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Generation of new protein kinase inhibitors utilizing cytochrome p450 mutant enzymes for indigoid synthesis.
Guengerich FP, Sorrells JL, Schmitt S, Krauser JA, Aryal P, Meijer L
(2004) J Med Chem 47: 3236-41
MeSH Terms: Aryl Hydrocarbon Hydroxylases, CDC2 Protein Kinase, Chromatography, High Pressure Liquid, Cyclin-Dependent Kinase 5, Cyclin-Dependent Kinases, Cytochrome P-450 CYP2A6, Escherichia coli, Glycogen Synthase Kinase 3, Humans, Indoles, Mixed Function Oxygenases, Mutation, Protein Kinase Inhibitors
Show Abstract · Added March 5, 2014
Indigoids, a class of bis-indoles, represent a promising protein kinase inhibitor scaffold. Oxidation of indole by cytochrome P450 (P450) has been shown to generate species (indoxyl, isatin) that couple to yield indigo and indirubin. Escherichia coli-expressed human P450 2A6 mutants isolated from a randomized library were incubated with 27 substituted indole derivatives. Extracts of the cultures were screened for inhibition of human cyclin-dependent kinases (CDK)-1 and -5 and glycogen synthase kinase-3 (GSK3). The extracts from cultures incubated with 5-methoxyindole were the most inhibitory. High-performance liquid chromatography (HPLC) separation yielded a mixture of seven colored indigoids. These indigoids included indigo, indirubin, the di(5-methoxy) derivatives of indigo and indirubin, and both of the possible mono 5-methoxy derivatives of indirubin, which were all identified by visible, mass, and NMR spectra. Cultures with 5-methylindole added to the media also yielded inhibitory material, and 5- and 5'-methylindirubin were characterized. The most inhibitory of these indigoids were the monosubstituted indirubins and 5,5'-dimethoxyindirubin, which was > or =10x more active than indirubin. Thus, the overall approach involves the use of a library of randomized enzyme mutants to activate component moieties of a desired set of larger molecules, thus yielding a library of drug candidates that can be screened and characterized. The general strategy may have additional applications.
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13 MeSH Terms
Over-expression of cyclin D1 regulates Cdk4 protein synthesis.
Parker MA, Deane NG, Thompson EA, Whitehead RH, Mithani SK, Washington MK, Datta PK, Dixon DA, Beauchamp RD
(2003) Cell Prolif 36: 347-60
MeSH Terms: Animals, Cell Line, Transformed, Cyclin D1, Cyclin-Dependent Kinase 4, Cyclin-Dependent Kinases, Gene Expression Regulation, Enzymologic, Hepatocytes, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Proto-Oncogene Proteins, RNA, Messenger, Transduction, Genetic
Show Abstract · Added June 14, 2013
Increased Cdk4 expression occurs coincident with over-expression of cyclin D1 in many human tumours and tumourigenic mouse models. Here, we investigate both in vivo and in vitro the mechanism by which Cdk4 expression is regulated in the context of cyclin D1 over-expression. Cdk4 mRNA levels in cyclin D1-over-expressing tissue and cultured cells were unchanged compared with controls. In contrast, Cdk4 protein levels were increased in cyclin D1-over-expressing tissue and cells versus their respective controls. This increase was not due to altered protein stability, but appeared to be due to an increase in Cdk4 protein synthesis. We also performed immunoprecipitation and in vitro kinase assays to demonstrate an increase in cyclin D1-Cdk4 complex formation and associated kinase activity. Blocking cyclin D1 expression resulted in diminished Cdk4 protein but not mRNA levels. These findings suggest a mechanism by which Cdk4 expression is increased in the context of cyclin D1 over-expression during tumourigenesis.
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14 MeSH Terms
Genetic rescue of Cdk4 null mice restores pancreatic beta-cell proliferation but not homeostatic cell number.
