together our results identify the Chk1 kinase and the Cdc25C pathway as potential targets of G2 checkpoint abrogation by UCN-01.

Bernard Ducommun - One of the best experts on this subject based on the ideXlab platform.

  • development of novel thiazolopyrimidines as cdc25b phosphatase inhibitors
    ChemMedChem, 2009
    Co-Authors: Odile Mondesert, Marylorene Goddard, Stephanie Kolb, Bruno O Villoutreix, Denis Jullien, Bernard Ducommun
    Abstract:

    The development of CDC25 phosphatase inhibitors is an interesting approach toward new antitumor agents, as CDC25 play key roles in cell-cycle regulation and are overexpressed in numerous cancers. We previously reported a novel compound belonging to the thiazolopyrimidine family that inhibits CDC25 activity with an IC(50) value of 13 microM and displays cytotoxic properties against HeLa cells. Structural modifications were subsequently conducted on this new pharmacophore which led to a library of 45 thiazolopyrimidines. Regarding the in vitro effects, 14 compounds inhibit CDC25B with IC(50)<20 microM, with the most efficient inhibitor 44 improving the potency to 4.5 microM. Steady-state kinetics were performed and showed a mixed inhibition pattern for all tested compounds. Furthermore, 44 was able to revert the bypass of genotoxicity-induced G(2) arrest upon CDC25B overexpression, indicating that this compound targets the dual-specificity phosphatase in cultured cells. Finally, the cytotoxic activities of the compounds were determined against two human cancer cell lines. The results indicate that the prostatic LNCaP cell line is more sensitive to these derivatives than the pancreatic adenocarcinoma MiaPaCa-2 line. With its interesting enzymatic and cellular properties, compound 44 appears to be a promising CDC25B inhibitor for further development.

  • cell cycle control by the cdc25 phosphatases
    Anti-cancer Agents in Medicinal Chemistry, 2008
    Co-Authors: Bernadette Aressy, Bernard Ducommun
    Abstract:

    : Cell division cycle 25 (CDC25) phosphatases are key actors in eukaryotic cell cycle control. They are responsible for the dephosphorylations that activate the cyclin-dependent kinases (CDK) at specific stages of the cell cycle. Human CDC25A, CDC25B and CDC25C are also central targets and regulators of the G2/M checkpoint mechanisms activated in response to DNA injury. The expression and activity of these enzymes is finely regulated by multiple mechanisms including post-translational modifications, interactions with regulatory partners, control of their intracellular localization, and cell cycle-regulated degradation. Altered expression of these phosphatases is associated with checkpoint bypass and genetic instability. Accordingly, increased expression of CDC25A and CDC25B is found in many high-grade tumors and is correlated with poor prognosis in human cancers. This review summarizes our current knowledge within this domain and discusses the data that support therapeutic strategies targeting CDC25 activity in the treatment of cancer.

  • receptor based virtual ligand screening for the identification of novel cdc25 phosphatase inhibitors
    Journal of Chemical Information and Modeling, 2008
    Co-Authors: Matthieu Montes, Mariepriscille Brun, Odile Mondesert, Marylorene Goddard, Maria A Miteva, Stephanie Kolb, Emmanuelle Braud, Christiane Garbay, Bernard Ducommun, Bruno O Villoutreix
    Abstract:

    CDC25 phosphatases play critical roles in cell cycle regulation and are attractive targets for anticancer therapies. Several small non-peptide molecules are known to inhibit CDC25, but many of them appear to form a covalent bond with the enzyme or act through oxidation of the thiolate group of the catalytic cysteine. Structure-based virtual ligand screening computations were performed with FRED, Surflex, and LigandFit, a compound collection of over 310 000 druglike molecules and the crystal structure of CDC25B in order to identify novel classes of ligands. In vitro experiments carried out on a selected list of 1500 molecules led to the discovery of 99 compounds able to inhibit CDC25B activity at 100 μM. Further docking computations were applied, allowing us to propose a binding mode for the most potent molecule (IC50 = 13 μM). Our best compounds represent promising new classes of CDC25 inhibitors that also exhibit antiproliferative properties.

  • inhibition of human tumor cell growth in vivo by an orally bioavailable inhibitor of cdc25 phosphatases
    Molecular Cancer Therapeutics, 2005
    Co-Authors: Mariechristine Brezak, Odile Mondesert, Muriel Quaranta, Marieodile Contourgalcera, Olivier Lavergne, Philip G. Kasprzyk, P. Auvray, Gregoire Prevost, Bernard Ducommun
    Abstract:

    Cell cycle regulators, such as the CDC25 phosphatases, are potential targets for the development of new anticancer drugs. Here we report the identification and the characterization of BN82685, a quinone-based CDC25 inhibitor that is active in vitro and in vivo . BN82685 inhibits recombinant CDC25A, B, and C phosphatases in vitro . It inhibits the growth of human tumor cell lines with an IC50 in the submicromolar range, independently of their resistance to chemotherapeutic agents. This inhibitory effect is irreversible on both the purified CDC25 enzyme in vitro and on tumor cell proliferation. The specificity of BN82685 towards the CDC25 phosphatases is shown by an increase in cyclin-dependent kinase 1 tyrosine 15 phosphorylation, by the reversion of the mitosis-inducing effect of CDC25B overexpression in HeLa cells, and by the lack of a growth inhibitory effect in an assay based on the use of a CDC25-independent fission yeast model. Finally, when administered p.o., BN82685 is shown to inhibit the growth of the human pancreatic tumor Mia PaCa-2 xenografted in athymic nude mice. BN82685 is therefore a promising new compound targeting CDC25, which confirms the interest of the inhibition of these enzymes as an anticancer therapeutic strategy.

  • design synthesis and biological evaluation of novel naphthoquinone derivatives with cdc25 phosphatase inhibitory activity
    Bioorganic & Medicinal Chemistry, 2005
    Co-Authors: Mariepriscille Brun, Delphine Angotti, Odile Mondesert, Muriel Quaranta, Matthieu Montes, Nohad Gresh, Maria A Miteva, Emmanuelle Braud, Bernard Ducommun, Christiane Garbay
    Abstract:

    CDC25 dual-specificity phosphatases are essential key regulators of eukaryotic cell cycle progression and the CDC25A and B isoforms are over-expressed in different tumors and related cancer cell lines. CDC25s are now considered to be interesting targets in the search for novel anticancer agents. We describe new compounds derived from vitamin K3 that inhibit CDC25B activity with IC50 values in the low micromolar range. These naphthoquinone derivatives also display antiproliferative activity on HeLa cells as expected for CDC25 inhibitors and inhibit cell growth in a clonogenic assay at submicromolar concentrations. They increase inhibitory tyrosine 15 phosphorylation of CDK and induce the cleavage of PARP, a hallmark of apoptosis.