Martín J, Hunt SL, Dubus P, Sotillo R, Néhmé-Pélluard F, Magnuson MA, Parlow AF, Malumbres M, Ortega S, Barbacid M
(2003) Oncogene 22: 5261-9
MeSH Terms: Animals, Body Constitution, Cell Division, Cyclin-Dependent Kinase 4, Cyclin-Dependent Kinases, Islets of Langerhans, Mice, Mutation, Phenotype, Proto-Oncogene Proteins
Show Abstract · Added February 23, 2011
Lack of Cdk4 expression in mice leads to insulin-deficient diabetes and female infertility owing to a reduced number of pancreatic beta cells and prolactin-producing pituitary lactotrophs, respectively. Cdk4 null mice display also reduced body and organ size. Here, we show that Cdk4 is essential for the postnatal proliferation of pancreatic beta cells but not for embryonic neogenesis from ductal epithelial cells. Re-expression of endogenous Cdk4 in beta cells and in the pituitary gland of Cdk4 null mice restores cell proliferation and results in fertile and normoglycemic animals, thus, demonstrating that the proliferation defects in these cellular populations are cell autonomous because of the lack of Cdk4 expression. However, these mice remain small in size, indicating that this phenotype is not because of pancreatic- or pituitary-mediated endocrine defects. This phenotype is a consequence of reduced cell numbers rather than reduced cell size. Thus, mammalian Cdk4 is not only involved in controlling proliferation of specific cell types but may play a wider role in establishing homeostatic cell numbers.
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10 MeSH Terms
An anillin homologue, Mid2p, acts during fission yeast cytokinesis to organize the septin ring and promote cell separation.
Tasto JJ, Morrell JL, Gould KL
(2003) J Cell Biol 160: 1093-103
MeSH Terms: Actins, Amino Acid Sequence, Calcium-Binding Proteins, Cell Division, Contractile Proteins, Cyclin-Dependent Kinases, DNA-Binding Proteins, Fungal Proteins, Gene Deletion, Gene Expression Regulation, Fungal, Genes, Fungal, Green Fluorescent Proteins, Humans, Intracellular Signaling Peptides and Proteins, Luminescent Proteins, Membrane Glycoproteins, Membrane Proteins, Molecular Sequence Data, Mutation, Protein Structure, Tertiary, Recombinant Fusion Proteins, Saccharomyces cerevisiae Proteins, Schizosaccharomyces, Sequence Homology, Amino Acid
Show Abstract · Added March 5, 2014
Anillin is a conserved protein required for cell division (Field, C.M., and B.M. Alberts. 1995. J. Cell Biol. 131:165-178; Oegema, K., M.S. Savoian, T.J. Mitchison, and C.M. Field. 2000. J. Cell Biol. 150:539-552). One fission yeast homologue of anillin, Mid1p, is necessary for the proper placement of the division site within the cell (Chang, F., A. Woollard, and P. Nurse. 1996. J. Cell Sci. 109(Pt 1):131-142; Sohrmann, M., C. Fankhauser, C. Brodbeck, and V. Simanis. 1996. Genes Dev. 10:2707-2719). Here, we identify and characterize a second fission yeast anillin homologue, Mid2p, which is not orthologous with Mid1p. Mid2p localizes as a single ring in the middle of the cell after anaphase in a septin- and actin-dependent manner and splits into two rings during septation. Mid2p colocalizes with septins, and mid2 Delta cells display disorganized, diffuse septin rings and a cell separation defect similar to septin deletion strains. mid2 gene expression and protein levels fluctuate during the cell cycle in a sep1- and Skp1/Cdc53/F-box (SCF)-dependent manner, respectively, implying that Mid2p activity must be carefully regulated. Overproduction of Mid2p depolarizes cell growth and affects the organization of both the septin and actin cytoskeletons. In the presence of a nondegradable Mid2p fragment, the septin ring is stabilized and cell cycle progression is delayed. These results suggest that Mid2p influences septin ring organization at the site of cell division and its turnover might normally be required to permit septin ring disassembly.
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24 MeSH Terms
Rapamycin potentiates transforming growth factor beta-induced growth arrest in nontransformed, oncogene-transformed, and human cancer cells.
Law BK, Chytil A, Dumont N, Hamilton EG, Waltner-Law ME, Aakre ME, Covington C, Moses HL
(2002) Mol Cell Biol 22: 8184-98
MeSH Terms: Animals, Antibiotics, Antineoplastic, CDC2-CDC28 Kinases, Carcinoma, Cell Cycle Proteins, Cell Division, Cell Line, Cell Transformation, Neoplastic, Cyclin-Dependent Kinase 2, Cyclin-Dependent Kinase Inhibitor p21, Cyclin-Dependent Kinase Inhibitor p27, Cyclin-Dependent Kinases, Cyclins, DNA-Binding Proteins, E2F4 Transcription Factor, Enzyme Inhibitors, Epithelial Cells, Genes, Reporter, Growth Inhibitors, Humans, Nuclear Proteins, Phosphoproteins, Protein Binding, Protein-Serine-Threonine Kinases, Proteins, Retinoblastoma Protein, Retinoblastoma-Like Protein p107, Retinoblastoma-Like Protein p130, Signal Transduction, Sirolimus, Tacrolimus Binding Proteins, Transcription Factors, Transforming Growth Factor beta, Tumor Suppressor Proteins
Show Abstract · Added February 17, 2014
Transforming growth factor beta (TGF-beta) induces cell cycle arrest of most nontransformed epithelial cell lines. In contrast, many human carcinomas are refractory to the growth-inhibitory effect of TGF-beta. TGF-beta overexpression inhibits tumorigenesis, and abolition of TGF-beta signaling accelerates tumorigenesis, suggesting that TGF-beta acts as a tumor suppressor in mouse models of cancer. A screen to identify agents that potentiate TGF-beta-induced growth arrest demonstrated that the potential anticancer agent rapamycin cooperated with TGF-beta to induce growth arrest in multiple cell lines. Rapamycin also augmented the ability of TGF-beta to inhibit the proliferation of E2F1-, c-Myc-, and (V12)H-Ras-transformed cells, even though these cells were insensitive to TGF-beta-mediated growth arrest in the absence of rapamycin. Rapamycin potentiation of TGF-beta-induced growth arrest could not be explained by increases in TGF-beta receptor levels or rapamycin-induced dissociation of FKBP12 from the TGF-beta type I receptor. Significantly, TGF-beta and rapamycin cooperated to induce growth inhibition of human carcinoma cells that are resistant to TGF-beta-induced growth arrest, and arrest correlated with a suppression of Cdk2 kinase activity. Inhibition of Cdk2 activity was associated with increased binding of p21 and p27 to Cdk2 and decreased phosphorylation of Cdk2 on Thr(160). Increased p21 and p27 binding to Cdk2 was accompanied by decreased p130, p107, and E2F4 binding to Cdk2. Together, these results indicate that rapamycin and TGF-beta cooperate to inhibit the proliferation of nontransformed cells and cancer cells by acting in concert to inhibit Cdk2 activity.
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34 MeSH Terms
PKB/Akt mediates cell-cycle progression by phosphorylation of p27(Kip1) at threonine 157 and modulation of its cellular localization.
Shin I, Yakes FM, Rojo F, Shin NY, Bakin AV, Baselga J, Arteaga CL
(2002) Nat Med 8: 1145-52
MeSH Terms: Breast Neoplasms, CDC2-CDC28 Kinases, Carcinoma, Cell Cycle, Cell Cycle Proteins, Cell Fractionation, Cell Line, Cell Nucleus, Cyclin E, Cyclin-Dependent Kinase 2, Cyclin-Dependent Kinase Inhibitor p27, Cyclin-Dependent Kinases, Enzyme Inhibitors, Female, Humans, MAP Kinase Kinase 1, Mitogen-Activated Protein Kinase Kinases, Phosphorylation, Protein-Serine-Threonine Kinases, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-akt, Recombinant Fusion Proteins, Threonine, Tumor Suppressor Proteins
Show Abstract · Added March 5, 2014
We have shown a novel mechanism of Akt-mediated regulation of the CDK inhibitor p27(kip1). Blockade of HER2/neu in tumor cells inhibits Akt kinase activity and upregulates nuclear levels of the CDK inhibitor (Kip1). Recombinant Akt and Akt precipitated from tumor cells phosphorylated wild-type p27 in vitro. p27 contains an Akt consensus RXRXXT(157)D within its nuclear localization motif. Active (myristoylated) Akt phosphorylated wild-type p27 in vivo but was unable to phosphorylate a T157A-p27 mutant. Wild-type p27 localized in the cytosol and nucleus, whereas T157A-p27 localized exclusively in the nucleus and was resistant to nuclear exclusion by Akt. T157A-p27 was more effective than wild-type p27 in inhibiting cyclin E/CDK2 activity and cell proliferation; these effects were not rescued by active Akt. Expression of Ser(473) phospho Akt in primary human breast cancers statistically correlated with expression of p27 in tumor cytosol. These data indicate that Akt may contribute to tumor-cell proliferation by phosphorylation and cytosolic retention of p27, thus relieving CDK2 from p27-induced inhibition.
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24 MeSH Terms
Cyclin D3 is essential for intestinal epithelial cell proliferation.
Ko TC, Pan F, Sheng H, Brown DB, Thompson EA, Beauchamp RD
(2002) World J Surg 26: 812-8
MeSH Terms: Animals, Cell Culture Techniques, Cyclin D3, Cyclin-Dependent Kinases, Cyclins, Food Deprivation, Immunoblotting, Interphase, Intestinal Mucosa, Male, Microinjections, Models, Animal, Rats, Rats, Sprague-Dawley
Show Abstract · Added May 3, 2013
Intestinal epithelium is a complex organ that undergoes continuous proliferation. D-type cyclins bind cyclin-dependent kinases (Cdk4 and Cdk6) and are expressed during the transition from G0 into the S phase. Previously, we reported that cyclins D1 and D3 are induced by growth factors in two rat intestinal epithelial cell lines, IEC-6 and RIE-1. However, transforming growth factor beta induces G1 arrest in both intestinal cell lines without inhibiting cyclin D3, suggesting that cyclin D3 may not have essential functions in the gut. In the present study, we determined whether cyclin D3 is required for the transition from G0 into the S phase in intestinal epithelial cells. Microinjection of anti-cyclin D3 antiserum inhibited quiescent IEC-6 and RIE-1 cells from entering the S phase, while cells microinjected with a nonspecific mouse immunoglobin G continued to progress into the S phase. We also examined the expression of cyclin D3 in rat jejunal mucosa after fasting and refeeding. Cyclin D3 levels were not altered by fasting and refeeding; however, Cdk4 expression was suppressed by fasting and returned to control levels after refeeding. Our results suggest that cyclin D3 is essential for intestinal epithelial cell proliferation, although its expression is not regulated by dietary restriction.
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14 MeSH Terms
ErbB2/Neu-induced, cyclin D1-dependent transformation is accelerated in p27-haploinsufficient mammary epithelial cells but impaired in p27-null cells.
Muraoka RS, Lenferink AE, Law B, Hamilton E, Brantley DM, Roebuck LR, Arteaga CL
(2002) Mol Cell Biol 22: 2204-19
MeSH Terms: Active Transport, Cell Nucleus, Amino Acid Sequence, Animals, Apoptosis, Base Sequence, Cell Cycle Proteins, Cell Division, Cell Nucleus, Cell Transformation, Neoplastic, Cyclin D1, Cyclin-Dependent Kinase 4, Cyclin-Dependent Kinase Inhibitor p27, Cyclin-Dependent Kinases, Flow Cytometry, Gene Deletion, Gene Dosage, Mammary Glands, Animal, Mammary Neoplasms, Experimental, Mice, Mice, Knockout, Molecular Sequence Data, Proto-Oncogene Proteins, RNA, Messenger, Receptor, ErbB-2, Tumor Cells, Cultured, Tumor Suppressor Proteins
Show Abstract · Added March 5, 2014
ErbB2/Neu destabilizes the cyclin-dependent kinase (Cdk) inhibitor p27 and increases expression of cyclin D1. Therefore, we studied the roles of p27 and cyclin D1 in ErbB2-mediated mammary epithelial cell transformation. Overexpression of ErbB2 or cyclin D1 in p27(+/-) primary murine mammary epithelial cells resulted in increased proliferation, cyclin D1 nuclear localization, and colony formation in soft agar compared to those in p27(+/+) cells. In contrast, ErbB2- or cyclin D1-overexpressing p27(-/-) cells displayed reduced proliferation, anchorage-independent growth, Cdk4 activity, cyclin D1 expression, and cyclin D1 nuclear localization compared to wild-type cells. A cyclin D1 mutation in its nuclear export sequence (T286A) partially rescued nuclear localization of cyclin D1 in p27(-/-) cells but did not increase proliferation or Cdk4 kinase activity. Overexpression of E2F1, however, increased proliferation to the same degree in p27(+/+), p27(+/-), and p27(-/-) cells. Mammary glands from MMTV (mouse mammary tumor virus)-neu/p27(+/-) mice exhibited alveolar hyperplasia, enhanced proliferation, decreased apoptosis, and accelerated tumor formation compared to MMTV-neu/p27(+/+) glands. However, MMTV-neu/p27(-/-) glands showed decreased proliferation, cyclin D1 expression, and Cdk4 activity, as well as markedly prolonged tumor latency, compared to MMTV-neu/p27(+/+) glands. These results suggest that p27(+/-) mammary epithelium may be more susceptible to oncogene-induced tumorigenesis, whereas p27-null glands, due to severely impaired cyclin D1/Cdk4 function, are more resistant to transformation.
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26 MeSH Terms
Mammary epithelial cell-cycle progression via the alpha(2)beta(1) integrin: unique and synergistic roles of the alpha(2) cytoplasmic domain.
Klekotka PA, Santoro SA, Ho A, Dowdy SF, Zutter MM
(2001) Am J Pathol 159: 983-92
MeSH Terms: Breast, CDC2-CDC28 Kinases, Cell Cycle, Collagen, Cyclin E, Cyclin-Dependent Kinase 2, Cyclin-Dependent Kinases, Cytoplasm, Drug Synergism, Epithelial Cells, Female, Humans, Integrins, Protein-Serine-Threonine Kinases, Receptor, Insulin, Receptors, Collagen, S Phase, Signal Transduction, Transfection
Show Abstract · Added February 16, 2014
The alpha(2)beta(1) integrin supports cell-cycle progression of mammary epithelial cells adherent to type I collagen matrices. Integrin collagen receptors containing the alpha(2) cytoplasmic domain stimulated expression of cyclin E and cyclin-dependent kinase (cdk)2, resulting in cyclin E/cdk2 activation in the absence of growth factors other than insulin. Integrin collagen receptors in which the alpha(2) cytoplasmic domain was replaced by the alpha(1) cytoplasmic domain or an alpha(2) subunit cytoplasmic domain truncated after the GFFKR sequence failed to stimulate cyclin E/cdk2 activation or entry into S phase in the absence of growth factors. Although overexpression of cyclins D or E or cdk2 in cells expressing the integrin collagen receptor with the alpha(1)-integrin cytoplasmic domain did not restore G(1) progression when mammary epithelial cells adhered to type I collagen, co-expression of cyclin E and cdk2 did rescue the ability of the transfectants to enter S phase. Activation of cyclin E/cdk2 complex by mammary epithelial cells required synergy between adhesion mediated by an integrin collagen receptor containing the alpha(2)-integrin subunit cytoplasmic domain and the insulin receptor.
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19 MeSH Terms
ErbB2/neu kinase modulates cellular p27(Kip1) and cyclin D1 through multiple signaling pathways.
Lenferink AE, Busse D, Flanagan WM, Yakes FM, Arteaga CL
(2001) Cancer Res 61: 6583-91
MeSH Terms: Breast Neoplasms, CDC2-CDC28 Kinases, Cell Cycle, Cell Cycle Proteins, Cell Division, Cyclin D1, Cyclin-Dependent Kinase 2, Cyclin-Dependent Kinase 4, Cyclin-Dependent Kinase Inhibitor p27, Cyclin-Dependent Kinases, Enzyme Activation, Enzyme Inhibitors, G1 Phase, Humans, MAP Kinase Signaling System, Mitogen-Activated Protein Kinases, Phosphatidylinositol 3-Kinases, Phosphorylation, Protein-Serine-Threonine Kinases, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-akt, Quinazolines, Receptor, ErbB-2, Signal Transduction, Transcription, Genetic, Transfection, Tumor Cells, Cultured, Tumor Suppressor Proteins, Tyrphostins, Up-Regulation
Show Abstract · Added March 5, 2014
It is well established that ErbB1 and ErbB2 can cooperate in mammary epithelial cell transformation. Therefore, to understand how ErbB1/ErbB2 signaling contributes to this process, we used the ErbB kinase inhibitor AG1478in ErbB2-dependent BT-474 and SKBR-3 human breast cancer cells. These cells overexpress ErbB2 and also display moderate levels of ErbB1. Treatment with AG1478 resulted in rapid ErbB2 dephosphorylation, reversible G(1) arrest, and interruption of constitutive mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling. Consequently, both MAPK-dependent transcription of cyclin D1 and phosphorylation of the cyclin-dependent kinase (Cdk) inhibitor p27 were inhibited. The inhibition of PI3K/Akt resulted in increased activity of glycogen synthase kinase-3beta, which phosphorylated cyclin D1, potentially reducing its steady-state levels. The loss of cyclin D1 reduced the amount of cyclin D1/Cdk4 complexes that can sequester p27 in the cytosol. This plus the reduced phosphorylation of p27 by MAPK enhanced the stability of p27 that associated with nuclear Cdk2 at high stoichiometry and inhibited its kinase activity. Antisense p27 oligonucleotides decreased p27 levels and abrogated the G(1) arrest induced by AG1478. Similarly, infection with an adenovirus encoding inducible cyclin D1 also counteracted the antiproliferative effect of AG1478. These data imply that: (a) modulation of both p27 and cyclin D1 are required for the growth arrest that results from blockade of the ErbB2 kinase; and (b) ErbB2 overexpressing cells use both MAPK and PI3K/Akt to modulate p27 and cyclin D1 and, hence, subvert the G(1)-to-S transition.
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30 MeSH Terms